SMART MODE:

NORMAL GENOMIC

• Buchwald G et al.
• Conformational switch and role of phosphorylation in PAK activation.
• Mol Cell Biol. 2001; 21: 5179-89
• Display abstract

• p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K(d) 0.6 microM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K(d) 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.

• Senadheera D, Haataja L, Groffen J, Heisterkamp N
• The small GTPase Rac interacts with ubiquitination complex proteins Cullin-1 and CDC23.
• Int J Mol Med. 2001; 8: 127-33
• Display abstract

• Racs are involved in the regulation of important cellular processes including mitogenesis. We found that the E3 ubiquitination ligase subunit Cullin-1 interacts with constitutively active Rac3 but not with wild-type Rac3 in yeast. In mammalian cell lysates, Cullin-1 bound to V12Rac3, effector domain mutants V12L37Rac3 and V12H40Rac3, and insert domain deletion mutant V12Rac3DeltaIns(124-135). Cullin-1 also formed a clearly detectable complex with other activated Rac3-related proteins including Rac1, Rac2, Cdc42 and RhoA but not with the distantly related small GTPase Rap1. Since the proteasome is involved in cell cycle control through the programmed degradation of cell cycle proteins, the possible regulation of Rac levels during the cell cycle was examined. However, Rac was expressed at constant levels throughout the cell cycle, and a specific proteasome inhibitor had no effect on Rac protein levels. These combined results indicate that the binding of activated Rac to Cullin-1 does not affect Rac protein levels, nor does it mediate the regulation of mitogenesis by Rac. However, Rac-Cullin-1 interactions may serve to regulate other E3 ligase functions such as subcellular localization. Indeed, activated Rac3 and Cullin-1 co-localized to the perinuclear region of the cell. We also detected complex formation between Rac and the APC component CDC23. These results indicate that Rac may regulate specific proteolytic processes through directed subcellular localization of SCF or APC complexes.

• Tan I, Seow KT, Lim L, Leung T
• Intermolecular and intramolecular interactions regulate catalytic activity of myotonic dystrophy kinase-related Cdc42-binding kinase alpha.
• Mol Cell Biol. 2001; 21: 2767-78
• Display abstract

• Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is a Cdc42-binding serine/threonine kinase with multiple functional domains. We had previously shown MRCKalpha to be implicated in Cdc42-mediated peripheral actin formation and neurite outgrowth in HeLa and PC12 cells, respectively. Here we demonstrate that native MRCK exists in high-molecular-weight complexes. We further show that the three independent coiled-coil (CC) domains and the N-terminal region preceding the kinase domain are responsible for intermolecular interactions leading to MRCKalpha multimerization. N terminus-mediated dimerization and consequent transautophosphorylation are critical processes regulating MRCKalpha catalytic activities. A region containing the two distal CC domains (CC2 and CC3; residues 658 to 930) was found to interact intramolecularly with the kinase domain and negatively regulates its activity. Its deletion also resulted in an active kinase, confirming a negative autoregulatory role. We provide evidence that the N terminus-mediated dimerization and activation of MRCK and the negative autoregulatory kinase-distal CC interaction are two mutually exclusive events that tightly regulate the catalytic state of the kinase. Disruption of this interaction by a mutant kinase domain resulted in increased kinase activity. MRCK kinase activity was also elevated when cells were treated with phorbol ester, which can interact directly with a cysteine-rich domain next to the distal CC domain. We therefore suggest that binding of phorbol ester to MRCK releases its autoinhibition, allowing N-terminal dimerization and subsequent kinase activation.

• Kuo CJ et al.
• Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain.
• J Cell Biol. 2001; 152: 1233-46
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• Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase-stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.

• Wendland J, Philippsen P
• Cell polarity and hyphal morphogenesis are controlled by multiple rho-protein modules in the filamentous ascomycete Ashbya gossypii.
• Genetics. 2001; 157: 601-10
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• Polarized cell growth requires a polarized organization of the actin cytoskeleton. Small GTP-binding proteins of the Rho-family have been shown to be involved in the regulation of actin polarization as well as other processes. Hyphal growth in filamentous fungi represents an ideal model to investigate mechanisms involved in generating cell polarity and establishing polarized cell growth. Since a potential role of Rho-proteins has not been studied so far in filamentous fungi we isolated and characterized the Ashbya gossypii homologs of the Saccharomyces cerevisiae CDC42, CDC24, RHO1, and RHO3 genes. The AgCDC42 and AgCDC24 genes can both complement conditional mutations in the S. cerevisiae CDC42 and CDC24 genes and both proteins are required for the establishment of actin polarization in A. gossypii germ cells. Agrho1 mutants show a cell lysis phenotype. Null mutant strains of Agrho3 show periodic swelling of hyphal tips that is overcome by repolarization and polar hyphal growth in a manner resembling the germination pattern of spores. Thus different Rho-protein modules are required for distinct steps during polarized hyphal growth of A. gossypii.

• Mosch HU, Kohler T, Braus GH
• Different domains of the essential GTPase Cdc42p required for growth and development of Saccharomyces cerevisiae.
• Mol Cell Biol. 2001; 21: 235-48
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• In budding yeast, the Rho-type GTPase Cdc42p is essential for cell division and regulates pseudohyphal development and invasive growth. Here, we isolated novel Cdc42p mutant proteins with single-amino-acid substitutions that are sufficient to uncouple functions of Cdc42p essential for cell division from regulatory functions required for pseudohyphal development and invasive growth. In haploid cells, Cdc42p is able to regulate invasive growth dependent on and independent of FLO11 gene expression. In diploid cells, Cdc42p regulates pseudohyphal development by controlling pseudohyphal cell (PH cell) morphogenesis and invasive growth. Several of the Cdc42p mutants isolated here block PH cell morphogenesis in response to nitrogen starvation without affecting morphology or polarity of yeast form cells in nutrient-rich conditions, indicating that these proteins are impaired for certain signaling functions. Interaction studies between development-specific Cdc42p mutants and known effector proteins indicate that in addition to the p21-activated (PAK)-like protein kinase Ste20p, the Cdc42p/Rac-interactive-binding domain containing Gic1p and Gic2p proteins and the PAK-like protein kinase Skm1p might be further effectors of Cdc42p that regulate pseudohyphal and invasive growth.

• Haddad E et al.
• The interaction between Cdc42 and WASP is required for SDF-1-induced T-lymphocyte chemotaxis.
• Blood. 2001; 97: 33-8
• Display abstract

• In studies aimed at further characterizing the cellular immunodeficiency of the Wiskott-Aldrich syndrome (WAS), we found that T lymphocytes from WAS patients display abnormal chemotaxis in response to the T-cell chemoattractant stromal cell-derived factor (SDF)-1. The Wiskott- Aldrich syndrome protein (WASP), together with the Rho family GTPase Cdc42, control stimulus-induced actin cytoskeleton rearrangements that are involved in cell motility. Because WASP is an effector of Cdc42, we further studied how Cdc42 and WASP are involved in SDF-1-induced chemotaxis of T lymphocytes. We provide here direct evidence that SDF-1 activates Cdc42. We then specifically investigated the role of the interaction between Cdc42 and WASP in SDF-1-responsive cells. This was achieved by abrogating this interaction with a recombinant polypeptide (TAT-CRIB), comprising the Cdc42/Rac interactive binding (CRIB) domain of WASP and a human immunodeficiency virus-TAT peptide that renders the fusion protein cell-permeant. This TAT-CRIB protein was shown to bind specifically to Cdc42-GTP and to inhibit the chemotactic response of a T-cell line to SDF-1. Altogether, these data demonstrate that Cdc42-WASP interaction is critical for SDF-1-induced chemotaxis of T cells.

• Gladfelter AS, Moskow JJ, Zyla TR, Lew DJ
• Isolation and characterization of effector-loop mutants of CDC42 in yeast.
• Mol Biol Cell. 2001; 12: 1239-55
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• The highly conserved small GTPase Cdc42p is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. Multiple effectors of Cdc42p have been identified, although it is unclear how their activities are coordinated to produce particular cell behaviors. One strategy used to address the contributions made by different effector pathways downstream of small GTPases has been the use of "effector-loop" mutants of the GTPase that selectively impair only a subset of effector pathways. We now report the generation and preliminary characterization of a set of effector-loop mutants of Saccharomyces cerevisiae CDC42. These mutants define genetically separable pathways influencing actin or septin organization. We have characterized the phenotypic defects of these mutants and the binding defects of the encoded proteins to known yeast Cdc42p effectors in vitro. The results suggest that these effectors cannot account for the observed phenotypes, and therefore that unknown effectors exist that affect both actin and septin organization. The availability of partial function alleles of CDC42 in a genetically tractable system serves as a useful starting point for genetic approaches to identify such novel effectors.

• Hong-Geller E, Holowka D, Siraganian RP, Baird B, Cerione RA
• Activated Cdc42/Rac reconstitutes Fcepsilon RI-mediated Ca2+ mobilization and degranulation in mutant RBL mast cells.
• Proc Natl Acad Sci U S A. 2001; 98: 1154-9
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• Antigen stimulation of mast cells via FcepsilonRI, the high-affinity receptor for IgE, triggers a signaling cascade that requires Ca(2+) mobilization for exocytosis of secretory granules during an allergic response. This study investigates critical signaling components by using mutant RBL mast cells that are defective in antigen-stimulated phospholipase Cgamma (PLCgamma) activation, as well as other signaling activities downstream of stimulated tyrosine phosphorylation. We show that the expression of activated versions of the Cdc42 or Rac1 GTPase restores antigen-stimulated Ca(2+) mobilization necessary for degranulation in these mutant cells. Wild-type Cdc42 and Rac1, as well as activated Cdc42 containing effector domain mutations, all fail to restore antigen-stimulated signaling leading to exocytosis. Expression of oncogenic Dbl, a guanine nucleotide exchange factor for Cdc42 and Rac1, partially restores sustained Ca(2+) mobilization and degranulation, suggesting that activation of endogenous Cdc42 and/or Rac1 is impaired in the mutant cells. Overexpression of PLCgamma1 with either activated Cdc42 or Rac1 synergistically stimulates degranulation, consistent with a critical defect in PLCgamma activation in these cells. Thus, our results point to activation of Cdc42 and/or Rac1 playing an essential role in antigen stimulation of early events that culminate in mast cell degranulation.

• Drees BL et al.
• A protein interaction map for cell polarity development.
• J Cell Biol. 2001; 154: 549-71
• Display abstract

• Many genes required for cell polarity development in budding yeast have been identified and arranged into a functional hierarchy. Core elements of the hierarchy are widely conserved, underlying cell polarity development in diverse eukaryotes. To enumerate more fully the protein-protein interactions that mediate cell polarity development, and to uncover novel mechanisms that coordinate the numerous events involved, we carried out a large-scale two-hybrid experiment. 68 Gal4 DNA binding domain fusions of yeast proteins associated with the actin cytoskeleton, septins, the secretory apparatus, and Rho-type GTPases were used to screen an array of yeast transformants that express approximately 90% of the predicted Saccharomyces cerevisiae open reading frames as Gal4 activation domain fusions. 191 protein-protein interactions were detected, of which 128 had not been described previously. 44 interactions implicated 20 previously uncharacterized proteins in cell polarity development. Further insights into possible roles of 13 of these proteins were revealed by their multiple two-hybrid interactions and by subcellular localization. Included in the interaction network were associations of Cdc42 and Rho1 pathways with proteins involved in exocytosis, septin organization, actin assembly, microtubule organization, autophagy, cytokinesis, and cell wall synthesis. Other interactions suggested direct connections between Rho1- and Cdc42-regulated pathways; the secretory apparatus and regulators of polarity establishment; actin assembly and the morphogenesis checkpoint; and the exocytic and endocytic machinery. In total, a network of interactions that provide an integrated response of signaling proteins, the cytoskeleton, and organelles to the spatial cues that direct polarity development was revealed.

• Hirsch DS, Pirone DM, Burbelo PD
• A new family of Cdc42 effector proteins, CEPs, function in fibroblast and epithelial cell shape changes.
• J Biol Chem. 2001; 276: 875-83
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• Cdc42, a Rho GTPase, regulates the organization of the actin cytoskeleton by its interaction with several distinct families of downstream effector proteins. Here, we report the identification of four new Cdc42-binding proteins that, along with MSE55, constitute a new family of effector proteins. These molecules, designated CEPs, contain three regions of homology, including a Cdc42 binding domain and two unique domains called CI and CII. Experimentally, we have verified that CEP2 and CEP5 bind Cdc42. Expression of CEP2, CEP3, CEP4, and CEP5 in NIH-3T3 fibroblasts induced pseudopodia formation. Fibroblasts coexpressing dominant negative Cdc42 with CEP2 or expressing a Cdc42/Rac interactive binding domain mutant of CEP2 did not induce pseudopodia formation. In primary keratinocytes, CEP2- and CEP5-expressing cells showed reduced F-actin localization at the adherens junctions with an increase in thin stress fibers that extended the length of the cell body. Keratinocytes expressing CEPs also showed an altered vinculin distribution and a loss of E-cadherin from adherens junctions. Similar effects were observed in keratinocytes expressing constitutively active Cdc42, but were not seen with a Cdc42/Rac interactive binding domain mutant of CEP2. These results suggest that CEPs act downstream of Cdc42 to induce actin filament assembly leading to cell shape changes.

• Kim S, Lee SH, Park D
• Leucine zipper-mediated homodimerization of the p21-activated kinase-interacting factor, beta Pix. Implication for a role in cytoskeletal reorganization.
• J Biol Chem. 2001; 276: 10581-4
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• Pix, a p21-activated kinase-interacting exchange factor, is known to be involved in the regulation of Cdc42/Rac GTPases. The 85-kDa betaPix-a protein contains an Src homology 3 domain, the tandem Dbl homology and Pleckstrin homology domains, a proline-rich region, and a GIT1-binding domain. In addition to those domains, betaPix-a also contains a putative leucine zipper domain at the C-terminal end. In this study, we demonstrate that the previously identified putative leucine zipper domain mediates the formation of betaPix-a homodimers. Using in vitro and in vivo methodologies, we show that deletion of the leucine zipper domain is sufficient to abolish betaPix-a homodimerization. In NIH3T3 fibroblast cells, expression of wild type betaPix-a induces the formation of membrane ruffles. However, cells expressing the leucine zipper domain deletion mutant could not form membrane ruffle structures. Moreover, platelet-derived growth factor-mediated cytoskeletal changes were completely blocked by the leucine zipper domain deletion mutant. The results suggest that the leucine zipper domain enables betaPix-a to homodimerize, and homodimerization is essential for betaPix-a signaling functions leading to the cytoskeletal reorganization.

• Terashima T et al.
• Expression of Rho-family GTPases (Rac, cdc42, RhoA) and their association with p-21 activated kinase in adult rat peripheral nerve.
• J Neurochem. 2001; 77: 986-93
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• To clarify the presence of the Rho family of small GTPases p21-activated kinase (pak) signaling pathway in the PNS, we have examined their expression, the association between the small GTPases and pak and the pak kinase activity in the PNS using immunoblot analysis, immunohistochemistry, co-immunoprecipitation study, and in vitro kinase assay. Immunoblot analysis showed the expression of Rac, cdc42, RhoA and pak in the dorsal root ganglion (DRG) and sciatic nerve. The localization of these proteins in the DRG neurons and axons and Schwann cells of the sciatic nerve was confirmed by immunohistochemistry. Co-immunoprecipitation studies indicated the in vivo associations of pak with Rac and cdc42, but not with RhoA, in both the DRG and sciatic nerve. The autophosphorylation of pak and phosphorylation of histone H4 by pak were also found in the DRG and sciatic nerve as well as in the CNS. These results suggest that the Rac/cdc42-pak signaling pathway exists and functions in the PNS and may mediate some intracellular signals.

• Hashimoto S, Tsubouchi A, Mazaki Y, Sabe H
• Interaction of paxillin with p21-activated Kinase (PAK). Association of paxillin alpha with the kinase-inactive and the Cdc42-activated forms of PAK3.
• J Biol Chem. 2001; 276: 6037-45
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• p21-activated kinases (PAKs) are implicated in integrin signalings, and have been proposed to associate with paxillin indirectly. We show here that paxillin can bind directly to PAK3. We examined several representative focal adhesion proteins, and found that paxillin is the sole protein that associates with PAK3. PAK3 associated with the alpha and beta isoforms of paxillin, but not with gamma. We also show that paxillin alpha associated with both the kinase-inactive and the Cdc42-activated forms of PAK3 in vivo, without affecting the activation states of the kinase. A number of different functions have been ascribed to PAKs; and PAKs can bind directly to growth factor signaling-adaptor molecule, Nck, and a guanine nucleotide exchanger, betaPIX. Our results revealed that paxillin alpha can compete with Nck and betaPIX in the binding of PAK3. Moreover, paxillin alpha can be phosphorylated by PAK3 at serine. Therefore, paxillin alpha, but not gamma, appears to be capable of linking both the kinase-inactive and activated forms of PAK3 to integrins independent of Nck and betaPIX, as Nck links PAK1 to growth factor receptors. Our results also revealed that paxillin is involved in highly complexed protein-protein interactions in integrin signaling.

• Pirone DM, Carter DE, Burbelo PD
• Evolutionary expansion of CRIB-containing Cdc42 effector proteins.
• Trends Genet. 2001; 17: 370-3
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• Cdc42, a small GTPase, regulates actin polymerization and other signaling pathways through interaction with many different downstream effector proteins. Most of these effector proteins contain a Cdc42-binding domain, called a CRIB domain. Here, we describe the evolutionary analysis of these CRIB-containing proteins in yeast, worms, flies and humans. The number of CRIB-containing effector proteins increases from yeast to humans, involving both an increase within families and the emergence of new families. These evolutionary changes correlate with the development of the more complex signaling pathways present in higher organisms.

• Boyce KJ, Hynes MJ, Andrianopoulos A
• The CDC42 homolog of the dimorphic fungus Penicillium marneffei is required for correct cell polarization during growth but not development.
• J Bacteriol. 2001; 183: 3447-57
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• The opportunistic human pathogenic fungus Penicillium marneffei is dimorphic and is thereby capable of growth either as filamentous multinucleate hyphae or as uninucleate yeast cells which divide by fission. The dimorphic switch is temperature dependent and requires regulated changes in morphology and cell shape. Cdc42p is a Rho family GTPase which in Saccharomyces cerevisiae is required for changes in polarized growth during mating and pseudohyphal development. Cdc42p homologs in higher organisms are also associated with changes in cell shape and polarity. We have cloned a highly conserved CDC42 homolog from P. marneffei named cflA. By the generation of dominant-negative and dominant-activated cflA transformants, we have shown that CflA initiates polarized growth and extension of the germ tube and subsequently maintains polarized growth in the vegetative mycelium. CflA is also required for polarization and determination of correct cell shape during yeast-like growth, and active CflA is required for the separation of yeast cells. However, correct cflA function is not required for dimorphic switching and does not appear to play a role during the generation of specialized structures during asexual development. In contrast, heterologous expression of cflA alleles in Aspergillus nidulans prevented conidiation.

• Ohtakara K et al.
• p21-activated kinase PAK phosphorylates desmin at sites different from those for Rho-associated kinase.
• Biochem Biophys Res Commun. 2000; 272: 712-6
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• p21-activated kinase (PAK) and Rho-associated kinase (Rho-kinase) have been shown to induce Ca(2+)-independent contraction of smooth muscle. PAK-induced contraction of Triton-skinned smooth muscle correlates with increased phosphorylation of caldesmon and desmin, although the role of desmin phosphorylation has remained obscure. Here we report that desmin serves as an excellent substrate for PAK in vitro. PAK phosphorylated desmin in a GTP. Cdc42/Rac-dependent manner. Phosphorylation of desmin by PAK dramatically inhibited its filament-forming ability. PAK phosphorylated mainly serine residues of the head domain of desmin, and the major phosphorylation sites differed from those for Rho-kinase. These results suggest that different site-specific phosphorylation of desmin via two divergent protein kinases downstream of Rho family GTPases would seem to increase the regulatory potential for organization of desmin filaments.

• Hoffman GR, Cerione RA
• Flipping the switch: the structural basis for signaling through the CRIB motif.
• Cell. 2000; 102: 403-6

• Symons M
• Adhesion signaling: PAK meets Rac on solid ground.
• Curr Biol. 2000; 10: 5357-5357
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• Interaction of cells with the extracellular matrix influences various aspects of cellular behavior. A recent study shows that cell-substrate adhesion is necessary for effective coupling of the small GTPase Rac to its effector PAK.

• Tanabe K et al.
• The small GTP-binding protein TC10 promotes nerve elongation in neuronal cells, and its expression is induced during nerve regeneration in rats.
• J Neurosci. 2000; 20: 4138-44
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• We have made a rat cDNA library using nerve-transected hypoglossal nuclei. Using this library, we performed expressed-sequence tag analysis coupled with in situ hybridization to identify genes whose expression is altered in response to nerve injury. In this gene screening, a member of Rho family GTPases, TC10, which had not yet been characterized in neuronal cells, was identified. TC10 mRNA expression was very low in normal motor neurons; however, axotomy induced its expression dramatically. Other family members such as RhoA, Rac1, and Cdc42 were moderately expressed in normal motor neurons and showed slight upregulation after axotomy. The expression level of TC10 mRNA was low in the embryonic brain and gradually increased with development. However, the expression of TC10 mRNA in the adult brain was lower and more restricted than that of RhoA, Rac1, and Cdc42. Cultured dorsal root ganglia exhibited dramatic neurite extension secondary to adenovirus-mediated expression of TC10. It can be concluded that although TC10 expression is lower in developing and mature motor neurons compared with other Rho family members, TC10 expression is induced by nerve injury to play a crucial role in nerve regeneration, particularly neurite elongation, in cooperation with other family members.

• Kozminski KG, Chen AJ, Rodal AA, Drubin DG
• Functions and functional domains of the GTPase Cdc42p.
• Mol Biol Cell. 2000; 11: 339-54
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• Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae. Cytological and genetic analyses of these cdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo. In addition, mapping the functional data for each cdc42 mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily. This region overlaps with a region (alpha5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions.

• Morreale A et al.
• Structure of Cdc42 bound to the GTPase binding domain of PAK.
• Nat Struct Biol. 2000; 7: 384-8
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• The Rho family GTPases, Cdc42, Rac and Rho, regulate signal transduction pathways via interactions with downstream effector proteins. We report here the solution structure of Cdc42 bound to the GTPase binding domain of alphaPAK, an effector of both Cdc42 and Rac. The structure is compared with those of Cdc42 bound to similar fragments of ACK and WASP, two effector proteins that bind only to Cdc42. The N-termini of all three effector fragments bind in an extended conformation to strand beta2 of Cdc42, and contact helices alpha1 and alpha5. The remaining residues bind to switches I and II of Cdc42, but in a significantly different manner. The structure, together with mutagenesis data, suggests reasons for the specificity of these interactions and provides insight into the mechanism of PAK activation.

• Longenecker KL et al.
• Structure of the BH domain from graf and its implications for Rho GTPase recognition.
• J Biol Chem. 2000; 275: 38605-10
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• Cellular signaling by small G-proteins is down-regulated by GTPase-activating proteins (GAPs), which increase the rate of GTP hydrolysis. The GTPase regulator associated with focal adhesion kinase (Graf) exhibits GAP activity toward the RhoA and Cdc42 GTPases, but is only weakly active toward the closely related Rac1. We determined the crystal structure of a 231-residue fragment of Graf (GrafGAP), a domain containing the GAP activity, at 2.4-A resolution. The structure clarifies the boundaries of the functional domain and yields insight to the mechanism of substrate recognition. Modeling its interaction with substrate suggested that a favorable interaction with Glu-95 of Cdc42 (Glu-97 of RhoA) would be absent with the corresponding Ala-95 of Rac1. Indeed, GrafGAP activity is diminished approximately 40-fold toward a Cdc42 E95A mutant, whereas a approximately 10-fold increase is observed for a Rac1 A95E mutant. The GrafGAP epitope that apparently interacts with Glu-95(Glu-97) contains Asn-225, which was recently found mutated in some myeloid leukemia patients. We conclude that position 95 of the GTPase is an important determinant for GrafGAP specificity in cellular function and tumor suppression.

• King CC, Reilly AM, Knaus UG
• Purification and in vitro activities of p21-activated kinases.
• Methods Enzymol. 2000; 325: 155-66

• Denker SP, Yan W, Barber DL
• Effect of Rho GTPases on Na-H exchanger in mammalian cells.
• Methods Enzymol. 2000; 325: 334-48

• Chamberlain CE, Kraynov VS, Hahn KM
• Imaging spatiotemporal dynamics of Rac activation in vivo with FLAIR.
• Methods Enzymol. 2000; 325: 389-400

• Vignal E et al.
• Characterization of TCL, a new GTPase of the rho family related to TC10 andCcdc42.
• J Biol Chem. 2000; 275: 36457-64
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• GTPases of the Rho family control a wide variety of cellular processes such as cell morphology, motility, proliferation, differentiation, and apoptosis. We report here the characterization of a new Rho member, which shares 85% and 78% amino acid similarity to TC10 and Cdc42, respectively. This GTPase, termed as TC10-like (TCL) is encoded by an unexpectedly large locus, made of five exons spanning over 85 kilobases on human chromosome 14. TCL mRNA is 2.5 kilobases long and is mainly expressed in heart. In vitro, TCL shows rapid GDP/GTP exchange and displays higher GTP dissociation and hydolysis rates than TC10. Using the yeast two-hybrid system and GST pull-down assays, we show that GTP-bound but not GDP-bound TCL protein directly interacts with Cdc42/Rac interacting binding domains, such as those found in PAK and WASP. Despite its overall similarity to TC10 and Cdc42, the constitutively active TCL mutant displays distinct morphogenic activity in REF-52 fibroblasts, producing large and dynamic F-actin-rich ruffles on the dorsal cell membrane. Interestingly, TCL morphogenic activity is blocked by dominant negative Rac1 and Cdc42 mutants, suggesting a cross-talk between these three Rho GTPases.

• Murata T, Goshima F, Daikoku T, Takakuwa H, Nishiyama Y
• Expression of herpes simplex virus type 2 US3 affects the Cdc42/Rac pathway and attenuates c-Jun N-terminal kinase activation.
• Genes Cells. 2000; 5: 1017-27
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• BACKGROUND: Although the US3 gene product of herpes simplex virus (HSV) has been identified as a serine/threonine protein kinase (PK), the functions are poorly understood. RESULTS: We found that US3 PK of HSV-2 induced disruption of actin filaments, cell rounding and accumulation of binucleate cells in HEp2 cells. Cell rounding was abrogated by expression of either kinase-dead forms of US3 PK or a mutant protein lacking the acidic cluster in the kinase regulatory domain. Co-expression of dominant active forms of Cdc42/Rac inhibited cell rounding, suggesting that a signal transduction pathway involving Cdc42/Rac may play a role in the morphological changes induced by the kinase. Cdc42 and Rac, members of the Rho family of small GTPases, function as molecular switches changing actin cytoskeletal organization, influencing transcription and controlling apoptotic cell death. By computed homology search, we noticed significant similarities between US3 PK and p21-activated kinase (PAK), which is activated by the Cdc42 or Rac. We also found that the expression of US3 suppressed the activation of c-Jun N-terminal kinase (JNK), a kinase that is downstream of PAK. CONCLUSIONS: These observations suggest that the US3 PK affects the Cdc42/Rac pathway and can act as an upstream suppressor of JNK in the stress-activated signalling pathway.

• Moskow JJ, Gladfelter AS, Lamson RE, Pryciak PM, Lew DJ
• Role of Cdc42p in pheromone-stimulated signal transduction in Saccharomyces cerevisiae.
• Mol Cell Biol. 2000; 20: 7559-71
• Display abstract

• CDC42 encodes a highly conserved GTPase of the Rho family that is best known for its role in regulating cell polarity and actin organization. In addition, various studies of both yeast and mammalian cells have suggested that Cdc42p, through its interaction with p21-activated kinases (PAKs), plays a role in signaling pathways that regulate target gene transcription. However, recent studies of the yeast pheromone response pathway suggested that prior results with temperature-sensitive cdc42 mutants were misleading and that Cdc42p and the Cdc42p-PAK interaction are not involved in signaling. To clarify this issue, we have identified and characterized novel viable pheromone-resistant cdc42 alleles that retain the ability to perform polarity-related functions. Mutation of the Cdc42p residue Val36 or Tyr40 caused defects in pheromone signaling and in the localization of the Ste20p PAK in vivo and affected binding to the Ste20p Cdc42p-Rac interactive binding (CRIB) domain in vitro. Epistasis analysis suggested that they affect the signaling step at which Ste20p acts, and overproduction of Ste20p rescued the defect. These results suggest that Cdc42p is in fact required for pheromone response and that interaction with the PAK Ste20p is critical for that role. Furthermore, the ste20DeltaCRIB allele, previously used to disrupt the Cdc42p-Ste20p interaction, behaved as an activated allele, largely bypassing the signaling defect of the cdc42 mutants. Additional observations lead us to suggest that Cdc42p collaborates with the SH3-domain protein Bem1p to facilitate signal transduction, possibly by providing a cell surface scaffold that aids in the local concentration of signaling kinases, thus promoting activation of a mitogen-activated protein kinase cascade by Ste20p.

• Richman TJ, Johnson DI
• Saccharomyces cerevisiae cdc42p GTPase is involved in preventing the recurrence of bud emergence during the cell cycle.
• Mol Cell Biol. 2000; 20: 8548-59
• Display abstract

• The Saccharomyces cerevisiae Cdc42p GTPase interacts with multiple regulators and downstream effectors through an approximately 25-amino-acid effector domain. Four effector domain mutations, Y32K, F37A, D38E, and Y40C, were introduced into Cdc42p and characterized for their effects on these interactions. Each mutant protein showed differential interactions with a number of downstream effectors and regulators and various levels of functionality. Specifically, Cdc42(D38E)p showed reduced interactions with the Cla4p p21-activated protein kinase and the Bem3p GTPase-activating protein and cdc42(D38E) was the only mutant allele able to complement the Deltacdc42 null mutant. However, the mutant protein was only partially functional, as indicated by a temperature-dependent multibudded phenotype seen in conjunction with defects in both septin ring localization and activation of the Swe1p-dependent morphogenetic checkpoint. Further analysis of this mutant suggested that the multiple buds emerged consecutively with a premature termination of bud enlargement preceding the appearance of the next bud. Cortical actin, the septin ring, Cla4p-green fluorescent protein (GFP), and GFP-Cdc24p all predominantly localized to one bud at a time per multibudded cell. These data suggest that Cdc42(D38E)p triggers a morphogenetic defect post-bud emergence, leading to cessation of bud growth and reorganization of the budding machinery to another random budding site, indicating that Cdc42p is involved in prevention of the initiation of supernumerary buds during the cell cycle.

• Royal I, Lamarche-Vane N, Lamorte L, Kaibuchi K, Park M
• Activation of cdc42, rac, PAK, and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation.
• Mol Biol Cell. 2000; 11: 1709-25
• Display abstract

• Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, processes that play crucial roles in tumor development, invasion, and metastasis. Little is known about the Met-dependent proximal signals that regulate these events. We show that HGF stimulation of epithelial cells leads to activation of the Rho GTPases, Cdc42 and Rac, concomitant with the formation of filopodia and lamellipodia. Notably, HGF-dependent activation of Rac but not Cdc42 is dependent on phosphatidylinositol 3-kinase. Moreover, HGF-induced lamellipodia formation and cell spreading require phosphatidylinositol 3-kinase and are inhibited by dominant negative Cdc42 or Rac. HGF induces activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase, and translocation of Rac, PAK, and Rho-dependent Rho-kinase to membrane ruffles. Use of dominant negative and activated mutants reveals an essential role for PAK but not Rho-kinase in HGF-induced epithelial cell spreading, whereas Rho-kinase activity is required for the formation of focal adhesions and stress fibers in response to HGF. We conclude that PAK and Rho-kinase play opposing roles in epithelial-mesenchymal transition induced by HGF, and provide new insight regarding the role of Cdc42 in these events.

• Qiu RG, Abo A, Steven Martin G
• A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation.
• Curr Biol. 2000; 10: 697-707
• Display abstract

• BACKGROUND: Rac and Cdc42 are members of the Rho family of small GTPases. They modulate cell growth and polarity, and contribute to oncogenic transformation by Ras. The molecular mechanisms underlying these functions remain elusive, however. RESULTS: We have identified a novel effector of Rac and Cdc42, hPar-6, which is the human homolog of a cell-polarity determinant in Caenorhabditis elegans. hPar-6 contains a PDZ domain and a Cdc42/Rac interactive binding (CRIB) motif, and interacts with Rac1 and Cdc42 in a GTP-dependent manner. hPar-6 also binds directly to an atypical protein kinase C isoform, PKCzeta, and forms a stable ternary complex with Rac1 or Cdc42 and PKCzeta. This association results in stimulation of PKCzeta kinase activity. Moreover, hPar-6 potentiates cell transformation by Rac1/Cdc42 and its interaction with Rac1/Cdc42 is essential for this effect. Cell transformation by hPar-6 involves a PKCzeta-dependent pathway distinct from the pathway mediated by Raf. CONCLUSIONS: These findings indicate that Rac/Cdc42 can regulate cell growth through Par-6 and PKCzeta, and suggest that deregulation of cell-polarity signaling can lead to cell transformation.

• Bi E, Chiavetta JB, Chen H, Chen GC, Chan CS, Pringle JR
• Identification of novel, evolutionarily conserved Cdc42p-interacting proteins and of redundant pathways linking Cdc24p and Cdc42p to actin polarization in yeast.
• Mol Biol Cell. 2000; 11: 773-93
• Display abstract

• In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p-Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures.

• Lei M et al.
• Structure of PAK1 in an autoinhibited conformation reveals a multistage activation switch.
• Cell. 2000; 102: 387-97
• Display abstract

• The p21-activated kinases (PAKs), stimulated by binding with GTP-liganded forms of Cdc42 or Rac, modulate cytoskeletal actin assembly and activate MAP-kinase pathways. The 2.3 A resolution crystal structure of a complex between the N-terminal autoregulatory fragment and the C-terminal kinase domain of PAK1 shows that GTPase binding will trigger a series of conformational changes, beginning with disruption of a PAK1 dimer and ending with rearrangement of the kinase active site into a catalytically competent state. An inhibitory switch (IS) domain, which overlaps the GTPase binding region of PAK1, positions a polypeptide segment across the kinase cleft. GTPase binding will refold part of the IS domain and unfold the rest. A related switch has been seen in the Wiskott-Aldrich syndrome protein (WASP).

• del Pozo MA, Price LS, Alderson NB, Ren XD, Schwartz MA
• Adhesion to the extracellular matrix regulates the coupling of the small GTPase Rac to its effector PAK.
• EMBO J. 2000; 19: 2008-14
• Display abstract

• The small GTPase Rac regulates cytoskeletal organization, cell cycle progression, gene expression and oncogenic transformation, processes that depend upon both soluble growth factors and adhesion to the extracellular matrix (ECM). We now show that growth factors and adhesion to the ECM both contribute independently and approximately equally to Rac activation. However, activated Rac in non-adherent cells failed to stimulate the Rac effector PAK. V12 Rac or Rac activated by serum translocated to the membrane fraction of adherent cells but remained mainly cytoplasmic in suspended cells. An activated Rac mutant lacking a membrane-targeting sequence did not activate PAK in adherent cells, while mutations that forced membrane targeting restored PAK activation in suspended cells. In vitro, V12 Rac showed greater binding to membranes from adherent relative to suspended cells, indicating that cell adhesion regulated membrane binding sites for Rac. These results show that ECM regulates the ability of Rac to couple with PAK via an effect on membrane binding sites that facilitate their interaction.

• Bock BC, Vacratsis PO, Qamirani E, Gallo KA
• Cdc42-induced activation of the mixed-lineage kinase SPRK in vivo. Requirement of the Cdc42/Rac interactive binding motif and changes in phosphorylation.
• J Biol Chem. 2000; 275: 14231-41
• Display abstract

• Src homology 3 domain (SH3)-containing proline-rich protein kinase (SPRK)/mixed-lineage kinase (MLK)-3 is a serine/threonine kinase that upon overexpression in mammalian cells activates the c-Jun NH(2)-terminal kinase pathway. The mechanisms by which SPRK activity is regulated are not well understood. The small Rho family GTPases, Rac and Cdc42, have been shown to bind and modulate the activities of signaling proteins, including SPRK, which contain Cdc42/Rac interactive binding motifs. Coexpression of SPRK and activated Cdc42 increases SPRKs activity. SPRKs Cdc42/Rac interactive binding-like motif contains six of the eight consensus residues. Using a site-directed mutagenesis approach, we show that SPRK contains a functional Cdc42/Rac interactive binding motif that is required for SPRKs association with and activation by Cdc42. However, experiments using a SPRK variant that lacks the COOH-terminal zipper region/basic stretch suggest that this region may also contribute to Cdc42 binding. Unlike the PAK family of protein kinases, we find that the activation of SPRK by Cdc42 cannot be recapitulated in an in vitro system using purified, recombinant proteins. Comparative phosphopeptide mapping demonstrates that coexpression of activated Cdc42 with SPRK alters the in vivo serine/threonine phosphorylation pattern of SPRK suggesting that the mechanism by which Cdc42 increases SPRKs catalytic activity involves a change in the in vivo phosphorylation of SPRK. This is, to the best of our knowledge, the first demonstrated example of a Cdc42-mediated change in the in vivo phosphorylation of a protein kinase. These studies suggest an additional component or cellular environment is required for SPRK activation by Cdc42.

• Lippincott J, Li R
• Involvement of PCH family proteins in cytokinesis and actin distribution.
• Microsc Res Tech. 2000; 49: 168-72
• Display abstract

• Pombe Cdc15 homology (PCH) proteins constitute an extensive protein family whose members have been found in diverse eukaryotic organisms. These proteins are characterized by the presence of several conserved sequence and structural motifs. Recent studies in yeast and mammalian cultured cells have implicated these proteins in actin-based processes, in particular, cytokinesis. Here we review the recent findings on the in vivo localization, function, and binding partners of PCH family members. We also provide new microscopy data regarding the in vivo dynamics of a budding yeast PCH protein involved in cytokinesis.

• Owen D, Mott HR, Laue ED, Lowe PN
• Residues in Cdc42 that specify binding to individual CRIB effector proteins.
• Biochemistry. 2000; 39: 1243-50
• Display abstract

• Cdc42 is a member of the Rho family of small G proteins. Signal transduction events emanating from Cdc42 lead to cytoskeletal rearrangements, cell proliferation, and cell differentiation. Many effector proteins have been identified for Cdc42; however, it is not clear how certain effectors specifically recognize and bind to Cdc42, as opposed to Rac or Rho, or in many cases, which effector controls what cellular events. Mutations were introduced into Cdc42 at residues: Met1, Val8, Phe28, Tyr32, Val33, Thr35, Val36, Phe37, Asp38, Tyr40, Val42, Met45, Ile46, Glu127, Ala130, Asn132, Gln134, Lys135, and Leu174. Measurements were made of their equilibrium binding constants to the Cdc42 binding domains of the CRIB effectors ACK, PAK, and WASP and to the GTPase-activating protein Rho GAP. Generally, mutations in the effector loop have an equally deleterious effect on binding to all CRIB proteins tested, though the F37A mutation resulted in significant selectivity. Residues outside the effector loop were found to be important for binding of Cdc42 to CRIB containing proteins and also to contribute to selectivity. Mutations such as V42A and L174A resulted in large, selective changes in binding to specific CRIB effectors. Neither mutation resulted in alteration in PAK binding, whereas both severely disrupt binding to ACK and only L174A disrupted binding to WASP. These mutations are interpreted using the structures of the Cdc42/ACK and Cdc42/WASP complexes to give insight into how effectors can specifically recognize Cdc42. Those mutations in Cdc42 that inhibit certain interactions, while retaining others, should aid investigations of the role of specific effectors in Cdc42 signaling in vivo.

• Zenke FT, King CC, Bohl BP, Bokoch GM
• Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity.
• J Biol Chem. 1999; 274: 32565-73
• Display abstract

• p21-activated kinases (Pak)/Ste20 kinases are regulated in vitro and in vivo by the small GTP-binding proteins Rac and Cdc42 and lipids, such as sphingosine, which stimulate autophosphorylation and phosphorylation of exogenous substrates. The mechanism of Pak activation by these agents remains unclear. We investigated Pak kinase activation in more detail to gain insight into the interplay between the GTPase/sphingosine binding, an intramolecular inhibitory interaction, and autophosphorylation. We present biochemical evidence that an autoinhibitory domain (ID) contained within amino acid residues 67-150 of Pak1 interacts with the carboxyl-terminal kinase domain and that this interaction is regulated in a GTPase-dependent fashion. Cdc42- and sphingosine-stimulated Pak1 activity can be inhibited in trans by recombinant ID peptide, indicating similarities in their mode of activation. However, Pak1, which was autophosphorylated in response to either GTPase or sphingosine, is highly active and is insensitive to inhibition by the ID peptide. We identified phospho-acceptor site threonine 423 in the kinase activation loop as a critical determinant for the sensitivity to autoinhibition and enzymatic activity. Phosphorylation studies suggested that the stimulatory effect of both GTPase and sphingosine results in exposure of the activation loop, making it accessible for intermolecular phosphorylation.

• Chang E et al.
• Direct binding and In vivo regulation of the fission yeast p21-activated kinase shk1 by the SH3 domain protein scd2.
• Mol Cell Biol. 1999; 19: 8066-74
• Display abstract

• The Ste20/p21-activated kinase homolog Shk1 is essential for viability and required for normal morphology, mating, and cell cycle control in the fission yeast Schizosaccharomyces pombe. Shk1 is regulated by the p21 G protein Cdc42, which has been shown to form a complex with the SH3 domain protein Scd2 (also called Ral3). In this study, we investigated whether Scd2 plays a role in regulating Shk1 function. We found that recombinant Scd2 and Shk1 interact directly in vitro and that they interact in vivo, as determined by the two-hybrid assay and genetic analyses in fission yeast. The second of two N-terminal SH3 domains of Scd2 is both necessary and sufficient for interaction with Shk1. While full-length Scd2 interacted with only the R1 N-terminal regulatory subdomain of Shk1, a C-terminal deletion mutant of Scd2 interacted with both the R1 and R3 subdomains of Shk1, suggesting that the non-SH3 C-terminal domain of Scd2 may be involved in defining specificity in SH3 binding domain recognition. Overexpression of Scd2 stimulated the autophosphorylation activity of wild-type Shk1 in fission yeast but, consistent with results of genetic analyses, did not stimulate the activity of a Shk1 protein lacking the R1 subdomain. Results of additional two-hybrid experiments suggest that Scd2 may stimulate Shk1 catalytic function, at least in part, by positively modulating protein-protein interaction between Cdc42 and Shk1. We propose that Scd2 functions as an organizing center, or scaffold, for the Cdc42 complex in fission yeast and that it acts in concert with Cdc42 to positively regulate Shk1 function.

• Li R, Debreceni B, Jia B, Gao Y, Tigyi G, Zheng Y
• Localization of the PAK1-, WASP-, and IQGAP1-specifying regions of Cdc42.
• J Biol Chem. 1999; 274: 29648-54
• Display abstract

• The Rho family small GTPase Cdc42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization. Potential effectors of Cdc42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1. To investigate the determinants of Cdc42-effector specificity, we utilized recombinant Cdc42 mutants and chimeras made between Cdc42 and RhoA to map the regions of Cdc42 contributing to specific effector p21-binding domain (PBD) interaction. Site-directed mutants of the switch I domain and neighboring regions of Cdc42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by Cdc42 involves a distinct mechanism. Differing from Rac1, the switch I domain and the surrounding region (amino acids 29 to 55) of Cdc42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157-191 and 84-120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique "insert region," residues 122-134, of Cdc42 to achieve high affinity binding. Microinjection of the constitutively active Cdc42/RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP-binding activity could retain the peripheral actin microspike (PAM)-inducing activity of Cdc42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by Cdc42 was insufficient for PAM-induction. Thus, Cdc42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.

• Tang Y, Yu J, Field J
• Signals from the Ras, Rac, and Rho GTPases converge on the Pak protein kinase in Rat-1 fibroblasts.
• Mol Cell Biol. 1999; 19: 1881-91
• Display abstract

• Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase), which, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak (p65(Pak)). Here we show that Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. We tested agents that activate or block specific components downstream of Ras and demonstrate a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggested that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins which fail to activate PI 3-kinase, did not activate Pak when tested alone but activated Pak when they were cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperated with Raf to activate Pak. A dominant negative Rho mutant also inhibited Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulated ERK. Cooperation was Pak dependent, since all combinations were inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho.

• Ho YD, Joyal JL, Li Z, Sacks DB
• IQGAP1 integrates Ca2+/calmodulin and Cdc42 signaling.
• J Biol Chem. 1999; 274: 464-70
• Display abstract

• Calmodulin regulates diverse Ca2+-dependent cellular processes, including cell cycle progression and cytoskeletal rearrangement. A recently identified calmodulin-binding protein, IQGAP1, interacts with both actin and Cdc42. In this study, evidence is presented that, in the absence of Ca2+, IQGAP1 bound to Cdc42, which maintained Cdc42 in the active GTP-bound state. Addition of Ca2+ both directly abrogated the effect of IQGAP1 on the intrinsic GTPase activity of Cdc42 and, in the presence of calmodulin, dissociated Cdc42 from IQGAP1. In addition, in vitro binding assays revealed that calmodulin associated with both the calponin homology domain and the IQ motifs of IQGAP1. Moreover, F-actin competed with Ca2+/calmodulin for binding to the calponin homology domain, but not the IQ motifs, of IQGAP1. Analysis of cell lysates revealed that calmodulin bound to IQGAP1 in a ternary complex with Cdc42. Increasing the Ca2+ concentration enhanced the interaction between calmodulin and IQGAP1, with a concomitant decrease in the association of IQGAP1 with Cdc42. Our data suggest that IQGAP1 functions as a scaffolding protein, providing a molecular link between Ca2+/calmodulin and Cdc42 signaling.

• Gatti A, Huang Z, Tuazon PT, Traugh JA
• Multisite autophosphorylation of p21-activated protein kinase gamma-PAK as a function of activation.
• J Biol Chem. 1999; 274: 8022-8
• Display abstract

• p21-activated protein kinase (PAK) is a family of serine/threonine kinases whose activity is stimulated by binding to small G-proteins such as Cdc42 and subsequent autophosphorylation. Focusing on the ubiquitous gamma-isoform of PAK in this study, baculovirus-infected insect cells were used to obtain recombinant gamma-PAK, while native gamma-PAK was isolated from rabbit reticulocytes. Two-dimensional gel electrophoresis of gamma-PAK followed by immunoblot analysis revealed a similar profile for native and recombinant gamma-PAK, both consisting of multiple protein spots. Following Cdc42-stimulated autophosphorylation, the two-dimensional profiles of native and recombinant gamma-PAK were characterized by a similar acidic shift, suggesting a common response to Cdc42. To understand the effect of differential phosphorylation on its activation status, gamma-PAK autophosphorylation was conducted in the presence or absence of activators such as Cdc42 and histone II-AS, followed by tryptic digestion and comparative two-dimensional phosphopeptide mapping. The major phosphopeptides were subjected to a combination of manual and automated amino acid sequencing. Overall, eight autophosphorylation sites were identified in Cdc42-activated gamma-PAK, six of which are in common with those previously reported in alpha-PAK, while Ser-19 and Ser-165 appear to be uniquely phosphorylated in the gamma-form. Further, the phosphorylation of Ser-141, Ser-165, and Thr-402 was found to correlate with gamma-PAK activation.

• Drgonova J, Drgon T, Roh DH, Cabib E
• The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell.
• J Cell Biol. 1999; 146: 373-87
• Display abstract

• Previous work showed that the GTP-binding protein Rho1p is required in the yeast, Saccharomyces cerevisiae, for activation of protein kinase C (Pkc1p) and for activity and regulation of beta(1-->3)glucan synthase. Here we demonstrate a hitherto unknown function of Rho1p required for cell cycle progression and cell polarization. Cells of mutant rho1(E45I) in the G1 stage of the cell cycle did not bud at 37 degrees C. In those cells actin reorganization and recruitment to the presumptive budding site did not take place at the nonpermissive temperature. Two mutants in adjacent amino acids, rho1(V43T) and rho1(F44Y), showed a similar behavior, although some budding and actin polarization occurred at the nonpermissive temperature. This was also the case for rho1(E45I) when placed in a different genetic background. Cdc42p and Spa2p, two proteins that normally also move to the bud site in a process independent from actin organization, failed to localize properly in rho1(E45I). Nuclear division did not occur in the mutant at 37 degrees C, although replication of DNA proceeded slowly. The rho1 mutants were also defective in the formation of mating projections and in congregation of actin at the projections in the presence of mating pheromone. The in vitro activity of beta(1-->3)glucan synthase in rho1 (E45I), although diminished at 37 degrees C, appeared sufficient for normal in vivo function and the budding defect was not suppressed by expression of a constitutively active allele of PKC1. Reciprocally, when Pkc1p function was eliminated by the use of a temperature-sensitive mutation and beta(1-->3)glucan synthesis abolished by an echinocandin-like inhibitor, a strain carrying a wild-type RHO1 allele was able to produce incipient buds. Taken together, these results reveal a novel function of Rho1p that must be executed in order for the yeast cell to polarize.

• Machesky LM, Insall RH
• Signaling to actin dynamics.
• J Cell Biol. 1999; 146: 267-72

• Abdul-Manan N et al.
• Structure of Cdc42 in complex with the GTPase-binding domain of the 'Wiskott-Aldrich syndrome' protein.
• Nature. 1999; 399: 379-83
• Display abstract

• The Rho-family GTP-hydrolysing proteins (GTPases), Cdc42, Rac and Rho, act as molecular switches in signalling pathways that regulate cytoskeletal architecture, gene expression and progression of the cell cycle. Cdc42 and Rac transmit many signals through GTP-dependent binding to effector proteins containing a Cdc42/Rac-interactive-binding (CRIB) motif. One such effector, the Wiskott-Aldrich syndrome protein (WASP), is postulated to link activation of Cdc42 directly to the rearrangement of actin. Human mutations in WASP cause severe defects in haematopoletic cell function, leading to clinical symptoms of thrombocytopenia, immunodeficiency and eczema. Here we report the solution structure of a complex between activated Cdc42 and a minimal GTPase-binding domain (GBD) from WASP. An extended amino-terminal GBD peptide that includes the CRIB motif contacts the switch I, beta2 and alpha5 regions of Cdc42. A carboxy-terminal beta-hairpin and alpha-helix pack against switch II. The Phe-X-His-X2-His portion of the CRIB motif and the alpha-helix appear to mediate sensitivity to the nucleotide switch through contacts to residues 36-40 of Cdc42. Discrimination between the Rho-family members is likely to be governed by GBD contacts to the switch I and alpha5 regions of the GTPases. Structural and biochemical data suggest that GBD-sequence divergence outside the CRIB motif may reflect additional regulatory interactions with functional domains that are specific to individual effectors.

• Mott HR et al.
• Structure of the small G protein Cdc42 bound to the GTPase-binding domain of ACK.
• Nature. 1999; 399: 384-8
• Display abstract

• The proteins Cdc42 and Rac are members of the Rho family of small GTPases (G proteins), which control signal-transduction pathways that lead to rearrangements of the cell cytoskeleton, cell differentiation and cell proliferation. They do so by binding to downstream effector proteins. Some of these, known as CRIB (for Cdc42/Rac interactive-binding) proteins, bind to both Cdc42 and Rac, such as the PAK1-3 serine/threonine kinases, whereas others are specific for Cdc42, such as the ACK tyrosine kinases and the Wiscott-Aldrich-syndrome proteins (WASPs). The effector loop of Cdc42 and Rac (comprising residues 30-40, also called switch I), is one of two regions which change conformation on exchange of GDP for GTP. This region is almost identical in Cdc42 and Racs, indicating that it does not determine the specificity of these G proteins. Here we report the solution structure of the complex of Cdc42 with the GTPase-binding domain ofACK. Both proteins undergo significant conformational changes on binding, to form a new type of G-protein/effector complex. The interaction extends the beta-sheet in Cdc42 by binding an extended strand from ACK, as seen in Ras/effector interactions, but it also involves other regions of the G protein that are important for determining the specificity of effector binding.

• Johnson DI
• Cdc42: An essential Rho-type GTPase controlling eukaryotic cell polarity.
• Microbiol Mol Biol Rev. 1999; 63: 54-105
• Display abstract

• Cdc42p is an essential GTPase that belongs to the Rho/Rac subfamily of Ras-like GTPases. These proteins act as molecular switches by responding to exogenous and/or endogenous signals and relaying those signals to activate downstream components of a biological pathway. The 11 current members of the Cdc42p family display between 75 and 100% amino acid identity and are functional as well as structural homologs. Cdc42p transduces signals to the actin cytoskeleton to initiate and maintain polarized gorwth and to mitogen-activated protein morphogenesis. In the budding yeast Saccharomyces cerevisiae, Cdc42p plays an important role in multiple actin-dependent morphogenetic events such as bud emergence, mating-projection formation, and pseudohyphal growth. In mammalian cells, Cdc42p regulates a variety of actin-dependent events and induces the JNK/SAPK protein kinase cascade, which leads to the activation of transcription factors within the nucleus. Cdc42p mediates these processes through interactions with a myriad of downstream effectors, whose number and regulation we are just starting to understand. In addition, Cdc42p has been implicated in a number of human diseases through interactions with its regulators and downstream effectors. While much is known about Cdc42p structure and functional interactions, little is known about the mechanism(s) by which it transduces signals within the cell. Future research should focus on this question as well as on the detailed analysis of the interactions of Cdc42p with its regulators and downstream effectors.

• Rinckel LA, Faris SL, Hitt ND, Kleinberg ME
• Rac1 disrupts p67phox/p40phox binding: a novel role for Rac in NADPH oxidase activation.
• Biochem Biophys Res Commun. 1999; 263: 118-22
• Display abstract

• Phagocytic cells possess a tightly regulated multicomponent enzyme complex, the NADPH oxidase, which produces superoxide, a reactive oxygen molecule that is an essential component of host defense against infection. Upon stimulation, a functional NADPH oxidase is assembled when the cytosolic proteins, Rac, p67phox, p47phox, and possibly p40phox, associate with the gp91phox and p22phox transmembrane proteins. Rac is a GTPase that in the GTP-bound state binds p67phox to activate NADPH oxidase. The function of p40phox is not known; it is believed to have a regulatory function in sequestering p67phox and p47phox in a cytosolic complex. We investigated binding interactions between p40phox, p67phox, and Rac and found that Rac1-GTP displaced p67phox bound to p40phox. In contrast, Cdc42, a GTPase homologous to Rac, did not displace p67phox from p40phox. A synthetic peptide corresponding to p67phox amino acids 170-199, a region identified previously as a Rac binding domain, significantly reduced the ability of Rac1-GTP to disrupt p67phox/p40phox binding. We hypothesize that Rac-GTP binds the p67phox N-terminal domain encompassing amino acids 170-199 that transmits a conformational change which causes p40phox to dissociate from its binding site in the p67phox C-terminus.

• Ridley A
• Rac and Rho.
• Curr Biol. 1999; 9: 156-156

• Kuroda S, Fukata M, Nakagawa M, Kaibuchi K
• Cdc42, Rac1, and their effector IQGAP1 as molecular switches for cadherin-mediated cell-cell adhesion.
• Biochem Biophys Res Commun. 1999; 262: 1-6
• Display abstract

• Cell-cell adhesion is a dynamic process in various cellular and developmental situations. Cadherins, well-known Ca(2+)-dependent adhesion molecules, are thought to play a major role in the regulation of cell-cell adhesion. However, the molecular mechanism underlying the rearrangement of cadherin-mediated cell-cell adhesion is largely unknown. Cdc42 and Rac1, belonging to the Rho small GTPase family, have recently been shown to be involved in the regulation of cell-cell adhesion. In addition, IQGAP1, an effector for Cdc42 and Rac1, has been shown to regulate the cadherin function through interaction with beta-catenin, a molecule associated with cadherin. In this review, we will summarize the mode of action of Cdc42 and Rac1 as well as IQGAP1 as molecular switches for the cadherin function, and then discuss physiological processes in which the Cdc42/Rac1/IQGAP1 system may be involved.

• Tu H, Wigler M
• Genetic evidence for Pak1 autoinhibition and its release by Cdc42.
• Mol Cell Biol. 1999; 19: 602-11
• Display abstract

• Pak1 protein kinase of Schizosaccharomyces pombe, a member of the p21-GTPase-activated protein kinase (PAK) family, participates in signaling pathways including sexual differentiation and morphogenesis. The regulatory domain of PAK proteins is thought to inhibit the kinase catalytic domain, as truncation of this region renders kinases more active. Here we report the detection in the two-hybrid system of the interaction between Pak1 regulatory domain and the kinase catalytic domain. Pak1 catalytic domain binds to the same highly conserved region on the regulatory domain that binds Cdc42, a GTPase protein capable of activating Pak1. Two-hybrid, mutant, and genetic analyses indicated that this intramolecular interaction rendered the kinase in a closed and inactive configuration. We show that Cdc42 can induce an open configuration of Pak1. We propose that Cdc42 interaction disrupts the intramolecular interactions of Pak1, thereby releasing the kinase from autoinhibition.

• Plemenitas A, Lu X, Geyer M, Veranic P, Simon MN, Peterlin BM
• Activation of Ste20 by Nef from human immunodeficiency virus induces cytoskeletal rearrangements and downstream effector functions in Saccharomyces cerevisiae.
• Virology. 1999; 258: 271-81
• Display abstract

• The negative factor (Nef) from human and simian immunodeficiency viruses is important for the pathogenesis of acquired immune deficiency syndrome. Among other targets, it activates the Nef-associated kinase, which is related to the p21-activated kinase. In this study, we demonstrate that Nef activates Ste20, the homolog of p21-activated kinase in Saccharomyces cerevisiae. Nef binds to the adaptor proteins Bem1 and Ste20 via its proline-rich (PXXP) and diarginine (RR) motifs, respectively. These interactions induce the mitogen-activated protein kinase and increase the rates of budding, sizes of cells, and patterns of mating projections. These effects of Nef depend on the small GTPase Cdc42 and guanine nucleotide exchange factor Cdc24. Thus, studies in S. cerevisiae identified specific interactions between Nef and cellular proteins and their associated signaling cascade.

• Donnenberg MS
• Salmonella strikes a balance.
• Nature. 1999; 401: 218-9

• Daniels RH, Bokoch GM
• p21-activated protein kinase: a crucial component of morphological signaling?
• Trends Biochem Sci. 1999; 24: 350-5
• Display abstract

• The mechanisms by which Rho family GTPases (Rho, Rac and Cdc42) regulate coordinated changes to the actin cytoskeleton are being elucidated. This review will focus on the current evidence that the p21-activated kinases (PAKs) are involved in regulating some of the diverse cytoskeletal changes induced by Rac and Cdc42. PAKs have been shown to be required for processes including neurite formation and axonal guidance, development of cell polarity and motile responses. Signaling molecules interacting with PAKs that might contribute to the regulation of such processes have recently been identified.

• Fu Y, Galan JE
• A salmonella protein antagonizes Rac-1 and Cdc42 to mediate host-cell recovery after bacterial invasion.
• Nature. 1999; 401: 293-7
• Display abstract

• An essential feature of the bacterial pathogen Salmonella spp. is its ability to enter cells that are normally non-phagocytic, such as those of the intestinal epithelium. The bacterium achieves entry by delivering effector proteins into the host-cell cytosol by means of a specialized protein-secretion system (termed type III), which causes reorganization of the cell's actin cytoskeleton and ruffling of its membrane. One of the bacterial effectors that stimulates these cellular responses is SopE, which acts as a guanyl-nucleotide-exchange factor on Rho GTPase proteins such as Cdc42 and Rac. As the actin-cytoskeleton reorganization induced by Salmonella is reversible and short-lived, infected cells regain their normal architecture after bacterial internalization. We show here that the S. Typhimurium effector protein SptP, which is delivered to the host-cell cytosol by the type-III secretion system, is directly responsible for the reversal of the actin cytoskeletal changes induced by the bacterium. SptP exerts this function by acting as a GTPase-activating protein (GAP) for Rac-1 and Cdc42.

• Lerm M, Selzer J, Hoffmeyer A, Rapp UR, Aktories K, Schmidt G
• Deamidation of Cdc42 and Rac by Escherichia coli cytotoxic necrotizing factor 1: activation of c-Jun N-terminal kinase in HeLa cells.
• Infect Immun. 1999; 67: 496-503
• Display abstract

• Recently, Escherichia coli cytotoxic necrotizing factor 1 (CNF1) was shown to activate the low-molecular-mass GTPase RhoA by deamidation of Gln63, thereby inhibiting intrinsic and GTPase-activating protein (GAP)-stimulated GTPase activities (G. Schmidt, P. Sehr, M. Wilm, J. Selzer, M. Mann, and K. Aktories, Nature 387:725-729, 1997; G. Flatau, E. Lemichez, M. Gauthier, P. Chardin, S. Paris, C. Fiorentini, and P. Boquet, Nature 387:729-733, 1997). Here we report that in addition to RhoA, Cdc42 and Rac also are targets for CNF1 in vitro and in intact cells. Treatment of HeLa cells with CNF1 induced a transient formation of microspikes and formation of membrane ruffles. CNF1 caused a transient 10- to 50-fold increase in the activity of the c-Jun N-terminal kinase. Tryptic peptides of Cdc42 obtained from CNF1-treated cells by immunoprecipitation exhibited an increase in mass of 1 Da compared to control peptides, indicating the deamidation of glutamine 61 by the toxin. The same increase in mass was observed with the respective peptides obtained from CNF1-modified recombinant Cdc42 and Rac1. Modification of recombinant Cdc42 and Rac1 by CNF1 inhibited intrinsic and GAP-stimulated GTPase activities and retarded binding of 2'(3')-O-(N-methylanthraniloyl)GDP. The data suggest that recombinant as well as cellular Cdc42 and Rac are substrates for CNF1.

• Burbelo PD, Snow DM, Bahou W, Spiegel S
• MSE55, a Cdc42 effector protein, induces long cellular extensions in fibroblasts.
• Proc Natl Acad Sci U S A. 1999; 96: 9083-8
• Display abstract

• Cdc42 is a member of the Rho GTPase family that regulates multiple cellular activities, including actin polymerization, kinase-signaling activation, and cell polarization. MSE55 is a nonkinase CRIB (Cdc42/Rac interactive-binding) domain-containing molecule of unknown function. Using glutathione S-transferase-capture experiments, we show that MSE55 binds to Cdc42 in a GTP-dependent manner. MSE55 binding to Cdc42 required an intact CRIB domain, because a MSE55 CRIB domain mutant no longer interacted with Cdc42. To study the function of MSE55 we transfected either wild-type MSE55 or a MSE55 CRIB mutant into mammalian cells. In Cos-7 cells, wild-type MSE55 localized at membrane ruffles and increased membrane actin polymerization, whereas expression of the MSE55 CRIB mutant showed fewer membrane ruffles. In contrast to these results, MSE55 induced the formation of long, actin-based protrusions in NIH 3T3 cells as detected by immunofluorescence and live-cell video microscopy. MSE55-induced protrusion formation was blocked by expression of dominant-negative N17Cdc42, but not by expression of dominant-negative N17Rac. These findings indicate that MSE55 is a Cdc42 effector protein that mediates actin cytoskeleton reorganization at the plasma membrane.

• Boettner B, Van Aelst L
• Rac and Cdc42 effectors.
• Prog Mol Subcell Biol. 1999; 22: 135-58

• Lu W, Mayer BJ
• Mechanism of activation of Pak1 kinase by membrane localization.
• Oncogene. 1999; 18: 797-806
• Display abstract

• Pak kinases are a family of serine/threonine protein kinases homologous to Ste20p of yeast. Paks can be activated in vivo and in vitro by binding to GTP-bound Cdc42 and Rac1, members of the Rho family of small GTPases implicated in regulating the organization of the actin cytoskeleton. We have previously reported that the SH2/SH3-containing adaptor protein Nck binds Pak kinase through its second SH3 domain. Pak1 can be targeted to the membrane by Nck in response to tyrosine phosphorylation, and membrane association of Pak1 is sufficient to increase its specific activity. The mechanism whereby Pak is activated by membrane localization, however, is unknown. We show here that expression of three proteins that inhibit Rho-family GTPases by different mechanisms (RhoGDI, Bcr and D57Y Cdc42) all block the activation of Pak by a membrane-targeted Nck SH3 domain, demonstrating that the in vivo activation of Pak1 induced by membrane localization is dependent on Rho-family GTPases. This implies that Pak activity can be regulated in cells both by the level of GTP loading of various Rho-family GTPases and the local concentration of Pak relative to these GTPases. Our data also suggest the existence of Rho-family GTPases in addition to Cdc42 and Rac1 that can activate Pak on membranes.

• Mosch HU, Kubler E, Krappmann S, Fink GR, Braus GH
• Crosstalk between the Ras2p-controlled mitogen-activated protein kinase and cAMP pathways during invasive growth of Saccharomyces cerevisiae.
• Mol Biol Cell. 1999; 10: 1325-35
• Display abstract

• The two highly conserved RAS genes of the budding yeast Saccharomyces cerevisiae are redundant for viability. Here we show that haploid invasive growth development depends on RAS2 but not RAS1. Ras1p is not sufficiently expressed to induce invasive growth. Ras2p activates invasive growth using either of two downstream signaling pathways, the filamentation MAPK (Cdc42p/Ste20p/MAPK) cascade or the cAMP-dependent protein kinase (Cyr1p/cAMP/PKA) pathway. This signal branch point can be uncoupled in cells expressing Ras2p mutant proteins that carry amino acid substitutions in the adenylyl cyclase interaction domain and therefore activate invasive growth solely dependent on the MAPK cascade. Both Ras2p-controlled signaling pathways stimulate expression of the filamentation response element-driven reporter gene depending on the transcription factors Ste12p and Tec1p, indicating a crosstalk between the MAPK and the cAMP signaling pathways in haploid cells during invasive growth.

• Bagrodia S, Cerione RA
• Pak to the future.
• Trends Cell Biol. 1999; 9: 350-5
• Display abstract

• Members of the Pak family of serine/threonine kinases serve as targets for the small GTP-binding proteins Cdc42 and Rac and have been implicated in a wide range of biological activities. Recently, some exciting developments help elaborate the regulation of Pak activity and identify downstream signalling targets. These include the discovery of the Cool/Pix and Cat proteins, which modulate Pak signalling, and downstream kinases that modulate the organization of the actin cytoskeleton or gene expression. We present these recent findings and consider how these new regulators and targets could explain some of the cellular effects that have been attributed to Pak family members.

• Stevens WK, Vranken W, Goudreau N, Xiang H, Xu P, Ni F
• Conformation of a Cdc42/Rac interactive binding peptide in complex with Cdc42 and analysis of the binding interface.
• Biochemistry. 1999; 38: 5968-75
• Display abstract

• Most of the putative effectors for the Rho-family small GTPases Cdc42 and Rac share a common sequence motif referred to as the Cdc42/Rac interactive binding (CRIB) motif. This sequence, with a consensus of I-S-x-P-(x)2-4-F-x-H-x-x-H-V-G [Burbelo, P. D., et al. (1995) J. Biol. Chem. 270, 29071-29074], has been shown to be essential for the functional interactions between these effector proteins and Cdc42. We have characterized the interactions of a 22-residue CRIB peptide derived from human PAK2 [PAK2(71-92)] with Cdc42 using proton and heteronuclear NMR spectroscopy. This CRIB peptide binds to GTP-gammaS-loaded Cdc42 in a saturable manner, with an apparent Kd of 0.6 &mgr;M, as determined by fluorescence titration using sNBD-labeled Cdc42. Interaction of the 22-residue peptide PAK2(71-92) with GTP-gammaS-loaded Cdc42 causes resonance perturbations in the 1H-15N HSQC spectrum of Cdc42 that are similar to those observed for a longer (46-amino acid) CRIB-containing protein fragment [Guo, W., et al. (1998) Biochemistry 37, 14030-14037]. Proton NMR studies of PAK2(71-92) demonstrate structuring of PAK2(71-92) in the presence of GTP-gammaS-loaded Cdc42, through the observation of many nonsequential transferred NOEs. Structure calculations based on the observed transferred NOEs show that the central portion of the Cdc42-bound CRIB peptide assumes a loop conformation in which the side chains of consensus residues Phe80, His82, Ile84, His85, and Val86 are brought into proximity. The CRIB motif may therefore represent a minimal interfacial region in the complexes between Cdc42 and its effector proteins.

• Joberty G, Perlungher RR, Macara IG
• The Borgs, a new family of Cdc42 and TC10 GTPase-interacting proteins.
• Mol Cell Biol. 1999; 19: 6585-97
• Display abstract

• The Rho family of GTPases plays key roles in the regulation of cell motility and morphogenesis. They also regulate protein kinase cascades, gene expression, and cell cycle progression. This multiplicity of roles requires that the Rho GTPases interact with a wide variety of downstream effector proteins. An understanding of their functions at a molecular level therefore requires the identification of the entire set of such effectors. Towards this end, we performed a two-hybrid screen using the TC10 GTPase as bait and identified a family of putative effector proteins related to MSE55, a murine stromal and epithelial cell protein of 55 kDa. We have named this family the Borg (binder of Rho GTPases) proteins. Complete open reading frames have been obtained for Borg1 through Borg3. We renamed MSE55 as Borg5. Borg1, Borg2, Borg4, and Borg5 bind both TC10 and Cdc42 in a GTP-dependent manner. Surprisingly, Borg3 bound only to Cdc42. An intact CRIB (Cdc42, Rac interactive binding) domain was required for binding. No interaction of the Borgs with Rac1 or RhoA was detectable. Three-hemagglutinin epitope (HA(3))-tagged Borg3 protein was mostly cytosolic when expressed ectopically in NIH 3T3 cells, with some accumulation in membrane ruffles. The phenotype induced by Borg3 was reminiscent of that caused by an inhibition of Rho function and was reversed by overexpression of Rho. Surprisingly, it was independent of the ability to bind Cdc42. Borg3 also inhibited Jun kinase activity by a mechanism that was independent of Cdc42 binding. HA(3)-Borg3 expression caused substantial delays in the spreading of cells on fibronectin surfaces after replating, and the spread cells lacked stress fibers. We propose that the Borg proteins function as negative regulators of Rho GTPase signaling.

• Richman TJ, Sawyer MM, Johnson DI
• The Cdc42p GTPase is involved in a G2/M morphogenetic checkpoint regulating the apical-isotropic switch and nuclear division in yeast.
• J Biol Chem. 1999; 274: 16861-70
• Display abstract

• The Cdc42p GTPase is involved in the signal transduction cascades controlling bud emergence and polarized cell growth in S. cerevisiae. Cells expressing the cdc42(V44A) effector domain mutant allele displayed morphological defects of highly elongated and multielongated budded cells indicative of a defect in the apical-isotropic switch in bud growth. In addition, these cells contained one, two, or multiple nuclei indicative of a G2/M delay in nuclear division and also a defect in cytokinesis and/or cell separation. Actin and chitin were delocalized, and septin ring structure was aberrant and partially delocalized to the tips of elongated cdc42(V44A) cells; however, Cdc42(V44A)p localization was normal. Two-hybrid protein analyses showed that the V44A mutation interfered with Cdc42p's interactions with Cla4p, a p21(Cdc42/Rac)-activated kinase (PAK)-like kinase, and the novel effectors Gic1p and Gic2p, but not with the Ste20p or Skm1p PAK-like kinases, the Bni1p formin, or the Iqg1p IQGAP homolog. Furthermore, the cdc42(V44A) morphological defects were suppressed by deletion of the Swe1p cyclin-dependent kinase inhibitory kinase and by overexpression of Cla4p, Ste20p, the Cdc12 septin protein, or the guanine nucleotide exchange factor Cdc24p. In sum, these results suggest that proper Cdc42p function is essential for timely progression through the apical-isotropic switch and G2/M transition and that Cdc42(V44A)p differentially interacts with a number of effectors and regulators.

• Cope MJ, Yang S, Shang C, Drubin DG
• Novel protein kinases Ark1p and Prk1p associate with and regulate the cortical actin cytoskeleton in budding yeast.
• J Cell Biol. 1999; 144: 1203-18
• Display abstract

• Ark1p (actin regulating kinase 1) was identified as a yeast protein that binds to Sla2p, an evolutionarily conserved cortical actin cytoskeleton protein. Ark1p and a second yeast protein, Prk1p, contain NH2-terminal kinase domains that are 70% identical. Together with six other putative kinases from a number of organisms, these proteins define a new protein kinase family that we have named the Ark family. Lack of both Ark1p and Prk1p resulted in the formation of large cytoplasmic actin clumps and severe defects in cell growth. These defects were rescued by wild-type, but not by kinase-dead versions of the proteins. Elevated levels of either Ark1p or Prk1p caused a number of actin and cell morphological defects that were not observed when the kinase-dead versions were overexpressed instead. Ark1p and Prk1p were shown to localize to actin cortical patches, making these two kinases the first signaling proteins demonstrated to be patch components. These results suggest that Ark1p and Prk1p may be downstream effectors of signaling pathways that control actin patch organization and function. Furthermore, results of double-mutant analyses suggest that Ark1p and Prk1p function in overlapping but distinct pathways that regulate the cortical actin cytoskeleton.

• Chen LM, Bagrodia S, Cerione RA, Galan JE
• Requirement of p21-activated kinase (PAK) for Salmonella typhimurium-induced nuclear responses.
• J Exp Med. 1999; 189: 1479-88
• Display abstract

• Salmonella typhimurium has sustained a long-standing association with its host and therefore has evolved sophisticated strategies to multiply and survive within this environment. Central to Salmonella pathogenesis is the function of a dedicated type III secretion system that delivers bacterial effector proteins into the host cell cytoplasm. These effectors stimulate nuclear responses and actin cytoskeleton reorganization leading to the production of proinflammatory cytokines and bacterial internalization. The stimulation of these responses requires the function of Cdc42, a member of the Rho family of small molecular weight GTPases, and SopE, a bacterial effector protein that stimulates guanine nucleotide exchange on Rho GTPases. However, nothing is known about the role of Cdc42 effector proteins in S. typhimurium-induced responses. We showed here that S. typhimurium infection of cultured epithelial cells results in the activation of p21-activated kinase (PAK), a serine/threonine kinase that is an effector of Cdc42-dependent responses. Transient expression of a kinase-defective PAK blocked both S. typhimurium- and SopE-induced c-Jun NH2-terminal kinase (JNK) activation but did not interfere with bacteria-induced actin cytoskeleton rearrangements. Similarly, expression of SH3-binding mutants of PAK did not block actin-mediated S. typhimurium entry into cultured cells. However, expression of an effector loop mutant of Cdc42Hs (Cdc42HsC40) unable to bind PAK and other CRIB (for Cdc42/Rac interacting binding)-containing target proteins resulted in abrogation of both S. typhimurium-induced nuclear and cytoskeletal responses. These results show that PAK kinase activity is required for bacteria-induced nuclear responses but it is not required for cytoskeletal rearrangements, indicating that S. typhimurium stimulates cellular responses through different Cdc42 downstream effector activities. In addition, these results demonstrate that the effector loop of Cdc42 implicated in the binding of PAK and other CRIB-containing target proteins is required for both responses.

• Redowicz MJ
• Rho-associated kinase: involvement in the cytoskeleton regulation.
• Arch Biochem Biophys. 1999; 364: 122-4

• Kyriakis JM
• Signaling by the germinal center kinase family of protein kinases.
• J Biol Chem. 1999; 274: 5259-62

• Holly SP, Blumer KJ
• PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae.
• J Cell Biol. 1999; 147: 845-56
• Display abstract

• During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.

• Kato M et al.
• Wiskott-Aldrich syndrome protein induces actin clustering without direct binding to Cdc42.
• J Biol Chem. 1999; 274: 27225-30
• Display abstract

• WASP (Wiskott-Aldrich syndrome protein) was identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome. WASP contains many functional domains and has been shown to induce the formation of clusters of actin filaments in a manner dependent on Cdc42. However, there has been no report investigating what domain(s) is(are) important for the function. Here we present for the first time the results of detailed analyses on the domain-function relationship of WASP. First, the C-terminal verprolin-cofilin-acidic domain was shown to be essential for the regulation of actin cytoskeleton. In addition, we found that the clustering of WASP itself is distinct from actin clustering. The partial protein containing the region from the N-terminal pleckstrin homology domain to the basic residue-rich region also clustered especially around the nucleus as wild type WASP without inducing actin clustering. Finally, we obtained the quite unexpected result that a WASP mutant deficient in binding to Cdc42 still induced actin cluster formation, indicating that direct interaction between Cdc42 and WASP is not required for the regulation of actin cytoskeleton. This result may explain why no Wiskott-Aldrich syndrome patients have been identified with a missense mutation in the Cdc42-binding site.

• Fackler OT, Luo W, Geyer M, Alberts AS, Peterlin BM
• Activation of Vav by Nef induces cytoskeletal rearrangements and downstream effector functions.
• Mol Cell. 1999; 3: 729-39
• Display abstract

• Nef of primate lentiviruses is critical for high levels of viremia and the progression to AIDS. Nef associates with and activates a serine/threonine kinase (Nef-associated kinase [NAK]) via the small GTPases Rac1 and Cdc42. We identified the protooncogene and guanine nucleotide exchange factor Vav as the specific binding partner of Nef proteins from HIV-1 and SIV. The interaction between Nef and Vav led to increased activity of Vav and its downstream effectors. Both cytoskeletal changes and the activation of c-Jun N-terminal kinase (JNK) were observed. Furthermore, a dominant-negative Vav protein inhibited NAK activation and viral replication. Thus, the interaction between Nef and Vav initiates a signaling cascade that changes structural and physiological parameters in the infected cell.

• Benard V, Bohl BP, Bokoch GM
• Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases.
• J Biol Chem. 1999; 274: 13198-204
• Display abstract

• A major function of Rac2 in neutrophils is the regulation of oxidant production important in bacterial killing. Rac and the related GTPase Cdc42 also regulate the dynamics of the actin cytoskeleton, necessary for leukocyte chemotaxis and phagocytosis of microorganisms. Although these GTPases appear to be critical downstream components of chemoattractant receptor signaling in human neutrophils, the pathways involved in direct control of Rac/Cdc42 activation remain to be determined. We describe an assay that measures the formation of Rac-GTP and Cdc42-GTP based on their specific binding to the p21-binding domain of p21-activated kinase 1. A p21-binding domain glutathione S-transferase fusion protein specifically binds Rac and Cdc42 in their GTP-bound forms both in vitro and in cell samples. Binding is selective for Rac and Cdc42 versus RhoA. Using this assay, we investigated Rac and Cdc42 activation in neutrophils and differentiated HL-60 cells. The chemoattractant fMet-Leu-Phe and the phorbol ester phorbol myristate acetate stimulate formation of Rac-GTP and Cdc42-GTP with distinct time courses that parallel cell activation. We also show that the signaling pathways leading to Rac and Cdc42 activation in HL-60 cells involve G proteins sensitive to pertussis toxin, as well as tyrosine kinase and phosphatidylinositol 3-kinase activities.

• Carlier MF, Ducruix A, Pantaloni D
• Signalling to actin: the Cdc42-N-WASP-Arp2/3 connection.
• Chem Biol. 1999; 6: 23540-23540
• Display abstract

• The molecular link between the signalling pathway regulating the formation of filopodia and the initiation of local actin polymerization has been elucidated: N-WASP, a close homologue of WASP, which is the product of the gene responsible for the Wiskott-Aldrich syndrome, mediates a direct connection between the small G-protein Cdc42 and the Arp2/3 complex.

• Hoshino M et al.
• Identification of the stef gene that encodes a novel guanine nucleotide exchange factor specific for Rac1.
• J Biol Chem. 1999; 274: 17837-44
• Display abstract

• The Rho family GTPases are involved in a variety of cellular events by changing the organization of actin cytoskeletal networks in response to extracellular signals. However, it is not clearly known how their activities are spatially and temporally regulated. Here we report the identification of a novel guanine nucleotide exchange factor for Rac1, STEF, which is related in overall amino acid sequence and modular structure to mouse Tiam1 and Drosophila SIF proteins. STEF protein contains two pleckstrin homology domains, a PDZ domain and a Dbl homology domain. The in vitro assay showed that STEF protein specifically enhanced the dissociation of GDP from Rac1 but not that from either RhoA or Cdc42. Expression of a truncated STEF protein in culture cells induced membrane ruffling with altered actin localization, which implies that this protein also activates Rac1 in vivo. The stef transcript was observed in restricted parts of mice, including cartilaginous tissues and the cortical plate of the central nervous system during embryogenesis. These findings suggested that STEF protein participates in the control of cellular events in several developing tissues, possibly changing the actin cytoskeletal network by activating Rac1.

• Brzeska H, Young R, Knaus U, Korn ED
• Myosin I heavy chain kinase: cloning of the full-length gene and acidic lipid-dependent activation by Rac and Cdc42.
• Proc Natl Acad Sci U S A. 1999; 96: 394-9
• Display abstract

• Acanthamoeba myosin I heavy chain kinase (MIHCK) phosphorylates the heavy chains of amoeba myosins I, increasing their actin-activated ATPase activities. The activity of MIHCK is increased by binding to acidic phospholipids or membranes and by autophosphorylation at multiple sites. Phosphorylation at a single site is necessary and sufficient for full activation of the expressed catalytic domain. The rate of autophosphorylation of native MIHCK is controlled by a region N-terminal to the catalytic domain. By its substrate specificity and the sequence of its C-terminal catalytic domain, MIHCK was identified as a p21-activated kinase (PAK). We have now cloned the full-length genomic DNA and cDNA of MIHCK and have shown it to contain the conserved p21-binding site common to many members of the PAK family. Like some mammalian PAKs, MIHCK is activated by Rac and Cdc42, and this activation is GTP-dependent and accompanied by autophosphorylation. In contrast to mammalian PAKs, activation of MIHCK by Rac and Cdc42 requires the presence of acidic lipids. Also unlike mammalian PAK, MIHCK is not activated by sphingosine or other non-negatively charged lipids. The acidic lipid-binding site is near the N terminus followed by the p21-binding region. The N-terminal regulatory domain of MIHCK contains alternating strongly positive and strongly negative regions. and the extremely Pro-rich middle region of MIHCK has a strongly acidic N-terminal segment and a strongly basic C-terminal segment. We propose that autophosphorylation activates MIHCK by neutralizing the basic segment of the Pro-rich region, thus unfolding the regulatory domain and abolishing its inhibition of the catalytic domain.

• Burridge K
• Crosstalk between Rac and Rho.
• Science. 1999; 283: 2028-9

• Fujita A et al.
• Hsl7p, a negative regulator of Ste20p protein kinase in the Saccharomyces cerevisiae filamentous growth-signaling pathway.
• Proc Natl Acad Sci U S A. 1999; 96: 8522-7
• Display abstract

• In the budding yeast, Saccharomyces cerevisiae, protein kinases Ste20p (p21(Cdc42p/Rac)-activated kinase), Ste11p [mitogen-activated protein kinase (MAPK) kinase kinase], Ste7p (MAPK kinase), Fus3p, and Kss1p (MAPKs) are utilized for haploid mating, invasive growth, and diploid filamentous growth. Members of the highly conserved Ste20p/p65(PAK) protein kinase family regulate MAPK signal transduction pathways from yeast to man. We describe here a potent negative regulator of Ste20p in the yeast filamentous growth-signaling pathway. We identified a mutant, hsl7, that exhibits filamentous growth on rich medium. Hsl7p belongs to a highly conserved protein family in eukaryotes. Hsl7p associates with the noncatalytic region within the amino-terminal half of Ste20p as well as Cdc42p. Deletions of HSL7 in haploid and diploid strains led to cell elongation and enhancement of both haploid invasive growth and diploid pseudohyphal growth. However, deletions of STE20 in haploid and diploid greatly diminished these hsl7-associated phenotypes. In addition, overexpression of HSL7 inhibited pseudohyphal growth. Thus, Hsl7p may inhibit the activity of Ste20p in the S. cerevisiae filamentous growth-signaling pathway. Our genetic analyses suggest the possibility that Cdc42p and Hsl7p compete for binding to Ste20p for pseudohyphal development when starved for nitrogen.

• Edwards DC, Sanders LC, Bokoch GM, Gill GN
• Activation of LIM-kinase by Pak1 couples Rac/Cdc42 GTPase signalling to actin cytoskeletal dynamics.
• Nat Cell Biol. 1999; 1: 253-9
• Display abstract

• Extracellular signals regulate actin dynamics through small GTPases of the Rho/Rac/Cdc42 (p21) family. Here we show that p21-activated kinase (Pak1) phosphorylates LIM-kinase at threonine residue 508 within LIM-kinase's activation loop, and increases LIM-kinase-mediated phosphorylation of the actin-regulatory protein cofilin tenfold in vitro. In vivo, activated Rac or Cdc42 increases association of Pak1 with LIM-kinase; this association requires structural determinants in both the amino-terminal regulatory and the carboxy-terminal catalytic domains of Pak1. A catalytically inactive LIM-kinase interferes with Rac-, Cdc42- and Pak1-dependent cytoskeletal changes. A Pak1-specific inhibitor, corresponding to the Pak1 autoinhibitory domain, blocks LIM-kinase-induced cytoskeletal changes. Activated GTPases can thus regulate actin depolymerization through Pak1 and LIM-kinase.

• Hing H, Xiao J, Harden N, Lim L, Zipursky SL
• Pak functions downstream of Dock to regulate photoreceptor axon guidance in Drosophila.
• Cell. 1999; 97: 853-63
• Display abstract

• The SH2/SH3 adaptor protein Dock has been proposed to transduce signals from guidance receptors to the actin cytoskeleton in Drosophila photoreceptor (R cell) growth cones. Here, we demonstrate that Drosophila p21-activated kinase (Pak) is required in a Dock pathway regulating R cell axon guidance and targeting. Dock and Pak colocalize to R cell axons and growth cones, physically interact, and their loss-of-function phenotypes are indistinguishable. Normal patterns of R cell connectivity require Pak's kinase activity and binding sites for both Dock and Cdc42/Rac. A membrane-tethered form of Pak (Pak(myr) acts as a dominant gain-of-function protein. Retinal expression of Pak(myr) rescues the R cell connectivity phenotype in dock mutants. These data establish Pak as a critical regulator of axon guidance and a downstream effector of Dock in vivo.

• van Leeuwen FN, van Delft S, Kain HE, van der Kammen RA, Collard JG
• Rac regulates phosphorylation of the myosin-II heavy chain, actinomyosin disassembly and cell spreading.
• Nat Cell Biol. 1999; 1: 242-8
• Display abstract

• GTPases of the Rho family regulate actinomyosin-based contraction in non-muscle cells. Activation of Rho increases contractility, leading to cell rounding and neurite retraction in neuronal cell lines. Activation of Rac promotes cell spreading and interferes with Rho-mediated cell rounding. Here we show that activation of Rac may antagonize Rho by regulating phosphorylation of the myosin-II heavy chain. Stimulation of PC12 cells or N1E-115 neuroblastoma cells with bradykinin induces phosphorylation of threonine residues in the myosin-II heavy chain; this phosphorylation is Ca2+ dependent and regulated by Rac. Both bradykinin-mediated and constitutive activation of Rac promote cell spreading, accompanied by a loss of cortical myosin II. Our results identify the myosin-II heavy chain as a new target of Rac-regulated kinase pathways, and implicate Rac as a Rho antagonist during myosin-II-dependent cell-shape changes.

• Nomanbhoy T, Cerione RA
• Fluorescence assays of Cdc42 interactions with target/effector proteins.
• Biochemistry. 1999; 38: 15878-84
• Display abstract

• The goal of these studies was to examine the interactions between the GTP-binding protein Cdc42 and its target/effectors by fluorescence spectroscopy. We have inserted fluorescent reporter groups at two distinct sites on Cdc42: N-methylanthraniloyl- (Mant-) derivatized nucleotides were complexed to the nucleotide-binding site of Cdc42, while a fluorescent succinimidyl ester was covalently attached to lysine 150. These two sites are separated by about 30 A on the Cdc42 molecule. Thus, the attachment of reporter groups to these sites enables the effects of target/effector binding to be viewed over a significant portion of the GTP-binding protein surface. We have taken advantage of fluorescence changes occurring at both sites to compare the interactions of activated Cdc42 with the limit binding domains from the following target/effectors: the serine/threonine kinase PAK, the tyrosine kinase ACK-2, and the RasGAP-related protein IQGAP. In addition, a unique lysine residue on the Cdc42-binding domain of ACK-2 (GBD-ACK) was covalently modified with a fluorescent succinimidyl ester. The distances separating this reactive lysine from the nucleotide binding site and lysine 150 of Cdc42 were determined by fluorescence resonance energy transfer and yielded a picture for Cdc42/GBD-ACK interactions that is consistent with recent NMR structural determinations for Cdc42/effector complexes. The changes in reporter group fluorescence at the reactive lysine of GBD-ACK, which were induced by the binding of activated Cdc42, were also examined. Overall, the results of these studies suggest not only that Cdc42 can induce conformational changes within an effector but also that in a reciprocal fashion the target/effectors induce conformational changes that span a significant distance on the GTP-binding protein.

• Chen XQ, Tan I, Leung T, Lim L
• The myotonic dystrophy kinase-related Cdc42-binding kinase is involved in the regulation of neurite outgrowth in PC12 cells.
• J Biol Chem. 1999; 274: 19901-5
• Display abstract

• The myotonic dystrophy kinase-related Cdc42-binding kinase (MRCKalpha) has been implicated in the morphological activities of Cdc42 in nonneural cells. Both MRCKalpha and the kinase-related Rho-binding kinase (ROKalpha) are involved in nonmuscle myosin light-chain phosphorylation and associated actin cytoskeleton reorganization. We now show that in PC12 cells, overexpression of the kinase domain of MRCKalpha and ROKalpha resulted in retraction of neurites formed on nerve growth factor (NGF) treatment, as observed with RhoA. However, introduction of kinase-dead MRCKalpha did not result in NGF-independent neurite outgrowth as observed with dominant negative kinase-dead ROKalpha or the Rho inhibitor C3. Neurite outgrowth induced by NGF or kinase-dead ROKalpha was inhibited by dominant negative Cdc42(N17), Rac1(N17), and the Src homology 3 domain of c-Crk, indicating the participation of common downstream components. Neurite outgrowth induced by either agent was blocked by kinase-dead MRCKalpha lacking the p21-binding domain or by a minimal C-terminal regulatory region consisting of the cysteine-rich domain/pleckstrin homology domain plus a region with homology to citron. The latter region alone was an effective blocker of NGF-induced outgrowth. These results suggest that although ROKalpha is involved in neurite retraction promoted by RhoA, the related MRCKalpha is conversely involved in neurite outgrowth promoted by Cdc42 and Rac.

• Li S et al.
• Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress.
• J Clin Invest. 1999; 103: 1141-50
• Display abstract

• Shear stress, the tangential component of hemodynamic forces, plays an important role in endothelial remodeling. In this study, we investigated the role of Rho family GTPases Cdc42 and Rho in shear stress-induced signal transduction and cytoskeleton reorganization. Our results showed that shear stress induced the translocation of Cdc42 and Rho from cytosol to membrane. Although both Cdc42 and Rho were involved in the shear stress-induced transcription factor AP-1 acting on the 12-O-tetradecanoyl-13-phorbol-acetate-responsive element (TRE), only Cdc42 was sufficient to activate AP-1/TRE. Dominant-negative mutants of Cdc42 and Rho, as well as recombinant C3 exoenzyme, attenuated the shear stress activation of c-Jun NH2-terminal kinases (JNKs), suggesting that Cdc42 and Rho regulate the shear stress induction of AP-1/TRE activity through JNKs. Shear stress-induced cell alignment and stress fiber formation were inhibited by the dominant-negative mutants of Rho and p160ROCK, but not by the dominant-negative mutant of Cdc42, indicating that the Rho-p160ROCK pathway regulates the cytoskeletal reorganization in response to shear stress.

• Lu Y, Settleman J
• The Drosophila Pkn protein kinase is a Rho/Rac effector target required for dorsal closure during embryogenesis.
• Genes Dev. 1999; 13: 1168-80
• Display abstract

• The PKN family of PKC-related protein kinases constitutes the major Rho GTPase-associated protein kinase activities detected in mammalian tissues. However, the biological functions of these kinases are unknown. We have identified a closely related PKN homolog in Drosophila (Pkn) that binds specifically to GTP-activated Rho1 and Rac1 GTPases through distinct binding sites on Pkn. The interaction of Pkn with either of these GTPases results in increased kinase activity, suggesting that Pkn is a shared Rho/Rac effector target. Characterization of a loss-of-function mutant of Drosophila Pkn revealed that this kinase is required specifically for the epidermal cell shape changes during the morphogenetic process of dorsal closure of the developing embryo. Moreover, Pkn, as well as the Rho1 GTPase, mediate a pathway for cell shape changes in dorsal closure that is independent of the previously reported Rac GTPase-mediated Jun amino (N)-terminal kinase (JNK) cascade that regulates gene expression required for dorsal closure. Thus, it appears that distinct but coordinated Rho- and Rac-mediated signaling pathways regulate the cell shape changes required for dorsal closure and that Pkn provides a GTPase effector function for cell shape changes in vivo, which acts together with a Rac-JNK transcriptional pathway in the morphogenesis of the Drosophila embryo.

• Sanders LC, Matsumura F, Bokoch GM, de Lanerolle P
• Inhibition of myosin light chain kinase by p21-activated kinase.
• Science. 1999; 283: 2083-5
• Display abstract

• p21-activated kinases (PAKs) are implicated in the cytoskeletal changes induced by the Rho family of guanosine triphosphatases. Cytoskeletal dynamics are primarily modulated by interactions of actin and myosin II that are regulated by myosin light chain kinase (MLCK)-mediated phosphorylation of the regulatory myosin light chain (MLC). p21-activated kinase 1 (PAK1) phosphorylates MLCK, resulting in decreased MLCK activity. MLCK activity and MLC phosphorylation were decreased, and cell spreading was inhibited in baby hamster kidney-21 and HeLa cells expressing constitutively active PAK1. These data indicate that MLCK is a target for PAKs and that PAKs may regulate cytoskeletal dynamics by decreasing MLCK activity and MLC phosphorylation.

• Leung T, Chen XQ, Tan I, Manser E, Lim L
• Myotonic dystrophy kinase-related Cdc42-binding kinase acts as a Cdc42 effector in promoting cytoskeletal reorganization.
• Mol Cell Biol. 1998; 18: 130-40
• Display abstract

• The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These approximately 190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C and n-chimaerin and a putative pleckstrin homology domain. MRCK alpha and Cdc42V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCK alpha resulted in actin and myosin reorganization. Expression of kinase-dead MRCK alpha blocked Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCK alpha mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCK alpha plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCK alpha mutants. These results suggest that MRCK alpha may act as a downstream effector of Cdc42 in cytoskeletal reorganization.

• Li E, Stupack D, Bokoch GM, Nemerow GR
• Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases.
• J Virol. 1998; 72: 8806-12
• Display abstract

• Adenovirus (Ad) endocytosis via alphav integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K). Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton. Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia). We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis. Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein. Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation. Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.

• Zhou K, Wang Y, Gorski JL, Nomura N, Collard J, Bokoch GM
• Guanine nucleotide exchange factors regulate specificity of downstream signaling from Rac and Cdc42.
• J Biol Chem. 1998; 273: 16782-6
• Display abstract

• The Rac and Cdc42 GTPases regulate diverse cellular behaviors involving the actin cytoskeleton, gene transcription, and the activity of multiple protein and lipid kinases. All of these pathways can potentially become activated when GTP-Rac or GTP-Cdc42 is formed in response to external cell signals, yet it is evident that each activity must also be able to be controlled individually. The mechanisms by which such specificity of GTPase signaling in response to upstream stimuli is achieved remains unclear. We investigated the action of several well characterized guanine nucleotide exchange factors (GEFRho) to activate Rac- and/or Cdc42-dependent kinase pathways. Coexpression studies in COS-7 cells revealed that the ability of individual guanine nucleotide exchange factors (GEFs) to activate the p21-activated kinase PAK1 could be dissociated from activation of c-Jun amino-terminal kinase, even though activation of both pathways requires the action of the GEFs on Rac and/or Cdc42. In contrast, expression of constitutively active forms of Rac or Cdc42 effectively stimulated both downstream kinases. We conclude that GEFs can be important determinants of downstream signaling specificity for members of the Rho GTPase family.

• Abo A et al.
• PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia.
• EMBO J. 1998; 17: 6527-40
• Display abstract

• The GTPases Rac and Cdc42Hs control diverse cellular functions. In addition to being mediators of intracellular signaling cascades, they have important roles in cell morphogenesis and mitogenesis. We have identified a novel PAK-related kinase, PAK4, as a new effector molecule for Cdc42Hs. PAK4 interacts only with the activated form of Cdc42Hs through its GTPase-binding domain (GBD). Co-expression of PAK4 and the constitutively active Cdc42HsV12 causes the redistribution of PAK4 to the brefeldin A-sensitive compartment of the Golgi membrane and the subsequent induction of filopodia and actin polymerization. Importantly, the reorganization of the actin cytoskeleton is dependent on PAK4 kinase activity and on its interaction with Cdc42Hs. Thus, unlike other members of the PAK family, PAK4 provides a novel link between Cdc42Hs and the actin cytoskeleton. The cellular locations of PAK4 and Cdc42Hs suggest a role for the Golgi in cell morphogenesis.

• Bagrodia S, Taylor SJ, Jordon KA, Van Aelst L, Cerione RA
• A novel regulator of p21-activated kinases.
• J Biol Chem. 1998; 273: 23633-6
• Display abstract

• Proteins of the p21-activated kinase (Pak) family have been implicated in the regulation of gene expression, cytoskeletal architecture, and apoptosis. Although the ability of Cdc42 and Rac GTPases to activate Pak is well established, relatively little else is known about Pak regulation or the identity of Pak cellular targets. Here we report the identification of two closely related Pak3-binding proteins, possibly arising from alternative splicing, designated p50 and p85(Cool-1) (cloned out of library). Both isoforms of Cool contain a Src homology 3 domain that directly mediates interaction with Pak3 and tandem Dbl homology and pleckstrin homology domains. Despite the presence of the Dbl homology-pleckstrin homology motif, a characteristic of Rho family activators, activation of Cdc42 or Rac by Cool is not detectable. Instead binding of p50(Cool-1), but not p85(Cool-1), to Pak3 represses its activation by upstream activators such as the Dbl oncoprotein, indicating a novel mechanism of regulation of Pak signaling.

• Nagata Ki et al.
• The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3.
• EMBO J. 1998; 17: 149-58
• Display abstract

• The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.

• Bellanger JM et al.
• [Role of the multifunctional Trio protein in the control of the Rac1 and RhoA gtpase signaling pathways]
• C R Seances Soc Biol Fil. 1998; 192: 367-74
• Display abstract

• The small GTPases Cdc42, Rac and RhoA have important regulatory roles in mediating cytoskeletal rearrangements, MAP kinase cascades and induction of G1 cell cycle progression. The activity of the GTPases is regulated by guanine nucleotide exchange factors (GEFs) which accelerate their GDP/GTP exchange rate, and thereby activate them. All the GEFs for the Rho-GTPases family share two conserved domains: the DH domain (for Dbl-homology domain) responsible for the enzymatic activity, and the PH domain, probably responsible for the proper localization of the molecule. Trio is a multifunctional protein that is comprised of two functional Rho-GEFs domains and a serine/threonine kinase domain. We have shown in vitro and in vivo that the first GEF domain (GEFD1) activates Rac1, while the second GEF domain (GEFD2) acts on RhoA. Moreover, the co-expression of both domains induces simultaneously the activation of both GTPases. To our knowledge, this is the first example of a member of the Rho-GEF family, that contains two functional exchange factor domains, with restricted and different specificity. We are currently investigating how these GEF domains are activated, by addressing the role of the PH domains in GTPases activation by Trio. We have shown that: 1) the PH1 of Trio is necessary for Rac activation by the GEFD1; 2) the PH1 of Trio targets the molecule to the cytoskeleton; 3) the GEFD1 domain of Trio binds, in a two-hybrid screen, the actin binding protein filamin. These data suggest that the PH1 targets Trio to the cytoskeleton close to Rac and its effectors, probably via interaction with the actin-binding protein filamin, consistent with a role of Trio in actin cytoskeleton remodeling.

• Cobellis G, Missero C, Di Lauro R
• Concomitant activation of MEK-1 and Rac-1 increases the proliferative potential of thyroid epithelial cells, without affecting their differentiation.
• Oncogene. 1998; 17: 2047-57
• Display abstract

• Activating point mutations in the Ras oncogene occur in a large number of human tumors, especially of epithelial origin. In thyroid follicular cells, ectopic expression of oncogenic H-Ras results in growth factor-independent proliferation, loss of differentiation and tumor formation in nude mice. In fibroblasts concomitant activation of the MAP kinase cascade and the small GTPase Rac-1 leads to full malignant transformation. We have tested the effects of these key downstream mediators of Ras in thyroid epithelial cells, by stably expressing either a constitutively active form of MEK-1 (MEK(deltaN3/S218E/S222D)), a constitutively active form of Rac-1 (Val12-Rac), or both. While the activation of one molecule or the other results in a weak phenotype, concomitant activation of both MEK-1 and Rac-1 in thyroid cells leads to growth factor-independent proliferation, morphological transformation and anchorage-independent growth. However, in contrast to Ras-transformed thyroid cells, the ones expressing the constitutively active forms of MEK-1 and Rac-1 maintain their differentiate phenotype and fail to form tumors when injected into nude mice. Thus, in thyroid epithelial cells, concomitant activation of MEK-1 and Rac-1 can reproduce only a subset of the Ras-induced effects and is not sufficient to cause full malignant transformation. Significantly, Ras-mediated increased proliferation and loss of differentiation can be dissociated in these cells.

• Luca FC, Winey M
• MOB1, an essential yeast gene required for completion of mitosis and maintenance of ploidy.
• Mol Biol Cell. 1998; 9: 29-46
• Display abstract

• Mob1p is an essential Saccharomyces cerevisiae protein, identified from a two-hybrid screen, that binds Mps1p, a protein kinase essential for spindle pole body duplication and mitotic checkpoint regulation. Mob1p contains no known structural motifs; however MOB1 is a member of a conserved gene family and shares sequence similarity with a nonessential yeast gene, MOB2. Mob1p is a phosphoprotein in vivo and a substrate for the Mps1p kinase in vitro. Conditional alleles of MOB1 cause a late nuclear division arrest at restrictive temperature. MOB1 exhibits genetic interaction with three other yeast genes required for the completion of mitosis, LTE1, CDC5, and CDC15 (the latter two encode essential protein kinases). Most haploid mutant mob1 strains also display a complete increase in ploidy at permissive temperature. The mechanism for the increase in ploidy may occur through MPS1 function. One mob1 strain, which maintains stable haploidy at both permissive and restrictive temperature, diploidizes at permissive temperature when combined with the mps1-1 mutation. Strains containing mob2Delta also display a complete increase in ploidy when combined with the mps1-1 mutation. Perhaps in addition to, or as part of, its essential function in late mitosis, MOB1 is required for a cell cycle reset function necessary for the initiation of the spindle pole body duplication.

• Massol P, Montcourrier P, Guillemot JC, Chavrier P
• Fc receptor-mediated phagocytosis requires CDC42 and Rac1.
• EMBO J. 1998; 17: 6219-29
• Display abstract

• At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined. We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization. This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins. Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse. These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases. Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis. Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.

• Verde F, Wiley DJ, Nurse P
• Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle.
• Proc Natl Acad Sci U S A. 1998; 95: 7526-31
• Display abstract

• The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34(cdc2) mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

• Oehlen LJ, Cross FR
• The role of Cdc42 in signal transduction and mating of the budding yeast Saccharomyces cerevisiae.
• J Biol Chem. 1998; 273: 8556-9
• Display abstract

• The small G-protein Cdc42 functions in many eukaryotic signal transduction pathways. In the budding yeast Saccharomyces cerevisiae, cells with defective Cdc42 fail to induce mating-specific genes in response to mating factor and to adopt the proper morphology for conjugation. Here we show that the failure of mating factor-induced transcription is largely the indirect result of arrest at a specific cell cycle position and/or the accumulation of high levels of the Cln1/2-Cdc28 kinase, a known repressor of mating factor signal transduction. Cdc42-defective cells with restored transcriptional induction have a partially restored mating ability but are still defective in the morphological response to mating factor. These results show that Cdc42 is not required for transduction of the mating factor signal per se but that it is essential for proper mating factor-induced morphogenesis.

• Ben-Ami G et al.
• Agents that inhibit Rho, Rac, and Cdc42 do not block formation of actin pedestals in HeLa cells infected with enteropathogenic Escherichia coli.
• Infect Immun. 1998; 66: 1755-8
• Display abstract

• Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.

• Lamarche-Vane N, Hall A
• CdGAP, a novel proline-rich GTPase-activating protein for Cdc42 and Rac.
• J Biol Chem. 1998; 273: 29172-7
• Display abstract

• Cdc42 mediates several signaling pathways leading to actin reorganization, transcriptional activation, and cell cycle control. Mutational analysis of Cdc42 has revealed that actin reorganization and transcriptional activation are induced through independent signaling pathways. The Y40C effector mutant of Cdc42 no longer interacts with many of its known target proteins, such as p65(PAK) and WASP, yet this mutant can still induce filopodia formation. To identify Cdc42 targets involved in actin rearrangements, we have screened a yeast two-hybrid cDNA library using the Y40C mutant of Cdc42 as a bait. We report here the identification of a novel serine- and proline-rich GTPase-activating protein, CdGAP, which is active in vitro on both Cdc42 and Rac. Microinjection of CdGAP into serum-starved fibroblasts inhibits both platelet-derived growth factor-induced lamellipodia and bradykinin-induced filopodia mediated by Rac and Cdc42, respectively. CdGAP does not show in vitro activity toward Rho, and it has no effect on lysophosphatidic acid-induced stress fiber formation when microinjected into fibroblasts. The carboxyl terminus of CdGAP reveals potential protein kinase C phosphorylation sites and five SH3 binding motifs. Thus, CdGAP is a novel GAP that is likely to participate in Cdc42- and Rac-induced signaling pathways leading to actin reorganization.

• Wu WJ, Lin R, Cerione RA, Manor D
• Transformation activity of Cdc42 requires a region unique to Rho-related proteins.
• J Biol Chem. 1998; 273: 16655-8
• Display abstract

• The Rho subfamily GTP-binding protein Cdc42 mediates actin cytoskeletal rearrangements and cell cycle progression and is essential for Ras transformation. Expression of a Cdc42 mutant (Cdc42(F28L)) that undergoes spontaneous activation (guanine nucleotide exchange) results in transformation of NIH3T3 fibroblasts. In this report, we show that deletion of residues 120-139 from Cdc42(F28L), which comprise an insert region unique to Rho subfamily proteins but is missing in other GTP-binding proteins, yields a Cdc42 molecule that still undergoes spontaneous GTP-GDP exchange and stimulates both actin cytoskeletal changes and the activation of the cellular targets p21-activated kinase and the c-Jun kinase (JNK1). However, this Cdc42 mutant is unable to transform cells. These findings indicate that the Rho subfamily insert region is dispensable for many of the known signaling pathways initiated by activated Cdc42 but is essential for its regulation of cell growth.

• Chabre M, Antonny B, Paris S
• PIP2: activator ... or terminator of small G proteins?
• Trends Biochem Sci. 1998; 23: 98-100

• Tjandra H, Compton J, Kellogg D
• Control of mitotic events by the Cdc42 GTPase, the Clb2 cyclin and a member of the PAK kinase family.
• Curr Biol. 1998; 8: 991-1000
• Display abstract

• BACKGROUND: Cyclins and cyclin-dependent kinases induce and coordinate the events of the cell cycle, although the mechanisms by which they do so remain largely unknown. In budding yeast, a pathway used by the Clb2 cyclin to control bud growth during mitosis provides a good model system in which to understand how cyclin-dependent kinases control cell-cycle events. In this pathway, Clb2 initiates a series of events that lead to the mitosis-specific activation of the Gin4 protein kinase. A protein called Nap1 is required in vivo for the activation of Gin4, and is able to bind to both Gin4 and Clb2. We have used a simple genetic screen to identify additional proteins that function in this pathway. RESULTS: We have found that the Cdc42 GTPase and a member of the PAK kinase family called Cla4 both function in the pathway used by Clb2 to control bud growth during mitosis. Cdc42 and Cla4 interact genetically with Gin4 and Nap1, and both are required in vivo for the mitosis-specific activation of the Gin4 kinase. Furthermore, Cla4 undergoes a dramatic hyperphosphorylation in response to the combined activity of Nap1, the Clb2-Cdc28 kinase complex, and the GTP-bound form of Cdc42. Evidence is presented which suggests that the hyperphosphorylated form of Cla4 is responsible for relaying the signal to activate Gin4. CONCLUSIONS: Previous studies have suggested that cyclin-dependent kinases control the cell cycle by directly phosphorylating proteins involved in specific events, such as nuclear lamins, microtubule-associated proteins and histones. In contrast, our results demonstrate that the Clb2-Cdc28 cyclin-dependent kinase complex controls specific cell-cycle events through a pathway that involves a GTPase and at least two different kinases. This suggests that cyclin-dependent kinases may control many cell-cycle events through GTPase-linked signaling pathways that resemble the intricate signaling pathways known to control many other cellular events.

• Zhang B, Chernoff J, Zheng Y
• Interaction of Rac1 with GTPase-activating proteins and putative effectors. A comparison with Cdc42 and RhoA.
• J Biol Chem. 1998; 273: 8776-82
• Display abstract

• The intrinsic GTPase activity of the Rho family GTP-binding protein Rac1 is drastically stimulated upon interaction with its GTPase-activating proteins (GAPs) and is significantly inhibited when coupled to certain effector targets such as the p21-activated kinases (PAKs) and IQGAPs. Here we have characterized the interaction of Rac1 with a panel of mammalian GAPs and putative effectors by measuring the kinetic and binding parameters involved and made comparisons with similar interactions for Cdc42 and RhoA. In contrast with Cdc42 (for which the GAP domain of p50RhoGAP is 50-fold more efficient than those of p190, Bcr, and 3BP-1) and with RhoA (toward which only p50RhoGAP and p190 displayed high efficiencies), the catalytic efficiencies (Kcat/Km) of the GAP domains of p50RhoGAP, p190, Bcr, and 3BP-1 on Rac1 are found to be comparable in a range between 0.9 and 2.6 min-1 microM-1. However, similar to the cases of Cdc42 and RhoA, the Km values of the GAP domains on Rac1 compare well to the binding affinity to the guanylyl imidodiphosphate-bound Rac1, which ranges from 10.5 to 40.5 microM, suggesting a rapid equilibrium reaction mechanism. The dissociation constants of the p21-binding domains of PAK1, PAK2, and the RasGAP-related domain of IQGAP1, which all cause significant reduction of the intrinsic rate of GTP hydrolysis upon binding to Rac1-GTP, are found to be 0.71, 0.26, and 2.13 microM for Rac1-GTP, compared with that determined for Cdc42-GTP at 2.9, 20.5, and 0.39 microM, respectively, under similar conditions. These results suggest that p50RhoGAP, p190, Bcr, and 3BP-1 are all capable of acting as a negative regulator for Rac1-mediated signaling, and that, although PAK1 and IQGAP1 can couple tightly with both Rac1 and Cdc42, PAK2 is likely to be a specific effector for Rac1 instead of Cdc42.

• Guo W, Sutcliffe MJ, Cerione RA, Oswald RE
• Identification of the binding surface on Cdc42Hs for p21-activated kinase.
• Biochemistry. 1998; 37: 14030-7
• Display abstract

• The Ras superfamily of GTP-binding proteins is involved in a number of cellular signaling events including, but not limited to, tumorigenesis, intracellular trafficking, and cytoskeletal organization. The Rho subfamily, of which Cdc42Hs is a member, is involved in cell morphogenesis through a GTPase cascade which regulates cytoskeletal changes. Cdc42Hs has been shown to stimulate DNA synthesis as well as to initiate a protein kinase cascade that begins with the activation of the p21-activated serine/threonine kinases (PAKs). We have determined previously the solution structure of Cdc42Hs [Feltham et al. (1997) Biochemistry 36, 8755-8766] using NMR spectroscopy. A minimal-binding domain of 46 amino acids of PAK was identified (PBD46), which binds Cdc42Hs with a KD of approximately 20 nM and inhibits GTP hydrolysis. The binding interface was mapped by producing a fully deuterated sample of 15N-Cdc42Hs bound to PBD46. A 1H,15N-NOESY-HSQC spectrum demonstrated that the binding surface on Cdc42Hs consists of the second beta-strand (beta2) and a portion of the loop between the first alpha-helix (alpha1) and beta2 (switch I). A complex of PBD46 bound to 15N-Cdc42Hs.GMPPCP exhibited extensive chemical shift changes in the 1H,15N-HSQC spectrum. Thus, PBD46 likely produces structural changes in Cdc42Hs which are not limited to the binding interface, consistent with its effects on GTP hydrolysis. These results suggest that the kinase-binding domain on Cdc42Hs is similar to, but more extensive than, the c-Raf-binding domain on the Ras antagonist, Rap1 [Nassar et al. (1995) Nature 375, 554-560)].

• Nassar N, Hoffman GR, Manor D, Clardy JC, Cerione RA
• Structures of Cdc42 bound to the active and catalytically compromised forms of Cdc42GAP.
• Nat Struct Biol. 1998; 5: 1047-52
• Display abstract

• The Rho-related small GTP-binding protein Cdc42 has a low intrinsic GTPase activity that is significantly enhanced by its specific GTPase-activating protein, Cdc42GAP. In this report, we present the tertiary structure for the aluminum fluoride-promoted complex between Cdc42 and a catalytically active domain of Cdc42GAP as well as the complex between Cdc42 and the catalytically compromised Cdc42GAP(R305A) mutant. These structures, which mimic the transition state for the GTP hydrolytic reaction, show the presence of an AIF3 molecule, as was seen for the corresponding Ras-p120RasGAP complex, but in contrast to what has been reported for the Rho-Cdc42GAP complex or for heterotrimeric G protein alpha subunits, where AIF4- was observed. The Cdc42GAP stabilizes both the switch I and switch II domains of Cdc42 and contributes a highly conserved arginine (Arg 305) to the active site. Comparison of the structures for the wild type and mutant Cdc42GAP complexes provides important insights into the GAP-catalyzed GTP hydrolytic reaction.

• Nagata K, Driessens M, Lamarche N, Gorski JL, Hall A
• Activation of G1 progression, JNK mitogen-activated protein kinase, and actin filament assembly by the exchange factor FGD1.
• J Biol Chem. 1998; 273: 15453-7
• Display abstract

• Cdc42 has been shown to control bifurcating pathways leading to filopodia formation/G1 cell cycle progression and to JNK mitogen-activated protein kinase activation. To dissect these pathways further, the cellular effects induced by a Cdc42 guanine nucleotide exchange factor, FGD1, have been examined. All exchange factors acting on the Rho GTPase family have juxtaposed Dbl homology (DH) and pleckstrin homology (PH) domains. We report here that FGD1 triggers G1 cell cycle progression and filopodia formation in Swiss 3T3 fibroblasts as well as JNK mitogen-activated protein kinase activation in COS cell transfection assays. FGD1-induced filopodia formation is Cdc42-dependent, and both the DH and PH domains are essential. Although expression of the FGD1 DH domain alone does not activate Cdc42 and induce filopodia, it does trigger both the JNK cascade in COS cells and G1 progression in quiescent Swiss 3T3 cells. We conclude that FGD1 can trigger G1 progression independently of actin polymerization or integrin adhesion complex assembly. Furthermore, since FGD1 activates JNK and G1 progression in a Cdc42-independent manner, it must have additional, as yet unidentified, targets.

• Ahmed S et al.
• Cryptic Rac-binding and p21(Cdc42Hs/Rac)-activated kinase phosphorylation sites of NADPH oxidase component p67(phox).
• J Biol Chem. 1998; 273: 15693-701
• Display abstract

• Rac1 is a member of the Rho family of small molecular mass GTPases that act as molecular switches to control actin-based cell morphology as well as cell growth and differentiation. Rac1 and Rac2 are specifically required for superoxide formation by components of the NADPH oxidase. In binding assays, Rac1 interacts directly with p67(phox), but not with the other oxidase components: cytochrome b, p40(phox), or p47(phox) (Prigmore, E., Ahmed, S., Best, A., Kozma, R. , Manser, E., Segal, A. W., and Lim, L. (1995) J. Biol. Chem. 270, 10717-10722). Here, the Rac1/2 interaction with p67(phox) has been characterized further. Rac1 and Rac2 can bind to p67(phox) amino acid residues 170-199, and the N terminus (amino acids 1-192) of p67(phox) can be used as a specific inhibitor of Rac signaling. Deletion of p67(phox) C-terminal sequences (amino acids 193-526), the C-terminal SH3 domain (amino acids 470-526), or the polyproline-rich motif (amino acids 226-236) stimulates Rac1 binding by approximately 8-fold. p21(Cdc42Hs/Rac)-activated kinase (PAK) phosphorylates p67(phox) amino acid residues adjacent to the Rac1/2-binding site, and this phosphorylation is stimulated by deletion of the C-terminal SH3 domain or the polyproline-rich motif. These data suggest a role for cryptic Rac-binding and PAK phosphorylation sites of p67(phox) in control of the NADPH oxidase.

• Nern A, Arkowitz RA
• A GTP-exchange factor required for cell orientation.
• Nature. 1998; 391: 195-8
• Display abstract

• The Rho-family of GTPases and their regulators are essential for cytoskeletal reorganization and transcriptional activation in response to extracellular signals. Little is known about what links these molecules to membrane receptors. In the budding yeast Saccharomyces cerevisiae, haploid cells respond to mating pheromone through a G-protein-coupled receptor and the betagamma subunit of the G protein, resulting in arrest of the cell cycle, transcriptional activation, and polarized growth towards a mating partner. The Rho-family GTPase Cdc42 and its exchange factor Cdc24 have been implicated in the mating process, but their specific role is unknown. Here we report the identification of cdc24 alleles that do not affect vegetative growth but drastically reduce the ability of yeast cells to mate. When exposed to mating pheromone, these mutants arrest growth, activate transcription, and undergo characteristic morphological and actin-cytoskeleton polarization. However, the mutants are unable to orient towards a pheromone gradient, and instead position their mating projection adjacent to their previous bud site. The mutants are specifically defective in the binding of Cdc24 to the G-protein betagamma subunit. Our results demonstrate that the association of an exchange factor and the betagamma subunit of a hetero-trimeric G protein links receptor-mediated activation to oriented cell growth.

• Hardt WD, Chen LM, Schuebel KE, Bustelo XR, Galan JE
• S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells.
• Cell. 1998; 93: 815-26
• Display abstract

• S. typhimurium stimulates signaling pathways leading to membrane ruffling, actin cytoskeleton rearrangements, and nuclear responses. The stimulation requires a protein secretion system (type III) that translocates bacterial proteins into the host cell. We show that SopE, a substrate of this secretion system, stimulates cytoskeletal reorganization and JNK activation in a CDC42- and Rac-1-dependent manner. A lambda gt11 cDNA library screen for proteins that interact with SopE identified Rac-1 and CDC42. Furthermore, purified SopE was shown to stimulate GDP/GTP nucleotide exchange in several Rho GTPases in vitro, including Rac-1 and CDC42. These findings establish a paradigm for microbial stimulation of cellular responses in which the pathogen induces signaling events by directly engaging the signaling machinery within the host cell.

• Yang P, Kansra S, Pimental RA, Gilbreth M, Marcus S
• Cloning and characterization of shk2, a gene encoding a novel p21-activated protein kinase from fission yeast.
• J Biol Chem. 1998; 273: 18481-9
• Display abstract

• We describe the characterization of a novel gene, shk2, encoding a second p21(cdc42/rac)-activated protein kinase (PAK) homolog in fission yeast. Like other known PAKs, Shk2 binds to Cdc42 in vivo and in vitro. While overexpression of either shk2 or cdc42 alone does not impair growth of wild type fission yeast cells, cooverexpression of the two genes is toxic and leads to highly aberrant cell morphology, providing evidence for functional interaction between Cdc42 and Shk2 proteins in vivo. Fission yeast shk2 null mutants are viable and exhibit no obvious phenotypic defects. Overexpression of shk2 restores viability and normal morphology but not full mating competence to fission yeast cells carrying a shk1 null mutation. Additional genetic data suggest that Shk2, like Cdc42 and Shk1, participates in Ras-dependent morphological control and mating response pathways in fission yeast. We also show that overexpression of byr2, a gene encoding a Ste11/MAPK kinase kinase homolog, suppresses the mating defect of cells partially defective for Shk1 function, providing evidence of a link between PAKs and mitogen-activated protein kinase signaling in fission yeast. Taken together, our results suggest that Shk2 is partially overlapping in function with Shk1, with Shk1 being the dominant protein in function.

• Fujisawa K et al.
• Different regions of Rho determine Rho-selective binding of different classes of Rho target molecules.
• J Biol Chem. 1998; 273: 18943-9
• Display abstract

• Based on their Rho binding motifs several Rho target molecules can be classified into three groups; class I includes the protein kinase PKN, rhophilin, and rhotekin, class II includes the protein kinases, Rho-associated coiled-coil containing protein kinases, ROCK-I and ROCK-II, and class III includes citron. Taking advantage of the selectivity in recognition by these targets between Rho and Rac, we examined the regions in Rho required for selective binding of each class of Rho target molecules. Yeast two-hybrid assays were performed using Rho/Rac chimeras and either rhophilin, ROCK-I, or citron. This study showed the existence of at least two distinct regions in Rho (amino acids 23-40 and 75-92) that are critical for the selective binding of these targets. The former was required for binding to citron, whereas the latter was necessary for binding to rhophilin. On the other hand, either region showed affinity to ROCK-I. This was further confirmed by ligand overlay assay using both recombinant ROCK-I and ROCK-II proteins. Consistently, Rho/Rac chimeras containing either region can induce stress fibers in transfected HeLa cells, and this induction is suppressed by treatment with Y-27632, a specific inhibitor of ROCK kinases. These results suggest that the selective binding of different classes of Rho targets to Rho is determined by interaction between distinct Rho-binding motifs of the targets and different regions of Rho.

• Moreau V, Way M
• Cdc42 is required for membrane dependent actin polymerization in vitro.
• FEBS Lett. 1998; 427: 353-6
• Display abstract

• In vitro actin based motility assays with bacterial pathogens have provided powerful systems to both understand and dissect actin dynamics as well as cell motility. Taking advantage of endogenous membrane vesicles in Xenopus extracts we have developed an in vitro assay to study membrane dependent actin polymerization. Our results demonstrate that membrane dependent actin polymerization, in contrast to Listeria stimulated actin filament assembly, is dependent on small GTPases of the Rho family. Using a combination of depletion and reconstitution experiments we have shown that Cdc42 but not Rac or Rho is required to stimulate actin polymerization from membranes. The in vitro system we have described here is amenable to identification of the downstream effectors of Cdc42 required for membrane dependent actin polymerization.

• Sells MA, Barratt JT, Caviston J, Ottilie S, Leberer E, Chernoff J
• Characterization of Pak2p, a pleckstrin homology domain-containing, p21-activated protein kinase from fission yeast.
• J Biol Chem. 1998; 273: 18490-8
• Display abstract

• p21-activated kinases (PAKs) bind to and are activated by Rho family GTPases such as Cdc42 and Rac. Since these GTPases play key roles in regulating cell polarity, stress responses, and cell cycle progression, the ability of PAK to affect these processes has been examined. We previously showed that fission yeast pak1+ encodes an essential protein that affects mating and cell polarity. Here, we characterize a second pak gene (pak2+) from Schizosaccharomyces pombe. Like the Saccharomyces cerevisiae proteins Cla4p and Skm1p, fission yeast Pak2p contains an N-terminal pleckstrin homology domain in addition to a p21-binding domain and a protein kinase domain that are common to other members of the PAK family. Unlike pak1+, pak2(+) is not essential for vegetative growth or for mating in S. pombe. Overexpression of the wild-type pak2+ allele suppresses the lethal growth defect associated with deletion of pak1+, and this suppression requires both the pleckstrin homology- and the p21-binding domains of Pak2p, as well as kinase activity. A substantial fraction of Pak2p is associated with membranous components, an association mediated both by the pleckstrin homology- and by the p21-binding domains. These results show that S. pombe encodes at least two pak genes with distinct functions and suggest that the membrane localization of Pak2p, directed by its interactions with membrane lipids and Cdc42p, is critical to its biological activity.

• Izawa I, Amano M, Chihara K, Yamamoto T, Kaibuchi K
• Possible involvement of the inactivation of the Rho-Rho-kinase pathway in oncogenic Ras-induced transformation.
• Oncogene. 1998; 17: 2863-71
• Display abstract

• Recent evidence has strongly suggested the involvement of Rho family small guanosine triphosphatases (GTPases) in Ras-induced transformation. To further clarify the role of Rho family GTPases in Ras-induced transformation, we examined the effects of dominant active or dominant negative forms of Rho family GTPases on the morphological changes induced by oncogenic Ras (RasV12) in Rat1 fibroblasts. The cells expressing RasV12 showed the severe disruption of actin stress fibers and cell adhesions. The coexpression of dominant active form of Rho (RhoV14) reverted not only the formation of stress fibers and focal adhesions but also cell-cell adhesions in Ras-transformed Rat1 cells. In addition, the coexpression of constitutively activated Rho-kinase, a downstream effector of Rho, restored the assembly of stress fibers and focal adhesions. Treatment of Ratl cells with lysophosphatidic acid, which is known to activate the Rho-Rho-kinase pathway, enhanced the stress fiber formation, whereas it failed to induce the stress fiber formation in the cells expressing RasV12. These results suggest that the Rho-Rho-kinase pathway may be inactivated in the cells expressing RasV12, and this may contribute to oncogenic Ras-induced transformation.

• Neudauer CL, Joberty G, Tatsis N, Macara IG
• Distinct cellular effects and interactions of the Rho-family GTPase TC10.
• Curr Biol. 1998; 8: 1151-60
• Display abstract

• BACKGROUND: Rho-family GTPases have central roles in cytoskeletal organization, proliferation, differentiation and apoptosis. Multiple factors possessing overlapping specificities for Rho GTPases have been identified. The Rho GTPases Cdc42 and Rac share many regulators and effectors, yet produce different phenotypes when expressed as gain-of-function mutants in cells. The Rho-family member TC10 has remained almost completely uncharacterized, so it was of interest to determine whether TC10 has unique cellular effects and interacts with the same targets as Cdc42 and Rac. RESULTS: A gain-of-function TC10 mutant protein expressed in fibroblasts induced cell rounding, loss of stress fibers and formation of peripheral extensions. The extensions were longer than those induced by the analogous Cdc42 mutant protein. Cells expressing TC10 also possessed fewer membrane ruffles and stress fibers than those expressing Cdc42. TC10 mRNA was most highly expressed in heart and skeletal muscle. The GTPase activity of TC10 was lower than that of Cdc42, and TC10 possessed a lower affinity for, but greater responsiveness to, the p50Rho GTPase-activating protein (p50RhoGAP) than did Cdc42. TC10 stimulated Jun N-terminal kinase (JNK) and p21-activated kinase (PAK) activities and interacted with a set of effectors (alpha-, beta- and gammaPAK, MRCKalpha/beta, MLK2, N-WASP and MSE55) that overlaps with those for Cdc42 and Rac. TC10 did not interact with MLK3 or WASP, and interacted only weakly with ACK-1. CONCLUSIONS: TC10 possesses distinct features, but exhibits a phenotype most closely related to that of Cdc42. It interacts with a similar subset of effectors to Cdc42 but not with MLK3, WASP or ACK-1. It is regulated differentially by p50RhoGAP.

• Yang N et al.
• Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization.
• Nature. 1998; 393: 809-12
• Display abstract

• Rac is a small GTPase of the Rho family that mediates stimulus-induced actin cytoskeletal reorganization to generate lamellipodia. Little is known about the signalling pathways that link Rac activation to changes in actin filament dynamics. Cofilin is known to be a potent regulator of actin filament dynamics, and its ability to bind and depolymerize actin is abolished by phosphorylation of serine residue at 3; however, the kinases responsible for this phosphorylation have not been identified. Here we show that LIM-kinase 1 (LIMK-1), a serine/threonine kinase containing LIM and PDZ domains, phosphorylates cofilin at Ser 3, both in vitro and in vivo. When expressed in cultured cells, LIMK-1 induces actin reorganization and reverses cofilin-induced actin depolymerization. Expression of an inactive form of LIMK-1 suppresses lamellipodium formation induced by Rac or insulin. Furthermore, insulin and an active form of Rac increase the activity of LIMK-1. Taken together, our results indicate that LIMK-1 participates in Rac-mediated actin cytoskeletal reorganization, probably by phosphorylating cofilin.

• Han JS, Kim HC, Chung JK, Kang HS, Donaldson J, Koh JK
• The potential role for CDC42 protein from rat brain cytosol in phospholipase D activation.
• Biochem Mol Biol Int. 1998; 45: 1089-103
• Display abstract

• Phospholipase D (PLD) has been extracted from rat brain membranes and chromatographically enriched 70-fold. From the rat brain cytosol, Cdc42 with a Mr of about 24,000 and ADP-ribosylation Factor (Arf) with a Mr of about 18,000 have been purified to near homogeneity. PLD was activated better by purified cytosolic Arf than by the other small G proteins tested. Cdc42 purified from rat brain cytosol showed 70% of PLD activation activity exerted by cytosolic Arf, suggesting that Cdc42 may be one of the major G proteins involved in the activation of membrane-associated PLD. While Cdc42 or RhoA exhibited synergistic activation of PLD when administered in conjunction with Arf, Cdc42 and RhoA showed an additive effect when used together. It is possible that Arf and Rho family proteins may have different interaction sites on PLD. These findings support a role for GTP-binding proteins of the Rho family as well as Arf in the activation of membrane-associated PLD and further suggest that Cdc42 may be a major G protein involved in the PLD activation in rat brain.

• Caron E, Hall A
• Identification of two distinct mechanisms of phagocytosis controlled by different Rho GTPases.
• Science. 1998; 282: 1717-21
• Display abstract

• The complement and immunoglobulin receptors are the major phagocytic receptors involved during infection. However, only immunoglobulin-dependent uptake results in a respiratory burst and an inflammatory response in macrophages. Rho guanosine triphosphatases (molecular switches that control the organization of the actin cytoskeleton) were found to be essential for both types of phagocytosis. Two distinct mechanisms of phagocytosis were identified: Type I, used by the immunoglobulin receptor, is mediated by Cdc42 and Rac, and type II, used by the complement receptor, is mediated by Rho. These results suggest a molecular basis for the different biological consequences that are associated with phagocytosis.

• Pryciak PM, Huntress FA
• Membrane recruitment of the kinase cascade scaffold protein Ste5 by the Gbetagamma complex underlies activation of the yeast pheromone response pathway.
• Genes Dev. 1998; 12: 2684-97
• Display abstract

• In the Saccharomyces cerevisiae pheromone response pathway, the Gbetagamma complex activates downstream responses by an unknown mechanism involving a MAP kinase cascade, the PAK-like kinase Ste20, and a Rho family GTPase, Cdc42. Here we show that Gbetagamma must remain membrane-associated after release from Galpha to activate the downstream pathway. We also show that pheromone stimulates translocation of the kinase cascade scaffold protein Ste5 to the cell surface. This recruitment requires Gbetagamma function and the Gbetagamma-binding domain of Ste5, but not the kinases downstream of Gbetagamma, suggesting that it is mediated by Gbetagamma itself. Furthermore, this event has functional significance, as artificial targeting of Ste5 to the plasma membrane, but not intracellular membranes, activates the pathway in the absence of pheromone or Gbetagamma. Remarkably, although independent of Gbetagamma, activation by membrane-targeted Ste5 requires Ste20, Cdc42, and Cdc24, indicating that their participation in this pathway does not require them to be activated by Gbetagamma. Thus, membrane recruitment of Ste5 defines a molecular activity for Gbetagamma. Moreover, our results suggest that this event promotes kinase cascade activation by delivering the Ste5-associated kinases to the cell surface kinase Ste20, whose function may depend on Cdc42 and Cdc24.

• Rudolph MG, Bayer P, Abo A, Kuhlmann J, Vetter IR, Wittinghofer A
• The Cdc42/Rac interactive binding region motif of the Wiskott Aldrich syndrome protein (WASP) is necessary but not sufficient for tight binding to Cdc42 and structure formation.
• J Biol Chem. 1998; 273: 18067-76
• Display abstract

• Wiskott Aldrich syndrome is a rare hereditary disease that affects cell morphology and signal transduction in hematopoietic cells. Different size fragments of the Wiskott Aldrich syndrome protein, W4, W7 and W13, were expressed in Escherichia coli or obtained from proteolysis. All contain the GTPase binding domain (GBD), also called Cdc42/Rac interactive binding region (CRIB), found in many putative downstream effectors of Rac and Cdc42. We have developed assays to measure the binding interaction between these fragments and Cdc42 employing fluorescent N-methylanthraniloyl-guanine nucleotide analogues. The fragments bind with submicromolar affinities in a GTP-dependent manner, with the largest fragment having the highest affinity, showing that the GBD/CRIB motif is necessary but not sufficient for tight binding. Rate constants for the interaction with W13 have been determined via surface plasmon resonance, and the equilibrium dissociation constant obtained from their ratio agrees with the value obtained by fluorescence measurements. Far UV circular dichroism spectra show significant secondary structure only for W13, supported by fluorescence studies using intrinsic protein fluorescence and quenching by acrylamide. Proton and 15N NMR measurements show that the GBD/CRIB motif has no apparent secondary structure and that the region C-terminal to the GBD/CRIB region is alpha-helical. The binding of Cdc42 induces a structural rearrangement of residues in the GBD/CRIB motif, or alternatively, the Wiskott Aldrich syndrome protein fragments have an ensemble of conformations, one of which is stabilized by Cdc42 binding. Thus, in contrast to Ras effectors, which have no conserved sequence elements but a defined domain structure with ubiquitin topology, Rac/Cdc42 effectors have a highly conserved binding region but no defined domain structure in the absence of the GTP-binding protein. Deviating from common belief GBD/CRIB is neither a structural domain nor sufficient for tight binding as regions outside this motif are necessary for structure formation and tight interaction with Rho/Rac proteins.

• Zhao ZS, Manser E, Chen XQ, Chong C, Leung T, Lim L
• A conserved negative regulatory region in alphaPAK: inhibition of PAK kinases reveals their morphological roles downstream of Cdc42 and Rac1.
• Mol Cell Biol. 1998; 18: 2153-63
• Display abstract

• AlphaPAK in a constitutively active form can exert morphological effects (E. Manser, H.-Y. Huang, T.-H. Loo, X.-Q. Chen, J.-M. Dong, T. Leung, and L. Lim, Mol. Cell. Biol. 17:1129-1143, 1997) resembling those of Cdc42G12V. PAK family kinases, conserved from yeasts to humans, are directly activated by Cdc42 or Rac1 through interaction with a conserved N-terminal motif (corresponding to residues 71 to 137 in alphaPAK). alphaPAK mutants with substitutions in this motif that resulted in severely reduced Cdc42 binding can be recruited normally to Cdc42G12V-driven focal complexes. Mutation of residues in the C-terminal portion of the motif (residues 101 to 137), though not affecting Cdc42 binding, produced a constitutively active kinase, suggesting this to be a negative regulatory region. Indeed, a 67-residue polypeptide encoding alphaPAK83-149 potently inhibited GTPgammaS-bound Cdc42-mediated kinase activation of both alphaPAK and betaPAK. Coexpression of this PAK inhibitor with Cdc42G12V prevented the formation of peripheral actin microspikes and associated loss of stress fibers normally induced by the p21. Coexpression of PAK inhibitor with Rac1G12V also prevented loss of stress fibers but not ruffling induced by the p21. Coexpression of alphaPAK83-149 completely blocked the phenotypic effects of hyperactive alphaPAKL107F in promoting dissolution of focal adhesions and actin stress fibers. These results, coupled with previous observations with constitutively active PAK, demonstrate that these kinases play an important role downstream of Cdc42 and Rac1 in cytoskeletal reorganization.

• Osman MA, Cerione RA
• Iqg1p, a yeast homologue of the mammalian IQGAPs, mediates cdc42p effects on the actin cytoskeleton.
• J Cell Biol. 1998; 142: 443-55
• Display abstract

• The Rho-type GTPase Cdc42p has been implicated in diverse cellular functions including cell shape, cell motility, and cytokinesis, all of which involve the reorganization of the actin cytoskeleton. Targets of Cdc42p that interface the actin cytoskeleton are likely candidates for mediating cellular activities. In this report, we identify and characterize a yeast homologue for the mammalian IQGAP, a cytoskeletal target for Cdc42p. The yeast IQGAP homologue, designated Iqg1p, displays a two-hybrid interaction with activated Cdc42p and coimmunoprecipitates with actin filaments. Deletion of IQG1 results in a temperature-sensitive lethality and causes aberrant morphologies including elongated and round multinucleated cells. This together with its localization at the mother-bud neck, suggest that Iqg1p promotes budding and cytokinesis. At restrictive temperatures, the vacuoles of the mutant cells enlarge and vesicles accumulate in the bud. Interestingly, Iqg1p shows two-hybrid interactions with the ankyrin repeat-containing protein, Akr1p (Kao, L.-R., J. Peterson, J. Ruiru, L. Bender, and A. Bender. 1996. Mol. Cell. Biol. 16:168-178), which inhibits pheromone signaling and appears to promote cytokinesis and/or trafficking. We also show two-hybrid interactions between Iqg1p and Afr1p, a septin-binding protein involved in projection formation (Konopka, J.B., C. DeMattei, and C. Davis. 1995. Mol. Cell. Biol. 15:723-730). We propose that Iqg1p acts as a scaffold to recruit and localize a protein complex involved in actin-based cellular functions and thus mediates the regulatory effects of Cdc42p on the actin cytoskeleton.

• Madaule P et al.
• Role of citron kinase as a target of the small GTPase Rho in cytokinesis.
• Nature. 1998; 394: 491-4
• Display abstract

• During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures, is known to be essential. Protein kinases are also thought to participate in cytokinesis. We now show that a splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK14 and ROK, which regulate myosin-based contractility. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. We find that overexpression of citron mutants results in the production of multinucleate cells and that a kinase-active mutant causes abnormal contraction during cytokinesis. We propose that citron kinase regulates cytokinesis at a step after Rho in the contractile process.

• Butty AC, Pryciak PM, Huang LS, Herskowitz I, Peter M
• The role of Far1p in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating.
• Science. 1998; 282: 1511-6
• Display abstract

• Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) determine tissue and cell polarity in a variety of organisms. In yeast, cells orient polarized growth toward the mating partner along a pheromone gradient by a mechanism that requires Far1p and Cdc24p. Far1p bound Gbetagamma and interacted with polarity establishment proteins, which organize the actin cytoskeleton. Cells containing mutated Far1p unable to bind Gbetagamma or polarity establishment proteins were defective for orienting growth toward their mating partner. In response to pheromones, Far1p moves from the nucleus to the cytoplasm. Thus, Far1p functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling marked by Gbetagamma to polarize assembly of the cytoskeleton in a morphogenetic gradient.

• Knaus UG, Wang Y, Reilly AM, Warnock D, Jackson JH
• Structural requirements for PAK activation by Rac GTPases.
• J Biol Chem. 1998; 273: 21512-8
• Display abstract

• The Rho family GTPases, Rac1 and Rac2, regulate a variety of cellular functions including cytoskeletal reorganization, the generation of reactive oxygen species, G1 cell cycle progression and, in concert with Ras, oncogenic transformation. Among the many putative protein targets identified for Rac (and/or Cdc42), the Ser/Thr kinase p21-activated kinase (PAK) is a prime candidate for mediating some of Rac's cellular effects. This report shows that Rac1 binds to and stimulates the kinase activity of PAK1 approximately 2- and 4-5-fold, respectively, better than Rac2. Mutational analysis was employed to determine the structural elements on Rac and PAK that are important for optimal binding and activation. The most notable difference between the highly homologous Rac isomers is the composition of their C-terminal polybasic domains. Mutation of these six basic residues in Rac1 to neutral amino acids dramatically decreased the ability of Rac1 to bind PAK1 and almost completely abolished its ability to stimulate PAK activity. Moreover, replacing the highly charged polybasic domain of Rac1 with the less charged domain of Rac2 (and vice versa) completely reversed the PAK binding/activation properties of the two Rac isomers. Thus, polybasic domain differences account for the disparate abilities of Rac1 and Rac2 to activate PAK. PAK proteins also contain a basic region, consisting of three contiguous lysine residues (Lys66-Lys67-Lys68), which lies outside of the previously identified Cdc42/Rac-binding domain. Mutation of these Lys residues to neutral residues decreased PAK binding to activated Rac1 and Rac2 (but not Cdc42) and greatly reduced PAK1 activation by Rac1, Rac2, and Cdc42 proteins in vivo. In contrast, mutation of lysines 66-68 to basic Arg residues did not decrease (and in some cases enhanced) the ability of Rac1, Rac2, and Cdc42 to bind and activate PAK1. Our studies suggest that the polybasic domain of Rac is a novel effector domain that may allow the two Rac isomers to activate different effector proteins. In addition, our results indicate that a basic region in PAK is required for PAK activation and that binding of Rac/Cdc42 to PAK is not sufficient for kinase activation.

• Frost JA, Khokhlatchev A, Stippec S, White MA, Cobb MH
• Differential effects of PAK1-activating mutations reveal activity-dependent and -independent effects on cytoskeletal regulation.
• J Biol Chem. 1998; 273: 28191-8
• Display abstract

• PAKs are serine/threonine protein kinases that are activated by binding to Rac or Cdc42hs. Different forms of activated PAK1 have been reported to either promote membrane ruffling and focal adhesion assembly or cause focal adhesion disassembly and stress fiber dissolution. To understand the basis for these distinct morphological effects, we have examined the mechanism of mutational activation of PAK1, and characterized the effects of different active PAK1 proteins on cytoskeletal structure in vivo. We find that PAK1 contains an autoinhibitory domain that overlaps with its small G protein binding domain and that two separate activating mutations within this regulatory region each decrease autoinhibitory activity. Because only one of these mutations affects Cdc42hs binding activity, this indicates that activation of PAK1 by these mutations results from interference with the function of the autoinhibitory domain and not with small G protein binding activity. When we examined the morphological effects of these different forms of PAK1 in vivo, we found that PAK1 kinase activity was associated with disassembly of focal adhesions and actin stress fibers and that this may require interaction with potential SH3 domain-containing proteins. Lamellipodia formation and membrane ruffling caused by active PAK1 expression, however, was independent of PAK1 catalytic activity and likely requires interaction among multiple proteins binding to the PAK1 regulatory domain.

• Zhang B, Zheng Y
• Negative regulation of Rho family GTPases Cdc42 and Rac2 by homodimer formation.
• J Biol Chem. 1998; 273: 25728-33
• Display abstract

• The Rho family GTPases are tightly regulated between the active GTP-bound state and the inactive GDP-bound state in a variety of signal transduction processes. Here the Rho family members Cdc42, Rac2, and RhoA were found to form reversible homodimers in both the GTP- and the GDP-bound states. The homophilic interaction of Cdc42 and Rac2, but not RhoA, in the GTP-bound state, caused a significant stimulation of the intrinsic GTPase activity, i.e. the activated form of Cdc42 and Rac2 acts as GTPase-activating proteins toward Cdc42-GTP or Rac2-GTP. The dimerization of the GTPases appeared to be mediated by the carboxyl-terminal polybasic domain, and the specific GTPase-activating effects of Cdc42 and Rac2 were also attributed to the structural determinant(s) in the same region of the molecules. Moreover, similar to the case of Cdc42 and Cdc42GAP interaction, Cdc42-GDP interacted with tetrafluoroaluminate and Cdc42-GTPgammaS (guanosine 5'-3-O-(thio)triphosphate) to form a transition state complex of the GTPase-activating reaction in which the carboxyl-terminal determinant(s) of the GTPgammaS-bound Cdc42 plays a critical role. These results provide a rationale for the fast rate of intrinsic GTP hydrolysis by Cdc42 and Rac and suggest that dimerization may play a role in the negative regulation of specific Rho family GTPases mediated by the carboxyl-terminal polybasic domain.

• Reif K, Cantrell DA
• Networking Rho family GTPases in lymphocytes.
• Immunity. 1998; 8: 395-401

• Tuazon PT, Chinwah M, Traugh JA
• Autophosphorylation and protein kinase activity of p21-activated protein kinase gamma-PAK are differentially affected by magnesium and manganese.
• Biochemistry. 1998; 37: 17024-9
• Display abstract

• To examine the requirements for activation of the p21-activated protein kinase gamma-PAK (Pak2, PAK I) from rabbit reticulocytes by Cdc42(GTPgammaS), autophosphorylation with ATP(Mg) or ATP(Mn) and its effects on protein kinase activity were examined. Autophosphorylation with ATP(Mg) alone was minimal with negligible protein kinase activity; the rate of autophosphorylation was increased 3-4-fold upon binding of Cdc42(GTPgammaS), resulting in a 3-fold stimulation of protein kinase activity with peptide and protein substrates. The rate of autophosphorylation with ATP(Mn) was 4.7-fold faster than with ATP(Mg) alone and was stimulated 2-fold by Cdc42(GTPgammaS). However, gamma-PAK autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS) did not phosphorylate peptide or protein substrates in the presence of ATP(Mn), indicating that gamma-PAK can utilize ATP(Mn) for autophosphorylation but not for phosphorylation of exogenous substrates. Tryptic phosphopeptide maps of gamma-PAK autophosphorylated with ATP(Mg) alone showed 3 phosphopeptides, while with Cdc42(GTPgammaS) a total of 9 major phosphopeptides was observed. When gamma-PAK was autophosphorylated with ATP(Mn) in the presence or absence of Cdc42(GTPgammaS), 7 major phosphopeptides were observed, which were identical to peptides obtained with Cdc42(GTPgammaS) and ATP(Mg). Utilizing a recombinant mutant of gamma-PAK with alanine replacing threonine 402 in the catalytic region (T402A), it was determined that the two additional phosphopeptides observed in active PAK (peptides 7 and 8) were due to phosphorylation of threonine 402. These results show that Mn sustains autophosphorylation on serine but does not support autophosphorylation of threonine 402, which is required for activity toward exogenous substrates, or phosphorylation of these substrates.

• Leonard DA, Lin R, Cerione RA, Manor D
• Biochemical studies of the mechanism of action of the Cdc42-GTPase-activating protein.
• J Biol Chem. 1998; 273: 16210-5
• Display abstract

• The small GTP-binding proteins Rac, Rho, and Cdc42 were shown to mediate a variety of signaling pathways including cytoskeletal rearrangements, cell-cycle progression, and transformation. Key to the proper function of these GTP-binding proteins is an efficient shut-off mechanism that ensures the decay of the signal. Regulatory proteins termed GAPs (GTPase-activating proteins) enhance the intrinsic GTP hydrolysis of the GTP-binding proteins, thereby ensuring signal termination. We have used site-specific mutagenesis to elucidate the limit domain for GAP activity in Cdc42-GAP, and show that in addition to the known GAP-homology domain (three conserved boxes), a C-terminal region outside that domain is also essential for GAP activity. In addition, we have replaced the conserved arginine (Arg305), which was suggested by structural studies to be a key catalytic residue, with an alanine and found that the R305A Cdc42-GAP mutant has a greatly diminished catalytic capacity but is still able to bind Cdc42 with high affinity. Thus, a key catalytic role for this residue is confirmed. However, we also present evidence for the involvement of an additional residue(s), since the R305A Cdc42-GAP mutant still exhibits measurable activity. Some of this residual activity might result from a neighboring arginine, since a double mutant R305A/R306A shows a further decrease in catalytic activity.

• Eby JJ et al.
• Actin cytoskeleton organization regulated by the PAK family of protein kinases.
• Curr Biol. 1998; 8: 967-70
• Display abstract

• Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton.

• Price LS, Leng J, Schwartz MA, Bokoch GM
• Activation of Rac and Cdc42 by integrins mediates cell spreading.
• Mol Biol Cell. 1998; 9: 1863-71
• Display abstract

• Adhesion to ECM is required for many cell functions including cytoskeletal organization, migration, and proliferation. We observed that when cells first adhere to extracellular matrix, they spread rapidly by extending filopodia-like projections and lamellipodia. These structures are similar to the Rac- and Cdc42-dependent structures observed in growth factor-stimulated cells. We therefore investigated the involvement of Rac and Cdc42 in adhesion and spreading on the ECM protein fibronectin. We found that integrin-dependent adhesion led to the rapid activation of p21-activated kinase, a downstream effector of Cdc42 and Rac, suggesting that integrins activate at least one of these GTPases. Dominant negative mutants of Rac and Cdc42 inhibit cell spreading in such a way as to suggest that integrins activate Cdc42, which leads to the subsequent activation of Rac; both GTPases then contribute to cell spreading. These results demonstrate that initial integrin-dependent activation of Rac and Cdc42 mediates cell spreading.

• Nikolic M, Chou MM, Lu W, Mayer BJ, Tsai LH
• The p35/Cdk5 kinase is a neuron-specific Rac effector that inhibits Pak1 activity.
• Nature. 1998; 395: 194-8
• Display abstract

• Cyclin-dependent kinase 5 (Cdk5) and its neuron-specific regulator p35 are essential for neuronal migration and for the laminar configuration of the cerebral cortex. In addition, p35/Cdk5 kinase concentrates at the leading edges of axonal growth cones and regulates neurite outgrowth in cortical neurons in culture. The Rho family of small GTPases is implicated in a range of cellular functions, including cell migration and neurite outgrowth. Here we show that the p35/Cdk5 kinase co-localizes with Rac in neuronal growth cones. Furthermore, p35 associates directly with Rac in a GTP-dependent manner. Another Rac effector, Pak1 kinase, is also present in the Rac-p35/Cdk5 complexes and co-localizes with p35/Cdk5 and Rac at neuronal peripheries. The active p35/Cdk5 kinase causes Pak1 hyperphosphorylation in a Rac-dependent manner, which results in down-regulation of Pak1 kinase activity. Because the Rho family of GTPases and the Pak kinases are implicated in actin polymerization, the modification of Pak1, imposed by the p35/Cdk5 kinase, is likely to have an impact on the dynamics of the reorganization of the actin cytoskeleton in neurons, thus promoting neuronal migration and neurite outgrowth.

• Miki H, Suetsugu S, Takenawa T
• WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac.
• EMBO J. 1998; 17: 6932-41
• Display abstract

• Rac is a Rho-family small GTPase that induces the formation of membrane ruffles. However, it is poorly understood how Rac-induced reorganization of the actin cytoskeleton, which is essential for ruffle formation, is regulated. Here we identify a novel Wiskott-Aldrich syndrome protein (WASP)-family protein, WASP family Verprolin-homologous protein (WAVE), as a regulator of actin reorganization downstream of Rac. Ectopically expressed WAVE induces the formation of actin filament clusters that overlap with the expressed WAVE itself. In this actin clustering, profilin, a monomeric actin-binding protein that has been suggested to be involved in actin polymerization, was shown to be essential. The expression of a dominant-active Rac mutant induces the translocation of endogenous WAVE from the cytosol to membrane ruffling areas. Furthermore, the co-expression of a deltaVPH WAVE mutant that cannot induce actin reorganization specifically suppresses the ruffle formation induced by Rac, but has no effect on Cdc42-induced actin-microspike formation, a phenomenon that is also known to be dependent on rapid actin reorganization. The deltaVPH WAVE also suppresses membrane-ruffling formation induced by platelet-derived growth factor in Swiss 3T3 cells. Taken together, we conclude that WAVE plays a critical role downstream of Rac in regulating the actin cytoskeleton required for membrane ruffling.

• Andra K, Nikolic B, Stocher M, Drenckhahn D, Wiche G
• Not just scaffolding: plectin regulates actin dynamics in cultured cells.
• Genes Dev. 1998; 12: 3442-51
• Display abstract

• Plectin, a major linker and scaffolding protein of the cytoskeleton, has been shown to be essential for the mechanical integrity of skin, skeletal muscle, and heart. Studying fibroblast and astroglial cell cultures derived from plectin (-/-) mice, we found that their actin cytoskeleton, including focal adhesion contacts, was developed more extensively than in wild-type cells. Also it failed to show characteristic short-term rearrangments in response to extracellular stimuli activating the Rho/Rac/Cdc42 signaling cascades. As a consequence, cell motility, adherence, and shear stress resistance were altered, and morphogenic processes were delayed. Furthermore, we show that plectin interacts with G-actin in vitro in a phosphatidylinositol-4,5-biphosphate-dependent manner and associates with actin stress fibers in living cells. The actin stress fiber phenotype of plectin-deficient fibroblasts could be reversed to a large degree by transient transfection of full-length plectin or plectin fragments containing the amino-terminal actin-binding domain (ABD). These results reveal a novel role of plectin as regulator of cellular processes involving actin filament dynamics that goes beyond its proposed role in scaffolding and mechanical stabilization of cells.

• Manser E et al.
• PAK kinases are directly coupled to the PIX family of nucleotide exchange factors.
• Mol Cell. 1998; 1: 183-92
• Display abstract

• The PAK family of kinases are regulated through interaction with the small GTPases Cdc42 and Rac1, but little is known of the signaling components immediately upstream or downstream of these proteins. We have purified and cloned a new class of Rho-p21 guanine nucleotide exchange factor binding tightly through its N-terminal SH3 domain to a conserved proline-rich PAK sequence with a Kd of 24 nM. This PAK-interacting exchange factor (PIX), which is widely expressed and enriched in Cdc42- and Rac1-driven focal complexes, is required for PAK recruitment to these sites. PIX can induce membrane ruffling, with an associated activation of Rac1. Our results suggest a role for PIX in Cdc42-to-Rac1 signaling, involving the PIX/PAK complex.

• Gauthier-Rouviere C, Vignal E, Meriane M, Roux P, Montcourier P, Fort P
• RhoG GTPase controls a pathway that independently activates Rac1 and Cdc42Hs.
• Mol Biol Cell. 1998; 9: 1379-94
• Display abstract

• RhoG is a member of the Rho family of GTPases that shares 72% and 62% sequence identity with Rac1 and Cdc42Hs, respectively. We have expressed mutant RhoG proteins fused to the green fluorescent protein and analyzed subsequent changes in cell surface morphology and modifications of cytoskeletal structures. In rat and mouse fibroblasts, green fluorescent protein chimera and endogenous RhoG proteins colocalize according to a tubular cytoplasmic pattern, with perinuclear accumulation and local concentration at the plasma membrane. Constitutively active RhoG proteins produce morphological and cytoskeletal changes similar to those elicited by a simultaneous activation of Rac1 and Cdc42Hs, i.e., the formation of ruffles, lamellipodia, filopodia, and partial loss of stress fibers. In addition, RhoG and Cdc42Hs promote the formation of microvilli at the cell apical membrane. RhoG-dependent events are not mediated through a direct interaction with Rac1 and Cdc42Hs targets such as PAK-1, POR1, or WASP proteins but require endogenous Rac1 and Cdc42Hs activities: coexpression of a dominant negative Rac1 impairs membrane ruffling and lamellipodia but not filopodia or microvilli formation. Conversely, coexpression of a dominant negative Cdc42Hs only blocks microvilli and filopodia, but not membrane ruffling and lamellipodia. Microtubule depolymerization upon nocodazole treatment leads to a loss of RhoG protein from the cell periphery associated with a reversal of the RhoG phenotype, whereas PDGF or bradykinin stimulation of nocodazole-treated cells could still promote Rac1- and Cdc42Hs-dependent cytoskeletal reorganization. Therefore, our data demonstrate that RhoG controls a pathway that requires the microtubule network and activates Rac1 and Cdc42Hs independently of their growth factor signaling pathways.

• Antonarakis SE, Van Aelst L
• Mind the GAP, Rho, Rab and GDI.
• Nat Genet. 1998; 19: 106-8

• Abo A
• Identification and functional reconstitution of effector proteins for the GTPases Rac and CDC42Hs.
• Methods Mol Biol. 1998; 84: 163-72

• Nomanbhoy T, Cerione RA
• Use of fluorescence spectroscopy to study the regulation of small G proteins.
• Methods Mol Biol. 1998; 84: 237-47
• Display abstract

• At present, there are a number of questions concerning the abilities of different regulators (GEFs, GAPs) and target molecules to bind to Cdc42Hs and related GTP-binding proteins. The ability to label Cdc42Hs with an extrinsic-reporter group (sNBD) with a 1:1 stoichiometry of probe incorporation per protein molecule, and without any loss of functional activity, provides a powerful reagent for quantitative assays of regulatory protein and target binding. The expectation is that the sNBD-labeled Cdc42Hs will be useful in definitively determining whether GAPs and individual targets compete with one another for binding to Cdc42Hs, or if multiple-target molecules can complex simultaneously with a single GTP-binding protein. Given the success in labeling Cdc42Hs with an extrinsic-reporter group, it seems likely that similar labeling approaches would be successful with other members of the family, such as the Rac and Rho proteins.

• Manser E, Leung T, Lim L
• Identification and characterization of small GTPase-associated kinases.
• Methods Mol Biol. 1998; 84: 295-305

• Davis CR, Richman TJ, Deliduka SB, Blaisdell JO, Collins CC, Johnson DI
• Analysis of the mechanisms of action of the Saccharomyces cerevisiae dominant lethal cdc42G12V and dominant negative cdc42D118A mutations.
• J Biol Chem. 1998; 273: 849-58
• Display abstract

• The Saccharomyces cerevisiae Cdc42p GTPase is localized to the plasma membrane and involved in signal transduction mechanisms controlling cell polarity. The mechanisms of action of the dominant negative cdc42(D118A) mutant and the lethal, gain of function cdc42(G12V) mutant were examined. Cdc42(D118A,C188S)p and its guanine-nucleotide exchange factor Cdc24p displayed a temperature-dependent interaction in the two-hybrid system, which correlated with the temperature dependence of the cdc42(D118A) phenotype and supported a Cdc24p sequestration model for the mechanism of cdc42(D118A) action. Five cdc42 mutations were isolated that led to decreased interactions with Cdc24p. The isolation of one mutation (V44A) correlated with the observations that the T35A effector domain mutation could interfere with Cdc42(D118A, C188S)p-Cdc24p interactions and could suppress the cdc42(D118A) mutation, suggesting that Cdc24p may interact with Cdc42p through its effector domain. The cdc42(G12V) mutant phenotypes were suppressed by the intragenic T35A and K183-187Q mutations and in skm1Delta and cla4Delta cells but not ste20Delta cells, suggesting that the mechanism of cdc42(G12V) action is through the Skm1p and Cla4p protein kinases at the plasma membrane. Two intragenic suppressors of cdc42(G12V) were also identified that displayed a dominant negative phenotype at 16 degrees C, which was not suppressed by overexpression of Cdc24p, suggesting an alternate mechanism of action for these dominant negative mutations.

• Schafer DA, Welch MD, Machesky LM, Bridgman PC, Meyer SM, Cooper JA
• Visualization and molecular analysis of actin assembly in living cells.
• J Cell Biol. 1998; 143: 1919-30
• Display abstract

• Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM-kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP-CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

• Shellman YG, Schauer IE, Oshiro G, Dohrmann P, Sclafani RA
• Oligomers of the Cdc7/Dbf4 protein kinase exist in the yeast cell.
• Mol Gen Genet. 1998; 259: 429-36
• Display abstract

• Cdc7/Dbf4 protein kinase is required for the initiation of DNA replication in Saccharomyces cerevisiae. Cdc7/Dbf4 protein kinase is not a cyclin-dependent kinase (CDK), but is regulated in a similar fashion in that the Cdc7 kinase subunit is inactive in the absence of the regulatory subunit Dbf4. In contrast to what is known about CDKs, Cdc7/Dbf4 protein kinase is shown to be an oligomer in the cell in this report. Genetic data that support this claim include interallelic complementation between several cdc7ts alleles and the cdc7T281A allele and also the results of experiments using the two-hybrid system with Cdc7 in both DNA-binding and transactivation domain plasmids. A molecular interaction between two different Cdc7 molecules was shown by using a HA-tagged Cdc7 protein that differs in size from the wild-type Cdc7 protein: an anti-HA antibody immunoprecipitates both proteins in approximately equal stoichiometry. Analysis of the native molecular weight of Cdc7/Dbf4 protein kinase is consistent with oligomerization of the Cdc7 protein in that complexes of about 180 and 300 kDa were found. Oligomers of Cdc7 protein may exist for the purpose of allosteric regulation or to allow phosphorylation of multiple substrate protein molecules.

• Hoffman GR, Nassar N, Oswald RE, Cerione RA
• Fluoride activation of the Rho family GTP-binding protein Cdc42Hs.
• J Biol Chem. 1998; 273: 4392-9
• Display abstract

• Aluminum tetrafluoride (AlF4-) activation of heterotrimeric G-protein alpha-subunits is a well established aspect of the biochemistry of these proteins; however, until recently it has been thought that AlF4- does not mediate effects on the Ras superfamily of low molecular weight GTP-binding proteins. Recent work demonstrating aluminum fluoride-induced complex formation between Ras and its GTPase-activating proteins (RasGAP and NF1) has provided important insights into the mechanism of GAP-stimulated GTP hydrolysis. We have characterized the AlF4--induced complex formation between the GDP-bound form of the Rho subfamily G-protein Cdc42Hs and a limit functional domain of the Cdc42-GAP using a variety of biochemical techniques. Our results indicate that the apparent affinity of GAP for the AlF4--mediated complex is similar to the affinity observed for the activated (GTP-bound) form of Cdc42 and that beryllium (Be) can replace aluminum in mediating fluoride-induced complex formation. Additionally, the AlF4--induced interaction is weakened significantly by the catalytically compromised GAP(R305A) mutant, indicating that this arginine is critical in transition state stabilization. Unlike Ras, we find that AlF4- and BeF3- mediate complex formation between Cdc42Hs.GDP and downstream target/effector molecules, indicating that there are important differences in the mechanism of effector binding between the Ras and Rho subfamily G-proteins.

• Aronheim A, Broder YC, Cohen A, Fritsch A, Belisle B, Abo A
• Chp, a homologue of the GTPase Cdc42Hs, activates the JNK pathway and is implicated in reorganizing the actin cytoskeleton.
• Curr Biol. 1998; 8: 1125-8
• Display abstract

• The p21-activated protein kinases (PAKs) are activated through direct interaction with the GTPases Rac and Cdc42Hs, which are implicated in the control of the mitogen-activated protein kinase (MAP kinase) c-Jun N-terminal kinase (JNK) and the reorganization of the actin cytoskeleton [1-3]. The exact role of the PAK proteins in these signaling pathways is not entirely clear. To elucidate the biological function of Pak2 and to identify its molecular targets, we used a novel two-hybrid system, the Ras recruitment system (RRS), that aims to detect protein-protein interactions at the inner surface of the plasma membrane (described in the accompanying paper by Broder et al. [4]). The Pak2 regulatory domain (PakR) was fused at the carboxyl terminus of a RasL61 mutant protein and screened against a myristoylated rat pituitary cDNA library. Four clones were identified that interact specifically with PakR and three were subsequently shown to encode a previously unknown homologue of the GTPase Cdc42Hs. This approximately 36 kDa protein, designated Chp, exhibits an overall sequence identity to Cdc42Hs of approximately 52%. Chp contains two additional sequences at the amino and carboxyl termini that are not found in any known GTPase. The amino terminus contains a polyproline sequence, typically found in Src homology 3 (SH3)-binding domains, and the carboxyl terminus appears to be important for Pak2 binding. Results from the microinjection of Chp into cells implicated Chp in the induction of lamellipodia and showed that Chp activates the JNK MAP kinase cascade.

• Featherstone C
• The many faces of WAS protein.
• Science. 1997; 275: 27-8

• Qiu RG, Abo A, McCormick F, Symons M
• Cdc42 regulates anchorage-independent growth and is necessary for Ras transformation.
• Mol Cell Biol. 1997; 17: 3449-58
• Display abstract

• The Rho family members Cdc42, Rac, and Rho play a central role in the organization of the actin cytoskeleton and regulate transcription. Whereas Rac and Rho have been implicated in transformation by oncogenic Ras, the role of Cdc42 in this process remains unknown. In this study, we found that Rat1 fibroblasts expressing constitutively active V12-Cdc42 were anchorage independent and proliferated in nude mice but failed to show enhanced growth in low serum. Similar to V12-Rac1-expressing Rat1 fibroblasts, V12-Cdc42 lines displayed a high frequency of multinucleated cells. Interestingly, coexpression of dominant negative N17-Rac1 blocked the V12-Cdc42-induced multinucleated phenotype but not growth in soft agar, indicating that Cdc42 controls anchorage independence in a Rac-independent fashion. We also showed that dominant negative N17-Cdc42 inhibited Ras focus formation and anchorage-independent growth and caused reversion of the transformed morphology, indicating that Cdc42 is necessary for Ras transformation. N17-Cdc42 caused only partial inhibition of Ras-induced low-serum growth, however. In contrast, whereas N17-Rac1 also effectively inhibited Ras-induced anchorage independence, it did not revert the morphology of Ras-transformed cells. N17-Rac1 strongly inhibited low-serum growth of Ras-transformed cells, however. Together, these data provide a novel function for Cdc42 in cell proliferation and indicate that Cdc42 and Rac play distinct roles in growth control and Ras transformation.

• Tu H, Barr M, Dong DL, Wigler M
• Multiple regulatory domains on the Byr2 protein kinase.
• Mol Cell Biol. 1997; 17: 5876-87
• Display abstract

• Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2.

• Tang Y et al.
• Kinase-deficient Pak1 mutants inhibit Ras transformation of Rat-1 fibroblasts.
• Mol Cell Biol. 1997; 17: 4454-64
• Display abstract

• Among the mechanisms by which the Ras oncogene induces cellular transformation, Ras activates the mitogen-activated protein kinase (MAPK or ERK) cascade and a related cascade leading to activation of Jun kinase (JNK or SAPK). JNK is additionally regulated by the Ras-related G proteins Rac and Cdc42. Ras also regulates the actin cytoskeleton through an incompletely elucidated Rac-dependent mechanism. A candidate for the physiological effector for both JNK and actin regulation by Rac and Cdc42 is the serine/threonine kinase Pak (p65pak). We show here that expression of a catalytically inactive mutant Pak, Pak1(R299), inhibits Ras transformation of Rat-1 fibroblasts but not of NIH 3T3 cells. Typically, 90 to 95% fewer transformed colonies were observed in cotransfection assays with Rat-1 cells. Pak1(R299) did not inhibit transformation by the Raf oncogene, indicating that inhibition was specific for Ras. Furthermore, Rat-1 cell lines expressing Pak1(R299) were highly resistant to Ras transformation, while cells expressing wild-type Pak1 were efficiently transformed by Ras. Pak1(L83,L86,R299), a mutant that fails to bind either Rac or Cdc42, also inhibited Ras transformation. Rac and Ras activation of JNK was inhibited by Pak1(R299) but not by Pak1(L83,L86,R299). Ras activation of ERK was inhibited by both Pak1(R299) and Pak1(L83,L86,R299), while neither mutant inhibited Raf activation of ERK. These results suggest that Pak1 interacts with components essential for Ras transformation and that inhibition can be uncoupled from JNK but not ERK signaling.

• Perona R, Montaner S, Saniger L, Sanchez-Perez I, Bravo R, Lacal JC
• Activation of the nuclear factor-kappaB by Rho, CDC42, and Rac-1 proteins.
• Genes Dev. 1997; 11: 463-75
• Display abstract

• The Rho family of small GTPases are critical elements involved in the regulation of signal transduction cascades from extracellular stimuli to the cell nucleus, including the JNK/SAPK signaling pathway, the c-fos serum response factor, and the p70 S6 kinase. Here we report a novel signaling pathway activated by the Rho proteins that may be responsible for their biological activities, including cytoskeleton organization, transformation, apoptosis, and metastasis. The human RhoA, CDC42, and Rac-1 proteins efficiently induce the transcriptional activity of nuclear factor kappaB (NF-kappaB) by a mechanism that involves phosphorylation of Ikappa Balpha and translocation of p50/p50 and p50/p65 dimers to the nucleus, but independent of the Ras GTPase and the Raf-1 kinase. We also show that activation of NF-kappaB by TNFalpha depends on CDC42 and RhoA, but not Rac-1 proteins, because this activity is drastically inhibited by their respective dominant-negative mutants. In contrast, activation of NF-kappaB by UV light was not affected by Rho, CDC42, or Rac-1 dominant-negative mutants. Thus, members of the Rho family of GTPases are involved specifically in the regulation of NF-kappaB-dependent transcription.

• Lian JP, Badwey JA
• Activation of the p21-activated protein kinases from neutrophils with an antibody that reacts with the N-terminal region of Pak 1.
• FEBS Lett. 1997; 404: 211-5
• Display abstract

• Neutrophils contain two renaturable p21-activated protein kinases (Paks) with molecular masses of ca. 69 and 63 kDa that undergo rapid activation upon stimulation of these cells with the chemoattractant fMet-Leu-Phe. We now report that these kinases undergo a massive, ATP-dependent activation in lysates of unstimulated neutrophils during immunoprecipitation with an antibody generated to residues 2-21 of the N-terminal region of Pak1. This activation was specific as it was completely blocked by a peptide that corresponds to residues 2-21 of Pak1 and was not observed with an antibody generated to the C-terminal region of Pak 1. The properties of the Paks activated with the antibody were virtually identical to those observed for these kinases from stimulated neutrophils, or activated in vitro with Rac-GTPgammaS plus ATP. These data indicate that perturbation of the N-terminal region of Pak can trigger activation of this enzyme, and that both the 69 and 63 kDa kinases may represent forms of Pak 1 that differ in their content of phosphate.

• Keely PJ, Westwick JK, Whitehead IP, Der CJ, Parise LV
• Cdc42 and Rac1 induce integrin-mediated cell motility and invasiveness through PI(3)K.
• Nature. 1997; 390: 632-6
• Display abstract

• Transformation of mammary epithelial cells into invasive carcinoma results in alterations in their integrin-mediated responses to the extracellular matrix, including a loss of normal epithelial polarization and differentiation, and a switch to a more motile, invasive phenotype. Changes in the actin cytoskeleton associated with this switch suggest that the small GTPases Cdc42 and Rac, which regulate actin organization, might modulate motility and invasion. However, the role of Cdc42 and Rac1 in epithelial cells, especially with respect to integrin-mediated events, has not been well characterized. Here we show that activation of Cdc42 and Rac1 disrupts the normal polarization of mammary epithelial cells in a collagenous matrix, and promotes motility and invasion. This motility does not require the activation of PAK, JNK, p70 S6 kinase, or Rho, but instead requires phosphatidylinositol-3-OH kinase (PI(3)K). Further, direct PI(3)K activation is sufficient to disrupt epithelial polarization and induce cell motility and invasion. PI(3)K inhibition also disrupts actin structures, suggesting that activation of PI(3)K by Cdc42 and Rac1 alters actin organization, leading to increased motility and invasiveness.

• Tapon N, Hall A
• Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton.
• Curr Opin Cell Biol. 1997; 9: 86-92
• Display abstract

• Rho, Rac and Cdc42 are three Ras-related GTP-binding proteins that control the assembly and disassembly of the actin cytoskeleton in response to extracellular signals. During the past year, numerous candidate downstream targets for these GTPases have been identified using affinity chromatography and yeast two-hybrid techniques. These techniques have revealed that Rho regulates the myosin light chain phosphatase and that Rho and Rac control the synthesis of phosphatidylinositol 4,5-bisphosphate, two activities that might help to explain the effects of these GTPases on the actin cytoskeleton.

• Fanger GR, Johnson NL, Johnson GL
• MEK kinases are regulated by EGF and selectively interact with Rac/Cdc42.
• EMBO J. 1997; 16: 4961-72
• Display abstract

• MEK kinases (MEKKs) 1, 2, 3 and 4 are members of sequential kinase pathways that regulate MAP kinases including c-Jun NH2-terminal kinases (JNKs) and extracellular regulated kinases (ERKs). Confocal immunofluorescence microscopy of COS cells demonstrated differential MEKK subcellular localization: MEKK1 was nuclear and in post-Golgi vesicular-like structures; MEKK2 and 4 were localized to distinct Golgi-associated vesicles that were dispersed by brefeldin A. MEKK1 and 2 were activated by EGF, and kinase-inactive mutants of each MEKK partially inhibited EGF-stimulated JNK activity. Kinase-inactive MEKK1, but not MEKK2, 3 or 4, strongly inhibited EGF-stimulated ERK activity. In contrast to MEKK2 and 3, MEKK1 and 4 specifically associated with Rac and Cdc42 and kinase-inactive mutants blocked Rac/Cdc42 stimulation of JNK activity. Inhibitory mutants of MEKK1-4 did not affect p21-activated kinase (PAK) activation of JNK, indicating that the PAK-regulated JNK pathway is independent of MEKKs. Thus, in different cellular locations, specific MEKKs are required for the regulation of MAPK family members, and MEKK1 and 4 are involved in the regulation of JNK activation by Rac/Cdc42 independent of PAK. Differential MEKK subcellular distribution and interaction with small GTP-binding proteins provides a mechanism to regulate MAP kinase responses in localized regions of the cell and to different upstream stimuli.

• Thompson G, Chalk PA, Lowe PN
• Interaction of PAK with Rac: determination of a minimum binding domain on PAK.
• Biochem Soc Trans. 1997; 25: 509-509

• Kuroda S, Fukata M, Fujii K, Nakamura T, Izawa I, Kaibuchi K
• Regulation of cell-cell adhesion of MDCK cells by Cdc42 and Rac1 small GTPases.
• Biochem Biophys Res Commun. 1997; 240: 430-5
• Display abstract

• Rac1, a member of the Rho small GTPases family, has recently been shown to be involved in the regulation of cell-cell adhesion mediated by cadherin. Here we showed that Cdc42, another member of Rho family, accumulated at cell-cell contact sites. Microinjection of Rho GDI, a negative regulator of the Rho family members, into Madin-Darby canine kidney (MDCK) cells resulted in perturbation of epithelial cell morphology and of cell-cell and cell-substratum adhesions, and comicroinjection of dominant active Cdc42 or Rac1 reversed the action of Rho GDI, suggesting that the active form of Cdc42 or Rac1 is required for maintaining the cell-cell and cell-substratum adhesions. These observations suggest that Cdc42, in addition to Rac1, can regulate the cell-cell adhesion.

• Carpenter CL, Tolias KF, Couvillon AC, Hartwig JH
• Signal transduction pathways involving the small G proteins rac and Cdc42 and phosphoinositide kinases.
• Adv Enzyme Regul. 1997; 37: 377-90
• Display abstract

• We found that rac specifically binds to a type I PtdIns-4-P 5-kinase and that both rac and Cdc42 in the activated forms associate with PI 3-kinase. The association of PI 3-kinase with rac was stimulated by PDGF in vivo. Rac is constitutively associated with a PtdIns-4-P 5-kinase and stimulates PtdIns-4,5-P2 production in permeabilized platelets. These data suggest a model in which the initial step in the activation of rac is release from rho GDI (Fig. 7). Rac in the GDP bound form can associate with the PtdIns-4-P 5-kinase and also interact with an exchange factor. GTP bound rac may then localize to sites of actin reorganization, bringing the PtdIns-4-P 5-kinase with it. Locally synthesized PtdIns-4,5-P2 binds to actin capping proteins, leading to their release and the production of actin free ends. Actin polymerization can then occur from the free ends. Many other factors must be involved to regulate the type and extent of actin polymerization that is necessary in such complex processes as cell movement and membrane ruffling. The rac-associated PtdIns-4-P 5-kinase and its product PtdIns-4,5-P2 may act at a crucial regulatory point that permits polymerization to begin.

• Sells MA, Knaus UG, Bagrodia S, Ambrose DM, Bokoch GM, Chernoff J
• Human p21-activated kinase (Pak1) regulates actin organization in mammalian cells.
• Curr Biol. 1997; 7: 202-10
• Display abstract

• BACKGROUND: The Rho family GTPases Cdc42, Rac1 and RhoA regulate the reorganization of the actin cytoskeleton induced by extracellular signals such as growth factors. In mammalian cells, Cdc42 regulates the formation of filopodia, whereas Rac regulates lamellipodia formation and membrane ruffling, and RhoA regulates the formation of stress fibers. Recently, the serine/threonine protein kinase p65(pak) autophosphorylates, thereby increasing its catalytic activity towards exogenous substrates. This kinase is therefore a candidate effector for the changes in cell shape induced by growth factors. RESULTS: Here, we report that the microinjection of activated Pak1 protein into quiescent Swiss 3T3 cells induces the rapid formation of polarized filopodia and membrane ruffles. The prolonged overexpression of Pak1 amino-terminal mutants that are unable to bind Cdc42 or Rac1 results in the accumulation of filamentous actin in large, polarized membrane ruffles and the formation of vinculin-containing focal complexes within these structures. This phenotype resembles that seen in motile fibroblasts. The amino-terminal Pak1 mutant displays enhanced binding to the adaptor protein Nck, which contains three Src-homology 3 (SH3) domains. Mutation of a proline residue within a conserved SH3-binding region at the amino terminus of Pak1 interferes with SH3-protein binding and alters the effects of Pak1 on the cytoskeleton. CONCLUSIONS: These results indicate that Pak1, acting through a protein that contains an SH3 domain, regulates the structure of the actin cytoskeleton in mammalian cells, and may serve as an effector for Cdc42 and/or Rac1 in promoting cell motility.

• Wilk-Blaszczak MA et al.
• The monomeric G-proteins Rac1 and/or Cdc42 are required for the inhibition of voltage-dependent calcium current by bradykinin.
• J Neurosci. 1997; 17: 4094-100
• Display abstract

• Although regulation of voltage-dependent calcium current (ICa,V) by neurotransmitters is a ubiquitous mechanism among nerve cells, the signaling pathways involved are not well understood. We have determined previously that in a neuroblastoma-glioma hybrid cell line (NG108-15), the heterotrimeric G-protein G13 mediates the inhibition of ICa,V produced by bradykinin (BK) via an unknown mechanism. Various reports indicate that G13 can couple to RhoA, Rac1, and Cdc42, which are closely related members of the Rho family of monomeric G-proteins. We have investigated their role as signaling intermediates in the pathway used by BK to inhibit ICa,V. Using immunoblot analysis and the PCR, we found evidence that RhoA, Rac1, and Cdc42 all are expressed in NG108-15 cells. Intracellularly perfused recombinant Rho-GDI (an inhibitor of guanine nucleotide exchange specific for the Rho family) attenuated the inhibition of ICa,V by BK. These findings indicate that activation of RhoA, Rac1, or Cdc42 may be required for the response to BK. To determine whether any of these monomeric G-proteins mediate the response to BK, we have intracellularly applied blocking antibodies specific for each of the candidate proteins. Only the anti-Rac1 antibody blocked the response to BK. In parallel experiments, peptides corresponding to the C-terminal regions of Rac1 and Cdc42 blocked the same response. These data indicate a novel functional contribution of Rac1 and possibly also of Cdc42 to the inhibition of ICa,V by neurotransmitters.

• Wu WJ, Leonard DA, A-Cerione R, Manor D
• Interaction between Cdc42Hs and RhoGDI is mediated through the Rho insert region.
• J Biol Chem. 1997; 272: 26153-8
• Display abstract

• Members of the Rho subfamily of GTP-binding proteins contain a region of amino acid sequence (residues 122-134) that is absent from other Ras-like proteins and is termed the Rho insert region. To address the functional role of this domain, we have constructed a Cdc42Hs/Ras chimera in which loop 8 from Ha-Ras was substituted for the region in Cdc42Hs that contains the 13-amino acid insert region. Our data indicate that the insert region of Cdc42Hs is not essential for its interactions with various target/effector molecules or for interactions with the guanine nucleotide exchange factor, Dbl, or the Cdc42 GTPase-activating protein (GAP). However, the regulation of GDP dissociation and GTP hydrolysis on Cdc42Hs by the Rho GDP-dissociation inhibitor (GDI) is extremely sensitive to changes in the insert region, such that a Cdc42Hs/Ha-Ras chimera that lacks this insert is no longer susceptible to a GDI-induced inhibition of GDP dissociation and GTP hydrolysis. The insensitivity to GDI activity is not due to the inability of the GDI molecule to bind to the Cdc42Hs/Ha-Ras chimera, and in fact, the GDI is fully capable of stimulating the release of this chimera from membranes.

• Gosser YQ et al.
• C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases.
• Nature. 1997; 387: 814-9
• Display abstract

• The Rho GDP-dissociation inhibitors (GDIs) negatively regulate Rho-family GTPases. The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes, and from inhibition of GTPase cycling between the GTP- and GDP-bound states. Here we demonstrate that these binding and inhibitory functions of rhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 (M[r] 16K) binds strongly to the Rho-family member Cdc42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The solution structure of this domain shows a beta-sandwich motif with a narrow hydrophobic cleft that binds isoprenes, and an exposed surface that interacts with the protein portion of Cdc42. The amino-terminal region of rhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from Cdc42. These results lead to a model of rhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling.

• Luo L, Lee T, Tsai L, Tang G, Jan LY, Jan YN
• Genghis Khan (Gek) as a putative effector for Drosophila Cdc42 and regulator of actin polymerization.
• Proc Natl Acad Sci U S A. 1997; 94: 12963-8
• Display abstract

• The small GTPases Cdc42 and Rac regulate a variety of biological processes, including actin polymerization, cell proliferation, and JNK/mitogen-activated protein kinase activation, conceivably via distinct effectors. Whereas the effector for mitogen-activated protein kinase activation appears to be p65PAK, the identity of effector(s) for actin polymerization remains unclear. We have found a putative effector for Drosophila Cdc42, Genghis Khan (Gek), which binds to Dcdc42 in a GTP-dependent and effector domain-dependent manner. Gek contains a predicted serine/threonine kinase catalytic domain that is 63% identical to human myotonic dystrophy protein kinase and has protein kinase activities. It also possesses a large coiled-coil domain, a putative phorbol ester binding domain, a pleckstrin homology domain, and a Cdc42 binding consensus sequence that is required for its binding to Dcdc42. To study the in vivo function of gek, we generated mutations in the Drosophila gek locus. Egg chambers homozygous for gek mutations exhibit abnormal accumulation of F-actin and are defective in producing fertilized eggs. These phenotypes can be rescued by a wild-type gek transgene. Our results suggest that this multidomain protein kinase is an effector for the regulation of actin polymerization by Cdc42.

• Evangelista M et al.
• Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis.
• Science. 1997; 276: 118-22
• Display abstract

• The Saccharomyces cerevisiae BNI1 gene product (Bni1p) is a member of the formin family of proteins, which participate in cell polarization, cytokinesis, and vertebrate limb formation. During mating pheromone response, bni1 mutants showed defects both in polarized morphogenesis and in reorganization of the underlying actin cytoskeleton. In two-hybrid experiments, Bni1p formed complexes with the activated form of the Rho-related guanosine triphosphatase Cdc42p, with actin, and with two actin-associated proteins, profilin and Bud6p (Aip3p). Both Bni1p and Bud6p (like Cdc42p and actin) localized to the tips of mating projections. Bni1p may function as a Cdc42p target that links the pheromone response pathway to the actin cytoskeleton.

• Matsubara K, Hinoi T, Koyama S, Kikuchi A
• The post-translational modifications of Ral and Rac1 are important for the action of Ral-binding protein 1, a putative effector protein of Ral.
• FEBS Lett. 1997; 410: 169-74
• Display abstract

• Ral-binding protein 1 (RalBP1) is a putative effector protein of Ral and possesses the GTPase-activating activity for Rac1 and CDC42. We examined the roles of the post-translational modifications of Ral and Rac1 for the action of RalBP1. In COS cells, Ral(G23V), a constitutively active form, was mainly detected in the membrane fraction while most of Ral(G23V/C203S), a Ral mutant which is not post-translationally modified, was found in the cytosol fraction. When RalBP1 was expressed alone in COS cells, it was found in the cytosol but not in the membrane fraction. When RalBP1 was coexpressed with Ral(G23V), a part of RalBP1 was found in the membrane fraction. However, when RalBP1 was coexpressed with Ral(G23V/C203S), all of RalBP1 was recovered in the cytosol fraction. Although Ral bound to RalBP1 at a molar ratio of 1:1, the interaction of Ral with RalBP1 did not affect the GTPase-activating activity of RalBP1 for Rac1. Furthermore, RalBP1 was more active on the post-translationally modified form of Rac1 and CDC42 than the unmodified form. These results suggest that the post-translational modification of Ral is important for the subcellular localization of RalBP1 and that the interaction of Ral with RalBP1 is not essential for the activity of RalBP1 but plays a role in recruiting RalBP1 to the membrane where its substrates, Rac1 and CDC42, reside.

• Yang W, Cerione RA
• Cloning and characterization of a novel Cdc42-associated tyrosine kinase, ACK-2, from bovine brain.
• J Biol Chem. 1997; 272: 24819-24
• Display abstract

• Cdc42 plays an important role in intracellular signaling pathways that influence cell morphology and motility and stimulate DNA synthesis. In attempts to determine whether nonreceptor tyrosine kinases play a fundamental role in Cdc42 signaling, we have cloned and biochemically characterized a new Cdc42-associated tyrosine kinase (ACK) from bovine brain. This tyrosine kinase, named ACK-2, has a calculated molecular mass of 83 kDa and shares a number of primary structural domains with the 120-kDa ACK (ACK-1). The main differences between the primary structures of ACK-2 and ACK-1 occur in the amino- and carboxyl-terminal regions. Like ACK-1, ACK-2 binds exclusively to activated (GTP-bound) Cdc42 and does not bind to its closest homologs, e.g. activated Rac. ACK-2 could not be activated by addition of glutathione S-transferase (GST)-Cdc42(Q61L), a GTPase-defective mutant, or by GTPgammaS-loaded GST-Cdc42 in in vitro kinase assays. However, ACK-2 was activated when cotransfected with wild type Cdc42 or Cdc42(Q61L) and stably associated with Cdc42(Q61L) in vivo, indicating that ACK-2 interacts with active Cdc42 in cells. Furthermore, the tyrosine kinase activity of ACK-2 was stimulated both by epidermal growth factor and bradykinin, suggesting that ACK-2 may play a role in the signaling actions of both receptor tyrosine kinases or heterotrimeric G-protein-coupled receptors.

• Zigmond SH, Joyce M, Borleis J, Bokoch GM, Devreotes PN
• Regulation of actin polymerization in cell-free systems by GTPgammaS and Cdc42.
• J Cell Biol. 1997; 138: 363-74
• Display abstract

• We have established a cell-free system to investigate pathways that regulate actin polymerization. Addition of GTPgammaS to lysates of polymorphonuclear leukocytes (PMNs) or Dictyostelium discoideum amoeba induced formation of filamentous actin. The GTPgammaS appeared to act via a small G-protein, since it was active in lysates ofD. discoideum mutants missing either the alpha2- or beta-subunit of the heterotrimeric G-protein required for chemoattractant-induced actin polymerization in living cells. Furthermore, recombinant Cdc42, but not Rho or Rac, induced polymerization in the cell-free system. The Cdc42-induced increase in filamentous actin required GTPgammaS binding and was inhibited by a fragment of the enzyme PAK1 that binds Cdc42. In a high speed supernatant, GTPgammaS alone was ineffective, but GTPgammaS-loaded Cdc42 induced actin polymerization, suggesting that the response was limited by guanine nucleotide exchange. Stimulating exchange by chelating magnesium, by adding acidic phospholipids, or by adding the exchange factors Cdc24 or Dbl restored the ability of GTPgammaS to induce polymerization. The stimulation of actin polymerization did not correlate with PIP2 synthesis.

• Manser E et al.
• Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes.
• Mol Cell Biol. 1997; 17: 1129-43
• Display abstract

• The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mol. Cell. Biol. 16:1896-1908, 1996), where it colocalizes with structures resembling focal complexes. We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.

• Leonard DA, Satoskar RS, Wu WJ, Bagrodia S, Cerione RA, Manor D
• Use of a fluorescence spectroscopic readout to characterize the interactions of Cdc42Hs with its target/effector, mPAK-3.
• Biochemistry. 1997; 36: 1173-80
• Display abstract

• The family of p21-activated kinases (PAKs) has been shown to contain a domain that can independently bind to the Ras-like proteins Cdc42Hs and Rac. We have expressed a 72 amino acid recombinant form of this p21-binding domain (PBD) from mPAK-3 in Escherichia Coli for use in structure-function studies. The protein can be purified on a nickel affinity resin due to a hexa-His tag that is incorporated onto the amino terminus of the domain. PBD binds to Cdc42Hs in a guanine nucleotide-dependent manner as demonstrated by a novel fluorescence assay that takes advantage of the spectroscopic properties of N-methylanthraniloyl (Mant)-guanine nucleotides. Ionic strength has little effect on the affinity of PBD for Cdc42Hs, but alkaline pH values tend to weaken the interaction. We have shown that the inhibition of the GTPase activity of Cdc42Hs, as well as a previously undescribed inhibition of guanine nucleotide dissociation, is mediated by the PBD portion of the mPAK-3 molecule. These findings suggest that PBD binding alters the geometry of the guanine nucleotide binding site on Cdc42Hs, perhaps as an outcome of the target/effector molecule binding in close proximity to the nucleotide domain. We therefore tested if mutations in the effector region of Cdc42Hs (32-40), which in Ras are very close to the guanine nucleotide binding site, had any effect on PBD binding. Changing tyrosine 32 to lysine (Y32K) resulted in a small (5-fold) inhibition of PBD binding, but the very conservative mutation D38E yielded at least a 50-fold decrease in affinity. Finally, the catalytic domain of the GTPase activating protein, Cdc42-GAP, was shown to inhibit PBD binding in a competitive manner, indicating that this target molecule and the negative regulator (GAP) bind to overlapping sites on the Cdc42Hs molecule.

• Luo L, Jan LY, Jan YN
• Rho family GTP-binding proteins in growth cone signalling.
• Curr Opin Neurobiol. 1997; 7: 81-6
• Display abstract

• Rho family GTP-binding proteins regulate various aspects of the actin cytoskeleton in a wide variety of organisms. Recent evidence suggests that they may also be important components of the signalling pathways that link the reception of extracellular cues to the regulation of the cytoskeleton in neuronal growth cones.

• Miller PJ, Johnson DI
• Characterization of the Saccharomyces cerevisiae cdc42-1ts allele and new temperature-conditional-lethal cdc42 alleles.
• Yeast. 1997; 13: 561-72
• Display abstract

• Cdc42p is a highly conserved GTPase involved in controlling cell polarity and polarizing the actin cytoskeleton. The CDC42 gene was first identified by the temperature-sensitive cell-division-cycle mutant cdc42-1ts in Saccharomyces cerevisiae. We have determined the DNA and predicted amino-acid sequence of the cdc42-1ts allele and identified multiple mutations in the coding region and 5' promoter region, thereby limiting its usefulness in genetic screens. Therefore, we generated additional temperature-conditional-lethal alleles in highly conserved amino-acid residues of both S. cerevisiae and Schizosaccharomyces pombe Cdc42p. The cdc42W97R temperature-sensitive allele in S. cerevisiae displayed the same cell-division-cycle arrest phenotype (large, round unbudded cells) as the cdc42-1ts mutant. However, it exhibited a bud-site selection defect and abnormal bud morphologies at the permissive temperature of 23 degrees C. These phenotypes suggest that Cdc42p functions in bud-site selection early in the morphogenetic process and also in polarizing growth patterns leading to proper bud morphogenesis later in the process. In S. pombe, the cdc42W97R mutant displayed a cold-sensitive, los-of-function phenotype when expressed from the thiamine-repressible nmt1 promoter under repressing conditions. In addition, cdc42T58A and cdc42S71P mutants showed a temperature-sensitive loss-of-function phenotype when expressed in S. pombe: these mutants did not display a conditional phenotype when expressed in S. cerevisiae. These new conditional-lethal cdc42 alleles will be important reagents for the further dissection of the cell polarity pathway in both yeasts.

• Chen GC, Kim YJ, Chan CS
• The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae.
• Genes Dev. 1997; 11: 2958-71
• Display abstract

• BEM2 of Saccharomyces cerevisiae encodes a Rho-type GTPase-activating protein that is required for proper bud site selection at 26 degrees C and for bud emergence at elevated temperatures. We show here that the temperature-sensitive growth phenotype of bem2 mutant cells can be suppressed by increased dosage of the GIC1 gene. The Gic1 protein, together with its structural homolog Gic2, are required for cell size and shape control, bud site selection, bud emergence, actin cytoskeletal organization, mitotic spindle orientation/positioning, and mating projection formation in response to mating pheromone. Each protein contains a CRIB (Cdc42/Rac-interactive binding) motif and each interacts in the two-hybrid assay with the GTP-bound form of the Rho-type Cdc42 GTPase, a key regulator of polarized growth in yeast. The CRIB motif of Gic1 and the effector domain of Cdc42 are required for this association. Genetic experiments indicate that Gic1 and Gic2 play positive roles in the Cdc42 signal transduction pathway, probably as effectors of Cdc42. Subcellular localization studies with a functional green fluorescent protein-Gic1 fusion protein indicate that this protein is concentrated at the incipient bud site of unbudded cells, at the bud tip and mother-bud neck of budded cells, and at cortical sites on large-budded cells that may delimit future bud sites in the two progeny cells. The ability of Gic1 to associate with Cdc42 is important for its function but is apparently not essential for its subcellular localization.

• Brown JL, Jaquenoud M, Gulli MP, Chant J, Peter M
• Novel Cdc42-binding proteins Gic1 and Gic2 control cell polarity in yeast.
• Genes Dev. 1997; 11: 2972-82
• Display abstract

• Cdc42p, a Rho-related GTP-binding protein, regulates cytoskeletal polarization and rearrangements in eukaryotic cells, but the effectors mediating this control remain unknown. Through the use of the complete yeast genomic sequence, we have identified two novel Cdc42p targets, Gic1p and Gic2p, which contain consensus Cdc42/Rac interactive-binding (CRIB) domains and bind specifically to Cdc42p-GTP. Gic1p and Gic2p colocalize with Cdc42p as cell polarity is established during the cell cycle and during mating in response to pheromones. Cells deleted for both GIC genes exhibit defects in actin and microtubule polarization similar to those observed in cdc42 mutants. Finally, the interaction of the Gic proteins and Cdc42p is essential, as mutations in the CRIB domain of Gic2p that eliminate Cdc42p binding disrupt Gic2p localization and function. Thus, Gic1p and Gic2p define a novel class of Cdc42p targets that are specifically required for cytoskeletal polarization in vivo.

• Chen JM, Manolatos S, Brandt-Rauf PW, Murphy RB, Monaco R, Pincus MR
• Computed three-dimensional structures for the ras-binding domain of the raf-p74 protein complexed with ras-p21 and with its suppressor protein, rap-1A.
• J Protein Chem. 1996; 15: 511-8
• Display abstract

• The three-dimensional structures of the ras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to the ras-binding domain, RBD (residues 55-131), of the raf-p74 protein, a critical target protein of ras-p21 in the ras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of both ras-p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of the ras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains of ras-p21 and rap-1a, including residues 32-47, a domain that directly interacts with RBD, 60-66, 96-110, involved in the interaction of ras-p21 with jun kinase (JNK) and jun protein, and 115-126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its beta-2 binding loop (residues 63-71) and residues 89-91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK and jun.

• Kirchhausen T, Rosen FS
• Disease mechanism: unravelling Wiskott-Aldrich syndrome.
• Curr Biol. 1996; 6: 676-8
• Display abstract

• The gene responsible for Wiskott-Aldrich syndrome, a disease affecting platelets and lymphocytes, has been cloned and its protein product (WASp) found to interact with the GTPase Cdc42. WASp seems to provide a link between Cdc42 and the actin cytoskeleton, perhaps explaining the cellular defects underlying the disease.

• Brown JL, Stowers L, Baer M, Trejo J, Coughlin S, Chant J
• Human Ste20 homologue hPAK1 links GTPases to the JNK MAP kinase pathway.
• Curr Biol. 1996; 6: 598-605
• Display abstract

• BACKGROUND. The Rho-related GTP-binding proteins Cdc42 and Rac1 have been shown to regulate signaling pathways involved in cytoskeletal reorganization and stress-responsive JNK (Jun N-terminal kinase) activation. However, to date, the GTPase targets that mediate these effects have not been identified. PAK defines a growing family of mammalian kinases that are related to yeast Ste20 and are activated in vitro through binding to Cdc42 and Rac1 (PAK: p21 Cdc42-/Rac-activated kinase). Clues to PAK function have come from studies of Ste20, which controls the activity of the yeast mating mitogen-activated protein (MAP) kinase cascade, in response to a heterotrimeric G protein and Cdc42. RESULTS. To initiate studies of mammalian Ste20-related kinases, we identified a novel human PAK isoform, hPAK1. When expressed in yeast, hPAK1 was able to replace Ste20 in the pheromone response pathway. Chemical mutagenesis of a plasmid encoding hPAK1, followed by transformation into yeast, led to the identification of a potent constitutively active hPAK1 with a substitution of a highly conserved amino-acid residue (L107F) in the Cdc42-binding domain. Expression of the hPAK1(L107F) allele in mammalian cells led to specific activation of the Jun N-terminal kinase MAP kinase pathway, but not the mechanistically related extracellular signal-regulated MAP kinase pathway. CONCLUSIONS. These results demonstrate that hPAK1 is a GTPase effector controlling a downstream MAP kinase pathway in mammalian cells, as Ste20 does in yeast. Thus, PAK and Ste20 kinases play key parts in linking extracellular signals from membrane components, such as receptor-associated G proteins and Rho-related GTPases, to nuclear responses, such as transcriptional activation.

• Marks PW, Kwiatkowski DJ
• Genomic organization and chromosomal location of murine Cdc42.
• Genomics. 1996; 38: 13-8
• Display abstract

• cdc42 is a member of the rho family of small GTPases, which are implicated as regulators of cellular morphology. To date, one murine and two human cdc42 isoforms have been identified. Here we report the cloning of a second murine isoform and provide evidence that the two isoforms arise from a single gene by alternative splicing. In contrast with the previously identified murine cdc42 sequence, which is expressed in a wide variety of tissues, the second isoform appears to be expressed exclusively in brain. Using single-strand conformation polymorphism analysis of a mouse backcross panel, the gene encoding cdc42 has been localized to distal chromosome 4.

• Leung T, Chen XQ, Manser E, Lim L
• The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton.
• Mol Cell Biol. 1996; 16: 5313-27
• Display abstract

• The GTPase RhoA has been implicated in various cellular activities, including the formation of stress fibers, motility, and cytokinesis. We recently reported on a p150 serine/threonine kinase (termed ROK alpha) binding RhoA only in its active GTP-bound state and on its cDNA; introduction of RhoA into HeLa cells resulted in translocation of the cytoplasmic kinase to plasma membranes, consistent with ROK alpha being a target for RhoA (T. Leung, E. Manser, L. Tan, and L. Lim, J. Biol. Chem. 256:29051-29054, 1995). Reanalysis of the cDNA revealed that ROK alpha contains an additional N-terminal region. We also isolated another cDNA which encoded a protein (ROK beta) with 90% identity to ROK alpha in the kinase domain. Both ROK alpha and ROK beta, which had a molecular mass of 160 kDa, contained a highly conserved cysteine/histidine-rich domain located within a putative pleckstrin homology domain. The kinases bound RhoA, RhoB, and RhoC but not Rac1 and Cdc42. The Rho-binding domain comprises about 30 amino acids. Mutations within this domain caused partial or complete loss of Rho binding. The morphological effects of ROK alpha were investigated by microinjecting HeLa cells with DNA constructs encoding various forms of ROK alpha. Full-length ROK alpha promoted formation of stress fibers and focal adhesion complexes, consistent with its being an effector of RhoA. ROK alpha truncated at the C terminus promoted this formation and also extensive condensation of actin microfilaments and nuclear disruption. The proteins exhibited protein kinase activity which was required for stress fiber formation; the kinase-dead ROK alpha K112A and N-terminally truncated mutants showed no such promotion. The latter mutant instead induced disassembly of stress fibers and focal adhesion complexes, accompanied by cell spreading. These effects were mediated by the C-terminal region containing Rho-binding, cysteine/histidine-rich, and pleckstrin homology domains. Thus, the multidomained ROK alpha appears to be involved in reorganization of the cytoskeleton, with the N and C termini acting as positive and negative regulators, respectively, of the kinase domain whose activity is crucial for formation of stress fibers and focal adhesion complexes.

• Aspenstrom P, Lindberg U, Hall A
• Two GTPases, Cdc42 and Rac, bind directly to a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome.
• Curr Biol. 1996; 6: 70-5
• Display abstract

• BACKGROUND: Members of the Rho family of small GTPases play an essential role in controlling the motile behaviour of animal cells. Specifically, Cdc42 and Rac have been shown to induce the formation of filopodia and lamellipodia, respectively, at the cell periphery of Swiss 3T3 fibroblasts. In addition, both GTPases are required for progression through G1 phase of the cell cycle, possibly by regulating the activity of the Jun N-terminal kinase (JNK) signalling pathway. In order to examine more closely the mechanisms underlying the diverse functions of Rho GTPases in mammalian cells, we searched for downstream targets of these proteins. RESULTS: A yeast two-hybrid screen for proteins interacting with the human Cdc42 GTPase identified WASP, a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome (WAS). Recombinant WASP, expressed in Escherichia coli, also bound to Cdc42 and weakly to Rac, but not at all to Rho. The Cdc42/Rac-binding domain was identified in a region between amino acids 201-321 of WASP, and binding was dependent on Cdc42 and Rac being in the GTP-bound conformation. Furthermore, WASP did not catalyze GTPase activation or nucleotide exchange activity on Cdc42. CONCLUSIONS: Positional cloning has implicated WASP in causing WAS, and the protein is defective in patients suffering from the disease. WASP is expressed exclusively in cells of hematopoietic lineage, and lymphocytes from WAS patients have a distorted cell-surface and exhibit reduced proliferative capacity. WASP has recently been found to bind to the Src-homology 3 (SH3) domain of the adapter protein Nck. This observation, and the results presented here, suggest that WAS is the result of defects in signal transduction pathways regulated by Cdc42/Rac and Nck.

• Nomanbhoy TK, Cerione R
• Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy.
• J Biol Chem. 1996; 271: 10004-9
• Display abstract

• The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding proteins is capable of three different biochemical activities. These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes. In order to better understand how GDI interactions with Rho-like proteins mediate these different effects, we have set out to develop a direct fluorescence spectroscopic assay for the binding of the GDI to the Rho-like protein, Cdc42Hs. We show here that when the GDI interacts with Cdc42Hs that contains bound N-methylanthraniloyl GDP (Mant-GDP), there is an approximately 20% quenching of the Mant fluorescence. The GDI-induced quenching is only observed when Mant-GDP is bound to Spodoptera frugiperda-expressed Cdc42Hs and is not detected when the Mant nucleotide is bound to Escherichia coli-expressed Cdc42Hs and thus shows the same requirement for isoprenylated GTP-binding protein as that observed when assaying GDI activity. A truncated Cdc42Hs mutant that lacks 8 amino acids from the carboxyl terminus and is insensitive to GDI regulation also does not show changes in the fluorescence of its bound Mant-GDP upon GDI addition. Thus, the GDI-induced quenching of Mant-GDP provides a direct read-out for the binding of the GDI to Cdc42Hs. Titration profiles of the GDI-induced quenching of the Mant-GDP fluorescence are saturable and are well fit to a simple 1:1 binding model for Cdc42Hs-GDI interactions with an apparent Kd value of 30 nM. A very similar Kd value (28 nM) is measured when titrating the GDI-induced quenching of the fluorescence of Mant-guanylyl imidotriphosphate, bound to Cdc42Hs. These results suggest that the GDI can bind to the GDP-bound and GTP-bound forms of Cdc42Hs equally well. We also have used the fluorescence assay for GDI interactions to demonstrate that the differences in functional potency observed between the GDI molecule and a related human leukemic protein, designated LD4, are due to differences in their binding affinities for Cdc42Hs. This, together with the results from studies using GDI/LD4 chimeras, allow us to conclude that a limit region within the carboxyl-terminal domain of the GDI molecule is responsible for its ability to bind with higher affinity (compared with LD4) to Cdc42Hs.

• Feig LA, Urano T, Cantor S
• Evidence for a Ras/Ral signaling cascade.
• Trends Biochem Sci. 1996; 21: 438-41
• Display abstract

• It is becoming clear that Ras proteins mediate their diverse biological functions by binding to, and participating in, the activation of multiple downstream targets. Recent work has identified nucleotide-exchange factors for Ral-GTPases as the newest members of the set of putative Ras 'effector molecules'. This new work has also detected two potential downstream targets of Ral proteins, a novel CDC42/Rac GTPase-activating protein and a phospholipase D.

• Mack D et al.
• Identification of the bud emergence gene BEM4 and its interactions with rho-type GTPases in Saccharomyces cerevisiae.
• Mol Cell Biol. 1996; 16: 4387-95
• Display abstract

• The Rho-type GTPase Cdc42p is required for cell polarization and bud emergence in Saccharomyces cerevisiae. To identify genes whose functions are linked to CDC42, we screened for (i) multicopy suppressors of a Ts- cdc42 mutant, (ii) mutants that require multiple copies of CDC42 for survival, and (iii) mutations that display synthetic lethality with a partial-loss-of-function allele of CDC24, which encodes a guanine nucleotide exchange factor for Cdc42p. In all three screens, we identified a new gene, BEM4. Cells from which BEM4 was deleted were inviable at 37 degrees C. These cells became unbudded, large, and round, consistent with a model in which Bem4p acts together with Cdc42p in polarity establishment and bud emergence. In some strains, the ability of CDC42 to serve as a multicopy suppressor of the Ts- growth defect of deltabem4 cells required co-overexpression of Rho1p, which is an essential Rho-type GTPase necessary for cell wall integrity. This finding suggests that Bem4p also affects Rho1p function. Bem4p displayed two-hybrid interactions with Cdc42p, Rho1p, and two of the three other known yeast Rho-type GTPases, suggesting that Bem4p can interact with multiple Rho-type GTPases. Models for the role of Bem4p include that it serves as a chaperone or modulates the interaction of these GTPases with one or more of their targets or regulators.

• Cullen BR
• HIV-1: is Nef a PAK animal?
• Curr Biol. 1996; 6: 1557-9
• Display abstract

• HIV-1 Nef is clearly essential for efficient viral replication in vivo, but it has been difficult to determine why. Recent evidence that Nef specifically activates a PAK-dependent signalling cascade may be the first step in defining the mechanism of action of this enigmatic viral protein.

• McCallum SJ, Wu WJ, Cerione RA
• Identification of a putative effector for Cdc42Hs with high sequence similarity to the RasGAP-related protein IQGAP1 and a Cdc42Hs binding partner with similarity to IQGAP2.
• J Biol Chem. 1996; 271: 21732-7
• Display abstract

• Cdc42 is a Ras-related GTP-binding protein that has been implicated in the regulation of the actin cytoskeleton and cell morphology. In this study, we have identified a protein with a molecular mass approximately 180 kDa from rabbit liver cytosol (designated p180), which binds preferentially to the GTP- and guanosine 5'-3-O-(thio)triphosphate-bound forms of Cdc42. Binding of p180 to GTP-bound Cdc42 maintains it in the GTP-bound state. Another cytosolic protein, with an apparent molecular mass of 175 kDa (p175), was also found to interact with Cdc42, but this association showed less dependence on guanine nucleotides. Both p180 and p175 were capable of binding to Rac1 but not to RhoA or Ha-Ras. The limit functional domain of the Cdc42-GAP protein did not compete with p180 or p175 for binding to Cdc42. However, the Cdc42-binding domain from mPAK-3, a member of the PAK (p21 activated kinase) family of serine/threonine kinases, competed with both proteins. The binding of p180 or p175 was inhibited by mutations of the putative effector loop of Cdc42. p180 and p175 also bound less effectively to a Cdc42/Ras chimera in which loop 8 from Ras was substituted for the predicted loop 8 in Cdc42 that includes a 13-amino acid insert present in all Rho family members but absent in Ras. Microsequencing of a p180 peptide revealed 92% identity with the human IQGAP1 protein, while two peptides derived from p175 were 89 and 100% identical to human IQGAP2. These findings identify IQGAP1 and IQGAP2 as a new class of target/effectors that utilize both regions of the switch I domain and an insert region distinct to Rho proteins for binding to Cdc42.

• Shibata H et al.
• Characterization of the interaction between RhoA and the amino-terminal region of PKN.
• FEBS Lett. 1996; 385: 221-4
• Display abstract

• The yeast two-hybrid system and in vitro binding assay were carried out to characterize the interaction between PKN and a small GTP-binding protein, RhoA. It was revealed that the region corresponding to the amino acid residues 33-111 in the amino-terminal region of PKN was sufficient to confer the ability to associate with RhoA. Each synthetic peptide fragment corresponding to the amino acid residues 74-93 and 94-113 of PKN inhibited the interaction between PKN and RhoA in the in vitro binding assay, suggesting that this region is important in the association with RhoA. The endogenous and the GAP-stimulated GTPase activity of RhoA was inhibited by the interaction with PKN, suggesting the presence of a regulatory mechanism that sustains the GTP-bound active form of RhoA.

• Bussey H
• Rho returns: its targets in focal adhesions.
• Science. 1996; 273: 203-203

• Teramoto H, Crespo P, Coso OA, Igishi T, Xu N, Gutkind JS
• The small GTP-binding protein rho activates c-Jun N-terminal kinases/stress-activated protein kinases in human kidney 293T cells. Evidence for a Pak-independent signaling pathway.
• J Biol Chem. 1996; 271: 25731-4
• Display abstract

• Work from a number of laboratories has established a role for certain small GTP-binding proteins in controlling the enzymatic activity of a family of serine-threonine kinases known as mitogen-activated protein kinases (MAPKs). MAPKs have been classified into three subfamilies: extracellular signal-regulated kinases (ERKs), also known as MAPKs; c-Jun N-terminal kinases (JNKs); and p38 kinase. Whereas Ras controls the activation of MAPKs, we and others have recently observed that in certain cells, the small GTP-binding proteins Rac1 and Cdc42 but not Rho regulate the activity of JNKs. Furthermore, because Rac1 and Cdc42 but not Rho bind and activate a kinase known as Pak1, it has been suggested that Pak1 is the most upstream component of the pathway linking these GTPases to JNK. However, in both yeast and mammalian cells, Rho1p, a Rho homologue, and RhoA, respectively, directly interact with a number of proteins, including kinases related to protein kinase C. In addition, in yeast, Rho1p controls the activity of a MAPK cascade involved in bud formation. Considering this diversity of target molecules for small GTP-binding proteins, their likely tissue specific distribution, and the potential role for Rho in signaling to a kinase cascade, we decided to extend our initial analysis, exploring the ability of Ras and Rho-related GTP-binding proteins to activate MAPK or JNK in a variety of cell lines. We found that in the human kidney epithelial cell line, 293T, Cdc42 and all Rho proteins, RhoA, RhoB, and RhoC, but not Rac or Ras can induce activation of JNK. Furthermore, we provide evidence that signaling from Rho proteins to JNK in 293T cells does not involve Pak1. Taken together these findings demonstrate that Rho signals to JNK in a cell type-specific manner and suggest the existence of a novel, Pak1-independent signaling route communicating the Rho family of small GTP-binding proteins to the JNK pathway.

• Best A, Ahmed S, Kozma R, Lim L
• The Ras-related GTPase Rac1 binds tubulin.
• J Biol Chem. 1996; 271: 3756-62
• Display abstract

• The Ras-related Rho family are involved in controlling actin-based changes in cell morphology. Microinjection of Rac1, RhoA, and Cdc42Hs into Swiss 3T3 cells induces pinocytosis and membrane ruffling, stress fiber formation, and filopodia formation, respectively. To identify target proteins involved in these signaling pathways cell extracts immobilized on nitrocellulose have been probed with [gamma-32P]GTP-labeled Rac1, RhoA, and Cdc42Hs. We have identified two 55-kDa brain proteins which bind Rac1 but not RhoA or Cdc42Hs. These 55-kDa proteins were abundant, had pI values of around 5.5, and could be purified by Q-Sepharose chromatography. The characteristics on two-dimensional gel analysis suggested the proteins comprised alpha- and beta-tubulin. Indeed, beta-tubulin specific antibodies detected one of the purified 55-kDa proteins. Rac1 bound pure tubulin (purified by cycles of polymerization and depolymerization) only in the GTP-bound state. The GTPase negative Rac1 point mutants, G12V and Q61L, did not significantly affect the ability of Rac1 to interact with tubulin while the "effector-site" mutant D38A prevented interaction. These results suggest that the Rac1-tubulin interaction may play a role in Rac1 function.

• Freeman JL, Abo A, Lambeth JD
• Rac "insert region" is a novel effector region that is implicated in the activation of NADPH oxidase, but not PAK65.
• J Biol Chem. 1996; 271: 19794-801
• Display abstract

• The small GTPase Rac assembles with the cytosolic p47(phox) and p67(phox) and the membrane-associated flavocytochrome b558 to form the multicomponent respiratory burst oxidase. Mutation of amino acids in a region of Rac (residues 26-45), homologous to an effector region in Ras, was previously shown to interfere with Rac binding to the oxidase. Herein we have elucidated an additional region in Rac involved in regulating oxidase activity. Rho family small GTPases contain a 12-amino acid "insert" region (residues 124-135) that is not present in Ras. Point mutations in and deletion of this region were constructed and used for in vitro studies of the activation of PAK65 and NADPH oxidase. Apparent binding constants (based on EC50 values) of the mutant Rac proteins for the oxidase are at least 13-25-fold higher than for wild-type Rac. Mutations in the insert region versus the 26-45 effector region resulted in distinct kinetic consequences, pointing to different roles for these two protein regions: mutations in the insert region but not the 26-45 effector region resulted in an increase in the EC50 for p67(phox). Although mutations in the 26-45 amino acid effector region showed markedly diminished activation of both PAK and the NADPH oxidase, insert region mutations did not affect activation of PAK. We propose that the combinatorial use of the 26-45 effector region and the insert region provides the Rho family GTPases with versatility in their specificity for several downstream targets.

• Akada R, Kallal L, Johnson DI, Kurjan J
• Genetic relationships between the G protein beta gamma complex, Ste5p, Ste20p and Cdc42p: investigation of effector roles in the yeast pheromone response pathway.
• Genetics. 1996; 143: 103-17
• Display abstract

• The Saccharomyces cerevisiae G protein beta gamma dimer, Ste4p/Ste18p, acts downstream of the alpha subunit, Gpa1p, to activate the pheromone response pathway and therefore must interact with a downstream effector. Synthetic sterile mutants that exacerbate the phenotype of ste4-ts mutations were isolated to identify proteins that functionally interact with Ste4p. The identification of a ste18 mutant indicated that this screen could identify proteins that interact directly with Ste4p. The other mutations were in STE5 and the STE20 kinase gene, which act near Ste4p in the pathway, and a new gene called STE21. ste20 null mutants showed residual mating, suggesting that another kinase may provide some function. Overexpression of Ste5p under galactose control activated the pheromone response pathway. This activation was dependent on Ste4p and Ste18p and partially dependent on Ste20p. These results cannot be explained by the linear pathway of Ste4p-->Ste20p-->Ste5p. Overexpression of Cdc42p resulted in a slight increase in pheromone induction of a reporter gene, and overexpression of activated forms of Cdc42p resulted in a further twofold increase. Mutations in pheromone response pathway components did not suppress the lethality associated with the activated CDC42 mutations, suggesting that this effect is independent of the pheromone response pathway.

• Lu X et al.
• CDC42 and Rac1 are implicated in the activation of the Nef-associated kinase and replication of HIV-1.
• Curr Biol. 1996; 6: 1677-84
• Display abstract

• BACKGROUND: The negative factor (Nef) of human and simian immunodeficiency viruses (HIV-1, HIV-2 and SIV) is required for high levels of viremia and progression to AIDS. Additionally, Nef leads to cellular activation, increased viral infectivity and decreased expression of CD4 on the cell surface. Previously, we and others demonstrated that Nef associates with a cellular serine kinase (NAK) activity. Recently, it was demonstrated that NAK bears structural and functional similarity to p21-activated kinases (PAKs). RESULTS: In this study, we demonstrate that Nef not only binds to but also activates NAK via the small GTPases CDC42 and Rac1. First, the dominant-negative PAK (PAKR), via its GTPase-binding domain, and dominant-negative GTPases (CDC42Hs-N17 and Rac1-N17) block the ability of Nef to associate with and activate NAK. Second, constitutively active small GTPases (CDC42Hs-V12 and Rac1-V12) potentiate the effects of Nef. Third, interactions between Nef and NAK result in several cellular effector functions, such as activation of the serum-response pathway. And finally, PAKR, CDC42Hs-N17 and Rac1-N17 decrease levels of HIV-1 production to those of virus from which the nef gene is deleted. CONCLUSIONS: By activating NAK via small GTPases and their downstream effectors, Nef interacts with regulatory pathways required for cell growth, cytoskeletal rearrangement and endocytosis. Thus, NAK could participate in the budding of new virions, the modification of viral proteins and the increased endocytosis of surface molecules such as CD4. Moreover, blocking the activity of these GTPases could lead to new therapeutic interventions against AIDS.

• Lamarche N et al.
• Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade.
• Cell. 1996; 87: 519-29
• Display abstract

• Rac and Cdc42 regulate a variety of responses in mammalian cells including formation of lamellipodia and filopodia, activation of the JNK MAP kinase cascade, and induction of G1 cell cycle progression. Rac is also one of the downstream targets required for Ras-induced malignant transformation. Rac and Cdc42 containing a Y40C effector site substitution no longer intact with the Ser/Thr kinase p65PAK and are unable to activate the JNK MAP kinase pathway. However, they still induce cytoskeletal changes and G1 cell cycle progression. Rac containing an F37A effector site substitution, on the other hand, no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. We conclude that Rac and Cdc42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets.

• Harden N et al.
• A Drosophila homolog of the Rac- and Cdc42-activated serine/threonine kinase PAK is a potential focal adhesion and focal complex protein that colocalizes with dynamic actin structures.
• Mol Cell Biol. 1996; 16: 1896-908
• Display abstract

• Changes in cell morphology are essential in the development of a multicellular organism. The regulation of the cytoskeleton by the Rho subfamily of small GTP-binding proteins is an important determinant of cell shape. The Rho subfamily has been shown to participate in a variety of morphogenetic processes during Drosophila melanogaster development. We describe here a Drosophila homolog, DPAK, of the serine/threonine kinase PAK, a protein which is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK bound to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK was elevated in the leading edge of epidermal cells, whose morphological changes drive dorsal closure of the embryo. We have previously shown that the accumulation of cytoskeletal elements initiating cell shape changes in these cells could be inhibited by expression of a dominant-negative DRacA transgene. We show that leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. We propose that DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure.

• Featherstone C
• How does one gene cause Wiskott-Aldrich syndrome?
• Lancet. 1996; 348: 950-950

• Joneson T, McDonough M, Bar-Sagi D, Van Aelst L
• RAC regulation of actin polymerization and proliferation by a pathway distinct from Jun kinase.
• Science. 1996; 274: 1374-6
• Display abstract

• The RAC guanine nucleotide binding proteins regulate multiple biological activities, including actin polymerization, activation of the Jun kinase (JNK) cascade, and cell proliferation. RAC effector loop mutants were identified that separate the ability of RAC to interact with different downstream effectors. One mutant of activated human RAC protein, RACV12H40 (with valine and histidine substituted at position 12 and 40, respectively), was defective in binding to PAK3, a Ste20-related p21-activated kinase (PAK), but bound to POR1, a RAC-binding protein. This mutant failed to stimulate PAK and JNK activity but still induced membrane ruffling and mediated transformation. A second mutant, RACV12L37 (with leucine substituted at position 37), which bound PAK but not POR1, induced JNK activation but was defective in inducing membrane ruffling and transformation. These results indicate that the effects of RAC on the JNK cascade and on actin polymerization and cell proliferation are mediated by distinct effector pathways that diverge at the level of RAC itself.

• Brill S et al.
• The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases.
• Mol Cell Biol. 1996; 16: 4869-78
• Display abstract

• We previously described IQGAP1 as a human protein related to a putative Ras GTPase-activating protein (RasGAP) from the fission yeast Schizosaccharomyces pombe. Here we report the identification of a liver-specific human protein that is 62% identical to IQGAP1. Like IQGAP1, the novel IQGAP2 protein harbors an N-terminal calponin homology motif which functions as an F-actin binding domain in members of the spectrin, filamin, and fimbrin families. Both IQGAPs also harbor several copies of a novel 50- to 55-amino-acid repeat, a single WW domain, and four IQ motifs and have 25% sequence identity with almost the entire S. pombe sar1 RasGAP homolog. As predicted by the presence of IQ motifs, IQGAP2 binds calmodulin. However, neither full-length nor truncated IQGAP2 stimulated the GTPase activity of Ras or its close relatives. Instead, IQGAP2 binds Cdc42 and Racl but not RhoA. This interaction involves the C-terminal half of IQGAP2 and appears to be independent of the nucleotide binding status of the GTPases. Although IQGAP2 shows no GAP activity towards Cdc42 and Rac1, the protein did inhibit both the intrinsic and RhoGAP-stimulated GTP hydrolysis rates of Cdc42 and Rac1, suggesting an alternative mechanism via which IQGAPs might modulate signaling by these GTPases. Since IQGAPs harbor a potential actin binding domain, they could play roles in the Cdc42 and Rac1 controlled generation of specific actin structures.

• Ohya Y et al.
• Mutational analysis of the beta-subunit of yeast geranylgeranyl transferase I.
• Mol Gen Genet. 1996; 252: 1-10
• Display abstract

• The gene CAL1 (also known as CDC43) of Saccharomyces cerevisiae encodes the beta subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded by cal1-1, and cdc43-2 to cdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of the cal1/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. The cal1-1 mutation, located most proximal to the C-terminus of the protein, differs from the other cdc43 mutations in several respects. An increase in soluble Rho1p was observed in the cal1-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype of cal1-1 is most efficiently suppressed by overproduction of Rho1p. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious in cal1-1 cells, but not in other cdc43 mutants or the wild-type strains. The cdc43-5 mutant cells accumulate Cdc42p in soluble pools and cdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among the cal1/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.

• Peter M, Neiman AM, Park HO, van Lohuizen M, Herskowitz I
• Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast.
• EMBO J. 1996; 15: 7046-59
• Display abstract

• STE20 encodes a protein kinase related to mammalian p65Pak which functions in several signal transduction pathways in yeast, including those involved in pseudohyphal and invasive growth, as well as mating. In addition, Ste20 plays an essential role in cells lacking Cla4, a kinase with significant homology to Ste20. It is not clear how the activity of Ste20 is regulated in response to these different signals in vivo, but it has been demonstrated recently that binding of the small GTP binding protein Cdc42 is able to activate Ste20 in vitro. Here we show that Ste20 functionally interacts with Cdc42 in a GTP-dependent manner in vivo: Ste20 mutants that can no longer bind Cdc42 were unable to restore growth of ste20 cla4 mutant cells. They were also defective for pseudohyphal growth and agar invasion, and displayed reduced mating efficiency when mated with themselves. Surprisingly, however, the kinase activity of such Ste20 mutants was normal when assayed in vitro. Furthermore, these alleles were able to fully activate the MAP kinase pathway triggered by mating pheromones in vivo, suggesting that binding of Cdc42 and Ste20 was not required to activate Ste20. Wild-type Ste20 protein was visualized as a crescent at emerging buds during vegetative growth and at shmoo tips in cells arrested with alpha-factor. In contrast, a Ste20 mutant protein unable to bind Cdc42 was found diffusely throughout the cytoplasm, suggesting that Cdc42 is required to localize Ste20 properly in vivo.

• Bender L, Lo HS, Lee H, Kokojan V, Peterson V, Bender A
• Associations among PH and SH3 domain-containing proteins and Rho-type GTPases in Yeast.
• J Cell Biol. 1996; 133: 879-94
• Display abstract

• The src homology region 3 (SH3) domain-bearing protein Bem1p and the Rho-type GTPase Cdc42p are important for bud emergence in Saccharomyces cervisiae. Here, we present evidence that through its second SH3 domain, Bem1p binds to the structurally and functionally similar proteins Boi1p and Boi2p, each of which contain an SH3 and pleckstrin homology (PH) domain. Deletion of BOI1 and BO12 together leads to impaired morphogenesis and poor ability. A PH domain-bearing segment of Boi1p that lacks the Bem1p-binding site is necessary and sufficient for function. This segment of Boi1p displays a two-hybrid interaction with Cdc42p, suggesting that Boi1p either binds directly to or is part of a larger complex that contains Cdc42p. Consistent with these possibilities, overexpression of Boi1p inhibits bud emergence, but this inhibition is counteracted by cooverexpression of Cdc42p. Increased expression of the Rho-type GTPase Rho3p, which is implicated in bud growth defects of boil boi2 mutants, suggesting that Boi1p and Boi2p may also play roles in the activation or function of Rho3p. These findings provide an example of a tight coupling in function between PH domain-bearing proteins and both Rho-type GTPases and SH3 domain-containing proteins, and they raise the possibility that Boi1p and Boi2 play a role in linking the actions of Cdc42p and Rho3p.

• Lim L, Manser E, Leung T, Hall C
• Regulation of phosphorylation pathways by p21 GTPases. The p21 Ras-related Rho subfamily and its role in phosphorylation signalling pathways.
• Eur J Biochem. 1996; 242: 171-85
• Display abstract

• The oncogenic Ras p21 GTPases regulate phosphorylation pathways that underlie a wealth of activities, including growth and differentiation, in organisms ranging from yeast to human. In metazoa, growth factors trigger conversion of Ras from an inactive GDP-bound form to an active GTP-bound form. This activation of Ras leads to activation of Raf. Raf is one of the initial kinases in the cytoplasmic mitogen-activated protein kinase (MAPK) cascade, involving extracellular-signal-regulated kinases (ERK), which culminates in nuclear transcription. The Ras-related subfamily of Rho p21s, including Rho, Rac and Cdc42 are similarly active in their GTP-bound forms. These p21s mediate growth-factor-induced morphological changes involving actin-based cellular structures. For example, in mammalian fibroblasts, Rho mediates the formation of cytoskeletal stress fibres induced by lysophosphatidic acid, while Rac mediates the formation of membrane ruffles induced by platelet-derived growth factor, and Cdc42 mediates the formation of peripheral filopodia by bradykinin. In some cases, factor-induced Rac activation results in Rho activation, and factor-induced Cdc42 activation leads to Rac activation, as determined by specific morphological changes. Although separate Cdc42/Rac and Rac/Rho hierarchies exist, these might not extend into a linear form (i.e. Cdc42-->Rac-->Rho) since Cdc42 and Rho activities may be competitive or even antagonistic. Thus Cdc42-mediated formation of filopodia is accompanied by loss of stress fibres (whose formation is mediated by Rho). Recently, mammalian kinases that bind to the GTP-bound forms of Rho p21s have been isolated. These kinases include the p21-activated serine/threonine kinase (PAK), which is stimulated by binding to Cdc42 and Rac, and the Rho-binding serine/threonine kinase (ROK), which is not as strongly stimulated by binding. These kinases act as effectors for their p21 partners since they can directly affect the reorganization of the relevant actin-containing structures. ROK promotes the formation of Rho-induced actin-containing stress fibres and focal-adhesion complexes, to which the ends of the stress fibres attach. PAK stimulates the disassembly of stress fibres, which has been shown to accompany formation of Cdc42-induced peripheral-actin-containing structures, including filopodia, which with Rac-induced membrane ruffles play a role in cell movement. PAK also fosters loss of focal-adhesion complexes. Thus, there is cooperation between different Rho p21s as well as antagonism, with their associated kinases having a role in the integration of the reorganization of the actin cytoskeleton. The similarity of PAK to the Saccharomyces cerevisiae kinase Ste20p, which initiates the yeast mating/pheromone MAPK cascade, led to experiments showing that Cdc42 regulates Ste20p in this MAPK pathway. This similarity has also led to the demonstration that mammalian Cdc42 and Rac can signal to the nucleus through MAPK pathways. However, c-Jun N-terminal kinase (JNK, stress-activated protein kinase) rather than ERK, is involved. PAK have been implicated in the JNK pathway, but their exact roles are uncertain. Thus members of the Rho subfamily, and kinases that bind to these p21s are intimately involved in immediate morphological processes as well as long-term transcriptional events.

• Kolluri R, Tolias KF, Carpenter CL, Rosen FS, Kirchhausen T
• Direct interaction of the Wiskott-Aldrich syndrome protein with the GTPase Cdc42.
• Proc Natl Acad Sci U S A. 1996; 93: 5615-8
• Display abstract

• Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder with the most severe pathology in the T lymphocytes and platelets. The disease arises from mutations in the gene encoding the WAS protein. T lymphocytes of affected males with WAS exhibit a severe disturbance of the actin cytoskeleton, suggesting that the WAS protein could regulate its organization. We show here that WAS protein interacts with a member of the Rho family of GTPases, Cdc42. This interaction, which is guanosine 5'-triphosphate (GTP)-dependent, was detected in cell lysates, in transient transfections and with purified recombinant proteins. A weaker interaction was also detected with Rac1 using WAS protein from cell lysates. It was also found that different mutant WAS proteins from three affected males retained their ability to interact with Cdc42 and that the level of expression of the WAS protein in these mutants was only 2-5% of normal. Taken together these data suggest that the WAS protein might function as a signal transduction adaptor downstream of Cdc42, and in affected males, the cytoskeletal abnormalities may result from a defect in Cdc42 signaling.

• Ridley AJ
• Rho: theme and variations.
• Curr Biol. 1996; 6: 1256-64
• Display abstract

• In addition to their roles in organizing the actin cytoskeleton, members of the Rho family of GTP-binding proteins have recently been implicated in a plethora of other functions, including the activation of kinase cascades and transcription factors, and the control of endocytosis and secretion. Alongside this expansion is proposed functions has been the identification of multiple target proteins that interact directly with Rho, Rac or Cdc42. Molecular connections are now being made along the signalling pathways activated by members of the Rho family.

• Nomanbhoy TK, Leonard DA, Manor D, Cerione RA
• Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using extrinsic reporter group fluorescence.
• Biochemistry. 1996; 35: 4602-8
• Display abstract

• The overall goal of these studies was to examine the applicability of extrinsic reporter group fluorescence in monitoring the GTP-binding/GTPase cycle of a Ras-like GTP-binding protein. Toward this end, we have labeled the GTP-binding protein Cdc42Hs with the environmentally sensitive fluorophore succinimidyl 6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoate (sNBD) at a single reactive lysine residue. We find that the sNBD-labeled Cdc42Hs undergoes a fluorescence enhancement at 545 nm when Cdc42Hs exchanges bound GDP for GTP. This enhancement is then fully reversed upon GTP hydrolysis. The specific GTPase-activating protein for Cdc42Hs, the Cdc42Hs-GAP, strongly stimulates the rate of reversal of the fluorescence enhancement at 545 nm, consistent with its ability to fully catalyze the GTPase reaction of Cdc42Hs. Conversely, the specific guanine nucleotide exchange factor (GEF), Cdc24, strongly stimulates the fluorescence enhancement that accompanies GTP binding, consistent with its ability to stimulate the GDP-GTP exchange reaction on Cdc42Hs. Resonance energy transfer measurements yielded a distance of approximately 32 A for the sNBD moiety and the guanine nucleotide binding site occupied with either N-methylanthraniloyl- (Mant) dGDP or MantdGTP. Taken together, these results identify a conformationally sensitive reporter site on the Cdc42Hs molecule that is located some distance away from the guanine nucleotide binding site but nonetheless provides a highly sensitive monitor for GTP-binding, GTPase activity, and the interactions of key regulatory proteins.

• Kamada Y, Qadota H, Python CP, Anraku Y, Ohya Y, Levin DE
• Activation of yeast protein kinase C by Rho1 GTPase.
• J Biol Chem. 1996; 271: 9193-6
• Display abstract

• We have investigated the role of the essential Rho1 GTPase in cell integrity signaling in budding yeast. Conditional rho1 mutants display a cell lysis defect that is similar to that of mutants in the cell integrity signaling pathway mediated by protein kinase C (Pkc1), which is suppressed by overexpression of Pkc1.rho1 mutants are also impaired in pathway activation in response to growth at elevated temperature. Pkc1 co-immunoprecipitates with Rho1 in yeast extracts, and recombinant Rho1 associates with Pkc1 in vitro in a GTP-dependent manner. Recombinant Rho1 confers upon Pkc1 the ability to be stimulated by phosphatidylserine, indicating that Rho1 controls signal transmission through Pkc1.

• Chou MM, Blenis J
• The 70 kDa S6 kinase complexes with and is activated by the Rho family G proteins Cdc42 and Rac1.
• Cell. 1996; 85: 573-83
• Display abstract

• The 70 kDa ribosomol S6 kinase (pp70S6k) plays an important role in the progression of cells through G1 phase of the cell cycle. However, little is known of the signaling molecules that mediate its activation. We demonstrate that Rho family G proteins regulate pp70S6k activity in vivo. Activated alleles of Cdc42 and Rac1, but not RhoA, stimulate pp70S6k activity in multiple cell types. Activation requires an intact effector domain and isoprenylation of Cdc42 and Rac1. Coexpression of Dbl, an exchange factor for Cdc42, also activates pp70S6k. Growth factor-induced activation of pp70S6k is abrogated by dominant negative alleles of Cdc42 and Rac1. In addition, Cdc42 and Rac1 form GTP-dependent complex with the catalytically inactive form of pp70S6k in vitro and in vivo, suggesting a mechanism by which these G proteins activate pp70S6k.

• Hotta K, Tanaka K, Mino A, Kohno H, Takai Y
• Interaction of the Rho family small G proteins with kinectin, an anchoring protein of kinesin motor.
• Biochem Biophys Res Commun. 1996; 225: 69-74
• Display abstract

• The Rho family small G proteins are implicated in various cell functions, such as cell morphological change, cell motility, and cytokinesis. However, their modes of action in regulating these cell functions remain to be clarified. In the present study, we have isolated a cDNA encoding a protein which interacts with the GTP-bound form, but not with the GDP-bound form, of the Rho family members, including RhoA, Racl, and Cdc42, by the yeast two-hybrid method. This protein is kinectin, known to be a vesicle membrane anchoring protein of kinesin, which is an ATPase motor transporting vesicles along microtubules.

• Leberer E, Chenevert J, Leeuw T, Harcus D, Herskowitz I, Thomas DY
• Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity.
• Mol Gen Genet. 1996; 252: 608-21
• Display abstract

• The pheromone signal in the yeast Saccharomyces cerevisiae is transmitted by the beta and gamma subunits of the mating response G-protein. The STE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G beta mutation. The same genetic screen identified BEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designated MDG1 (multicopy suppressor of defective G-protein). The MDG1 gene was independently isolated in a search for multicopy suppressors of a bem1 mutation. The MDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein from Aequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion of MDG1 causes sterility in cells in which the wild-type G beta has been replaced by partly defective G beta derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted for STE20 is partially suppressed by multiple copies of BEM1 and CDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels of STE20 and BEM1 are capable of suppressing a temperature-sensitive mutation in CDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.

• Rooney RD et al.
• Cleavage arrest of early frog embryos by the G protein-activated protein kinase PAK I.
• J Biol Chem. 1996; 271: 21498-504
• Display abstract

• PAK I is a member of the PAK (p21-activated protein kinase) family and is activated by Cdc42 (Jakobi, R., Chen, C.-J., Tuazon, P. T., and Traugh, J. A. (1996) J. Biol. Chem. 271, 6206-6211). To examine the effects of PAK I on cleavage arrest, subfemtomole amounts of endogenously active (58 kDa) and inactive (60 kDa) PAK I and a tryptic peptide (37 kDa) containing the active catalytic domain were injected into one blastomere of 2-cell frog embryos. Active PAK I resulted in cleavage arrest in the injected blastomere at mitotic metaphase, whereas the uninjected blastomere progressed through mid- to late cleavage. Injection of other protein kinases at similar concentrations had no effect on cleavage. Endogenous PAK I was highly active in frog oocytes, and antibody to PAK I reacted specifically with protein of 58-60 kDa. PAK I protein was decreased at 60 min post-fertilization, with little or no PAK I protein or activity detectable at 80 min post-fertilization or in 2-cell embryos. At the 4-cell stage PAK I protein increased, but the protein kinase was present primarily as an inactive form. Rac2 and Cdc42, but not Rac 1, were identified in oocytes and throughout early embryo development. Thus, PAK I appears to be a potent cytostatic protein kinase involved in maintaining cells in a non-dividing state. PAK I activity is high in oocytes and appears to be regulated by degradation/synthesis and through autophosphorylation via binding of Cdc42. PAK I may act through regulation of the stress-activated protein kinase signaling pathway and/or by direct regulation of multiple metabolic pathways.

• Mosch HU, Roberts RL, Fink GR
• Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.
• Proc Natl Acad Sci U S A. 1996; 93: 5352-6
• Display abstract

• RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology.

• Lee SF, Egelhoff TT, Mahasneh A, Cote GP
• Cloning and characterization of a Dictyostelium myosin I heavy chain kinase activated by Cdc42 and Rac.
• J Biol Chem. 1996; 271: 27044-8
• Display abstract

• The motile activities of the small, single-headed class I myosins (myosin I) from the lower eukaryotes Acanthamoeba and Dictyostelium are activated by phosphorylation of a single serine or threonine residue in the head domain of the heavy chain. Recently, we purified a myosin I heavy chain kinase (MIHCK) from Dictyostelium based on its ability to activate the Dictyostelium myosin ID isozyme (Lee, S. -F., and Cote, G. P. (1995) J. Biol. Chem. 270, 11776-11782). The complete sequence of the Dictyostelium MIHCK has now been determined, revealing a protein of 98 kDa that is composed of an amino-terminal domain rich in proline, glutamine, and serine, a putative Cdc42/Rac binding motif, and a carboxyl-terminal kinase catalytic domain. MIHCK shares significant sequence identity with the Saccharomyces cerevisiae Ste20p kinase and the mammalian p21-activated kinase. Gel overlay assays and affinity chromatography experiments showed that MIHCK interacted with GTPgammaS (guanosine 5'-3-O-(thiotriphosphate))-labeled Cdc42 and Rac1 but not RhoA. In the presence of GTPgammaS-Rac1 MIHCK autophosphorylation increased from 1 to 9 mol of phosphate/mol, and the rate of Dictyostelium myosin ID phosphorylation was stimulated 10-fold. MIHCK may therefore provide a direct link between Cdc42/Rac signaling pathways and motile processes driven by myosin I molecules.

• Kuroda S et al.
• Identification of IQGAP as a putative target for the small GTPases, Cdc42 and Rac1.
• J Biol Chem. 1996; 271: 23363-7
• Display abstract

• Cdc42 and Rac1 have been implicated in the regulation of various cell functions such as cell morphology, polarity, and cell proliferation. We have partially purified a Cdc42- and Rac1-associated protein with molecular mass of about 170 kDa (p170) from bovine brain cytosol. This protein interacted with guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).glutathione S-transferase (GST)-Cdc42 and GTPgammaS++.GST-Rac1 but not with the GDP.GST-Cdc42, GDP.GST-Rac1, or GTPgammaS.GST-RhoA). We identified p170 as an IQGAP, which is originally identified as a putative Ras GTPase-activating protein. Recombinant IQGAP specifically interacted with GTPgammaS.Cdc42 and GTPgammaS.Rac1. The C-terminal fragment of IQGAP was responsible for their interactions. IQGAP was specifically immunoprecipitated with dominant-active Cdc42(Val12) or Rac1(Val12) from the COS7 cells expressing Cdc42(Val12) or Rac1(Val12), respectively. Immunofluorescence analysis revealed that IQGAP was accumulated at insulin- or Rac1-induced membrane ruffling areas. This accumulation of IQGAP was blocked by the microinjection of the dominant-negative Rac1(Asn17) or Cdc42(Asn17). Moreover, IQGAP was accumulated at the cell-cell junction in MDCK cells, where alpha-catenin and ZO-1 were localized. These results suggest that IQGAP is a novel target molecule for Cdc42 and Rac1.

• Hu MC, Qiu WR, Wang X, Meyer CF, Tan TH
• Human HPK1, a novel human hematopoietic progenitor kinase that activates the JNK/SAPK kinase cascade.
• Genes Dev. 1996; 10: 2251-64
• Display abstract

• The c-Jun amino-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs) play a crucial role in stress responses in mammalian cells. The mechanism underlying this pathway in the hematopoietic system is unclear, but it is a key in understanding the molecular basis of blood cell differentiation. We have cloned a novel protein kinase, termed hematopoietic progenitor kinase 1 (HPK1), that is expressed predominantly in hematopoietic cells, including early progenitor cells. HPK1 is related distantly to the p21(Cdc42/Rac1)-activated kinase (PAK) and yeast STE20 implicated in the mitogen-activated protein kinase (MAPK) cascade. Expression of HPK1 activates JNK1 specifically, and it elevates strongly AP-1-mediated transcriptional activity in vivo. HPK1 binds and phosphorylates MEKK1 directly, whereas JNK1 activation by HPK1 is inhibited by a dominant-negative MEKK1 or MKK4/SEK mutant. Interestingly, unlike PAK65, HPK1 does not contain the small GTPase Rac1/Cdc42-binding domain and does not bind to either Rac1 or Cdc42, suggesting that HPK1. activation is Rac1/Cdc42-independent. These results indicate that HPK1 is a novel functional activator of the JNK/SAPK signaling pathway.

• Stevenson BJ et al.
• Mutation of RGA1, which encodes a putative GTPase-activating protein for the polarity-establishment protein Cdc42p, activates the pheromone-response pathway in the yeast Saccharomyces cerevisiae.
• Genes Dev. 1995; 9: 2949-63
• Display abstract

• We have selected yeast mutants that exhibit a constitutively active pheromone-response pathway in the absence of the beta subunit of the trimeric G protein. Genetic analysis of one such mutant revealed that it contained recessive mutations in two distinct genes, both of which contributed to the constitutive phenotype. One mutation identifies the RGA1 locus (Rho GTPase activating protein), which encodes a protein with homology to GAP domains and to LIM domains. Deletion of RGA1 is sufficient to activate the pathway in strains lacking the G beta subunit. Moreover, in wild-type strains, deletion of RGA1 increases signaling in the pheromone pathway, whereas over-expression of RGA1 dampens signaling, demonstrating that Rga1p functions as a negative regulator of the pheromone response pathway. The second mutation present in the original mutant proved to be an allele of a known gene, PBS2, which encodes a putative protein kinase that functions in the high osmolarity stress pathway. The pbs2 mutation enhanced the rga1 mutant phenotype, but by itself did not activate the pheromone pathway. Genetic and two-hybrid analyses indicate that an important target of Rga1p is Cdc42p, a p21 GTPase required for polarity establishment and bud emergence. This finding coupled with recent experiments with mammalian and yeast cells indicating that Cdc42p can interact with and activate Ste20p, a protein kinase that operates in the pheromone pathway, leads us to suggest that Rga1p controls the activity of Cdc42p, which in turn controls the magnitude of signaling in the pheromone pathway via Ste20p.

• Cantor SB, Urano T, Feig LA
• Identification and characterization of Ral-binding protein 1, a potential downstream target of Ral GTPases.
• Mol Cell Biol. 1995; 15: 4578-84
• Display abstract

• Ral proteins constitute a distinct family of Ras-related GTPases. Although similar to Ras in amino acid sequence, Ral proteins are activated by a unique nucleotide exchange factor and inactivated by a distinct GTPase-activating protein. Unlike Ras, they fail to promote transformed foci when activated versions are expressed in cells. To identify downstream targets that might mediate a Ral-specific function, we used a Saccharomyces cerevisiae-based interaction assay to clone a novel cDNA that encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically to the active GTP-bound form of RalA and not to a mutant Ral with a point mutation in its putative effector domain. In addition to a Ral-binding domain, RalBP1 also contains a Rho-GTPase-activating protein domain that interacts preferentially with Rho family member CDC42. Since CDC42 has been implicated in bud site selection in S. cerevisiae and filopodium formation in mammalian cells, Ral may function to modulate the actin cytoskeleton through its interactions with RalBP1.

• Leung T, Manser E, Tan L, Lim L
• A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes.
• J Biol Chem. 1995; 270: 29051-4
• Display abstract

• We previously reported the cloning of a serine/threonine kinase, PAK (for p21 (Cdc42/Rac)-activated kinase), which binds to the Ras-related GTPases Cdc42Hs and Rac1 (Manser, E., Leung, T., Salihuddin, H., Zhao, Z-s., and Lim, L. (1994) Nature 367, 40-46). These p21 proteins together with RhoA comprise the Rho subfamily of proteins that are involved in morphological events. We now report the isolation of a rat cDNA encoding a 150-kDa protein, which specifically binds RhoA in its GTP form and contains an N-terminal serine/threonine kinase domain highly related to the human myotonic dystrophy kinase and a cysteine-rich domain toward the C terminus. The RhoA binding domain is unrelated to other p21 binding domains. Antibody raised against the kinase domain of the predicted protein, termed ROK alpha (for ROK alpha, RhoA-binding kinase), recognized a ubiquitous 150-kDa protein. The brain p150 purified by affinity chromatography with RhoA exhibited serine/threonine kinase activity. In cultured cells, immunoreactive p150 was recruited to membranes upon transfection with dominant positive RhoAV14 mutant and was localized with actin microfilaments at the cell periphery. These results are consistent with a role for the kinase ROK alpha as an effector for RhoA.

• Polverino A et al.
• Activation of mitogen-activated protein kinase cascades by p21-activated protein kinases in cell-free extracts of Xenopus oocytes.
• J Biol Chem. 1995; 270: 26067-70
• Display abstract

• In the evolutionarily distant yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, genetic evidence suggests that activation of pheromone-induced mitogen-activated protein kinase (MAPK) cascades involves the function of the p21cdc42/racl-activated protein kinases (PAKs) Ste20 and Shk1, respectively. In this report, we show that purified Ste20 and Shk1 were each capable of inducing p42MAPK activation in cell-free extracts of Xenopus laevis oocytes, while a mammalian Ste20/Shk1-related protein kinase, p65pak (Pak1), did not induce activation of p42MAPK. In contrast to p42MAPK, activation of JNK/SAPK in Xenopus oocyte extracts was induced by both the yeast Ste20 and Shk1 kinases, as well as by mammalian Pak1. Our results demonstrate that MAPK cascades that are responsive to PAKs are conserved in higher eukaryotes and suggest that distinct PAKs may regulate distinct MAPK modules.

• Teo M, Manser E, Lim L
• Identification and molecular cloning of a p21cdc42/rac1-activated serine/threonine kinase that is rapidly activated by thrombin in platelets.
• J Biol Chem. 1995; 270: 26690-7
• Display abstract

• The brain-enriched p21cdc42/rac1-activated serine/threonine kinase, p65PAK, was identified and purified on the basis of overlays with [gamma-32P]GTP-Cdc42 onto SDS-fractionated proteins (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). In this study, the ubiquitously expressed p21cdc42/rac1 binding protein with relative molecular weight of 62,000 was purified from rat testes and shown to contain peptides related to PAK. It has thus been designated as the gamma-PAK isoform (alpha- and beta-isoforms being brain enriched). Isolation of gamma-PAK cDNAs show that the kinase is highly conserved with alpha-PAK in both the p2 binding and kinase domains. The purified protein exhibited kinase activity that was activated by GTP-Cdc42 or GTP-Rac1 in vitro. In platelets, a p62 in situ renaturable kinase was recognized by antibodies raised against gamma-PAK. This thrombin-activated protein kinase appears to coprecipitate with another kinase of M(r) 86,000, suggesting that PAK may be part of a thrombin-responsive signaling complex.

• Marcus S, Polverino A, Chang E, Robbins D, Cobb MH, Wigler MH
• Shk1, a homolog of the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases, is a component of a Ras/Cdc42 signaling module in the fission yeast Schizosaccharomyces pombe.
• Proc Natl Acad Sci U S A. 1995; 92: 6180-4
• Display abstract

• We describe a protein kinase, Shk1, from the fission yeast Schizosaccharomyces pombe, which is structurally related to the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases. We provide genetic evidence for physical and functional interaction between Shk1 and the Cdc42 GTP-binding protein required for normal cell morphology and mating in S. pombe. We further show that expression of the STE20 gene complements the shk1 null mutation and that Shk1 is capable of signaling to the pheromone-responsive mitogen-activated protein kinase cascade in S. cerevisiae. Our results lead us to propose that signaling modules composed of small GTP-binding proteins and protein kinases related to Shk1, Ste20, and p65PAK, are highly conserved in evolution and participate in both cytoskeletal functions and mitogen-activated protein kinase signaling pathways.

• Kwong CH, Adams AG, Leto TL
• Characterization of the effector-specifying domain of Rac involved in NADPH oxidase activation.
• J Biol Chem. 1995; 270: 19868-72
• Display abstract

• Production of microbicidal oxidants by phagocytic leukocytes requires activation of a latent NADPH oxidase by the coordinated assembly of a membrane-associated flavocytochrome b558, with three cytosolic components, p47phox, p67phox, and the low molecular weight GTP-binding protein Rac. Rac1 and Rac2 have 92% sequence identity and are both active in supporting the oxidase, while CDC42Hs, the closest relative to Rac with 70% sequence identity, only weakly supports oxidase activation in vitro. We have used CDC42Hs as a foil to identify residues in Rac that are critical for oxidase activation. Most of the divergent sequences of CDC42Hs could be incorporated into Rac-CDC42Hs chimeric proteins without affecting cell-free NADPH oxidase activity. However, incorporation of the amino-terminal segment of CDC42Hs (residues 1-40), which differs from Rac1 by only four residues (positions 3, 27, 30, and 33), resulted in a marked loss of oxidase activation capacity. Point mutagenesis studies showed that this was due to changes at residues 27 and 30, but not residues 3 and 33. Conversely, incorporation of the amino terminus of Rac1 (residues 1-40) into CDC42Hs increased its activity to that of Rac1, indicating that this terminus contains the effector-specifying domain of Rac. Taken together, these studies show that the difference in the activity between CDC42Hs and Rac1 is due entirely to differences in amino acids at position 27 and 30.

• Coso OA et al.
• The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway.
• Cell. 1995; 81: 1137-46
• Display abstract

• c-Jun amino-terminal kinases (JNKs) and mitogen-activated protein kinases (MAPKs) are closely related; however, they are independently regulated by a variety of environmental stimuli. Although molecules linking growth factor receptors to MAPKs have been recently identified, little is known about pathways controlling JNK activation. Here, we show that in COS-7 cells, activated Ras effectively stimulates MAPK but poorly induces JNK activity. In contrast, mutationally activated Rac1 and Cdc42 GTPases potently activate JNK without affecting MAPK, and oncogenic guanine nucleotide exchange factors for these Rho-like proteins selectively stimulate JNK activity. Furthermore, expression of inhibitory molecules for Rho-related GTPases and dominant negative mutants of Rac1 and Cdc42 block JNK activation by oncogenic exchange factors or after induction by inflammatory cytokines and growth factors. Taken together, these findings strongly support a critical role for Rac1 and Cdc42 in controlling the JNK signaling pathway.

• Hill CS, Wynne J, Treisman R
• The Rho family GTPases RhoA, Rac1, and CDC42Hs regulate transcriptional activation by SRF.
• Cell. 1995; 81: 1159-70
• Display abstract

• The c-fos serum response element (SRE) forms a ternary complex with the transcription factors SRF (serum response factor) and TCF (ternary complex factor). By itself, SRF can mediate transcriptional activation induced by serum, lysophosphatidic acid, or intracellular activation of heterotrimeric G proteins. Activated forms of the Rho family GTPases RhoA, Rac1, and CDC42Hs also activate transcription via SRF and act synergistically at the SRE with signals that activate TCF. Functional Rho is required for signaling to SRF by several stimuli, but not by activated CDC42Hs or Rac1. Activation of the SRF-linked signaling pathway does not correlate with activation of the MAP kinases ERK, SAPK/JNK, or MPK2/p38. Functional Rho is required for regulated activity of the c-fos promoter. These results establish SRF as a nuclear target of a novel Rho-mediated signaling pathway.

• Zhao ZS, Leung T, Manser E, Lim L
• Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24.
• Mol Cell Biol. 1995; 15: 5246-57
• Display abstract

• Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein beta gamma subunits. A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1. The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p. Cdc42p is required for alpha-factor-induced activation of FUS1.cdc24ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p12V mutants. Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway. The two-hybrid system revealed that Ste4p interacts with Cdc24p. We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling.

• Diekmann D, Nobes CD, Burbelo PD, Abo A, Hall A
• Rac GTPase interacts with GAPs and target proteins through multiple effector sites.
• EMBO J. 1995; 14: 5297-305
• Display abstract

• Rac, a small GTPase in the ras superfamily, regulates at least two biological processes in animal cells: (i) the polymerization of actin and the assembly of integrin complexes to produce lamellipodia and ruffles; and (ii) the activity of an NADPH oxidase in phagocytic cells. NADPH oxidase activation is mediated through a rac effector protein, p67phox, and using chimeras made between rac and the closely related GTPase, rho, we have identified two distinct effector sites in rac, one N-terminal and one C-terminal, both of which are required for activation of p67phox. The same two effector sites are essential for rac-induced actin polymerization in fibroblasts. p65PAK, a ubiquitous serine/threonine kinase, interacts with rac at both the N- and C-terminal effector sites, but the GTPase-activating protein, bcr interacts with rac at a different region. This makes p65PAK, but not bcr, a candidate effector of rac-induced lamellipodium formation.

• Martin GA, Bollag G, McCormick F, Abo A
• A novel serine kinase activated by rac1/CDC42Hs-dependent autophosphorylation is related to PAK65 and STE20.
• EMBO J. 1995; 14: 4385-4385

• Jullien-Flores V et al.
• Bridging Ral GTPase to Rho pathways. RLIP76, a Ral effector with CDC42/Rac GTPase-activating protein activity.
• J Biol Chem. 1995; 270: 22473-7
• Display abstract

• Ra1A and Ra1B are GTPases of unknown function and are activated by proteins, Ra1GDS, that interact with the active form of another GTPase, Ras. To elucidate Ral function, we have searched for proteins interacting with an activated form of Ra1A using the two-hybrid method and a Jurkat cell library. We have identified a partial cDNA encoding a protein, RLIP1, which binds to activated Ra1A and this binding requires an intact effector domain of Ra1A. Biochemical data with purified Ra1A confirm the genetic results. This protein also bears a region of homology with GTPase-activating protein (GAP) domains that are involved in the regulation of GTPases of the Rho family and, indeed, RLIP1 displays a GAP activity acting upon Rac1 and CDC42, but not RhoA. This GAP region is not required for RLIP1 binding to Ra1. The whole cDNA was cloned, and it encodes a 76-kDa polypeptide, RLIP76, which also binds RalA. The Rho pathway is involved in membrane and cytoskeleton modifications after mitogenic stimulation and acts in parallel to and synergistically with the Ras pathway. We propose that these pathways are linked through a cascade composed of Ras --> Ra1GDS --> Ra1 --> RLIP76 --> CDC42/Rac1/Rho, allowing modulation of the Rho pathway by the Ras pathway.

• Leeuw T et al.
• Pheromone response in yeast: association of Bem1p with proteins of the MAP kinase cascade and actin.
• Science. 1995; 270: 1210-3
• Display abstract

• Haploid cells of the yeast Saccharomyces cerevisiae respond to mating pheromones with polarized growth toward the mating partner. This morphological response requires the function of the cell polarity establishment protein Bem1p. Immunochemical and two-hybrid protein interaction assays revealed that Bem1p interacts with two components of the pheromone-responsive mitogen-activated protein (MAP) kinase cascade, Ste20p and Ste5p, as well as with actin. Mutants of Bem1p that are associated with defective pheromone-induced polarized morphogenesis interacted with Ste5p and actin but not with Ste20p. Thus, the association of Bem1p with Ste20p and Ste5p may contribute to the conveyance of spatial information that regulates polarized rearrangement of the actin cytoskeleton during yeast mating.

• Prigmore E et al.
• A 68-kDa kinase and NADPH oxidase component p67phox are targets for Cdc42Hs and Rac1 in neutrophils.
• J Biol Chem. 1995; 270: 10717-22
• Display abstract

• Cdc42Hs and Rac1 are members of the Ras superfamily of small molecular weight (p21) GTP binding proteins. Cdc42Hs induces filopodia formation in Swiss 3T3 fibroblasts while Rac1 induces membrane ruffling. Rac1 also activates superoxide production by the components (cytochrome b, p40phox, p67phox, and p47phox) of the neutrophil oxidase. To isolate target proteins involved in these signaling pathways, we have probed proteins from neutrophil cytosol immobilized on nitrocellulose with Cdc42Hs labeled with [gamma-32P]GTP. Cdc42Hs probe detected binding protein(s) of 66-68 kDa in neutrophil cytosol. Rac1 probe also detected the 66-68-kDa proteins, suggesting the possibility that p67phox may be a binding protein for both of these p21 proteins. Indeed, Cdc42Hs and Rac1 were found to bind specifically to purified recombinant p67phox but not the other oxidase components. A 68-kDa Cdc42Hs binding protein was purified from neutrophil cytosol and found to be related to the recently described p65pak kinase from brain. These results suggest that the p68 kinase and p67phox are targets for Cdc42Hs and Rac1 in neutrophils.

• Simon MN, De Virgilio C, Souza B, Pringle JR, Abo A, Reed SI
• Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway.
• Nature. 1995; 376: 702-5
• Display abstract

• In the budding yeast Saccharomyces cerevisiae, the process of conjugation of haploid cells of genotype MATa and MAT alpha to form MATa/alpha diploids is triggered by pheromones produced by each mating type. These pheromones stimulate a cellular response by interaction with receptors linked to a heterotrimeric G protein. Although genetic analysis indicates that the pheromone signal is transmitted through the G beta gamma dimer, the initial target(s) of G protein activation remain to be determined. Temperature-sensitive cells with mutations of the CDC24 and CDC42 genes, which are incapable of budding and of generating cell polarity at the restrictive temperature, are also unable to mate. Cdc24 acts as a guanylyl-nucleotide-exchange factor for the Rho-type GTPase Cdc42, which has been shown to be a fundamental component of the molecular machinery controlling morphogenesis in eukaryotic cells. Therefore, the inability of cdc24 and cdc42 mutants to mate has been presumed to be due to a requirement for generation of cell polarity and related morphogenetic events during conjugation. But here we show that Cdc42 has a direct signalling role in the mating-pheromone response between the G protein and the downstream protein kinase cascade.

• Cerione RA, Leonard D, Zheng Y
• Purification of baculovirus-expressed Cdc42Hs.
• Methods Enzymol. 1995; 256: 11-5

• Manser E, Leung T, Lim L
• Purification and assay of kinases that interact with Rac/Cdc42.
• Methods Enzymol. 1995; 256: 215-27

• Posada J, Miller PJ, McCullough J, Ziman M, Johnson DI
• Genetic and biochemical analysis of Cdc42p function in Saccharomyces cerevisiae and Schizosaccharomyces pombe.
• Methods Enzymol. 1995; 256: 281-90

• Ghomashchi F, Zhang X, Liu L, Gelb MH
• Binding of prenylated and polybasic peptides to membranes: affinities and intervesicle exchange.
• Biochemistry. 1995; 34: 11910-8
• Display abstract

• The small GTP-binding protein G25K and the protein K-Ras 4B contain prenyl groups (geranylgeranyl and farnesyl, respectively) that are thioether linked to a C-terminal cysteine which is methylated on its alpha-carboxyl group. These proteins, like many other prenyl proteins, also have a string of basic residues near their C-termini. A series of prenylated peptides based on the C-terminal sequences of human brain G25K and human K-Ras 4B were synthesized and analyzed for their membrane binding affinities. G25K peptides containing an N-terminal N-acetyltryptophan group were studied because their binding to membranes containing a trace of dansylated phospholipid could be detected by fluorescence resonance energy transfer. The G25K peptide lacking a prenyl group and a C-terminal methyl ester did not detectably bind to vesicles, and binding was enhanced by more than 500-fold if the peptide was geranylgeranylated. For the farnesylated peptide, methylation of the C-terminus increased membrane affinity by at least 60-fold if the vesicles contained phosphatidylserine and by 3-fold if they lacked this acidic lipid. The geranylgeranylated and methylated G25K peptide remains irreversibly attached to vesicles over several minutes only if the vesicles contain phosphatidylserine, whereas the corresponding nonmethylated or farnesylated and methylated peptides dissociate rapidly (less than a few seconds) from neutral or anionic vesicles. Farnesylation of the nonmethylated K-Ras 4B peptide enhances its affinity to vesicles containing acidic phospholipids (phosphatidylglycerol or phosphatidylserine) by 70-fold, and methylation leads to an additional dramatic (150-fold) increase in membrane affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

• Stowers L, Yelon D, Berg LJ, Chant J
• Regulation of the polarization of T cells toward antigen-presenting cells by Ras-related GTPase CDC42.
• Proc Natl Acad Sci U S A. 1995; 92: 5027-31
• Display abstract

• The mechanisms by which cells rapidly polarize in the direction of external signals are not understood. Helper T cells, when contacted by an antigen-presenting cell, polarize their cytoskeletons toward the antigen-presenting cell within minutes. Here we show that, in T cells, the mammalian Ras-related GTPase CDC42 (the homologue of yeast CDC42, a protein involved in budding polarity) can regulate the polarization of both actin and microtubules toward antigen-presenting cells but is not involved in other T-cell signaling processes such as those which culminate in interleukin 2 production. Although T-cell polarization appears dispensable for signaling leading to interleukin 2 production, polarization may direct lymphokine secretion towards the correct antigen-presenting cell in a crowded cellular environment. Inhibitor experiments suggest that phosphatidylinositol 3-kinase is required for cytoskeletal polarization but that calcineurin activity, known to be important for other aspects of signaling, is not. Apparent conservation of CDC42 function between yeast and T cells suggests that this GTPase is a general regulator of cytoskeletal polarity in many cell types.

• Li R, Zheng Y, Drubin DG
• Regulation of cortical actin cytoskeleton assembly during polarized cell growth in budding yeast.
• J Cell Biol. 1995; 128: 599-615
• Display abstract

• We have established an in vitro assay for assembly of the cortical actin cytoskeleton of budding yeast cells. After permeabilization of yeast by a novel procedure designed to maintain the spatial organization of cellular constituents, exogenously added fluorescently labeled actin monomers assemble into distinct structures in a pattern that is similar to the cortical actin distribution in vivo. Actin assembly in the bud of small-budded cells requires a nucleation activity provided by protein factors that appear to be distinct from the barbed ends of endogenous actin filaments. This nucleation activity is lost in cells that lack either Sla1 or Sla2, proteins previously implicated in cortical actin cytoskeleton function, suggesting a possible role for these proteins in the nucleation reaction. The rate and the extent of actin assembly in the bud are increased in permeabilized delta cap2 cells, providing evidence that capping protein regulates the ability of the barbed ends of actin filaments to grow in yeast cells. Actin incorporation in the bud can be stimulated by treating the permeabilized cells with GTP-gamma S, and, significantly, the stimulatory effect is eliminated by a mutation in CDC42, a gene that encodes a Rho-like GTP-binding protein required for bud formation. Furthermore, the lack of actin nucleation activity in the cdc42 mutant can be complemented in vitro by a constitutively active Cdc42 protein. These results suggest that Cdc42 is closely involved in regulating actin assembly during polarized cell growth.

• Manser E et al.
• Molecular cloning of a new member of the p21-Cdc42/Rac-activated kinase (PAK) family.
• J Biol Chem. 1995; 270: 25070-8
• Display abstract

• A number of "target" proteins for the Rho family of small GTP-binding proteins have now been identified, including the protein kinases ACK and p65PAK (Manser, E., Leung, T., Salihuddin, H., Zhao, Z.-S., and Lim, L. (1994) Nature 367, 40-46). The purified serine/threonine kinase p65PAK has been shown to be directly activated by GTP-Rac1 or GTP-Cdc42. Here we report the cDNA sequence encoding a new brain-enriched PAK isoform beta-PAK, which shares 79% amino acid identity with the previously described alpha-isoform. Their mRNAs are differentially expressed in the brain, with alpha-PAK mRNA being particularly abundant in motor-associated regions. In vitro translation products of the alpha- and beta-PAK cDNAs exhibited relative molecular masses of 68,000 and 65,000, respectively, by SDS-polyacrylamide analysis. A specific beta-PAK peptide sequence was obtained from rat brain-purified p65PAK. Recombinant alpha- and beta-PAKs exhibited an increase in kinase activity mediated by GTP-p21 induced autophosphorylation. Cdc42 was a more potent activator in vitro of alpha-PAK kinase, and the fully activated enzyme is 300 times more active than the unphosphorylated form. Interestingly the down-regulation in the binding of p21s to recombinant beta-PAK and brain p65PAK, which is observed upon kinase activation does not occur with recombinant alpha-PAK.

• Simon JP et al.
• Regulation of post-Golgi vesicle production in an in vitro system.
• Cold Spring Harb Symp Quant Biol. 1995; 60: 179-95

• Peppelenbosch MP et al.
• Rac mediates growth factor-induced arachidonic acid release.
• Cell. 1995; 81: 849-56
• Display abstract

• Growth factor-induced stress fiber formation involves signal transduction through Rac and Rho proteins and production of leukotrienes from arachidonic acid metabolism. In exploring the relationship between these pathways, we found that Rac is essential for EGF-induced arachidonic acid production and subsequent generation of leukotrienes and that Rac V12, a constitutively activated mutant of Rac, generates leukotrienes in a growth factor-independent manner. Leukotrienes generated by EGF or Rac V12 are necessary and sufficient for stress fiber formation. Furthermore, leukotriene-dependent stress fiber formation requires Rho proteins. We have therefore identified elements of a pathway from growth factor receptors that includes Rac, arachidonic acid production, arachidonic acid metabolism to leukotrienes, and leukotriene-dependent Rho activation. This appears to be the major pathway by which Rac influences Rho-dependent cytoskeleton rearrangements.

• Ziman M, Johnson DI
• Genetic evidence for a functional interaction between Saccharomyces cerevisiae CDC24 and CDC42.
• Yeast. 1994; 10: 463-74
• Display abstract

• Cdc24p and Cdc42p are involved in the control of cell polarity during the Saccharomyces cerevisiae cell cycle. Cdc42p is a member of the Ras superfamily of GTPases and Cdc24p displays limited amino-acid sequence similarity with the Dbl proto-oncoprotein, which acts to stimulate guanine-nucleotide exchange on human Cdc42p. We have performed several genetic experiments to test whether Cdc24p and Cdc42p interact within the cell. First, overexpression of Cdc24p suppressed the dominant-negative cdc42D118A allele. Second, overexpression of wild-type CDC24 and CDC42 genes together was a lethal event resulting in a morphological phenotype of large, round, unbudded cells, indicating a loss of cell polarity. Third, a cdc24ts cdc42ts double mutant exhibited a synthetic-lethal phenotype at the semi-permissive temperature of 30 degrees C. These data suggest that Cdc24p and Cdc42p interact within the cell and that Cdc24p may be involved in the regulation of Cdc42p activity.

• Manser E, Leung T, Salihuddin H, Zhao ZS, Lim L
• A brain serine/threonine protein kinase activated by Cdc42 and Rac1.
• Nature. 1994; 367: 40-6
• Display abstract

• A new brain serine/threonine protein kinase may be a target for the p21ras-related proteins Cdc42 and Rac1. The kinase sequence is related to that of the yeast protein STE20, implicated in pheromone-response pathways. The kinase complexes specifically with activated (GTP-bound) p21, inhibiting p21 GTPase activity and leading to kinase autophosphorylation and activation. Autophosphorylated kinase has a decreased affinity for Cdc42/Rac, freeing the p21 for further stimulatory activities or downregulation by GTPase-activating proteins. This bimolecular interaction provides a model for studying p21 regulation of mammalian phosphorylation signalling pathways.

• Ohya Y, Qadota H, Anraku Y, Pringle JR, Botstein D
• Suppression of yeast geranylgeranyl transferase I defect by alternative prenylation of two target GTPases, Rho1p and Cdc42p.
• Mol Biol Cell. 1993; 4: 1017-25
• Display abstract

• Geranylgeranyl transferase I (GGTase I), which modifies proteins containing the sequence Cys-Ali-Ali-Leu (Ali: aliphatic) at their C-termini, is indispensable for growth in the budding yeast Saccharomyces cerevisiae. We report here that GGTase I is no longer essential when Rho1p and Cdc42p are simultaneously overproduced. The lethality of a GGTase I deletion is most efficiently suppressed by provision of both Rho1p and Cdc42p with altered C-terminal sequences (Cys-Ali-Ali-Met) corresponding to the C-termini of substrates of farnesyl transferase (FTase). Under these circumstances, the FTase, normally not essential for growth of yeast, becomes essential.

• Barfod ET et al.
• Cloning and expression of a human CDC42 GTPase-activating protein reveals a functional SH3-binding domain.
• J Biol Chem. 1993; 268: 26059-62
• Display abstract

• CDC42, a member of the Rho family of small GTP-binding proteins, regulates cytoskeletal rearrangements required for cell division. Activating mutations in CDC42 that are refractory to GTPase activation confer a phenotype of large, multinucleated cells. Like other small GTP-binding proteins, CDC42 is activated by a guanosine exchange factor and inactivated by a GTPase-activating protein (GAP). An unidentified 25-kDa platelet protein has been shown to function as a specific CDC42GAP. Here we report the cloning of a cDNA encoding this GAP from a human platelet-precursor cell line. Sequence analysis reveals the presence of three consensus box regions characteristic of rhoGAPs. A glutathione S-transferase fusion protein containing the three boxes derived from the new clone strongly stimulated the GTPase activity of CDC42 but was much less effective on other Rho proteins. This indicates that the cDNA clone encodes a specific GAP for CDC42. Sequence analysis also reveals a potential proline-rich Src homology 3 (SH3)-binding domain preceding the first consensus box. Binding experiments show that this motif can interact with the SH3 domains of p85 alpha and of c-Src. Thus, CDC42GAP may function as a link between CDC42 and other signaling pathways.

• Leberer E, Dignard D, Harcus D, Hougan L, Whiteway M, Thomas DY
• Cloning of Saccharomyces cerevisiae STE5 as a suppressor of a Ste20 protein kinase mutant: structural and functional similarity of Ste5 to Far1.
• Mol Gen Genet. 1993; 241: 241-54
• Display abstract

• The beta and gamma subunits of the mating response G-protein in the yeast Saccharomyces cerevisiae have been shown to transmit the mating pheromone signal to downstream components of the pheromone response pathway. A protein kinase homologue encoded by the STE20 gene has recently been identified as a potential G beta gamma target. We have searched multicopy plasmid genomic DNA libraries for high gene dosage suppressors of the signal transduction defect of ste20 mutant cells. This screen identified the STE5 gene encoding an essential component of the pheromone signal transduction pathway. We provide genetic evidence for a functional interrelationship between the STE5 gene product and the Ste20 protein kinase. We have sequenced the STE5 gene, which encodes a predicted protein of 917 amino acids and is specifically transcribed in haploid cells. Transcription is slightly induced by treatment of cells with pheromone. Ste5 has homology with Far1, a yeast protein required for efficient mating and the pheromone-inducible inhibition of a G1 cyclin, Cln2. A STE5 multicopy plasmid is able to suppress the signal transduction defect of far1 null mutant cells suggesting that Ste5, at elevated levels, is able functionally to replace Far1. The genetically predicted point of function of Ste5 within the pheromone signalling pathway suggests that Ste5 is involved in the regulation of a G beta gamma-activated protein kinase cascade which links a G-protein coupled receptor to yeast homologues of mitogen-activated protein kinases.

• Manser E, Leung T, Salihuddin H, Tan L, Lim L
• A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42.
• Nature. 1993; 363: 364-7
• Display abstract

• The Ras-related Rho subfamily of GTP-binding proteins (p21s), which includes Rho, Rac and Cdc42Hs, is implicated in different aspects of cytoskeletal organization. These proteins behave like Ras (p21ras) in that their active GTP-bound form is inactivated by intrinsic hydrolysis of the nucleotide gamma-phosphate, which can be stimulated by GTPase-activating proteins (GAPs). We have previously shown that there is a diversity of GAPs that recognize this subfamily, including n-chimaerin, which is enriched in the hippocampus; we also detected proteins that bind these p21 proteins and seem to inhibit GTP hydrolysis. We now report the characterization of a hippocampal complementary DNA encoding a tyrosine kinase that specifically binds Cdc42Hs in its GTP-bound form. This binding is mediated by a unique sequence of 47 amino acids C-terminal to an SH3 domain and inhibits both the intrinsic and GAP-stimulated GTPase activity of Cdc42Hs. Our findings indicate that there may be a regulatory mechanism that sustains the GTP-bound active form of Cdc42Hs and which is directly linked to a tyrosine phosphorylation pathway.

• Leberer E, Dignard D, Harcus D, Thomas DY, Whiteway M
• The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components.
• EMBO J. 1992; 11: 4815-24
• Display abstract

• In the yeast Saccharomyces cerevisiae the G-protein beta gamma subunits have been shown to trigger downstream events of the pheromone response pathway. We have identified a new gene, designated STE20, which encodes a protein kinase homologue with sequence similarity to protein kinase C, which is required to transmit the pheromone signal from G beta gamma to downstream components of the signalling pathway. Overproduction of the kinase suppresses the mating defect of dominant-negative G beta mutations providing genetic evidence for an interaction with G beta, and epistasis experiments show that this kinase functions after or at the same point as G beta gamma, but before any of the other currently identified components of the signalling pathway. This points to a potentially new mechanism of G-protein mediated signal transduction, the activation of a protein kinase through G beta gamma.

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