NORMAL GENOMIC

• Montague P, Kennedy PG, Barnett SC
• Subcellular localization of Mayven following expression of wild type andmutant EGFP tagged cDNAs.
• BMC Neurosci. 2010; 11: 63-63
• Display abstract

• BACKGROUND: Process formation by glial cells is crucial to their function.Mayven, an actin binding, multi-domain polypeptide, and member of theBTB-BACK-Kelch family have been shown to be important in oligodendrocyteprocess extension. To assess the role of Mayven in neural cell processextension we have tracked the subcellular distribution of exogenous Mayvenfollowing expression of a rat Mayven -EGFP cDNA in a variety of neuralcell backgrounds and specifically in OEC tranfectants following drugtreatment to disrupt the integrity of the cytoskeleton. A comparison wasmade between the subcellular localization following transient transfectionof OECs with full-length Mayven cDNA and a series of mutant domainconstructs. RESULTS: The subcellular location of Mayven in OECtransfectants showed a characteristic distribution with intense foci ofstaining towards the process tips corresponding to regions of accumulatedMayven overlapping in part with lammelipodial actin and was absent fromthe filipodia and the outer membrane. This signature pattern was alsoobserved in Schwann cells, Oli-Neu cells, astrocytes and the neuroblastomacell line B104 transfectants and resembled the exogenous and endogenousMayven distribution in oligodendrocytes. This contrasted with thelocalization pattern in non-neural cells. There was a re-localization ofMayven in OEC transfectants following drug treatment to challenge theintegrity of the actin cytoskeleton while breakdown of the microtubularcomponent had no discernible impact on the accumulation of Mayven in theprocess tips. Deletion of the first three amino acids of the SH3 motif ofthe putative Fyn Kinase binding domain at the amino terminus significantlycompromised this signature pattern as did the removal of the last Kelchrepeat unit of six unit Kelch domain comprising the carboxyl terminus. Inaddition, there was a reduction in process length in mutant transfectants.Co-expression studies with a haemagglutinin (HA) tagged wild type MayvencDNA and EGFP tagged mutant cDNAs suggested a homomeric interactionmediated by the BTB/POZ domain. CONCLUSIONS: Exogenous Mayven istransported to the lamellipodia in neural transfectants associating withthe actin cytoskeletal network. In addition to the importance of theinternal BTB/POZ domain, this subcellular distribution pattern isdependent on the presence of an intact amino and carboxyl terminus.

• Kugler JM, Chicoine J, Lasko P
• Bicaudal-C associates with a Trailer Hitch/Me31B complex and is requiredfor efficient Gurken secretion.
• Dev Biol. 2009; 328: 160-72
• Display abstract

• Bicaudal-C (Bic-C) is a multiple KH-domain RNA-binding protein requiredfor Drosophila oogenesis and, maternally, for embryonic patterning. Inearly oogenesis, Bic-C negatively regulates target mRNAs, including Bic-C,by recruiting the CCR4 deadenylase through a direct association with itsNOT3 subunit. Here, we identify a novel function for Bic-C in secretion ofthe TGF-alpha homolog Gurken (Grk). In Bic-C mutant egg chambers, Grk issequestered within actin-coated structures during mid-oogenesis. As aconsequence, Egfr signalling is not efficiently activated in thedorsal-anterior follicle cells. This phenotype is strikingly similar tothat of trailer hitch (tral) mutants. Consistent with the idea that Bic-Cand Tral act together in Grk secretion, Bic-C co-localizes with Tralwithin cytoplasmic granules, and can be co-purified with multiple proteincomponents of a Tral mRNP complex. Taken together, our results implicatetranslational regulation by Bic-C and Tral in the secretory pathway.

• Fukuoka M, Suetsugu S, Miki H, Fukami K, Endo T, Takenawa T
• A novel neural Wiskott-Aldrich syndrome protein (N-WASP) binding protein,WISH, induces Arp2/3 complex activation independent of Cdc42.
• J Cell Biol. 2001; 152: 471-82
• Display abstract

• We identified a novel adaptor protein that contains a Src homology (SH)3domain, SH3 binding proline-rich sequences, and a leucine zipper-likemotif and termed this protein WASP interacting SH3 protein (WISH). WISH isexpressed predominantly in neural tissues and testis. It bound Ash/Grb2through its proline-rich regions and neural Wiskott-Aldrich syndromeprotein (N-WASP) through its SH3 domain. WISH strongly enhancedN-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro,resulting in rapid actin polymerization. Furthermore, coexpression of WISHand N-WASP induced marked formation of microspikes in Cos7 cells, even inthe absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42still induced microspike formation when coexpressed with WISH. We alsoexamined the contribution of WISH to a rapid actin polymerization inducedby brain extract in vitro. Arp2/3 complex was essential for brainextract-induced rapid actin polymerization. Addition of WISH to extractsincreased actin polymerization as Cdc42 did. However, WISH unexpectedlycould activate actin polymerization even in N-WASP-depleted extracts.These findings suggest that WISH activates Arp2/3 complex throughN-WASP-dependent and -independent pathways without Cdc42, resulting in therapid actin polymerization required for microspike formation.

• Krugmann S, Jordens I, Gevaert K, Driessens M, Vandekerckhove J, Hall A
• Cdc42 induces filopodia by promoting the formation of an IRSp53:Menacomplex.
• Curr Biol. 2001; 11: 1645-55
• Display abstract

• BACKGROUND: The Rho GTPases Rho, Rac, and Cdc42 regulate the organizationof the actin cytoskeleton by interacting with multiple, distinctdownstream effector proteins. Cdc42 controls the formation of actinbundle-containing filopodia at the cellular periphery. The molecularmechanism for this remains as yet unclear. RESULTS: We report here thatCdc42 interacts with IRSp53/BAP2 alpha, an SH3 domain-containing scaffoldprotein, at a partial CRIB motif and that an N-terminal fragment of IRSp53binds, via an intramolecular interaction, to the CRIB motif-containingcentral region. Overexpression of IRSp53 in fibroblasts leads to theformation of filopodia, and both this and Cdc42-induced filopodia areinhibited by expression of the N-terminal IRSp53 fragment. Using affinitychromatography, we have identified Mena, an Ena/VASP family member, asinteracting with the SH3 domain of IRSp53. Mena and IRSp53 actsynergistically to promote filopodia formation. CONCLUSION: We concludethat the interaction of Cdc42 with the partial CRIB motif of IRSp53relieves an intramolecular, autoinhibitory interaction with the Nterminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. ThisIRSp53:Mena complex initiates actin filament assembly into filopodia.

• Benard V, Bohl BP, Bokoch GM
• Characterization of rac and cdc42 activation in chemoattractant-stimulatedhuman 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 oxidantproduction important in bacterial killing. Rac and the related GTPaseCdc42 also regulate the dynamics of the actin cytoskeleton, necessary forleukocyte chemotaxis and phagocytosis of microorganisms. Although theseGTPases appear to be critical downstream components of chemoattractantreceptor signaling in human neutrophils, the pathways involved in directcontrol of Rac/Cdc42 activation remain to be determined. We describe anassay that measures the formation of Rac-GTP and Cdc42-GTP based on theirspecific binding to the p21-binding domain of p21-activated kinase 1. Ap21-binding domain glutathione S-transferase fusion protein specificallybinds Rac and Cdc42 in their GTP-bound forms both in vitro and in cellsamples. Binding is selective for Rac and Cdc42 versus RhoA. Using thisassay, we investigated Rac and Cdc42 activation in neutrophils anddifferentiated HL-60 cells. The chemoattractant fMet-Leu-Phe and thephorbol ester phorbol myristate acetate stimulate formation of Rac-GTP andCdc42-GTP with distinct time courses that parallel cell activation. Wealso show that the signaling pathways leading to Rac and Cdc42 activationin HL-60 cells involve G proteins sensitive to pertussis toxin, as well astyrosine kinase and phosphatidylinositol 3-kinase activities.

• Ahmed S et al.
• Cryptic Rac-binding and p21(Cdc42Hs/Rac)-activated kinase phosphorylationsites 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 thatact as molecular switches to control actin-based cell morphology as wellas cell growth and differentiation. Rac1 and Rac2 are specificallyrequired for superoxide formation by components of the NADPH oxidase. Inbinding assays, Rac1 interacts directly with p67(phox), but not with theother 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 withp67(phox) has been characterized further. Rac1 and Rac2 can bind top67(phox) amino acid residues 170-199, and the N terminus (amino acids1-192) of p67(phox) can be used as a specific inhibitor of Rac signaling.Deletion of p67(phox) C-terminal sequences (amino acids 193-526), theC-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) aminoacid residues adjacent to the Rac1/2-binding site, and thisphosphorylation is stimulated by deletion of the C-terminal SH3 domain orthe polyproline-rich motif. These data suggest a role for crypticRac-binding and PAK phosphorylation sites of p67(phox) in control of theNADPH oxidase.

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

• BACKGROUND: The Rho family GTPases Cdc42, Rac1 and RhoA regulate thereorganization of the actin cytoskeleton induced by extracellular signalssuch as growth factors. In mammalian cells, Cdc42 regulates the formationof filopodia, whereas Rac regulates lamellipodia formation and membraneruffling, and RhoA regulates the formation of stress fibers. Recently, theserine/threonine protein kinase p65(pak) autophosphorylates, therebyincreasing its catalytic activity towards exogenous substrates. Thiskinase is therefore a candidate effector for the changes in cell shapeinduced by growth factors. RESULTS: Here, we report that themicroinjection of activated Pak1 protein into quiescent Swiss 3T3 cellsinduces the rapid formation of polarized filopodia and membrane ruffles.The prolonged overexpression of Pak1 amino-terminal mutants that areunable to bind Cdc42 or Rac1 results in the accumulation of filamentousactin in large, polarized membrane ruffles and the formation ofvinculin-containing focal complexes within these structures. Thisphenotype resembles that seen in motile fibroblasts. The amino-terminalPak1 mutant displays enhanced binding to the adaptor protein Nck, whichcontains three Src-homology 3 (SH3) domains. Mutation of a proline residuewithin a conserved SH3-binding region at the amino terminus of Pak1interferes with SH3-protein binding and alters the effects of Pak1 on thecytoskeleton. CONCLUSIONS: These results indicate that Pak1, actingthrough a protein that contains an SH3 domain, regulates the structure ofthe actin cytoskeleton in mammalian cells, and may serve as an effectorfor Cdc42 and/or Rac1 in promoting cell motility.

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

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

• Honda R, Ohba Y, Yasuda H
• 14-3-3 zeta protein binds to the carboxyl half of mouse wee1 kinase.
• Biochem Biophys Res Commun. 1997; 230: 262-5
• Display abstract

• To identify proteins which bind to mouse wee1 kinase, the yeast"two-hybrid" system was used with a mouse cDNA library. Using the carboxylhalf of weel kinase, the 14-3-3 zeta protein was isolated. Recombinant14-3-3 zeta was demonstrated to bind to wee1 kinase in vitro. The wee1kinase phosphorylated by cdc2 kinase also bound to 14-3-3 zeta protein.When both wee1 kinase and 14-3-3 zeta were transfected into COS-1 cells,they formed a complex in a cell. The sequence of wee1 kinase necessary forthe binding was tested by a two hybrid system expressing different lengthsof peptides derived from wee1 kinase. Both the entire kinase domain and asequence in the carboxyl terminus was thought to be necessary for thebinding. The function of 14-3-3 zeta protein remained to be elucidated inrelation to the regulation of G2 to M phase transition through wee1kinase.

• Hart MJ, Callow MG, Souza B, Polakis P
• IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is apotential effector for cdc42Hs.
• EMBO J. 1996; 15: 2997-3005
• Display abstract

• Proteins that associate with the GTP-bound forms of the Ras superfamily ofproteins are potential effector targets for these molecular switches. A195 kDa protein was purified from cell lysates by affinity chromatographyon immobilized cdc42Hs-GTP and a corresponding cDNA was isolated. Sequenceanalysis revealed localized identities to calponin, the WW domain,unconventional myosins and to the rasGAP-related domain (GRD) contained inIRA, NF-1, SAR1 and rasGAP. p195 was found to be identical to IQGAP1, aprotein previously reported to bind ras. Purified recombinant p195/IQGAP1bound to and inhibited the GTPase activity of cdc42Hs and rac whereas nointeraction with ras was detected. The C-terminal half of IQGAP1containing the GRD bound to cdc42 and rac in a GRD-dependent fashion, buta smaller fragment containing only the GRD did not. Cdc42 was alsoco-immunoprecipitated from cell lysates with antibody specific top195/IQGAP1. Calmodulin also co-immunoprecipitated with p195/IQGAP1 andwas found to associate with fragments containing the IQ domain. Expressionof a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway inyeast, but no effect on ras was observed. In mammalian cells, bothendogenous and ectopically expressed p195/IQGAP1 were localized tolamellipodia and ruffling cell membranes, where co-localization with actinwas apparent. These results suggest that IQGAP1 is an effector target forcdc42Hs and may mediate the effects of this GTPase on cell morphology.

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

• Ral proteins constitute a distinct family of Ras-related GTPases. Althoughsimilar to Ras in amino acid sequence, Ral proteins are activated by aunique nucleotide exchange factor and inactivated by a distinctGTPase-activating protein. Unlike Ras, they fail to promote transformedfoci when activated versions are expressed in cells. To identifydownstream targets that might mediate a Ral-specific function, we used aSaccharomyces cerevisiae-based interaction assay to clone a novel cDNAthat encodes a Ral-binding protein (RalBP1). RalBP1 binds specifically tothe active GTP-bound form of RalA and not to a mutant Ral with a pointmutation in its putative effector domain. In addition to a Ral-bindingdomain, RalBP1 also contains a Rho-GTPase-activating protein domain thatinteracts preferentially with Rho family member CDC42. Since CDC42 hasbeen implicated in bud site selection in S. cerevisiae and filopodiumformation in mammalian cells, Ral may function to modulate the actincytoskeleton through its interactions with RalBP1.

• Tolias KF, Cantley LC, Carpenter CL
• Rho family GTPases bind to phosphoinositide kinases.
• J Biol Chem. 1995; 270: 17656-9
• Display abstract

• Rho family GTPases appear to play an important role in the regulation ofthe actin cytoskeleton, but the mechanism of regulation is unknown. Sincephosphoinositide 3-kinase and phosphatidylinositol 4,5-bisphosphate havealso been implicated in actin reorganization, we investigated thepossibility that Rho family members interact with phosphoinositidekinases. We found that both GTP- and GDP-bound Rac1 associate withphosphatidylinositol-4-phosphate 5-kinase in vitro and in vivo.Phosphoinositide 3-kinase also bound to Rac1 and Cdc42Hs, and theseinteractions were GTP-dependent. Stimulation of Swiss 3T3 cells withplatelet-derived growth factor induced the association of PI 3-kinase withRac in immunoprecipitates. PI 3-kinase activity was also detected in Cdc42immunoprecipitates from COS7 cells. These results suggest thatphosphoinositide kinases are involved in Rho family signal transductionpathways and raise the possibility that the effects of Rho family memberson the actin cytoskeleton are mediated in part by phosphoinositidekinases.

• Peterson J, Zheng Y, Bender L, Myers A, Cerione R, Bender A
• Interactions between the bud emergence proteins Bem1p and Bem2p andRho-type GTPases in yeast.
• J Cell Biol. 1994; 127: 1395-406
• Display abstract

• The SH3 domain-containing protein Bem1p is needed for normal bud emergenceand mating projection formation, two processes that require asymmetricreorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae.To identify proteins that functionally and/or physically interact withBem1p, we screened for mutations that display synthetic lethality with amutant allele of the BEM1 gene and for genes whose products displaytwo-hybrid interactions with the Bem1 protein. CDC24, which is requiredfor bud emergence and encodes a GEF (guanine-nucleotide exchange factor)for the essential Rho-type GTPase Cdc42p, was identified during bothscreens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEFdomain, can interact with a portion of Bem1p that lacks both SH3 domains.Bacterially expressed Cdc24p and Bem1p bind to each other in vitro,indicating that no other yeast proteins are required for this interaction.The most frequently identified gene that arose from the bem1synthetic-lethal screen was the bud-emergence gene BEM2 (Bender andPringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2(increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Herewe show that Bem2p contains a GAP (GTPase-activating protein) domain forRho-type GTPases, and that this portion of Bem2p can stimulate in vitrothe GTPase activity of Rho1p, a second essential yeast Rho-type GTPase.Cells deleted for BEM2 become large and multinucleate. These and othergenetic, two-hybrid, biochemical, and phenotypic data suggest thatmultiple Rho-type GTPases control the reorganization of the corticalcytoskeleton in yeast and that the functions of these GTPases are tightlycoupled. Also, these findings raise the possibility that Bem1p mayregulate or be a target of action of one or more of these GTPases.