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• W2013121253 abstract "guanosine 5′-3-O-(thio)triphosphate WASp homology 2 WASp homology 1 phosphatidylinositol 4,5-bisphosphate WH2 and acidic acidic polyproline, WH2, and acidic GTPase-binding domain Actin polymerization is required for many types of cell motility, such as chemotaxis, nerve growth cone movement, cell spreading, and platelet activation (reviewed in Ref. 1Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1310) Google Scholar). In the lamellipodia that push forward the leading edge of motile cells, polymerizing filaments form a meshwork consisting of “Y branches” with the pointed end of one filament attached to the side of another filament (2Svitkina T.M. Verkhovsky A.B. McQuade K.M. Borisy G.G. J. Cell Biol. 1997; 139: 397-415Crossref PubMed Scopus (564) Google Scholar). This meshwork presumably may provide a rigid body against which polymerization can drive membrane protrusion (3Mogilner A. Oster G. Biophys. J. 1996; 71: 3030-3045Abstract Full Text PDF PubMed Scopus (714) Google Scholar). A major unanswered question is how cells integrate signals coming through a variety of pathways to control when and where actin polymerizes. The filaments grow from a huge pool of unpolymerized actin maintained by monomer-binding proteins at a concentration approximately 1000-fold higher than required for spontaneous polymerization of actin (reviewed in Ref. 4Pollard T.D. Cooper J.A. Annu. Rev. Biochem. 1986; 55: 987-1035Crossref PubMed Google Scholar). The monomer-binding protein profilin biases the direction of filament elongation, allowing growth at the fast growing barbed end but not the slow growing pointed end (reviewed in Ref. 4Pollard T.D. Cooper J.A. Annu. Rev. Biochem. 1986; 55: 987-1035Crossref PubMed Google Scholar). In cells capping proteins block the barbed end of most filaments, so some mechanism is required to start new filaments (5Schafer D.A. Jennings P.B. Cooper J.A. J. Cell Biol. 1996; 135: 169-179Crossref PubMed Scopus (337) Google Scholar). Cells might trigger actin polymerization in three ways: 1) de novo nucleation of filaments from monomeric actin; 2) severing existing filaments to create uncapped barbed ends; and 3) uncapping existing barbed ends. There is evidence for each of these mechanisms in various cellular processes, but new filaments are often created during cell motility (6Chan A.Y. Raft S. Bailly M. Wyckoff J.B. Segall J.E. Condeelis J.S. J. Cell Sci. 1998; 111: 199-211Crossref PubMed Google Scholar), placing emphasis on mechanisms 1 and 2. Although activation of de novo nucleation by cell stimulation has long been an attractive model (7Carson M. Weber A. Zigmond S.H. J. Cell Biol. 1986; 103: 2707-2714Crossref PubMed Scopus (61) Google Scholar), no barbed end nucleating factors were known until it was discovered that Arp2/3 complex promotes actin nucleation, creating filaments that grow at their barbed ends (8Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Crossref PubMed Scopus (1046) Google Scholar). Because nucleation is rate-limiting in actin polymerization and strongly suppressed by monomer-binding proteins, Arp2/3 complex may be a key mediator of actin polymerization in cells. Arp2/3 complex also cross-links actin filaments end-to-side, indistinguishable from the Y branches at the leading edge (8Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Crossref PubMed Scopus (1046) Google Scholar). Based on these biochemical activities, Mullins et al. (8Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Crossref PubMed Scopus (1046) Google Scholar) proposed the dendritic nucleation model, whereby Arp2/3 complex both creates new filaments and cross-links them into a branching meshwork. Cellular observations support this model. Arp2/3 complex is concentrated at the leading edge of motile cells (9Kelleher J.F. Atkinson S.J. Pollard T.D. J. Cell Biol. 1995; 131: 385-397Crossref PubMed Scopus (163) Google Scholar, 10Machesky L.M. Reeves E. Wientjes F. Mattheyse F.J. Grogan A. Totty N.F. Burlingame A.L. Hsuan J.J. Segal A.W. Biochem. J. 1997; 328: 105-112Crossref PubMed Scopus (178) Google Scholar, 11Schafer D.A. Welch M.D. Machesky L.M. Bridgman P.C. Meyer S.M. Cooper J.A. J. Cell Biol. 1998; 143: 1919-1930Crossref PubMed Scopus (152) Google Scholar, 12Bailly M. Macaluso F. Cammer M. Chan A. Segall J.E. Condeelis J.S. J. Cell Biol. 1999; 145: 331-345Crossref PubMed Scopus (175) Google Scholar, 13Svitkina T.M. Borisy G.G. J. Cell Biol. 1999; 145: 1009-1026Crossref PubMed Scopus (914) Google Scholar), specifically at the junctions of the Y branches (12Bailly M. Macaluso F. Cammer M. Chan A. Segall J.E. Condeelis J.S. J. Cell Biol. 1999; 145: 331-345Crossref PubMed Scopus (175) Google Scholar, 13Svitkina T.M. Borisy G.G. J. Cell Biol. 1999; 145: 1009-1026Crossref PubMed Scopus (914) Google Scholar). It exists in all eukaryotes examined, and ablation of Arp2/3 complex subunits inSaccharomyces cerevisiae and Schizosaccharomyces pombe is lethal or severely debilitating (14Schwob E. Martin R.P. Nature. 1992; 355: 179-182Crossref PubMed Scopus (98) Google Scholar, 15Lees-Miller J.P. Henry G. Helfman D.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 80-83Crossref PubMed Scopus (75) Google Scholar, 16Balasubramanian M.K. Feoktistova A. McCollum D. Gould K.L. EMBO J. 1996; 15: 6426-6437Crossref PubMed Scopus (76) Google Scholar, 17McCollum D. Feoktistova A. Morphew M. Balasubramanian M. Gould K.L. EMBO J. 1996; 15: 6438-6446Crossref PubMed Scopus (114) Google Scholar, 18Moreau V. Madania A. Martin R.P. Winsor B. J. Cell Biol. 1996; 134: 117-132Crossref PubMed Scopus (111) Google Scholar, 19Moreau V. Galan J.M. Devilliers G. Haguenauer-Tsapis R. Winsor B. Mol. Biol. Cell. 1997; 8: 1361-1375Crossref PubMed Scopus (126) Google Scholar, 20Winter D. Podtelejnikov A.V. Mann M. Li R. Curr. Biol. 1997; 7: 519-529Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 21Winter D.C. Choe E.Y. Li R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7288-7293Crossref PubMed Scopus (165) Google Scholar). The next breakthrough was the discovery that ActA, a cell surface protein from the pathogenic bacterium, Listeria monocytogenes, stimulates Arp2/3 complex to nucleate actinin vitro (22Welch M.D. Rosenblatt J. Skoble J. Portnoy D.A. Mitchison T.J. Science. 1998; 281: 105-108Crossref PubMed Scopus (421) Google Scholar). Listeria uses force generated by actin polymerization to propel itself around the cytoplasm of eukaryotic cells. ActA is the only bacterial protein required to induce polymerization, but ActA cannot stimulate actin filament formation by itself (reviewed in Ref. 23Cossart P. Lecuit M. EMBO J. 1998; 17: 3797-3806Crossref PubMed Scopus (249) Google Scholar). This work suggested that cellular factors might activate Arp2/3 complex to nucleate actin. This year WASp/Scar proteins were identified as the first example of such factors (24Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar, 25Machesky L.M. Mullins D.M. Higgs H.N. Kaiser D.A. Blanchoin L. May R.C. Hall M.E. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3739-3744Crossref PubMed Scopus (617) Google Scholar, 26Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar, 27Winter D. Lechler T. Li R. Curr. Biol. 1999; 9: 501-504Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 28Yarar D. To W. Abo A. Welch M.D. Curr. Biol. 1999; 9: 555-558Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). These proteins also interact with a variety of cell signaling molecules known to influence cytoskeletal dynamics, bringing us closer to forging a connection between surface receptor stimulation and actin polymerization. The rapid progress reviewed here depended upon groundwork from many laboratories. Analysis of Wiskott-Aldrich syndrome protein (WASp) and its neural homolog N-WASP revealed a binding site for Rho family GTPases and other domains that affect actin assembly in cells (29Ochs H.D. Semin. Hematol. 1998; 35: 332-345PubMed Google Scholar, 30Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (556) Google Scholar). Study of GTPγS1-stimulated actin polymerization in extracts of vertebrate cells (31Ma L. Cantley L.C. Janmey P.A. Kirschner M.W. J. Cell Biol. 1998; 140: 1125-1136Crossref PubMed Scopus (168) Google Scholar, 32Ma L. Rohatgi R. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15362-15367Crossref PubMed Scopus (154) Google Scholar, 33Zigmond S.H. Joyce M. Borleis J. Bokoch G.M. Devreotes P.N. J. Cell Biol. 1997; 138: 363-374Crossref PubMed Scopus (145) Google Scholar, 34Zigmond S.H. Joyce M. Yang C. Brown K. Huang M. Pring M. J. Cell Biol. 1998; 142: 1001-1012Crossref PubMed Scopus (65) Google Scholar),Dictyostelium (33Zigmond S.H. Joyce M. Borleis J. Bokoch G.M. Devreotes P.N. J. Cell Biol. 1997; 138: 363-374Crossref PubMed Scopus (145) Google Scholar), and Acanthamoeba (35Mullins R.D. Pollard T.D. Curr. Biol. 1999; 9: 405-415Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) demonstrated that the Rho family GTPase Cdc42 mediates the effect of GTP and that Arp2/3 complex is required. Similar experiments with extracted yeast suggested that Bee1p (a WASp homolog) and Arp2/3 complex are required for actin patch assembly (20Winter D. Podtelejnikov A.V. Mann M. Li R. Curr. Biol. 1997; 7: 519-529Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 36Lechler T. Li R. J. Cell Biol. 1997; 138: 95-103Crossref PubMed Scopus (51) Google Scholar, 37Li R. J. Cell Biol. 1997; 136: 649-658Crossref PubMed Scopus (219) Google Scholar). The Arp2/3 complex contains one copy of each of seven strongly associated protein subunits (Fig. 1; reviewed in detail in Ref. 38Mullins R.D. Pollard T.D. Curr. Opin. Struct. Biol. 1999; 9: 244-249Crossref PubMed Scopus (93) Google Scholar). Arp2 and Arp3 are actin-related proteins. The other five subunits are novel. The complex is very abundant, approximately 2 μm in the cytoplasm ofAcanthamoeba (9Kelleher J.F. Atkinson S.J. Pollard T.D. J. Cell Biol. 1995; 131: 385-397Crossref PubMed Scopus (163) Google Scholar). Analysis of molecular models of Arp2 and Arp3 first led to the hypothesis that they form a stable dimer that binds the pointed end of actin filaments and nucleates growth in the barbed direction (9Kelleher J.F. Atkinson S.J. Pollard T.D. J. Cell Biol. 1995; 131: 385-397Crossref PubMed Scopus (163) Google Scholar). Polymerization assays established that the complex binds the pointed end with nanomolar affinity and has modest nucleation activity (8Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Crossref PubMed Scopus (1046) Google Scholar). The mammalian WASp/Scar family currently consists of five members: WASp, N-WASP, and three Scar isoforms (Fig.2). Several lines of evidence implicate these proteins in actin polymerization. The gene encoding WASp, apparently expressed only in hematopoietic cells, is mutated in Wiskott-Aldrich syndrome, an X-linked human disease with selective defects in platelet development and lymphocytes (29Ochs H.D. Semin. Hematol. 1998; 35: 332-345PubMed Google Scholar). The presence of a binding motif (GBD) for activated Cdc42 and Rac hinted that WASp might regulate actin, because these Rho family GTPases influence actin dynamics and because transfection of WASp rearranges actin filaments in cultured cells (39Symons M. Derry J.M.J. Kariak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar). The WASp homologue N-WASP is expressed more widely in vertebrate cells than WASp (30Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (556) Google Scholar) and causes filopodial formation when co-expressed with Cdc42 in cultured cells (40Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (567) Google Scholar). Scar was discovered inDictyostelium where disruption of its gene rescues the developmental defect caused by disruption of a cyclic AMP receptor (41Bear J.E. Rawls J.F. Saxe C.L. J. Cell Biol. 1998; 142: 1325-1335Crossref PubMed Scopus (233) Google Scholar). Deletion of Scar in wild type cells causes cytoskeletal defects. A mammalian homologue of Scar, termed WAVE or Scar1, might be involved in Rac-induced membrane ruffling, although it does not contain a GBD (42Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (576) Google Scholar). No information is yet available on the functions of two other human Scar-related open reading frames (GenBank accession numbers BAA74923 and CAA18609). In S. cerevisiae, a WASp/Scar homologue, known as Las17p or Bee1p, is essential for cortical actin patch formation and for endocytosis (37Li R. J. Cell Biol. 1997; 136: 649-658Crossref PubMed Scopus (219) Google Scholar, 43Naqvi S.N. Zahn R. Mitchell D.A. Stevenson B.J. Munn A.L. Curr. Biol. 1998; 8: 959-962Abstract Full Text Full Text PDF PubMed Google Scholar). The C-terminal 65–105 amino acids of WASp/Scar proteins (Fig.3) enhance nucleation by Arp2/3 complex (25Machesky L.M. Mullins D.M. Higgs H.N. Kaiser D.A. Blanchoin L. May R.C. Hall M.E. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3739-3744Crossref PubMed Scopus (617) Google Scholar, 26Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar, 27Winter D. Lechler T. Li R. Curr. Biol. 1999; 9: 501-504Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 28Yarar D. To W. Abo A. Welch M.D. Curr. Biol. 1999; 9: 555-558Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). This region starts with a WASp homology 2 (WH2) motif, a 16–19-amino acid sequence that participates in actin monomer binding (24Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar). N-WASP has two tandem WH2 motifs. The C-terminal 15–20 residues, designated “A,” generally possess a strongly negative net charge and interact with Arp2/3 complex (24Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar). Between these two regions are 30–40 residues of unknown functional significance. Some have suggested that this region of WASp and N-WASP contains a short sequence similar to the actin monomer-binding protein cofilin (“cofilin homology domain”), although in our opinion the sequence similarity is too limited to indicate homology (Fig. 3). Furthermore, these short sequences cannot be a domain in the usual sense, because in ADF/cofilins they form part of a β-strand, a loop, and part of an α-helix rather than an independently folded structure (44Leonard S.A. Gittis A.G. Petrella E.C. Pollard T.D. Lattman E.E. Nat. Struct. Biol. 1997; 4: 369-373Crossref PubMed Scopus (62) Google Scholar). However, there is evidence that this region participates in binding actin monomers (45Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Crossref PubMed Scopus (116) Google Scholar). The C terminus of Las17p/Bee1p differs significantly from other WASp/Scar proteins. In its WH2 motif, it has two unique inserts of unknown significance. The region between WH2 and A motifs differs even more, with >50% of this sequence consisting of G, A, or P. Perhaps more importantly, Bee1p is the only WASp/Scar protein in which the C-terminal 20 residues have a net neutral charge (excluding the C-terminal carboxyl group, Fig. 3). These differences might explain some functional differences of Bee1p (27Winter D. Lechler T. Li R. Curr. Biol. 1999; 9: 501-504Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). N-terminal to this nucleation-activating region, WASp and N-WASP bind an impressive list of protein ligands (TableI): the Rho family GTPases Cdc42 and Rac, WASp-interacting protein, calmodulin, Src kinases, Tec kinases, Grb2, Nck, and profilin. These proteins provide a myriad of possibilities to regulate either the activity or location of WASp or N-WASP. No interacting proteins for Scar1 other than actin, profilin, and Arp2/3 complex have been identified.Table IMolecules that interact with WASp/Scar proteinsMoleculeWASp/Scar partnerBinding regionEvidenceEffect on nucleationRefs.Cdc42/RacWASp, N-WASPGBD, other?BO, BAStimulates26,39Symons M. Derry J.M.J. Kariak B. Jiang S. Lemahieu V. McCormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (749) Google Scholar, 45Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Crossref PubMed Scopus (116) Google ScholarPIP2N-WASPWH1?BAStimulates26Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar, 30Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (556) Google ScholarWIPWASp, Beelp101–151 (WASp)TH, GST, IP?46Rudolph M.G. Bayer P. Abo A. Kuhlman J. Vetter I.R. Wittinghofer A. J. Biol. Chem. 1998; 273: 18067-18076Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 47Ramesh N. Anton I.M. Hartwig J.H. Geha R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14671-14676Crossref PubMed Scopus (309) Google Scholar, 65Beckerle M.C. Cell. 1998; 95: 741-748Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 66Vaduva G. Martin N.C. Hopper A.K. J. Cell Biol. 1997; 139: 1821-1833Crossref PubMed Scopus (105) Google ScholarCalmodulinN-WASPIQGST, IP?30Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (556) Google ScholarSrc kinasesWASpPolyprolineGST, IP, P?48Stewart D.M. Tian L. Nelson D.L. J. Immunol. 1999; 162: 5019-5024PubMed Google Scholar, 49Banin S. Truong O. Katz D.R. Waterfield M.D. Brickell P.M. Cout I. Curr. Biol. 1996; 6: 981-989Abstract Full Text Full Text PDF PubMed Scopus (124) Google ScholarTec kinasesWASpPolyprolineGST, IP, P?49Banin S. Truong O. Katz D.R. Waterfield M.D. Brickell P.M. Cout I. Curr. Biol. 1996; 6: 981-989Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar–51Cory G.O.C. MacCarthy-Morrogh L. Banin S. Gout I. Brickell P.M. Levinsky R.J. Kinnon C. Lovering R.C. J. Immunol. 1996; 157: 3791-3795PubMed Google ScholarGrb2WASp, N-WASpPolyprolineGST, IP?50Guinamard R. Aspenstrom P. Fougereau M. Chavrier P. Guillemot J.C. FEBS Lett. 1998; 434: 431-436Crossref PubMed Scopus (94) Google Scholar, 52Baba Y. Nonoyama S. Matsushita M. Yamadori T. Hashimoto S. Imai K. Arai S. Kunikata T. Kurimoto M. Kurosaki T. Ochs H. Yata J. Kishimoto T. Tsukada S. Blood. 1999; 93: 2003-2012Crossref PubMed Google Scholar,53She H. Rockow S. Tang J. Nishimura R. Skolnik E.Y. Chen M. Margolis B. Li W. Mol. Biol. Cell. 1997; 8: 1709-1721Crossref PubMed Scopus (106) Google ScholarNckWASpPolyprolineGST, IP?52Baba Y. Nonoyama S. Matsushita M. Yamadori T. Hashimoto S. Imai K. Arai S. Kunikata T. Kurimoto M. Kurosaki T. Ochs H. Yata J. Kishimoto T. Tsukada S. Blood. 1999; 93: 2003-2012Crossref PubMed Google Scholar, 54Oda A. Ochs H. Druker D.J. Ozaki K. Watanabe C. Handa M. Miyakawa Y. Ikeda Y. Blood. 1998; 92: 1852-1858Crossref PubMed Google Scholar, 55Rivero-Lezcano O.M. Marcilla A. Sameshima J.H. Robbins K.C. Mol. Cell. Biol. 1995; 15: 5725-5731Crossref PubMed Scopus (280) Google ScholarProfilinN-WASP, Scar1PolyprolineGST, IP?48Stewart D.M. Tian L. Nelson D.L. J. Immunol. 1999; 162: 5019-5024PubMed Google Scholar,63Blanchoin L. Pollard T.D. J. Biol. Chem. 1998; 273: 25106-25111Abstract Full Text Full Text PDF PubMed Scopus (144) Google ScholarBO, blot overlay; BA, direct binding assay; GST, GST fusion pulldown; IP, co-immunoprecipitation; P, phosphorylates WASp/Scar; TH, two hybrid; WIP, WASp-interacting protein. Open table in a new tab BO, blot overlay; BA, direct binding assay; GST, GST fusion pulldown; IP, co-immunoprecipitation; P, phosphorylates WASp/Scar; TH, two hybrid; WIP, WASp-interacting protein. WASp and N-WASp also have an N-terminal WASp homology 1 domain (WH1). An atomic structure of an EVH1 domain, a homolog of the WH1 domain found in adapter proteins including Ena and VASP, shows that the fold of the domain is similar to a pleckstrin homology domain (57Prehoda K.E. Lee D.J. Lim W.A. Cell. 1999; (in press)PubMed Google Scholar). Ligands with the sequence FPPPP bind EVH1 in the place of an intrinsic α-helix found in pleckstrin homology domains, which can bind PIP2. PIP2 apparently binds an N-terminal construct of N-WASP (30Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (556) Google Scholar) and activates nucleation by full-length N-WASP (26Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar), but more detailed work defining the binding site is needed. Machesky and Insall (24Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar) found that human WASp and Scar1 interact with the p21 subunit in two-hybrid assay and with the whole complex in biochemical assays. Introduction of pieces of these proteins into cultured cells disrupts membrane ruffling and delocalizes Arp2/3 complex. The minimum Scar fragment sufficient for these effects on cells contains the WH2 and acidic (WA) motifs. The same Scar fragment greatly enhances actin nucleation by Arp2/3 complex, reducing the lag time and increasing the number of filaments (25Machesky L.M. Mullins D.M. Higgs H.N. Kaiser D.A. Blanchoin L. May R.C. Hall M.E. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3739-3744Crossref PubMed Scopus (617) Google Scholar). WASp/Scar proteins activate nucleation of actin filaments by Arp2/3 complex (Fig. 4). To understand the mechanism one must first appreciate how actin behaves by itself. Nucleation by actin monomers alone is very unfavorable because of the instability of actin dimers and trimers, obligate intermediates on the pathway to filaments (reviewed in Ref.4Pollard T.D. Cooper J.A. Annu. Rev. Biochem. 1986; 55: 987-1035Crossref PubMed Google Scholar). Unfavorable nucleation accounts for the lag at the onset of spontaneous polymerization. During the lag trimers form immediately, but the bulk rate of polymerization is low because the concentration of these nuclei is exceedingly low. Trimers have a lifetime of microseconds because of rapid dissociation to dimers and the rare addition of actin monomers to form a stable filament. The number of filaments accumulates during the reaction, mainly by nucleation but also by infrequent breaks into two filaments. Early in the reaction, end to end annealing consumes a significant number of filament ends. At any point in the reaction the concentration of filament barbed ends can be calculated from the polymerization rate (polymerization rate = [ends]((k +) [actin monomers] − (k −)). Arp2/3 complex alone promotes polymerization, but the reaction is inefficient (8Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Crossref PubMed Scopus (1046) Google Scholar). The kinetic data are best fit by a model where the complex captures actin dimers, which then grow into filaments by addition of actin subunits at the barbed end. The model accounts for the inefficiency by the low concentration of dimers and by dissociation of 98% of captured dimers from the complex before producing a filament. The initial lag persists even with high concentrations of Arp2/3 complex. Addition of C-terminal constructs of WASp/Scar proteins to Arp2/3 complex significantly decreases but does not eliminate the lag in polymerization (Fig. 4 A). Together they also increase the number of filament ends, as reflected by the large increase in slope of the polymerization curve. Addition of preformed actin filaments along with Arp2/3 complex and WASp/Scar constructs reduces the lag still further (Fig.4 A). This result suggests that the dendritic nucleation model is fundamentally correct; association of Arp2/3 complex with the side of an existing filament enhances its nucleation of a new filament. This hypothesis (Fig. 4 B) explains the persistent lag in experiments with actin, Arp2/3, and WASp/Scar. More detailed work in our laboratory suggests that the lag with Arp2/3 complex and WASp is due to the buildup of filaments by spontaneous polymerization, with a threshold of <100 nm actin in filaments needed to activate Arp2/3 complex and WASp. 2H. N. Higgs, L. Blanchoin, and T. D. Pollard, submitted for publication. The order of events is still unclear; Arp2/3 complex binds both the sides of actin filaments and WASp/Scar WA, so it could bind filaments either before or after association with WA and actin subunits. The WH2 and A regions of Scar1 are required to stimulate nucleation by Arp2/3 complex (25Machesky L.M. Mullins D.M. Higgs H.N. Kaiser D.A. Blanchoin L. May R.C. Hall M.E. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3739-3744Crossref PubMed Scopus (617) Google Scholar). The adjacent polyproline domain may favor one or more steps in the reaction but is not absolutely required either in the presence or absence of profilin. The A region of human Scar1 interacts directly with the p21 subunit (24Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar) but does not activate nucleation (25Machesky L.M. Mullins D.M. Higgs H.N. Kaiser D.A. Blanchoin L. May R.C. Hall M.E. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3739-3744Crossref PubMed Scopus (617) Google Scholar). The A regions of N-WASP and WASp are also insufficient for Arp2/3 complex activation (26Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar). 3H. N. Higgs and T. D. Pollard, unpublished observations. On the other hand, the A region (with an undefined N-terminal boundary) of yeast Bee1p appears to activate nucleation by yeast Arp2/3 complex (27Winter D. Lechler T. Li R. Curr. Biol. 1999; 9: 501-504Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Although unlikely given the conservation of these molecules, Bee1p may activate yeast Arp2/3 complex in a different way than other WASp/Scar proteins. More detailed dissection of this region is needed to determine the minimal functional WASp/Scar unit and the role of actin monomer binding by WASp/Scar proteins in nucleation. Two lines of evidence suggest that Arp2/3 complex plays a major role but do not rule out other pathways. First, most of the GTP-stimulated actin polymerization in cell extracts depends on Arp2/3 complex (32Ma L. Rohatgi R. Kirschner M.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15362-15367Crossref PubMed Scopus (154) Google Scholar, 35Mullins R.D. Pollard T.D. Curr. Biol. 1999; 9: 405-415Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), as does actin patch reconstitution in yeast (20Winter D. Podtelejnikov A.V. Mann M. Li R. Curr. Biol. 1997; 7: 519-529Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Second, most of the filaments at the leading edge of keratocytes and fibroblasts are incorporated into 70° branching networks with Arp2/3 complex localized at the branches (13Svitkina T.M. Borisy G.G. J. Cell Biol. 1999; 145: 1009-1026Crossref PubMed Scopus (914) Google Scholar). The affinities of Arp2/3 complex for both the pointed end and side of actin filaments allow the complex to form these branched networks (8Mullins R.D. Heuser J.A. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6181-6186Crossref PubMed Scopus (1046) Google Scholar, 58Mullins R.D. Kelleher J.F. Xu J. Pollard T.D. Mol. Biol. Cell. 1998; 9: 841-852Crossref PubMed Scopus (75) Google Scholar). WASp/Scar proteins are not required for branching by Arp2/3 complex but may stabilize either or both associations. On the other hand, experiments with budding yeast suggest alternative pathways. Neither Arp2 nor Arp3 is essential for viability, although null strains are extremely sick and the phenotype may depend on the genetic background of the particular strain (21Winter D.C. Choe E.Y. Li R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7288-7293Crossref PubMed Scopus (165) Google Scholar). A variety of stimuli regulates cellular actin polymerization, acting through receptor tyrosine kinase/mitogen-activated protein kinase pathways, seven helix receptors, and integrins. Details remain unclear, but an attractive hypothesis (24Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar) is that signals from different kinds of receptors and signaling pathways converge on particular members of WASp/Scar proteins, which funnel these signals through Arp2/3 complex as a final common pathway to actin filament formation. Any of these signals could, in principle, affect either the activity of Arp2/3 complex or its localization in cells. The only demonstrated activators for any WASp/Scar protein are Cdc42 and PIP2. Both activate N-WASP and Arp2/3 complex to nucleate actin filaments in vitro (26Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar). Participation of Cdc42 and PIP2 in activating WASp/Scar does not restrict the upstream pathways, because seven-helix receptors, tyrosine kinase receptors, and integrins can all influence these signaling molecules (59Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5230) Google Scholar, 60Pike L.J. Casey L. J. Biol. Chem. 1996; 271: 26453-26456Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). WASp-interacting protein, Grb2, Src kinases, Tec kinases, calmodulin, and Nck are other potential regulators of WASp/Scar proteins (Table I). Like Cdc42 and PIP2, multiple receptor classes might regulate most if not all of these proteins. These proteins could act in the same way as Cdc42 and PIP2, enabling WASp and N-WASP to activate Arp2/3 complex, but they might have other roles such as targeting WASp and N-WASP to particular parts of a cell. Any effect of WASp phosphorylation by Src or Tec kinases is entirely unclear. The identity of Scar activators is unknown, although the link between Scar and cAMP receptor in Dictyostelium suggests a role for seven-helix receptors and trimeric G-proteins. Given the large number of potential regulatory proteins for WASp/Scar proteins, they alone may regulate actin filament nucleation mediated by Arp2/3 complex. However, the existence of ActA establishes a precedent for other protein activators. Posttranslational modifications of Arp2/3 complex subunits also need to be considered along with differential expression of two p41 ARC isoforms in mammals (10Machesky L.M. Reeves E. Wientjes F. Mattheyse F.J. Grogan A. Totty N.F. Burlingame A.L. Hsuan J.J. Segal A.W. Biochem. J. 1997; 328: 105-112Crossref PubMed Scopus (178) Google Scholar, 61Welch M.D. DePace A.H. Verma S. Iwamatsu A. Mitchison T.J. J. Cell Biol. 1997; 138: 375-384Crossref PubMed Scopus (409) Google Scholar). It is not clear how WASp/Scar proteins and Arp2/3 complex interact with activators at the leading edge. Because the signal-activating nucleation often comes from a surface receptor and because many of the signal-transducing molecules that bind and/or activate WASp/Scar proteins are bound to receptors or membranes, they may activate Arp2/3 complex on the membrane (26Rohatgi R. Ma L. Miki H. Lopez M. Kirchhausen T. Takenawa T. Kirschner M.W. Cell. 1999; 97: 221-231Abstract Full Text Full Text PDF PubMed Scopus (1082) Google Scholar). However, Arp2/3 complex is concentrated in the cortical actin filament network rather than on membranes (9Kelleher J.F. Atkinson S.J. Pollard T.D. J. Cell Biol. 1995; 131: 385-397Crossref PubMed Scopus (163) Google Scholar, 10Machesky L.M. Reeves E. Wientjes F. Mattheyse F.J. Grogan A. Totty N.F. Burlingame A.L. Hsuan J.J. Segal A.W. Biochem. J. 1997; 328: 105-112Crossref PubMed Scopus (178) Google Scholar, 12Bailly M. Macaluso F. Cammer M. Chan A. Segall J.E. Condeelis J.S. J. Cell Biol. 1999; 145: 331-345Crossref PubMed Scopus (175) Google Scholar, 13Svitkina T.M. Borisy G.G. J. Cell Biol. 1999; 145: 1009-1026Crossref PubMed Scopus (914) Google Scholar, 61Welch M.D. DePace A.H. Verma S. Iwamatsu A. Mitchison T.J. J. Cell Biol. 1997; 138: 375-384Crossref PubMed Scopus (409) Google Scholar, 62Mullins R.D. Stafford W.F. Pollard T.D. J. Cell Biol. 1997; 136: 331-343Crossref PubMed Scopus (195) Google Scholar). Additional work is required to pinpoint the sites of Arp2/3 complex activation. The C terminus of WASp/Scar binds monomeric actin (24Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar,30Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (556) Google Scholar, 42Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (576) Google Scholar), which might stabilize the nascent filament during nucleation (Fig. 4 B). However, the way in which WASp/Scar C termini bind actin is not clear. In our hands, human WASp WA binds monomeric actin with a K d of about 0.5 μm. It inhibits elongation at the pointed end but does not inhibit barbed end growth or sequester actin monomers.2 Scar1 behaves similarly (24Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar). Others find that N-WASP WA and Scar1 WA depolymerize actin filaments (30Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (556) Google Scholar, 40Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (567) Google Scholar, 42Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (576) Google Scholar, 45Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Crossref PubMed Scopus (116) Google Scholar). One hypothesis proposed for N-WASP WA is that it severs actin filaments, thereby exposing new barbed ends for rapid elongation (40Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (567) Google Scholar). However, the data for severing could be interpreted in other ways, such as actin monomer sequestration. In our hands, neither WASp WA nor Scar1 WA severs filaments or sequesters monomers.2 Profilin is thought to participate in actin dynamics as a nucleotide exchange factor (63Blanchoin L. Pollard T.D. J. Biol. Chem. 1998; 273: 25106-25111Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar) and carrier for subunits during elongation (64Pantaloni D. Carlier M.F. Cell. 1993; 75: 1007-1014Abstract Full Text PDF PubMed Scopus (460) Google Scholar), but the role of its interaction with proline-rich sequences of WASp/Scar proteins in nucleation by Arp2/3 complex is unclear. WASp/Scar family members contain at least four potential profilin-binding sites of 5 or more prolines (Fig. 2), and some evidence suggests that profilin enhances the cellular activities of N-WASP and Scar1 (42Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (576) Google Scholar, 56Suetsugu S. Miki H. Takenawa T. EMBO J. 1998; 17: 6516-6526Crossref PubMed Scopus (194) Google Scholar). In vitro profilin reduces background nucleation, increasing the signal to noise of the Arp2/3 complex/Scar trigger for new filament formation (25Machesky L.M. Mullins D.M. Higgs H.N. Kaiser D.A. Blanchoin L. May R.C. Hall M.E. Pollard T.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3739-3744Crossref PubMed Scopus (617) Google Scholar). Scar1 PWA (with potential profilin-binding sites) is more effective than Scar1 WA in the presence of profilin, but the rate is lower than without profilin. Thus profilin interaction with the P domain does not enhance the activation of Arp2/3 complex. One idea is that profilin bound to polyproline-containing ligands targets actin to nucleation or elongation sites (65Beckerle M.C. Cell. 1998; 95: 741-748Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). We question this theory, because participation of profilin could only slow down diffusion-limited actin filament elongation rate and profilin inhibits nucleation. The recent results summarized here show that WASp/Scar proteins stimulate the formation of new actin filaments by Arp2/3 complex. These fascinating WASp/Scar proteins may in turn be regulated by several signaling pathways. The enthusiasm for these new insights should not lessen the attention given to other complementary mechanisms of actin filament generation such as severing or uncapping. Work also needs to continue on the mechanisms by which actin networks are disassembled to recycle subunits to sites of growth." @default.
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• W2013121253 title "Regulation of Actin Polymerization by Arp2/3 Complex and WASp/Scar Proteins" @default.
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