SMART MODE:

NORMAL GENOMIC

• 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)3 domain, SH3 binding proline-rich sequences, and a leucine zipper-like motif and termed this protein WASP interacting SH3 protein (WISH). WISH is expressed predominantly in neural tissues and testis. It bound Ash/Grb2 through its proline-rich regions and neural Wiskott-Aldrich syndrome protein (N-WASP) through its SH3 domain. WISH strongly enhanced N-WASP-induced Arp2/3 complex activation independent of Cdc42 in vitro, resulting in rapid actin polymerization. Furthermore, coexpression of WISH and N-WASP induced marked formation of microspikes in Cos7 cells, even in the absence of stimuli. An N-WASP mutant (H208D) that cannot bind Cdc42 still induced microspike formation when coexpressed with WISH. We also examined the contribution of WISH to a rapid actin polymerization induced by brain extract in vitro. Arp2/3 complex was essential for brain extract-induced rapid actin polymerization. Addition of WISH to extracts increased actin polymerization as Cdc42 did. However, WISH unexpectedly could activate actin polymerization even in N-WASP-depleted extracts. These findings suggest that WISH activates Arp2/3 complex through N-WASP-dependent and -independent pathways without Cdc42, resulting in the rapid actin polymerization required for microspike formation.

• Fradelizi J et al.
• ActA and human zyxin harbour Arp2/3-independent actin-polymerization activity.
• Nat Cell Biol. 2001; 3: 699-707
• Display abstract

• The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin polymerization in a bead assay in vitro and in a mitochondrial-targeting assay in cells. We found that human zyxin and the related protein ActA of Listeria monocytogenes can generate new actin structures in a vasodilator-stimulated phosphoprotein-dependent (VASP) manner, but independently of the Arp2/3 complex. These results are consistent with the concept that there are multiple actin-polymerization machines in cells. With these simple tests it is possible to probe the specific function of proteins or identify novel molecules that act upon cellular actin polymerization.

• Ben-Yaacov S, Le Borgne R, Abramson I, Schweisguth F, Schejter ED
• Wasp, the Drosophila Wiskott-Aldrich syndrome gene homologue, is required for cell fate decisions mediated by Notch signaling.
• J Cell Biol. 2001; 152: 1-13
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• Wiskott-Aldrich syndrome proteins, encoded by the Wiskott-Aldrich syndrome gene family, bridge signal transduction pathways and the microfilament-based cytoskeleton. Mutations in the Drosophila homologue, Wasp (Wsp), reveal an essential requirement for this gene in implementation of cell fate decisions during adult and embryonic sensory organ development. Phenotypic analysis of Wsp mutant animals demonstrates a bias towards neuronal differentiation, at the expense of other cell types, resulting from improper execution of the program of asymmetric cell divisions which underlie sensory organ development. Generation of two similar daughter cells after division of the sensory organ precursor cell constitutes a prominent defect in the Wsp sensory organ lineage. The asymmetric segregation of key elements such as Numb is unaffected during this division, despite the misassignment of cell fates. The requirement for Wsp extends to additional cell fate decisions in lineages of the embryonic central nervous system and mesoderm. The nature of the Wsp mutant phenotypes, coupled with genetic interaction studies, identifies an essential role for Wsp in lineage decisions mediated by the Notch signaling pathway.

• Reinhard M, Jarchau T, Walter U
• Actin-based motility: stop and go with Ena/VASP proteins.
• Trends Biochem Sci. 2001; 26: 243-9
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• Proteins of the Ena/VASP (Enabled/vasodilator-stimulated phosphoprotein) family are involved in Abl and/or cyclic nucleotide-dependent protein kinase signaling pathways. These proteins are also crucial factors in regulating actin dynamics and associated processes such as cell-cell adhesion, platelet function and actin-based motility of both cytopathogenic Listeria and their eukaryotic host cells. Although biochemical mechanisms have emerged depicting Ena/VASP proteins as enhancers of actin filament formation, increasing evidence also suggests that these proteins have inhibitory functions in integrin regulation, cell motility and axon guidance.

• Ferreira PA
• Characterization of RanBP2-associated molecular components in neuroretina.
• Methods Enzymol. 2000; 315: 455-68

• Machesky LM
• Putting on the brakes: a negative regulatory function for Ena/VASP proteins in cell migration.
• Cell. 2000; 101: 685-8

• Pfeuffer T, Goebel W, Laubinger J, Bachmann M, Kuhn M
• LaXp180, a mammalian ActA-binding protein, identified with the yeast two-hybrid system, co-localizes with intracellular Listeria monocytogenes.
• Cell Microbiol. 2000; 2: 101-14
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• The Listeria monocytogenes surface protein ActA is an important virulence factor required for listerial intracellular movement by inducing actin polymerization. The only host cell protein known that directly interacts with ActA is the phosphoprotein VASP, which binds to the central proline-rich repeat region of ActA. To identify additional ActA-binding proteins, we applied the yeast two-hybrid system to search for mouse proteins that interact with ActA. A mouse cDNA library was screened for ActA-interacting proteins (AIPs) using ActA from strain L. monocytogenes EGD as bait. Three different AIPs were identified, one of which was identical to the human protein LaXp180 (also called CC1). Binding of LaXp180 to ActA was also demonstrated in vitro using recombinant histidine-tagged LaXp180 and recombinant ActA. Using an anti-LaXp180 antibody and fluorescence microscopy, we showed that LaXp180 co-localizes with a subset of intracellular, ActA-expressing L. monocytogenes but was never detected on intracellularly growing but ActA-deficient mutants. Furthermore, LaXp180 binding to intracellular L. monocytogenes was asymmetrical and mutually exclusive with F-actin polymerization on the bacterial surface. LaXp180 is a putative binding partner of stathmin, a protein involved in signal transduction pathways and in the regulation of microtubule dynamics. Using immunofluorescence, we showed that stathmin co-localizes with intracellular ActA-expressing L. monocytogenes.

• Hortsch M
• Structural and functional evolution of the L1 family: are four adhesion molecules better than one?
• Mol Cell Neurosci. 2000; 15: 1-10

• Bear JE, Loureiro JJ, Libova I, Fassler R, Wehland J, Gertler FB
• Negative regulation of fibroblast motility by Ena/VASP proteins.
• Cell. 2000; 101: 717-28
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• Ena/VASP proteins have been implicated in cell motility through regulation of the actin cytoskeleton and are found at focal adhesions and the leading edge. Using overexpression, loss-of-function, and inhibitory approaches, we find that Ena/VASP proteins negatively regulate fibroblast motility. A dose-dependent decrease in movement is observed when Ena/VASP proteins are overexpressed in fibroblasts. Neutralization or deletion of all Ena/VASP proteins results in increased cell movement. Selective depletion of Ena/VASP proteins from focal adhesions, but not the leading edge, has no effect on motility. Constitutive membrane targeting of Ena/VASP proteins inhibits motility. These results are in marked contrast to current models for Ena/VASP function derived mainly from their role in the actin-driven movement of Listeria monocytogenes.

• Nikolopoulos SN, Turner CE
• Actopaxin, a new focal adhesion protein that binds paxillin LD motifs and actin and regulates cell adhesion.
• J Cell Biol. 2000; 151: 1435-48
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• Paxillin is a focal adhesion adapter protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Paxillin LD motifs have been demonstrated to bind to several proteins associated with remodeling of the actin cytoskeleton including the focal adhesion kinase, vinculin, and a complex of proteins comprising p95PKL, PIX, and PAK (Turner, C.E., M. C. Brown, J.A. Perrotta, M.C. Riedy, S.N. Nikolopoulos, A.R. McDonald, S. Bagrodia, S. Thomas, and P.S. Leventhal. 1999. J. Cell Biol. 145:851-863). In this study, we report the cloning and initial characterization of a new paxillin LD motif-binding protein, actopaxin. Analysis of the deduced amino acid sequence of actopaxin reveals a 42-kD protein with two calponin homology domains and a paxillin-binding subdomain (PBS). Western blotting identifies actopaxin as a widely expressed protein. Actopaxin binds directly to both F-actin and paxillin LD1 and LD4 motifs. It exhibits robust focal adhesion localization in several cultured cell types but is not found along the length of the associated actin-rich stress fibers. Similar to paxillin, it is absent from actin-rich cell-cell adherens junctions. Also, actopaxin colocalizes with paxillin to rudimentary focal complexes at the leading edge of migrating cells. An actopaxin PBS mutant incapable of binding paxillin in vitro cannot target to focal adhesions when expressed in fibroblasts. In addition, ectopic expression of the PBS mutant and/or the COOH terminus of actopaxin in HeLa cells resulted in substantial reduction in adhesion to collagen. Together, these results suggest an important role for actopaxin in integrin-dependent remodeling of the actin cytoskeleton during cell motility and cell adhesion.

• Ball LJ et al.
• Dual epitope recognition by the VASP EVH1 domain modulates polyproline ligand specificity and binding affinity.
• EMBO J. 2000; 19: 4903-14
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• The Ena-VASP family of proteins act as molecular adaptors linking the cytoskeletal system to signal transduction pathways. Their N-terminal EVH1 domains use groups of exposed aromatic residues to specifically recognize 'FPPPP' motifs found in the mammalian zyxin and vinculin proteins, and ActA protein of the intracellular bacterium Listeria monocytogenes. Here, evidence is provided that the affinities of these EVH1-peptide interactions are strongly dependent on the recognition of residues flanking the core FPPPP motifs. Determination of the VASP EVH1 domain solution structure, together with peptide library screening, measurement of individual K(d)s by fluorescence titration, and NMR chemical shift mapping, revealed a second affinity-determining epitope present in all four ActA EVH1-binding motifs. The epitope was shown to interact with a complementary hydrophobic site on the EVH1 surface and to increase strongly the affinity of ActA for EVH1 domains. We propose that this epitope, which is absent in the sequences of the native EVH1-interaction partners zyxin and vinculin, may provide the pathogen with an advantage when competing for the recruitment of the host VASP and Mena proteins in the infected cell.

• Kanai F et al.
• TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins.
• EMBO J. 2000; 19: 6778-91
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• The highly conserved and ubiquitously expressed 14-3-3 proteins regulate differentiation, cell cycle progression and apoptosis by binding intracellular phosphoproteins involved in signal transduction. By screening in vitro translated cDNA pools for the ability to bind 14-3-3, we identified a novel transcriptional co-activator, TAZ (transcriptional co-activator with PDZ-binding motif) as a 14-3-3-binding molecule. TAZ shares homology with Yes-associated protein (YAP), contains a WW domain and functions as a transcriptional co-activator by binding to the PPXY motif present on transcription factors. 14-3-3 binding requires TAZ phosphorylation on a single serine residue, resulting in the inhibition of TAZ transcriptional co-activation through 14-3-3-mediated nuclear export. The C-terminus of TAZ contains a highly conserved PDZ-binding motif that localizes TAZ into discrete nuclear foci and is essential for TAZ-stimulated gene transcription. TAZ uses this same motif to bind the PDZ domain-containing protein NHERF-2, a molecule that tethers plasma membrane ion channels and receptors to cytoskeletal actin. TAZ may link events at the plasma membrane and cytoskeleton to nuclear transcription in a manner that can be regulated by 14-3-3.

• Rong SB, Vihinen M
• Structural basis of Wiskott-Aldrich syndrome causing mutations in the WH1 domain.
• J Mol Med. 2000; 78: 530-7
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• Wiskott-Aldrich syndrome (WAS) is an X-linked recessive immunodeficiency disease associated with eczema, hemorrhagic episodes, and recurrent severe infections. The N-terminus of the cytoplasmic WAS protein (WASP) has similarity to WH1 domains, which recognize proline-rich sequences and direct protein localization and formation of multicomponent assemblies. About one-half of the WAS-causing mutations affect the WH1 domain, but this forms only about one-fifth of the length of the protein. To understand the structural and functional effects of WAS-causing mutations within the WH1 domain, the three-dimensional model of the WASP WH1 domain was constructed based on the crystal structures of the Mena and Ev1 EVH1 (WH1) domains. Based on the model, the protein structural effects of the mutations were evaluated and putative ligand-binding regions identified. Mutations in the WASP WH1 domain were found to influence both the function and structure of the WASP. The amino acid substitutions cause general and local structural changes because of steric clashes and changes to the positions of adjacent strands and the fold of the protein. Some mutations alter the electrostatics and interactions with partners and other domains of WASP.

• Kortschak RD, Tucker PW, Saint R
• ARID proteins come in from the desert.
• Trends Biochem Sci. 2000; 25: 294-9
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• Members of the recently discovered ARID (AT-rich interaction domain) family of DNA-binding proteins are found in fungi and invertebrate and vertebrate metazoans. ARID-encoding genes are involved in a variety of biological processes including embryonic development, cell lineage gene regulation and cell cycle control. Although the specific roles of this domain and of ARID-containing proteins in transcriptional regulation are yet to be elucidated, they include both positive and negative transcriptional regulation and a likely involvement in the modification of chromatin structure.

• Lambrechts A et al.
• cAMP-dependent protein kinase phosphorylation of EVL, a Mena/VASP relative, regulates its interaction with actin and SH3 domains.
• J Biol Chem. 2000; 275: 36143-51
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• Proteins of the Ena/VASP family are implicated in processes that require dynamic actin remodeling such as axon guidance and platelet activation. In this work, we explored some of the pathways that likely regulate actin dynamics in part via EVL (Ena/VASP-like protein). Two isoforms, EVL and EVL-I, were highly expressed in hematopoietic cells of thymus and spleen. In CD3-activated T-cells, EVL was found in F-actin-rich patches and at the distal tips of the microspikes that formed on the activated side of the T-cells. Like the other family members, EVL localized to focal adhesions and the leading edge of lamellipodia when expressed in fibroblasts. EVL was a substrate for the cAMP-dependent protein kinase, and this phosphorylation regulated several of the interactions between EVL and its ligands. Unlike VASP, EVL nucleated actin polymerization under physiological conditions, whereas phosphorylation of both EVL and VASP decreased their nucleating activity. EVL bound directly to the Abl, Lyn, and nSrc SH3 domains; the FE65 WW domain; and profilin, likely via its proline-rich core. Binding of Abl and nSrc SH3 domains, but not profilin or other SH3 domains, was abolished by cAMP-dependent protein kinase phosphorylation of EVL. We show strong cooperative binding of two profilin dimers on the polyproline sequence of EVL. Additionally, profilin competed with the SH3 domains for binding to partially overlapping binding sites. These data suggest that the function of EVL could be modulated in a complex manner by its interactions with multiple ligands and through phosphorylation by cyclic nucleotide dependent kinases.

• Ishino K, Kaneyama, Shibanuma M, Nose K
• Specific decrease in the level of Hic-5, a focal adhesion protein, during immortalization of mouse embryonic fibroblasts, and its association with focal adhesion kinase.
• J Cell Biochem. 2000; 76: 411-9
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• Hic-5 is a paxillin homologue with four LIM domains in its C-terminal region, localized mainly in focal adhesions in normal fibroblasts. Hic-5 is also known to associate with focal adhesion kinase (FAK) or the related CAKbeta, and with vinculin. In the present study, we examined changes in Hic-5 and paxillin protein levels in primary mouse embryo fibroblasts (MEF) during mortal and immortal stages. The Hic-5 level was markedly decreased when cells became immortalized, whereas that of paxillin was increased. The vinculin level was not changed significantly. Hic-5 was mainly localized in focal adhesion plaques of mortal MEF but was localized in the nuclear periphery in the immortalized MEF; the number of focal adhesion plaques was decreased in these cells. Mouse Hic-5 contains three LD domains in its N-terminal half, and the first LD domain (LD1) appears to be involved in interaction with FAK. However, this interaction was not essential for recruitment of Hic-5 to focal adhesions, since its subcellular localization was similar in FAK(-/-) cells. Forced expression of Hic-5 decreased colony forming ability of MEF from FAK(+/+) mice, but not of FAK(-/-) cells. These observations suggested the involvement of Hic-5 in determination of cellular proliferative capacity in collaboration with other cytoskeletal components.

• Drees B, Friederich E, Fradelizi J, Louvard D, Beckerle MC, Golsteyn RM
• Characterization of the interaction between zyxin and members of the Ena/vasodilator-stimulated phosphoprotein family of proteins.
• J Biol Chem. 2000; 275: 22503-11
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• Zyxin contains a proline-rich N-terminal domain that is similar to the C-terminal domain in the ActA protein of the bacteria, Listeria monocytogenes. We screened the entire amino acid sequence of human zyxin for Mena-interacting peptides and found that, as with ActA, proline-rich sequences were the sole zyxin sequences capable of binding to Ena/vasodilator-stimulated phosphoprotein (VASP) family members in vitro. From this information, we tested zyxin mutants in which the proline-rich sequences were altered. The reduction in Mena/VASP binding was confirmed by peptide tests, immunoprecipitation, and ectopic expression of zyxin variants at the surface of mitochondria. By transfection assays we showed that zyxin interaction with Mena/VASP in vivo enhances the production of actin-rich structures at the apical surface of cells. Microinjection into cells of peptides corresponding to the first proline-rich sequence of zyxin caused the loss of Mena/VASP from focal contacts. Furthermore, these peptides reduced the degree of spreading of cells replated after trypsinization. We conclude that zyxin and proteins that harbor similar proline-rich repeats contribute to the positioning of Mena/VASP proteins. The positioning of Ena/VASP family members appears to be important when the actin cytoskeleton is reorganized, such as during spreading.

• Kim AS, Kakalis LT, Abdul-Manan N, Liu GA, Rosen MK
• Autoinhibition and activation mechanisms of the Wiskott-Aldrich syndrome protein.
• Nature. 2000; 404: 151-8
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• The Rho-family GTPase, Cdc42, can regulate the actin cytoskeleton through activation of Wiskott-Aldrich syndrome protein (WASP) family members. Activation relieves an autoinhibitory contact between the GTPase-binding domain and the carboxy-terminal region of WASP proteins. Here we report the autoinhibited structure of the GTPase-binding domain of WASP, which can be induced by the C-terminal region or by organic co-solvents. In the autoinhibited complex, intramolecular interactions with the GTPase-binding domain occlude residues of the C terminus that regulate the Arp2/3 actin-nucleating complex. Binding of Cdc42 to the GTPase-binding domain causes a dramatic conformational change, resulting in disruption of the hydrophobic core and release of the C terminus, enabling its interaction with the actin regulatory machinery. These data show that 'intrinsically unstructured' peptides such as the GTPase-binding domain of WASP can be induced into distinct structural and functional states depending on context.

• Skoble J, Portnoy DA, Welch MD
• Three regions within ActA promote Arp2/3 complex-mediated actin nucleation and Listeria monocytogenes motility.
• J Cell Biol. 2000; 150: 527-38
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• The Listeria monocytogenes ActA protein induces actin-based motility by enhancing the actin nucleating activity of the host Arp2/3 complex. Using systematic truncation analysis, we identified a 136-residue NH(2)-terminal fragment that was fully active in stimulating nucleation in vitro. Further deletion analysis demonstrated that this fragment contains three regions, which are important for nucleation and share functional and/or limited sequence similarity with host WASP family proteins: an acidic stretch, an actin monomer-binding region, and a cofilin homology sequence. To determine the contribution of each region to actin-based motility, we compared the biochemical activities of ActA derivatives with the phenotypes of corresponding mutant bacteria in cells. The acidic stretch functions to increase the efficiency of actin nucleation, the rate and frequency of motility, and the effectiveness of cell-cell spread. The monomer-binding region is required for actin nucleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer in host cytosol. The cofilin homology sequence is critical for stimulating actin nucleation with the Arp2/3 complex in vitro, and is essential for actin polymerization and motility in cells. These data demonstrate that each region contributes to actin-based motility, and that the cofilin homology sequence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.

• Huttelmaier S, Harbeck B, Steffens O, Messerschmidt T, Illenberger S, Jockusch BM
• Characterization of the actin binding properties of the vasodilator-stimulated phosphoprotein VASP.
• FEBS Lett. 1999; 451: 68-74
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• The vasodilator-stimulated phosphoprotein (VASP) colocalizes with the ends of stress fibers in cell-matrix and cell-cell contacts. We report here that bacterially expressed murine VASP directly interacts with skeletal muscle actin in several test systems including cosedimentation, viscometry and polymerization assays. It nucleates actin polymerization and tightly bundles actin filaments. The interaction with actin is salt-sensitive, indicating that the complex formation is primarily based on electrostatic interactions. Actin binding is confined to the C-terminal domain of VASP (EVH2). This domain, when expressed as a fusion protein with EGFP, associates with stress fibers in transiently transfected cells.

• Thanos CD, Bowie JU
• p53 Family members p63 and p73 are SAM domain-containing proteins.
• Protein Sci. 1999; 8: 1708-10
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• Homologs of the tumor suppressor p53, called p63 and p73, have been identified. The p63 and p73 family members possess a domain structure similar to p53, but contain variable C-terminal extensions. We find that some of the C-terminal extensions contain Sterile Alpha Motif (SAM) domains. SAM domains are protein modules that are involved in protein-protein interactions. Consistent with this role, the C-terminal SAM domains of the p63 and p73 may regulate function by recruiting other protein effectors.

• Marino M, Braun L, Cossart P, Ghosh P
• Structure of the lnlB leucine-rich repeats, a domain that triggers host cell invasion by the bacterial pathogen L. monocytogenes.
• Mol Cell. 1999; 4: 1063-72
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• The L. monocytogenes protein lnlB activates phosphoinositide 3-kinase and induces phagocytosis in several mammalian cell types. The 1.86 A resolution X-ray crystal structure of the leucine-rich repeat domain of lnlB that is both necessary and sufficient to induce phagocytosis is presented here. The structure supports a crucial role for calcium in host cell invasion by L. monocytogenes and supplies a rationale for its function. Calciums are bound to the protein in an unusually exposed manner that suggests that the metals may act as a bridge between lnlB and mammalian cell surface receptors. The structure also identifies surfaces on the curved and elongated molecule that may constitute additional interaction sites in forming a bacterial-mammalian signaling complex.

• Laurent V et al.
• Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes.
• J Cell Biol. 1999; 144: 1245-58
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• Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament.

• Mowbray SL, Bjorkman AJ
• Conformational changes of ribose-binding protein and two related repressors are tailored to fit the functional need.
• J Mol Biol. 1999; 294: 487-99
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• The structures and conformational changes of the periplasmic ribose-binding protein and two repressors, PurR and LacI, were compared. Although the closed, ligand-bound structures of the three proteins are very similar, they differ greatly in the degree and direction in which they open, as well as in the amount of internal rearrangement within the domains during that process. Water molecules and a relatively symmetrical inter-domain connection region assist in the large opening observed for the binding protein, while the design of the repressors appears to preclude such dramatic movements. The dimeric nature of the latter proteins, an important aspect in their binding of pseudo-symmetrical DNA sequences, also appears to be a determinant in the allowed motion. Slight differences in the structure of PurR and LacI explain how they can converge to a similar DNA-binding state in response to different binding states of their small molecule effector.

• Cicchetti G, Maurer P, Wagener P, Kocks C
• Actin and phosphoinositide binding by the ActA protein of the bacterial pathogen Listeria monocytogenes.
• J Biol Chem. 1999; 274: 33616-26
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• The surface protein ActA of the pathogenic bacterium Listeria monocytogenes induces actin-driven movement of bacteria in the cytoplasm of infected host cells and serves as a model for actin-based motility in general. We generated and purified soluble recombinant fragments of ActA and assessed their ability to interact with the acidic phospholipids phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, both implicated in the regulation of actin polymerization. Purified ActA consisted of biologically active, elongated molecules with an alpha-helix and beta-sheet content of 11 and 32%, respectively. In the presence of either phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate, but not phosphatidylcholine, ActA molecules underwent a structural change that raised the alpha-helix content to 19% and lowered the beta-sheet content to 27%. Co-sedimentation experiments with phosphatidylcholine vesicles containing different acidic phospholipids demonstrated that ActA binds preferentially to D-3 phosphoinositides. The D-3 phosphoinositide binding activity was mapped to a small subregion in the N-terminal domain of ActA. This subregion comprised 19 amino acids and showed homology to cecropins. In addition, we found that amino acids 33 to 74 of ActA mediated actin binding by the whole, folded ActA molecule. These findings shed new light on ActA function.

• Turner CE et al.
• Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.
• J Cell Biol. 1999; 145: 851-63
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• Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

• Bork P, Doerks T, Springer TA, Snel B
• Domains in plexins: links to integrins and transcription factors.
• Trends Biochem Sci. 1999; 24: 261-3

• Jonckheere V, Lambrechts A, Vandekerckhove J, Ampe C
• Dimerization of profilin II upon binding the (GP5)3 peptide from VASP overcomes the inhibition of actin nucleation by profilin II and thymosin beta4.
• FEBS Lett. 1999; 447: 257-63
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• Profilin II dimers bind the (GP5)3 peptide derived from VASP with an affinity of approximately 0.5 microM. The resulting profilin II-peptide complex overcomes the combined capacity of thymosin beta4 and profilin II to inhibit actin nucleation and restores the extent of filament formation. We do not observe such an effect when barbed filament ends are capped. Neither can profilin I, in the presence of the peptide, promote actin polymerization during its early phase consistent with a lower affinity. Since a Pro17 peptide-profilin II complex only partially restores actin polymerization, the glycine residues in the VASP peptide appear important.

• Purich DL, Southwick FS
• Actin-based motility of the intracellular pathogen Listeria monocytogenes: assessing the inhibitory specificity of ABM-1 peptide analogues.
• Mol Cell Biol Res Commun. 1999; 1: 176-81
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• Actin-Based Motility motifs [ABM-1 sequence = (D/E)FPPPPX(D/E), where X = P or T, and ABM-2 sequence = XPPPPP, where X denotes G, A, L, P, and S] facilitate assembly of an activated motility complex. Potent inhibition of intracellular motility of pathogens by ABM-1 and ABM-2 peptide analogues has served as a criterion for investigating actin-based motility. To assess the specificity of ABM-1 peptide inhibitors, we microinjected proline-rich peptides into Listeria-infected PtK2 host cells. Use of a combinatorial ABM-1 peptide library (empirical formula = D1E2F2P4T1) demonstrated that high-potency inhibition requires a precise sequence, and not merely a particular amino acid composition. Calculated concentrations of specific sequences in this library indicate that the entire (D/E)FPPPPX(D/E) motif is needed to achieve high-affinity inhibition in living cells. The failure of the well known proline-rich SH3 binding antagonists VSL-12 or APP-12 to inhibit Listeria motility also indicates that SH3 interactions are unlikely to control actin-based motility directly.

• May RC et al.
• The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes.
• Curr Biol. 1999; 9: 759-62
• Display abstract

• Actin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2] [3]; it is also believed to regulate actin dynamics in lamellipodia [4] [5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6] [7]. The Arp2/3 complex also interacts with members of the Wiskott-Aldrich syndrome protein (WASP) [8] family - Scar1 [9] [10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton.

• Yamada M et al.
• Flow cytometric analysis of Wiskott-Aldrich syndrome (WAS) protein in lymphocytes from WAS patients and their familial carriers.
• Blood. 1999; 93: 756-7

• Critchley DR, Holt MR, Barry ST, Priddle H, Hemmings L, Norman J
• Integrin-mediated cell adhesion: the cytoskeletal connection.
• Biochem Soc Symp. 1999; 65: 79-99
• Display abstract

• Members of the integrin family of cell adhesion molecules play a pivotal role in the interaction between animal cells and the extracellular matrix. This article reviews the evidence (i) that the integrin beta-subunit cytoplasmic domain is important in the localization of integrins to focal adhesions, and for integrin-mediated cell adhesion/spreading; and (ii) that the integrin beta-subunit can be linked to F-actin via the actin-binding proteins talin, alpha-actinin and filamin. Talin has two or more actin-binding sites, and three binding sites for the cytoskeletal protein vinculin. Because vinculin can also bind F-actin, it may cross-link talin and actin, thereby stabilizing the interaction. In addition, vinculin contains a binding site for VASP (vasodilator-stimulated phospho-protein), a protein which may serve to recruit a profilin/G-actin complex to talin, which has actin-nucleating activity. Evidence that talin, vinculin and alpha-actinin are important in the assembly of focal adhesions, obtained using antisense technology and protein microinjection, is reviewed. To analyse the role of talin in focal adhesions, we have disrupted both copies of the talin gene in mouse embryonic stem (ES) cells. Undifferentiated talin (-/-) ES cell mutants are unable to assemble focal adhesions when plated on fibronectin, whereas vinculin (-/-) ES cells are able to do so. Finally, the role of small GTP-binding proteins in the assembly of focal adhesions is discussed, along with our recent studies using streptolysin-O-permeabilized Swiss 3T3 cells which suggest that the GTP-binding protein ADP-ribosylation factor-1 (ARF-1) is important in targeting the protein paxillin to focal adhesions.

• Snapper SB, Rosen FS
• The Wiskott-Aldrich syndrome protein (WASP): roles in signaling and cytoskeletal organization.
• Annu Rev Immunol. 1999; 17: 905-29
• Display abstract

• The Wiskott-Aldrich Syndrome (WAS) is a rare X-linked primary immunodeficiency that is characterized by recurrent infections, hematopoietic malignancies, eczema, and thrombocytopenia. A variety of hematopoietic cells are affected by the genetic defect, including lymphocytes, neutrophils, monocytes, and platelets. Early studies noted both signaling and cytoskeletal abnormalities in lymphocytes from WAS patients. Following the identification of WASP, the gene mutated in patients with this syndrome, and the more generally expressed WASP homologue N-WASP, studies have demonstrated that WASP-family molecules associate with numerous signaling molecules known to alter the actin cytoskeleton. WASP/N-WASP may depolymerize actin directly and/or serve as an adaptor or scaffold for these signaling molecules in a complex cascade that regulates the cytoskeleton.

• Bachmann C, Fischer L, Walter U, Reinhard M
• The EVH2 domain of the vasodilator-stimulated phosphoprotein mediates tetramerization, F-actin binding, and actin bundle formation.
• J Biol Chem. 1999; 274: 23549-57
• Display abstract

• Vasodilator-stimulated phosphoprotein (VASP) is a member of the Ena/VASP family of proteins that are implicated in regulation of the actin cytoskeleton. All family members share a tripartite structural organization, comprising an N-terminal Ena/VASP homology (EVH) 1 domain, a more divergent proline-rich central part, and a common C-terminal EVH2 region of about 160-190 amino acids. Using chemical cross-linking, sucrose gradient sedimentation, and gel filtration analyses of different truncated VASP constructs, we demonstrate that the VASP EVH2 region is both necessary and sufficient for tetramerization. Moreover, co-sedimentation and fluorescent phalloidin staining showed that the EVH2 region binds and bundles F-actin in vitro and localizes to stress fibers in transfected cells. Analysis of the functional contribution of highly conserved blocks within this region indicated that residues 259-276 of human VASP are essential for the interaction with F-actin, whereas residues 343-380 are required for tetramerization, probably via coiled-coil formation. Interactions with F-actin are enhanced by VASP tetramerization. The results demonstrate that the C-terminal EVH2 segment is not only conserved in sequence but also forms a distinct functional entity. The data suggest that the EVH2 segment represents a novel oligomerization and F-actin binding domain.

• Hauser W et al.
• Megakaryocyte hyperplasia and enhanced agonist-induced platelet activation in vasodilator-stimulated phosphoprotein knockout mice.
• Proc Natl Acad Sci U S A. 1999; 96: 8120-5
• Display abstract

• Vasodilator-stimulated phosphoprotein (VASP), a substrate of cAMP- and cGMP-dependent protein kinases, is associated with focal adhesions, cell-cell contacts, microfilaments, and highly dynamic membrane regions. VASP, which is expressed in most cell types and in particularly high levels in human platelets, binds to profilin, zyxin, vinculin, F-actin, and the Listeria monocytogenes surface protein ActA. VASP is a member of the enabled (Ena)/VASP protein family and is thought to be involved in actin filament formation and integrin alphaIIbbeta3 inhibition in human platelets. To gain further insight into the in vivo function of this protein, VASP-deficient mice were generated by homologous recombination. VASP-/- mice demonstrated hyperplasia of megakaryocytes in bone marrow and spleen but exhibited no other macroscopic or microscopic abnormalities. Activation of platelets with thrombin induced a more than 2-fold higher surface expression of P-selectin and fibrinogen binding in VASP-deficient platelets in comparison to wild type. These data support the concept that VASP is a negative modulator of platelet and integrin alphaIIbbeta3 activation. Although the limited phenotypic differences between wild-type and VASP-/- mice suggested functional compensation of VASP by members of the Ena/VASP family, alterations in the expression levels of mammalian enabled (Mena) and Ena-VASP-like (Evl) protein were not detected. VASP-deficient mice may provide an interesting model system for diseases in which enhanced platelet activation plays a major role.

• Van Troys M, Vandekerckhove J, Ampe C
• Structural modules in actin-binding proteins: towards a new classification.
• Biochim Biophys Acta. 1999; 1448: 323-48
• Display abstract

• The number of actin binding proteins for which (part of) the three-dimensional structure is known, is steadily increasing. This has led to a picture in which defined structural modules with actin binding capacity are shared between different actin binding proteins. A classification of these based on their common three-dimensional modules appears a logical future step and in this review we provide an initial list starting from the currently known structures. The discussed cases illustrate that a comparison of the similarities and variations within the common structural actin binding unit of different members of a particular class may ultimately provide shortcuts for defining their actin target site and for understanding their effect on actin dynamics. Within this concept, the multitude of possible interactions by an extensive, and still increasing, list of actin binding proteins becomes manageable because they can be presented as variations upon a limited number of structural themes. We discuss the possible evolutionary routes that may have produced the present array of actin binding modules.

• Stewart DM, Tian L, Nelson DL
• Mutations that cause the Wiskott-Aldrich syndrome impair the interaction of Wiskott-Aldrich syndrome protein (WASP) with WASP interacting protein.
• J Immunol. 1999; 162: 5019-24
• Display abstract

• Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, eczema, immune deficiency, and a proclivity toward lymphoid malignancy. Lymphocytes of affected individuals show defects of activation, motility, and cytoskeletal structure. The disease gene encodes a 502-amino acid protein named the WAS protein (WASP). Studies have identified a number of important interactions that place WASP in a role of integrating signaling pathways with cytoskeletal function. We performed a two-hybrid screen to identify proteins interacting with WASP and cloned a proline-rich protein as a specific WASP interactor. Our clone of this protein, termed WASP interacting protein (WIP) by others, shows a difference in seven amino acid residues, compared with the previously published sequence revealing an additional profilin binding motif. Deletion mutant analysis reveals that WASP residues 101-151 are necessary for WASP-WIP interaction. Point mutant analyses in the two-hybrid system and in vitro show impairment of WASP-WIP interaction with three WASP missense mutants known to cause WAS. We conclude that impaired WASP-WIP interaction may contribute to WAS.

• Carl UD, Pollmann M, Orr E, Gertlere FB, Chakraborty T, Wehland J
• Aromatic and basic residues within the EVH1 domain of VASP specify its interaction with proline-rich ligands.
• Curr Biol. 1999; 9: 715-8
• Display abstract

• Short contiguous peptides harboring proline-rich motifs are frequently involved in protein-protein interactions, such as associations with Src homology 3 (SH3) and WW domains. Although patches of aromatic residues present in either domain interact with polyprolines, their overall structures are distinct, suggesting that additional protein families exist that use stacked aromatic amino acids (AA domains) to bind polyproline motifs [1] [2] [3]. A polyproline motif (E/DFPPPPTD/E in the single-letter amino-acid code), present in the ActA protein of the intracellular bacterial pathogen Listeria monocytogenes, serves as a ligand for the Ena/VASP protein family --the vasodilator-stimulated phosphoprotein (VASP), the murine protein Mena, Drosophila Enabled (Ena) and the Ena/VASP-like protein Evl [4] [5] [6] [7]. These share a similar overall structure characterized by the two highly conserved Ena/VASP homology domains (EVH1 and EVH2) [5]. Here, using three independent assays, we have delineated the minimal EVH1 domain. Mutations of aromatic and basic residues within two conserved hydrophilic regions of the EVH1 domain abolished binding to ActA. Binding of an EVH1 mutant with reversed charges could partially be rescued by introducing complementary mutations within the ligand. Like SH3 domains, aromatic residues within the EVH1 domain interacted with polyprolines, whereas the ligand specificity of either domain was determined by reciprocally charged residues. The EVH1 domain is therefore a new addition to the AA domain superfamily, which includes SH3 and WW domains.

• Nonoyama S, Ochs HD
• Characterization of the Wiskott-Aldrich syndrome protein and its role in the disease.
• Curr Opin Immunol. 1998; 10: 407-12
• Display abstract

• Wiskott-Aldrich syndrome is an X-linked disorder characterized by thrombocytopenia, eczema and immunodeficiency. The Wiskott-Aldrich syndrome protein and the gene that encodes it have been identified by positional cloning and the protein has been shown to contain a pleckstrin-homology domain, a GTPase-binding domain, a proline-rich region and a verprolin/cofilin homology domain. Subsequent studies suggest that the protein is involved in signal transduction and the regulation of the cytoskeleton.

• Brown MC, Curtis MS, Turner CE
• Paxillin LD motifs may define a new family of protein recognition domains.
• Nat Struct Biol. 1998; 5: 677-8

• Dowbenko D, Spencer S, Quan C, Lasky LA
• Identification of a novel polyproline recognition site in the cytoskeletal associated protein, proline serine threonine phosphatase interacting protein.
• J Biol Chem. 1998; 273: 989-96
• Display abstract

• Protein-protein interactions are often mediated by the recognition of proline-rich domains by SH3 or WW modules. Previously, we demonstrated that the PEST-type protein-tyrosine phosphatase, PTP HSCF (hematopoietic stem cell fraction), bound to a novel cytoskeletal associated protein, proline serine threonine phosphatase interacting protein (PST PIP), via an interaction between the proline-rich COOH terminus of the PTP and a site within the putative coiled-coil domain of PST PIP. Here we describe a more detailed analysis of this interaction. Earlier data suggested that the NH2 terminus of PST PIP was important for binding to the phosphatase, and deletion of the NH2-terminal 50 amino acids of the PST PIP resulted in an apparently misfolded protein that was incapable of binding PTP HSCF. To examine the region involved with binding to PTP HSCF, alanine-scanning mutants were produced at intervals throughout PST PIP. This analysis demonstrated that a tryptophan at position 232 was essential for binding in vitro. Transfection experiments demonstrated that the Trp232 mutant protein was capable of association with the cortical cytoskeleton but was not bound to PTP HSCF in vivo. Alanine scanning of a peptide derived from the COOH-terminal proline-rich domain of PTP HSCF revealed that a subset of prolines, as well as other residues, was required for efficient binding to PST PIP, and introduction of alanines at some of these positions in the protein resulted in decreased binding to PST PIP in vitro and in vivo. Analysis of in vivo tyrosine phosphorylation of the Trp232 mutant of PST PIP in the presence of v-Src revealed that this protein was phosphorylated more efficiently than the wild-type molecule. Thus, the interaction between PTP HSCF and PST PIP is mediated by a novel site in the cytoskeletal associated protein which interacts with residues within the proline-rich COOH terminus of the phosphatase.

• Wehland J, Carl UD
• The sophisticated survival strategies of the pathogen Listeria monocytogenes.
• Int Microbiol. 1998; 1: 11-8
• Display abstract

• The function of the ActA protein of Listeria monocytogenes has been partially elucidated. These results illustrate the sophistication with which intracellular pathogens like Listeria use the host cell to their advantage, and have provided new insights into some of the molecular mechanisms of complex cell functions such as actin-promoted cell motility. The clarification of these processes is of fundamental importance not only for understanding elementary processes such as development and growth, but also for the treatment of both diseases caused by cytopathogenic bacteria such as Listeria and pathophysiological processes arising from disorders in cell motility and cell adhesion.

• Holt MR, Critchley DR, Brindle NP
• The focal adhesion phosphoprotein, VASP.
• Int J Biochem Cell Biol. 1998; 30: 307-11
• Display abstract

• Vasodilator-stimulated phosphoprotein (VASP) is associated with focal adhesions and areas of dynamic membrane activity, where it is thought to have an important role in actin filament assembly and cell motility. VASP contains a central proline-rich sequence which recruits the G-actin binding protein profilin. Localization of VASP to the leading edge of a migrating cell can lead to local accumulation of profilin, which in turn can supply actin monomers to growing filament ends. VASP binds to the focal adhesion proteins vinculin and zyxin and this probably directs the phosphoprotein to focal adhesions and the leading edge of stimulated cells. VASP functions as a binding intermediate between profilin and focal adhesion proteins. Intracellular pathogens, including Listeria monocytogenes, have coat proteins which bind VASP. This is one way in which these pathogens use VASP, and other proteins from the host cell, to assemble the actin filaments they require to move around the cytoplasm of infected cells and enter neighbouring cells. Understanding the role of VASP and other proteins in cell and bacterial motility is likely to lead to development of new therapeutic strategies for diseases including atherosclerosis and tumour growth, and for limiting the spread of intracellular pathogens.

• Lipsky BP, Beals CR, Staunton DE
• Leupaxin is a novel LIM domain protein that forms a complex with PYK2.
• J Biol Chem. 1998; 273: 11709-13
• Display abstract

• We have identified a novel cytoplasmic protein, leupaxin, that is preferentially expressed in hematopoietic cells and is most homologous to the focal adhesion protein, paxillin. Leupaxin possesses two types of protein interaction domains. There are four carboxyl-terminal LIM domains in leupaxin that share 70% amino acid identity and 80% similarity with those in paxillin. Paxillin LIM domains mediate localization to focal contacts. In the amino-terminal region of leupaxin there are three short stretches of approximately 13 amino acids that share 70-90% similarity with paxillin LD motifs. Paxillin LD motifs have been implicated in focal adhesion kinase (FAK) and vinculin binding resulting in the localization of FAK to focal adhesions. Leupaxin is expressed in cell types, such as macrophage, that lack FAK. We demonstrate here that leupaxin associates with a second FAK family member, PYK2. As leupaxin and PYK2 are both preferentially expressed in leukocytes they may therefore form a cell type-specific signaling complex. We also demonstrate that leupaxin is a substrate for a tyrosine kinase in lymphoid cells and thus may function in and be regulated by tyrosine kinase activity. Leupaxin is thus a phosphotyrosine protein with LD and LIM binding motifs most homologous to paxillin that may assemble and regulate PYK2 signaling complexes in leukocytes.

• MacCarthy-Morrogh L et al.
• Absence of expression of the Wiskott-Aldrich syndrome protein in peripheral blood cells of Wiskott-Aldrich syndrome patients.
• Clin Immunol Immunopathol. 1998; 88: 22-7
• Display abstract

• Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that is usually associated with thrombocytopenia and eczema. The very variable phenotype of WAS results from defects in the WAS protein (WASP), the function of which is not well understood. In many cases causative mutations have now been identified in the WAS gene. Attempts have been made to correlate the nature of the mutations with the severity of the disease. In this study we investigated mutations in 13 patients with WAS and analyzed the expression of WASP in patient blood samples by immunoblot analysis. We found that despite extensive variation in the nature of the mutations in patients with severe WAS symptoms, none express the protein. However, in 1 patient with a mild clinical phenotype WASP expression was detected. Such an analysis could be used as an initial screening procedure for the diagnosis of WAS prior to genotypic analysis.

• Ahern-Djamali SM et al.
• Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains.
• Mol Biol Cell. 1998; 9: 2157-71
• Display abstract

• Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

• Kirchhausen T
• Wiskott-Aldrich syndrome: a gene, a multifunctional protein and the beginnings of an explanation.
• Mol Med Today. 1998; 4: 300-4
• Display abstract

• Patients with Wiskott-Aldrich syndrome show various defects in the normal function of platelets and lymphocytes. The recent identification of the gene responsible for this syndrome has led to a surge of studies aimed at solving the puzzle posed by the varied phenotype observed in this disease. It is now known that WASP, the protein product of this gene, can interact with a large number of other proteins known to be involved in the regulation of signal transduction and cytoskeletal organization. Thus, WASP appears to integrate these two basic and fundamental cellular mechanisms. Several groups are now focusing on understanding the function of WASP in detail, and translating this new knowledge into improved therapies.

• Ochs HD
• The Wiskott-Aldrich syndrome.
• Springer Semin Immunopathol. 1998; 19: 435-58

• Andrews PA, Koffman CG
• Outcome of renal transplantation in Wiskott-Aldrich syndrome.
• Nephrol Dial Transplant. 1998; 13: 524-5

• Theriot JA, Fung DC
• Listeria monocytogenes-based assays for actin assembly factors.
• Methods Enzymol. 1998; 298: 114-22

• Ariga T, Yamada M, Sakiyama Y, Tatsuzawa O
• A case of Wiskott-Aldrich syndrome with dual mutations in exon 10 of the WASP gene: an additional de novo one-base insertion, which restores frame shift due to an inherent one-base deletion, detected in the major population of the patient's peripheral blood lymphocytes.
• Blood. 1998; 92: 699-701

• Purich DL, Southwick FS
• ABM-1 and ABM-2 homology sequences: consensus docking sites for actin-based motility defined by oligoproline regions in Listeria ActA surface protein and human VASP.
• Biochem Biophys Res Commun. 1997; 231: 686-91
• Display abstract

• Actin-based motility involves a cascade of binding interactions designed to assemble actin regulatory proteins into functional locomotory units. Listeria ActA surface protein contains a series of nearly identical EFPPPPTDE-type oligoproline sequences for binding vasodilator-stimulated phosphoprotein (VASP). The latter is a tetrameric protein with numerous GPP-PPP docking sites for profilin, a 15 kDa regulatory protein that promotes actin filament assembly. Analysis of known actin regulatory proteins led to the identification of distinct Actin-Based Motility homology sequences ABM-1; (D/E)FPPPPX(D/E); and ABM-2, XPPPPP (where X denotes G, A, L, and S).

• Ariga T, Yamada M, Sakiyama Y
• Mutation analysis of five Japanese families with Wiskott-Aldrich syndrome and determination of the family members' carrier status using three different methods.
• Pediatr Res. 1997; 41: 535-40
• Display abstract

• Mutation analysis for five families with Wiskottt-Aldrich syndrome was performed. The mutations found were two missense mutations, two one-base deletion mutations, and a large deletion mutation in the WASP gene. The three mutations had been reported before, but the remaining two were new. We used the mutation information to determine the carrier status of the female relatives of the patients. Three different approaches were taken depending on the type of mutation, and the carrier determination was successfully performed.

• Ariga T, Yamada M, Ito S, Iwamura M, Iseki M, Sakiyama Y
• Characterization of a deletion mutation involving exons 3-7 of the WASP gene detected in a patient with Wiskott-Aldrich syndrome.
• Hum Mutat. 1997; 10: 310-6
• Display abstract

• A case of Wiskott-Aldrich syndrome (WAS) suspected to have a deletion mutation in the WAS protein (WASP) gene had previously been reported (Ariga et al., 1997). Genomic polymerase chain reaction (PCR) suggested that exons 3-7 of the WASP gene were included in the deletion. Present Southern blot studies confirm that the deletion is approximately 2.0 kb in length, involving exons 3-7 and seemed to have been created by the fusion of introns 2 and 7. To characterize the deletion mutation in detail, we analyzed the PCR-amplified fragments of introns 2 and 7 from normal individuals and the fragment suspected of including the deletion junction from the patient. Sequencing of the patient fragment revealed that the deletion mutation involving exons 3-7 of the WASP gene did, indeed, result from the fusion of introns 2 and 7.

• Kang F, Laine RO, Bubb MR, Southwick FS, Purich DL
• Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP): implications for actin-based Listeria motility.
• Biochemistry. 1997; 36: 8384-92
• Display abstract

• Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers [consensus sequence = FE(D)FPPPPTD(E)E(D)] to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP5) registers to localize the actin-regulatory protein profilin to promote actin polymerization. We now report that fluorescence titration showed that GP5GP5GP5 peptide binds to profilin (KD of 84 microM), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin. Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 microM. This inhibition was completely neutralized when equimolar concentrations of profilin and GP5GP5GP5 were simultaneously microinjected. Fluorescence studies with [His-133-Ser]-profilin, a site-directed mutant previously shown to be defective in binding poly-l-proline [Bjorkegren, C., Rozycki, M., Schutt, C. E., Lindberg, U., & Karlsson, R. (1993) FEBS Lett. 333, 123-126], exhibits little or no evidence of saturable GP5GP5GP5 binding. When an equimolar concentration of this [His-133-Ser]-profilin mutant was co-injected with GP5GP5GP5, the peptide's inhibitory action remained completely unaffected, indicating that GP5GP5GP5 binding to wild-type profilin represents a key step in actin-based pathogen motility. We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.

• Welch MD, Iwamatsu A, Mitchison TJ
• Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenes.
• Nature. 1997; 385: 265-9
• Display abstract

• The pathogenic bacterium Listeria monocytogenes is capable of directed movement within the cytoplasm of infected host cells. Propulsion is thought to be driven by actin polymerization at the bacterial cell surface, and moving bacteria leave in their wake a tail of actin filaments. Determining the mechanism by which L. monocytogenes polymerizes actin may aid the understanding of how actin polymerization is controlled in the cell. Actin assembly by L. monocytogenes requires the bacterial surface protein ActA and protein components present in host cell cytoplasm. We have purified an eight-polypeptide complex that possesses the properties of the host-cell actin polymerization factor. The pure complex is sufficient to initiate ActA-dependent actin polymerization at the surface of L. monocytogenes, and is required to mediate actin tail formation and motility. Two subunits of this protein complex are actin-related proteins (ARPs) belonging to the Arp2 and Arp3 subfamilies. The Arp3 subunit localizes to the surface of stationary bacteria and the tails of motile bacteria in tissue culture cells infected with L. monocytogenes; this is consistent with a role for the complex in promoting actin assembly in vivo. The activity and subunit composition of the Arp2/3 complex suggests that it forms a template that nucleates actin polymerization.

• Machesky LM
• Cell motility: complex dynamics at the leading edge.
• Curr Biol. 1997; 7: 1647-1647
• Display abstract

• The intracellular pathogen Listeria monocytogenes is a useful model for general actin-based cell motility, because it recruits host actin and associated proteins for movement. Recent data have shown that these associated proteins include the Ena/VASP family of proteins and the actin-related proteins Arp2 and Arp3.

• Smith GA, Portnoy DA
• How the Listeria monocytogenes ActA protein converts actin polymerization into a motile force.
• Trends Microbiol. 1997; 5: 272-6
• Display abstract

• The ActA protein is an essential determinant of pathogenicity that is responsible for the actin-based motility of Listeria monocytogenes in mammalian cells and cell-free extracts. ActA appears to control at least four functions that collectively lead to actin-based motility: (1) initiation of actin polymerization, (2) polarization of ActA function, (3) transformation of actin polymerization into a motile force and (4) acceleration of movement mediated by the host protein profilin.

• Kondoh T, Matsumoto T, Tsuji Y
• Wiskott-Aldrich syndrome in two sisters.
• Am J Med Genet. 1997; 74: 218-9

• Lambrechts A, Verschelde JL, Jonckheere V, Goethals M, Vandekerckhove J, Ampe C
• The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences.
• EMBO J. 1997; 16: 484-94
• Display abstract

• We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.

• Beckerle MC
• Zyxin: zinc fingers at sites of cell adhesion.
• Bioessays. 1997; 19: 949-57
• Display abstract

• Zyxin is a low abundance phosphoprotein that is localized at sites of cell-substratum adhesion in fibroblasts. Zyxin displays the architectural features of an intracellular signal transducer. The protein exhibits an extensive proline-rich domain, a nuclear export signal and three copies of the LIM motif, a double zinc-finger domain found in many proteins that play central roles in regulation of cell differentiation. Zyxin interacts with alpha-actinin, members of the cysteine-rich protein (CRP) family, proteins that display Src homology 3 (SH3) domains and Ena/VASP family members. Zyxin and its partners have been implicated in the spatial control of actin filament assembly as well as in pathways important for cell differentiation. Based on its repertoire of binding partners and its behavior, zyxin may serve as a scaffold for the assembly of multimeric protein machines that function in the nucleus and at sites of cell adhesion.

• Zhu Q et al.
• Wiskott-Aldrich syndrome/X-linked thrombocytopenia: WASP gene mutations, protein expression, and phenotype.
• Blood. 1997; 90: 2680-9
• Display abstract

• Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), caused by mutations of the WAS protein (WASP) gene, represent different phenotypes of the same disease. To demonstrate a phenotype/genotype correlation, we determined WASP gene mutations in 48 unrelated WAS families. Mutations included missense (20 families) and nonsense (eight) mutations located mostly in exons 1 to 4, and splice-site mutations (seven) and deletions and insertions (13) located preferentially in exons 7 to 11. Both genomic DNA and cDNA were sequenced and WASP expression was measured in cell lysates using peptide-specific rabbit anti-WASP antibodies. WASP was expressed in hematopoietic cell lines including bone marrow-derived CD34+ cells. Missense mutations located in exons 1 to 3 caused mild disease in all but one family and permitted WASP expression, although frequently at decreased concentration. Missense mutations affecting exon 4 were associated with classic WAS and, with one exception, barely detectable WASP. Nonsense mutations caused classic WAS and lack of protein. Insertions, deletions, and splice-site mutations resulted in classic WAS and absent, unstable, truncated, or multiply spliced protein. Using affinity precipitation, WASP was found to bind to Src SH3-containing proteins Fyn, Lck, PLC-gamma, and Grb2, and mutated WASP, if expressed, was able to bind to Fyn-glutathione S-transferase (GST) fusion protein. We conclude that missense mutations affecting the PH domain (exons 1 to 3) of WASP inhibit less important functions of the protein and result in a mild phenotype, and that missense mutations affecting exon 4 and complex mutations affecting the 3' portion of WASP interfere with crucial functions of the protein and cause classic WAS.

• Ramesh N, Anton IM, Hartwig JH, Geha RS
• WIP, a protein associated with wiskott-aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells.
• Proc Natl Acad Sci U S A. 1997; 94: 14671-6
• Display abstract

• Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency caused by mutations that affect the WAS protein (WASP) and characterized by cytoskeletal abnormalities in hematopoietic cells. By using the yeast two-hybrid system we have identified a proline-rich WASP-interacting protein (WIP), which coimmunoprecipitated with WASP from lymphocytes. WIP binds to WASP at a site distinct from the Cdc42 binding site and has actin as well as profilin binding motifs. Expression of WIP in human B cells, but not of a WIP truncation mutant that lacks the actin binding motif, increased polymerized actin content and induced the appearance of actin-containing cerebriform projections on the cell surface. These results suggest that WIP plays a role in cortical actin assembly that may be important for lymphocyte function.

• Ireton K, Cossart P
• Host-pathogen interactions during entry and actin-based movement of Listeria monocytogenes.
• Annu Rev Genet. 1997; 31: 113-38
• Display abstract

• Listeria monocytogenes is a pathogenic bacterium that induces its own uptake into mammalian cells, and spreads from one cell to another by an actin-based motility process. Entry into host cells involves the bacterial surface proteins InlA (internalin) and InlB. The receptor for InlA is the cell adhesion molecule E-cadherin. InlB-mediated entry requires activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor. Actin-based movement of L. monocytogenes is mediated by the bacterial surface protein ActA. The N-terminal region of this protein is necessary and sufficient for polymerization of host cell actin. Other host proteins involved in bacterial motility include profilin, Vasodilator-Stimulated Phosphoprotein (VASP), the Arp2/Arp3 complex, and cofilin. Studies of entry and intracellular movement of L. monocytogenes could lead to a better understanding of receptor-ligand signaling and dynamics of actin polymerization in mammalian cells.

• Goldberg MB
• Shigella actin-based motility in the absence of vinculin.
• Cell Motil Cytoskeleton. 1997; 37: 44-53
• Display abstract

• Reports on the role of vasodilator-stimulated phosphoprotein (VASP) and proline-rich sequences in actin-based motility of Listeria and potentially of Shigella flexneri have led to the suggestion that vinculin might be an essential docking protein on the surface O2 motile Shigella. Therefore, whether vinculin had a functional role in Shigella actin-based motility was tested by examining Shigella infection of the vinculin-deficient F9 cell line variant 5.51. Shigella are able to form actin tails and surface protrusions in 5.51 cells that are indistinguishable from those they produce in F9 cells, and Shigella rates of intracellular movement and protrusion formation are similar in the two cell lines. These data disprove the model of Shigella actin-based motility in which vinculin is an essential docking protein for either the formation of actin tails or the acceleration of motile bacteria.

• Schindelhauer D et al.
• Wiskott-Aldrich syndrome: no strict genotype-phenotype correlations but clustering of missense mutations in the amino-terminal part of the WASP gene product.
• Hum Genet. 1996; 98: 68-76
• Display abstract

• The Wiskott-Aldrich syndrome protein (WASP) gene was found to be mutated in patients presenting with WAS and in patients showing X-linked thrombocytopenia. Mutation analysis in 19 families of German, Swiss and Turkish descent by single-strand conformation polymorphism and sequencing resulted in the detection of seven novel and 10 known mutations. A striking clustering of missense mutations in the first four exons contrasted with a random distribution of nonsense mutations. More than 85% of all known missense mutations were localized in the amino-terminal stretch of the WASP gene product; this region contained a mutational hot spot at codon 86. No genotype-phenotype correlation emerged after a comparison of the identified mutations with the resulting clinical picture for a classical WAS phenotype. A substitution at codon 86 resulted in an extremely variable expression of the disease in a large Swiss family. An extended homology search revealed a distant relationship of this stretch to the vasodilator-stimulated phosphoprotein (VASP), which is involved in the maintenance of cyto-architecture by interacting with actin-like filaments.

• Schwartz M et al.
• Mutation spectrum in patients with Wiskott-Aldrich syndrome and X-linked thrombocytopenia: identification of twelve different mutations in the WASP gene.
• Thromb Haemost. 1996; 75: 546-50
• Display abstract

• Twelve different mutations in the WASP gene were found in twelve unrelated families with Wiskott-Aldrich syndrome (WAS) or X-linked thrombocytopenia (XLT). Four frameshift, one splice, one nonsense mutation, and one 18-base-pair deletion were detected in seven patients with WAS. Only missense mutations were found in five patients diagnosed as having XLT. One of the nucleotide substitutions in exon 2 (codon 86) results in an Arg to Cys replacement. Two other nucleotide substitutions in this codon, R86L and R86H, have been reported previously, both giving rise to typical WAS symptoms, indicating a mutational hot spot in this codon. The finding of mutations in the WASP gene in both WAS and XLT gives further evidence of these syndromes being allelic. The relatively small size of the WASP gene facilitates the detection of mutations and a reliable diagnosis of both carriers and affected fetuses in families with WAS or XLT.

• Koonin EV
• Human choroideremia protein contains a FAD-binding domain.
• Nat Genet. 1996; 12: 237-9

• Smith GA, Theriot JA, Portnoy DA
• The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin.
• J Cell Biol. 1996; 135: 647-60
• Display abstract

• The ActA protein is responsible for the actin-based movement of Listeria monocytogenes in the cytosol of eukaryotic cells. Analysis of mutants in which we varied the number of proline-rich repeats (PRR; consensus sequence DFPPPPTDEEL) revealed a linear relationship between the number of PRRs and the rate of movement, with each repeat contributing approximately 2-3 microns/min. Mutants lacking all functional PRRs (generated by deletion or point mutation) moved at rates 30% of wild-type. Indirect immunofluorescence indicated that the PRRs were directly responsible for binding of vasodilator-stimulated phosphoprotein (VASP) and for the localization of profilin at the bacterial surface. The long repeats, which are interdigitated between the PRRs, increased the frequency with which actin-based motility occurred by a mechanism independent of the PRRs, VASP, and profilin. Lastly, a mutant which expressed low levels of ActA exhibited a phenotype indicative of a threshold; there was a very low percentage of moving bacteria, but when movement did occur, it was at wild-type rates. These results indicate that the ActA protein directs at least three separable events: (1) initiation of actin polymerization that is independent of the repeat region; (2) initiation of movement dependent on the long repeats and the amount of ActA; and (3) movement rate dependent on the PRRs.

• Wolfsberg TG, White JM
• ADAMs in fertilization and development.
• Dev Biol. 1996; 180: 389-401

• Rosen FS
• The enigma of the Wiskott-Aldrich syndrome begins to unravel.
• J Clin Invest. 1996; 97: 2407-2407

• Chakraborty T
• The molecular mechanisms of actin-based intracellular motility by Listeria monocytogenes.
• Microbiologia. 1996; 12: 237-44
• Display abstract

• A key virulence trait of bacteria and viruses that multiply in the cytoplasm of the infected cell is their ability to direct movement intracellularly and to spread from cell-to-cell. Intracellular movement is effected by harnessing components of the host microfilament system. This mode of locomotion by intracytoplasmic parasites has recently gained much interest as a model to examine microfilament assembly and function. Of the intracellular bacteria employing association with the host cytoskeleton to effect movement, the Gram-positive pathogen Listeria monocytogenes is the most well studied. This review summarizes the current state of the understanding, at the molecular level, of how L. monocytogenes subverts the host cell contractile machinery to meet its own need to move and spread within infected host cells.

• Gerstel B, Grobe L, Pistor S, Chakraborty T, Wehland J
• The ActA polypeptides of Listeria ivanovii and Listeria monocytogenes harbor related binding sites for host microfilament proteins.
• Infect Immun. 1996; 64: 1929-36
• Display abstract

• The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes acts as a nucleator protein, generating the actin cytoskeleton around intracellularly motile bacteria. In this work, we examined the functional similarity of ActA from Listeria ivanovii (iActA) ATCC 19119 to its L. monocytogenes counterpart. The amino acid sequence of iActA predicts a molecular mass of 123 kDa and harbors eight proline-rich repeats. For functional analysis, various iActA derivatives and hybrid constructs of L. ivanovii and L. monocytogenes ActA polypeptides were transiently expressed in epithelial cells and examined for recruitment of host microfilament proteins by a mitochondrial targeting assay. As has been demonstrated with ActA, iActA also spontaneously inserted into the surface of mitochondria and induced recruitment of actin, alpha-actinin, and the vasodilator-stimulated phosphoprotein (VASP) to these subcellular organelles. By comparison of amino-terminally truncated iActA derivatives for their ability to recruit cytoskeletal proteins, a region essential for actin filament accumulation was identified between amino acid residues 290 and 325. Such derivatives, however, retained their ability to bind VASP. Replacement of the proline-rich repeats in ActA with those of iActA also resulted in VASP recruitment. Hence, despite the limited overall sequence homology between ActA and iActA, the two molecules consist of at least two similar domains: a highly positively charged N-terminal domain that is directly involved in actin filament recruitment and a proline-rich repeat region required for VASP binding.

• Reinhard M, Rudiger M, Jockusch BM, Walter U
• VASP interaction with vinculin: a recurring theme of interactions with proline-rich motifs.
• FEBS Lett. 1996; 399: 103-7
• Display abstract

• VASP (vasodilator-stimulated phosphoprotein), a protein associated with microfilaments at cellular contact sites, has been identified as a ligand for profilin and zyxin, two proteins also involved in microfilament dynamics and organization at these regions. Here, we report that VASP also directly binds to vinculin, another component of adherens junctions. Competition experiments with a vinculin-derived peptide showed that a proline-rich motif, located in the hinge region that connects vinculin's head and tail domains, is involved in VASP binding. The same motif is present in zyxin but the interactions of VASP with vinculin and zyxin differ in detail. Hence, this motif may be recognized by VASP in different ways when presented in distinct cellular sites.

• Kuska B
• Wiskott-Aldrich syndrome is a "wonderful mystery".
• J Natl Cancer Inst. 1996; 88: 1258-61

• Stewart DM, Treiber-Held S, Kurman CC, Facchetti F, Notarangelo LD, Nelson DL
• Studies of the expression of the Wiskott-Aldrich syndrome protein.
• J Clin Invest. 1996; 97: 2627-34
• Display abstract

• The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by thrombocytopenia, eczema, disorders in cell-mediated and humoral immunity, and a proclivity to lymphoproliferative disease. The gene responsible encodes a 53-kD proline-rich protein of unknown function (WASP). We produced a FLAG-WASP fusion protein that was used to immunize mice and produce mAbs against WASP. Using monoclonal anti-WASP in Western immunoblots, we have determined that WASP is present in the cytoplasmic but not nuclear fraction of normal human peripheral blood mononuclear cells, in normal human platelets, in T lymphocytes, non-T lymphocytes, and monocytes. The protein is produced in the B cell immunoblastic cell line DS-1, in normal EBV-transformed B cell lines, and in HEL92.1.7, but is barely detectable in MOLT-4 and not detectable in K562. WASP was present in two of four EBV-transformed cell lines from WAS patients. Splenic tissue immunostaining was performed in two patients, and the results correlated with the results of the Western blots. Sequence analysis of WASP cDNA from two patients who produce WASP show mutations causing amino acid substitutions. These studies establish a foundation for further studies aimed at understanding the function of WASP.

• Zeile WL, Purich DL, Southwick FS
• Recognition of two classes of oligoproline sequences in profilin-mediated acceleration of actin-based Shigella motility.
• J Cell Biol. 1996; 133: 49-59
• Display abstract

• The gram negative rod Shigella flexneri uses it surface protein IcsA to induce host cell actin assembly and to achieve intracellular motility. Yet, the IcsA protein lacks the oligoproline sequences found in ActA, the surface protein required for locomotion of the gram positive rod Listeria monocytogenes. Microinjection of a peptide matching the second ActA oligoproline repeat (FEFPPPPTDE) stops Listeria locomotion (Southwick, F.S., and D.L. Purich. 1994a. Proc. Natl. Acad. Sci. USA. 91:5168-5172), and submicromolar concentrations (intracellular concentration 80-800 nM) similarly arrest Shigella rocket-tail assembly and intracellular motility. Coinjection of a binary solution containing profilin and the ActA analogue increased the observed rates of intracellular motility by a factor of three (mean velocity 0.90 +/- 0.07 mu m/s, SD n=16 before injection vs 0.3 +/- 0.1 mu m/s, n=33 postinjection, intracellular concentration = 80 nM profilin plus 80 nM ActA analogue). Recent evidence suggests the ActA analogue may act by displacing the profilin-binding protein VASP (Pistor, S.C., T. Chakaborty, V. Walter, and J. Wehland. 1995. Curr. Biol. 5:517-525). At considerably higher intracellular concentrations (10 muM), the VASP oligoproline sequence (GPPPPP)3 thought to represent the profilin-binding site (Reinhard, M., K. Giehl, K. Abel, C. Haffner, T. Jarchau, V. Hoppe, B.M. Jockusch, and U. Walter. 1995. EMBO (Eur. Mol. Biol. Organ.) J. 14:1583-1589) also inhibited Shigella movement. A binary mixture of the VASP analogue and profilin (each 10 muM intracellular concentration) led to a doubling of Shigella intracellular migration velocity (0.09 +/- 0.06 mu m/s, n = 25 preinjection vs 0.18 +/- 0.10 mu m/s, n = 61 postinjection). Thus, the two structurally divergent bacteria, Listeria and Shigella, have adopted convergent mechanisms involving profilin recognition of VASP oligoproline sequences and VASP recognition of oligoproline sequences in ActA or an ActA-like host protein to induce host cell actin assembly and to provide the force for intracellular locomotion and cell-cell spread.

• Southwick FS, Purich DL
• Inhibition of Listeria locomotion by mosquito oostatic factor, a natural oligoproline peptide uncoupler of profilin action.
• Infect Immun. 1995; 63: 182-90
• Display abstract

• Mosquito oostatic factor, a naturally occurring decapeptide (YDPAPPPPPP), strikingly resembles the primary structure of oligoproline-rich regions within the protein ActA, a bacterial surface protein required for Listeria motility in host cells. When microinjected into Listeria-infected PtK2 cells, the insect oostatic factor rapidly blocks Listeria-induced actin rocket tail assembly as well as intracellular locomotion of this pathogen. At intracellular concentrations of about 90 nM, transient inhibition of rocket tail formation and bacterial locomotion occurs, followed by full recovery of tail length and motility. However, at 0.9 microM oostatic factor, both processes are permanently arrested. Introduction of oostatic factor by microinjection also causes PtK2 peripheral membrane retraction in both Listeria-infected and uninfected cells. Epifluorescence microscopy with bodipy-phallacidin reveals that cells microinjected with the insect factor lose all actin stress fibers and accumulate F-actin in regions of membrane retraction. When the insect peptide is combined with profilin as an equimolar binary solution (1 microM [final concentration] each), intracellular addition fails to inhibit Listeria rocket-tail formation, fails to block intracellular bacterial movement, and no longer causes marked membrane retraction. The ability of profilin to neutralize the inhibitory action of oostatic factor is consistent with complex formation, and this finding suggests that profilin may interact directly with ActA peptide as well as a host cell peripheral membrane component to promote actin filament assembly by locally generating ATP-actin. Dispersal of profilin from such sites by oligoproline-rich peptide inhibitors suggests that profilin is directly involved in intracellular pathogen locomotion and reorganization of actin cytoskeleton of the host cell peripheral membrane.

• Rivero-Lezcano OM, Marcilla A, Sameshima JH, Robbins KC
• Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains.
• Mol Cell Biol. 1995; 15: 5725-31
• Display abstract

• In the second of a series of experiments designed to identify p47nck-Src homology 3 (SH3)-binding molecules, we report the cloning of SAKAP II (Src A box Nck-associated protein II) from an HL60 cDNA expression library. This molecule has been identified as a cDNA encoding the protein product of WASP, which is mutated in Wiskott-Aldrich syndrome patients. Studies in vivo and in vitro demonstrated a highly specific interaction between the SH3 domains of p47nck and Wiskott-Aldrich syndrome protein. Furthermore, anti-Wiskott-Aldrich syndrome protein antibodies recognized a protein of 66 kDa by Western blot (immunoblot) analysis. In vitro translation studies identified the 66-kDa protein as the protein product of WASP, and subcellular fractionation experiments showed that p66WASP is mainly present in the cytosol fraction, although significant amounts are also present in membrane and nuclear fractions. The main p47nck region implicated in the association with p66WASP was found to be the carboxy-terminal SH3 domain.

• Bennett CP, Barnicoat AJ, Cotter F, Wang Q, Mathew CG
• A variant of Wiskott-Aldrich syndrome with nephropathy is linked to DXS255.
• J Med Genet. 1995; 32: 757-8

• Pollard TD
• Actin cytoskeleton. Missing link for intracellular bacterial motility?
• Curr Biol. 1995; 5: 837-40
• Display abstract

• Vasodilator-stimulated phosphoprotein (VASP) associates with virulence factors on the surface of intracellular bacteria; by binding to profilin, VASP may help direct the actin assembly that appears to drive bacterial motility.

• Derry JM et al.
• The mouse homolog of the Wiskott-Aldrich syndrome protein (WASP) gene is highly conserved and maps near the scurfy (sf) mutation on the X chromosome.
• Genomics. 1995; 29: 471-7
• Display abstract

• The mouse WASP gene, the homolog of the gene mutated in Wiskott-Aldrich syndrome, has been isolated and sequenced. the predicted amino acid sequence is 86% identical to the human WASP sequence. A distinct feature of the mouse gene is an expanded polymorphic GGA trinucleotide repeat that codes for polyglycine and varies from 15 to 17 triplets in different Mus musculus strains. The genomic structure of the mouse WASP gene is expressed as an approximately 2.4-kb mRNA in thymus and spleen. Chromosomal mapping in an interspecific M. Musculus/M. spretus backcross placed the Wasp locus near the centromere of the mouse X chromosome, inseparable from Gata1, Tcfe3, and scurfy (sf). This localization makes Wasp a candidate for involvement in scurfy, a T cell-mediated fatal lymphoreticular disease of mice that has previously been proposed as a mouse homolog of Wiskott-Aldrich syndrome. Northern analysis of sf tissue samples indicated the presence of WASP mRNA in liver and skin, presumably as a consequence of lymphocytic infiltration, but non abnormalities in the amount or size of mRNA present.

• Pistor S, Chakraborty T, Walter U, Wehland J
• The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins.
• Curr Biol. 1995; 5: 517-25
• Display abstract

• BACKGROUND: Several intracellular pathogens, including Listeria monocytogenes, use components of the host actin-based cytoskeleton for intracellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-based motility. Genetic analysis of L. monocytogenes led to the identification of the 90 kD surface-bound ActA polypeptide as the sole bacterial factor required for the initiation of recruitment of host actin filaments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is inside a mammalian cell, including alpha-actinin, fimbrin, filamin, villin, ezrin/radixin, profilin and the vasodilator-stimulated phosphoprotein, VASP. Only VASP is known to bind directly to ActA. We sought to determine which regions of the ActA molecule interact with VASP and other components of the host microfilament system. RESULTS: We used the previously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host actin-based cytoskeleton. By examining amino-terminally truncated ActA derivatives for their ability to recruit cytoskeletal proteins, an essential element for actin filament nucleation was identified between amino acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit filamentous actin, albeit poorly, but was unable to bind VASP. CONCLUSIONS: Our studies reveal the initial interactions that take place between invading Listeria and host microfilament proteins. The listerial ActA polypeptide contains at least two essential sites that are required for efficient microfilament assembly: an amino-terminal 23 amino-acid region for actin filament nucleation, and VASP-binding proline-rich repeats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important components of structures involved in cell motility.

• Zhu Q et al.
• The Wiskott-Aldrich syndrome and X-linked congenital thrombocytopenia are caused by mutations of the same gene.
• Blood. 1995; 86: 3797-804
• Display abstract

• The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, small platelets, eczema, recurrent infections, and immunodeficiency. Besides the classic WAS phenotype, there is a group of patients with congenital X-linked thrombocytopenia (XLT) who have small platelets but only transient eczema, if any, and minimal immune deficiency. Because the gene responsible for WAS has been sequenced, it was possible to correlate the WAS phenotypes with WAS gene mutations. Using a fingerprinting screening technique, we determined the approximate location of the mutation in 13 unrelated WAS patients with mild to severe clinical symptoms. Direct sequence analysis of cDNA and genomic DNA obtained from patient-derived cell lines showed 12 unique mutations distributed throughout the WAS gene, including insertions, deletions, and point mutations resulting in amino acid substitutions, termination, exon skipping, or splicing defects. Of 4 unrelated patients with the XLT phenotype, 3 had missense mutations affecting exon 2 and 1 had a splice-site mutation affecting exon 9. Patients with classic WAS had more complex mutations, resulting in termination codons, frameshift, and early termination. These findings provide direct evidence that XLT and WAS are caused by mutations of the same gene and suggest that severe clinical phenotypes are associated with complex mutations.

• al Mulhim I
• Wiskott-Aldrich syndrome.
• Indian Pediatr. 1995; 32: 1224-8

• Reinhard M et al.
• The proline-rich focal adhesion and microfilament protein VASP is a ligand for profilins.
• EMBO J. 1995; 14: 1583-9
• Display abstract

• Profilins are small proteins that form complexes with G-actin and phosphoinositides and are therefore considered to link the microfilament system to signal transduction pathways. In addition, they bind to poly-L-proline, but the biological significance of this interaction is not yet known. The recent molecular cloning of the vasodilator-stimulated phosphoprotein (VASP), an established in vivo substrate of cAMP- and cGMP-dependent protein kinases, revealed the presence of a proline-rich domain which prompted us to investigate a possible interaction with profilins. VASP is a microfilament and focal adhesion associated protein which is also concentrated in highly dynamic regions of the cell cortex. Here, we demonstrate that VASP is a natural proline-rich profilin ligand. Human platelet VASP bound directly to purified profilins from human platelets, calf thymus and birch pollen. Moreover, VASP and a novel protein were specifically extracted from total cell lysates by profilin affinity chromatography and subsequently eluted either with poly-L-proline or a peptide corresponding to a proline-rich VASP motif. Finally, the subcellular distributions of VASP and profilin suggest that both proteins also interact within living cells. Our data support the hypothesis that profilin and VASP act in concert to convey signal transduction to actin filament formation.

• Chakraborty T et al.
• A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.
• EMBO J. 1995; 14: 1314-21
• Display abstract

• The surface-bound ActA polypeptide of the intracellular bacterial pathogen Listeria monocytogenes is the sole listerial factor needed for recruitment of host actin filaments by intracellularly motile bacteria. Here we report that following Listeria infection the host vasodilator-stimulated phosphoprotein (VASP), a microfilament- and focal adhesion-associated substrate of both the cAMP- and cGMP-dependent protein kinases, accumulates on the surface of intracytoplasmic bacteria prior to the detection of F-actin 'clouds'. VASP remains associated with the surface of highly motile bacteria, where it is polarly located, juxtaposed between one extremity of the bacterial surface and the front of the actin comet tail. Since actin filament polymerization occurs only at the very front of the tail, VASP exhibits properties of a host protein required to promote actin polymerization. Purified VASP binds directly to the ActA polypeptide in vitro. A ligand-overlay blot using purified radiolabelled VASP enabled us to identify the ActA homologue of the related intracellular motile pathogen, Listeria ivanovii, as a protein with a molecular mass of approximately 150 kDa. VASP also associates with actin filaments recruited by another intracellularly motile bacterial pathogen, Shigella flexneri. Hence, by the simple expedient of expressing surface-bound attractor molecules, bacterial pathogens effectively harness cytoskeletal components to achieve intracellular movement.

• Wengler GS, Notarangelo LD, Giliani S, Pirastru MG, Ugazio AG, Parolini O
• Mutation analysis in Wiskott Aldrich syndrome on chorionic villus DNA.
• Lancet. 1995; 346: 641-2

• Sullivan KE
• Genetic and clinical advances in Wiskott-Aldrich syndrome.
• Curr Opin Pediatr. 1995; 7: 683-7
• Display abstract

• Wiskott-Aldrich syndrome is an immunodeficiency associated with thrombocytopenia, recurrent infections, eczema, and a predisposition to malignancy. Until this past year, the genetic defect was unknown, and our understanding of the disease was limited to defining the aberrant immunologic and hematologic functions in these patients. The identification of the genetic defect has already improved our understanding of the pathogenesis of this complex disease and has already resulted in the development of a more widely applicable prenatal diagnostic test. Other important developments this past year include the development of a diagnostic assay that should simplify the identification of Wiskott-Aldrich syndrome patients and a retrospective review that characterizes the natural history of the disease.

• Haffner C, Jarchau T, Reinhard M, Hoppe J, Lohmann SM, Walter U
• Molecular cloning, structural analysis and functional expression of the proline-rich focal adhesion and microfilament-associated protein VASP.
• EMBO J. 1995; 14: 19-27
• Display abstract

• The vasodilator-stimulated phosphoprotein (VASP), a substrate for cAMP- and cGMP-dependent protein kinases in vitro and in intact cells, is associated with actin filaments, focal adhesions and dynamic membrane regions. VASP, cloned here from human HL-60 and canine MDCK cells, is organized into three distinct domains. A central proline-rich domain contains a GPPPPP motif as a single copy and as a 3-fold tandem repeat, as well as three conserved phosphorylation sites for cyclic nucleotide-dependent protein kinases. A C-terminal domain contains a repetitive mixed-charge cluster which is predicted to form an alpha-helix. The hydrodynamic properties of purified human VASP together with the calculated molecular mass of cloned VASP suggest that the native protein is a homotetramer with an elongated structure. VASP over-expressed in transiently transfected BHK21 cells was predominantly detected at stress fibres, at focal adhesions and in F-actin-containing cell surface protrusions, whereas truncated VASP lacking the C-terminal domain was no longer concentrated at focal adhesions. These data indicate that the C-terminal domain is required for anchoring VASP at focal adhesion sites, whereas the central domain is suggested to mediate VASP interaction with profilin. Our results provide evidence for the structural basis by which VASP, both a target of the cAMP and cGMP signal transduction pathways and a component of the actin-based cytoskeleton, including the cytoskeleton-membrane interface, may be able to exchange signals between these networks.

• Reinhard M, Jouvenal K, Tripier D, Walter U
• Identification, purification, and characterization of a zyxin-related protein that binds the focal adhesion and microfilament protein VASP (vasodilator-stimulated phosphoprotein).
• Proc Natl Acad Sci U S A. 1995; 92: 7956-60
• Display abstract

• VASP (vasodilator-stimulated phosphoprotein), an established substrate of cAMP- and cGMP-dependent protein kinases in vitro and in living cells, is associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity. Here, the identification of an 83-kDa protein (p83) that specifically binds VASP in blot overlays of different cell homogenates is reported. With VASP overlays as a detection tool, p83 was purified from porcine platelets and used to generate monospecific polyclonal antibodies. VASP binding to purified p83 in solid-phase binding assays and the closely matching subcellular localization in double-label immunofluorescence analyses demonstrated that both proteins also directly interact as native proteins in vitro and possibly in living cells. The subcellular distribution, the biochemical properties, as well as microsequencing data revealed that porcine platelet p83 is related to chicken gizzard zyxin and most likely represents the mammalian equivalent of the chicken protein. The VASP-p83 interaction may contribute to the targeting of VASP to focal adhesions, microfilaments, and dynamic membrane regions. Together with our recent identification of VASP as a natural ligand of the profilin poly-(L-proline) binding site, our present results suggest that, by linking profilin to zyxin/p83, VASP may participate in spatially confined profilin-regulated F-actin formation.

• Kwan SP, Hagemann TL, Blaese RM, Knutsen A, Rosen FS
• Scanning of the Wiskott-Aldrich syndrome (WAS) gene: identification of 18 novel alterations including a possible mutation hotspot at Arg86 resulting in thrombocytopenia, a mild WAS phenotype.
• Hum Mol Genet. 1995; 4: 1995-8

• Southwick FS, Purich DL
• Arrest of Listeria movement in host cells by a bacterial ActA analogue: implications for actin-based motility.
• Proc Natl Acad Sci U S A. 1994; 91: 5168-72
• Display abstract

• Upon entering the host cell's cytoplasm, the pathogen Listeria monocytogenes can subvert the normal contractile system of the host cell; subsequent assembly of polar actin-filament structures is likely to provide the force for rapid intracellular bacterial movement and its cell-to-cell spread. We have now investigated the functional consequences of microinjecting Listeria-infected PtK2 cells with a synthetic peptide, CFEFPPPPTDE. This peptide represents one of four related oligoproline stretches in ActA, a bacterial surface protein necessary for Listeria-induced actin assembly. Over an estimated intracellular concentration range of 80 nM to 0.8 microM, this analogue rapidly blocks the formation of the actin-filament tails and arrests intracellular bacterial motility. Over the same time scale and concentration range, introduction of the ActA analogue also causes host cell membrane retraction. Bodipyphallacidin staining reveals that microinjection of the ActA analogue results in massive retraction of the actin cytoskeleton. Microinjection of 1-20 microM poly(L-proline) (intracellular concentration) fails to block Listeria intracellular movement or polar actin-filament assembly. As observed with ActA, however, poly(L-proline) does cause membrane retraction. Our findings demonstrate the efficacy of low molecular weight peptides in efforts to distinguish mechanistic features in Listeria motility and PtK2 host cell membrane reorganization. These observations also suggest that a cytoskeletal component sensitive to specific oligoproline peptides may participate in protein-protein interactions essential for both of these actin-associated processes.

• Hara T et al.
• The UDP-galactose translocator gene is mapped to band Xp11.23-p11.22 containing the Wiskott-Aldrich syndrome locus.
• Somat Cell Mol Genet. 1993; 19: 571-5
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• We have cloned a segment of the human gene encoding UDP-galactose translocator by genetic complementation of its defective mutant in mouse FM3A cells. Chromosome mapping using fluorescent in situ hybridization revealed that the cloned gene hybridized to the Xp11.23-11.23 region of the X chromosome. This region is shared by the locus of Wiskott-Aldrich syndrome, an X-linked recessive immunodeficiency disorder, characterized by defective sugar chains on cell surface components. Genetic and phenotypic similarities suggest a possible link between UDP-galactose translocator and the Wiskott-Aldrich syndrome (WAS).

• Somerville C, Forsyth KD
• Wiskott Aldrich syndrome: an immunodeficiency syndrome not rare in Western Australia.
• Pediatr Allergy Immunol. 1993; 4: 65-72
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• Wiskott Aldrich syndrome, a combined cellular and humoral X-linked immunodeficiency, is generally considered to be rare. The aim of this study was to ascertain the true prevalence in the paediatric population in Western Australia, describe the clinical features, and summarise the current literature on this unusual condition. All cases of Wiskott Aldrich syndrome presenting to Princess Margaret Hospital in Perth during the period from January 1960 to January 1990 were identified by a retrospective review of case records and by interviewing hospital immunology, haematology and general clinical staff. Nine cases of Wiskott Aldrich syndrome are described, demonstrating that the prevalence of Wiskott Aldrich syndrome in Western Australia is nine times that expected from previous reports. Death occurred in a number of patients before the correct diagnosis was recognised. The clinical features in this group are quite variable. Low isohaemagglutinins, elevated IgE, blunted DTH skin multitest, and very low CD8 numbers are however consistent features. Wiskott Aldrich syndrome may be more prevalent than previously recognised, and should be considered in males with thrombocytopenia and infection.

• Walters JP, Morais P
• Wiskott-Aldrich syndrome in a Jamaican.
• West Indian Med J. 1993; 42: 79-81
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• A 3-week-old boy presented with severe thrombocytopenia and recurrent infections. He was subsequently found to have Wiskott-Aldrich Syndrome, a rare X-linked disorder. Splenectomy was performed and he died of post-splenectomy sepsis despite prophylactic antibiotic therapy. This is the first recorded case in the English-speaking Caribbean.

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