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Wiskott-Aldrich Syndrome Protein Induces Actin Clustering without Direct Binding to Cdc42

1999; Elsevier BV; Volume: 274; Issue: 38 Linguagem: Inglês

10.1074/jbc.274.38.27225

ISSN

1083-351X

Autores

Masayoshi Kato, Hiroaki Miki, Kohsuke Imai, Shigeaki Nonoyama, Toshihiko Suzuki, Chihiro Sasakawa, Tadaomi Takenawa,

Tópico(s)

Cell Image Analysis Techniques

Resumo

WASP (Wiskott-Aldrichsyndrome protein) was identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome. WASP contains many functional domains and has been shown to induce the formation of clusters of actin filaments in a manner dependent on Cdc42. However, there has been no report investigating what domain(s) is(are) important for the function. Here we present for the first time the results of detailed analyses on the domain-function relationship of WASP. First, the C-terminal verprolin-cofilin-acidic domain was shown to be essential for the regulation of actin cytoskeleton. In addition, we found that the clustering of WASP itself is distinct from actin clustering. The partial protein containing the region from the N-terminal pleckstrin homology domain to the basic residue-rich region also clustered especially around the nucleus as wild type WASP without inducing actin clustering. Finally, we obtained the quite unexpected result that a WASP mutant deficient in binding to Cdc42 still induced actin cluster formation, indicating that direct interaction between Cdc42 and WASP is not required for the regulation of actin cytoskeleton. This result may explain why no Wiskott-Aldrich syndrome patients have been identified with a missense mutation in the Cdc42-binding site. WASP (Wiskott-Aldrichsyndrome protein) was identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome. WASP contains many functional domains and has been shown to induce the formation of clusters of actin filaments in a manner dependent on Cdc42. However, there has been no report investigating what domain(s) is(are) important for the function. Here we present for the first time the results of detailed analyses on the domain-function relationship of WASP. First, the C-terminal verprolin-cofilin-acidic domain was shown to be essential for the regulation of actin cytoskeleton. In addition, we found that the clustering of WASP itself is distinct from actin clustering. The partial protein containing the region from the N-terminal pleckstrin homology domain to the basic residue-rich region also clustered especially around the nucleus as wild type WASP without inducing actin clustering. Finally, we obtained the quite unexpected result that a WASP mutant deficient in binding to Cdc42 still induced actin cluster formation, indicating that direct interaction between Cdc42 and WASP is not required for the regulation of actin cytoskeleton. This result may explain why no Wiskott-Aldrich syndrome patients have been identified with a missense mutation in the Cdc42-binding site. pleckstrin homology Cdc42 and Rac interactive binding GTPase binding domain glutathione S-transferase proline-rich verprolin-cofilin-acidic wild type guanosine 5′-3-O-(thio)triphosphate WASP insert basic-rich region verprolin homology WASP (Wiskott-Aldrich syndromeprotein) was originally identified as the gene product whose mutation causes the human hereditary disease Wiskott-Aldrich syndrome (1Derry J.M. Ochs H.D. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (825) Google Scholar). WASP is composed of 502 amino acid residues and contains many functional domains such as the pleckstrin homology (PH)1 domain, GTPase binding domain (GBD)/Cdc42 and Rac interactive binding (CRIB) motif, proline-rich (PR) domain, verprolin homology (VPH) domain, cofilin homology domain, and C-terminal highly acidic region. Accumulating evidence has shown that WASP directly interacts with many signaling and/or cytoskeletal proteins such as Cdc42 (2Aspenstrom P. Lindberg U. Hall A. Curr. Biol. 1996; 6: 70-75Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, 3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar, 4Kolluri R. Tolias K.F. Carpenter C.L. Rosen F.S. Kirchhausen T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5615-5618Crossref PubMed Scopus (187) Google Scholar), WASP-interacting protein (5Ramesh N. Anton I.M. Hartwig J.H. Geha R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14671-14676Crossref PubMed Scopus (304) Google Scholar), Src family tyrosine kinases (6Banin S. Truong O. Katz D.R. Waterfield M.D. Brickell P.M. Gout I. Curr. Biol. 1996; 6: 981-988Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7Bunnell S.C. Henry P.A. Kolluri R. Kirchhausen T. Rickles R.J. Berg L.J. J. Biol. Chem. 1996; 271: 25646-25656Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 8Cory G.O. 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 Scholar, 9Guinamard R. Aspenstrom P. Fougereau M. Chavrier P. Guillemot J.C. FEBS Lett. 1998; 434: 431-436Crossref PubMed Scopus (94) Google Scholar), Nck (10Rivero-Lezcano O.M. Marcilla A. Sameshima J.H. Robbins K.C. Mol. Cell. Biol. 1995; 15: 5725-5731Crossref PubMed Scopus (280) Google Scholar), Grb2/Ash (6Banin S. Truong O. Katz D.R. Waterfield M.D. Brickell P.M. Gout I. Curr. Biol. 1996; 6: 981-988Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar,8Cory G.O. 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 Scholar, 11Miki H. Nonoyama S. Zhu Q. Aruffo A. Ochs H.D. Takenawa T. Cell Growth Differ. 1997; 8: 195-202PubMed Google Scholar), phospholipase Cγ (6Banin S. Truong O. Katz D.R. Waterfield M.D. Brickell P.M. Gout I. Curr. Biol. 1996; 6: 981-988Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 8Cory G.O. 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 Scholar), and the 85-kDa subunit of phosphatidylinositol 3-kinase (6Banin S. Truong O. Katz D.R. Waterfield M.D. Brickell P.M. Gout I. Curr. Biol. 1996; 6: 981-988Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) through the functional domains described above (mainly, through the PR domain). The binding to Cdc42 is thought to be of particular importance (3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). Cdc42 is a Rho family small GTPase that is known to play a critical role in the formation of actin microspikes in response to external stimuli (12Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3714) Google Scholar, 13Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (881) Google Scholar). Symons et al. (3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar) identified WASP as a Cdc42-binding protein in search of novel Cdc42 effector proteins. Indeed, it was shown that activated Cdc42 specifically and directly bound to the GBD/CRIB motif of WASP. They also demonstrated that ectopic expression of WASP induces the formation of actin filament (F-actin) clusters that overlap with the expressed WASP itself. Another WASP family protein, N-WASP, also has the PH domain, GBD/CRIB motif, PR domain, VPH domain, and C-terminal acidic region (17Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (547) Google Scholar). This protein has been shown to play important roles in Cdc42-dependent filopodium formation (14Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (559) Google Scholar), although in structure it is very similar to WASP. Both of these proteins cause actin polymerization, but with different features when they are expressed in cells; WASP mainly localizes at perinuclear areas and causes actin clustering (3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar), but most N-WASP is present at plasma membranes and induces filopodium formation (14Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (559) Google Scholar, 17Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (547) Google Scholar). In the case of N-WASP, direct binding of activated Cdc42 seems to unmask the folded inactive N-WASP (14Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (559) Google Scholar). With regard to N-WASP, the mechanism by which it induces filopodium has been well studied. However, it still remains unknown how WASP is regulated by Cdc42 and how WASP induces actin clustering. We here report the results of expression analyses of various WASP mutants. We identified the regions that are responsible for the regulation of actin cytoskeleton and the intracellular localization of WASP itself. The most unexpected and interesting result is that the direct binding between WASP and Cdc42 has no physiological function in the actin cluster formation induced by WASP, at least in these expression systems. Polyclonal anti-WASP antibody was prepared in rabbits immunized with bacterially expressed recombinant protein (amino acids 149–310). Antiserum was purified with protein A gel (Pierce). The monoclonal antibody specific for the c-Myc epitope tag was purchased from Santa Cruz Biotechnology, Inc. The secondary antibodies linked to alkaline phosphatase (used in Western blotting) and fluorescein (used in immunofluorescence microscopy) were from Promega and Capel, respectively. The polyclonal antibody specific for the Arp3 was prepared in rabbits according to the method of Welch et al. (18Welch M.D. Iwamatsu A. Mitchison T.J. Nature. 1997; 385: 265-269Crossref PubMed Scopus (490) Google Scholar). Construction of mutants of ΔPHWI (deletion of amino acids 1–193), ΔWI (deletion of amino acids 155–193), ΔPRVCA (deletion of amino acids 311–502), and ΔPR (deletion of amino acids 311–413) was done by using the polymerase chain reaction. ΔPH (107–502) was obtained by Acc I digestion and excision of the cDNA fragment coding for amino acids 1–106. PHWI (amino acids 1–222) and PHBR (amino acids 1–240) were obtained as cDNA truncated at the Pvu II site and Apa LI site, respectively. H246DΔSH was prepared as follows. The polymerase chain reaction fragment (1–247; H246D) obtained with mutagenized primer was ligated to the PmaC I site of the wild type cDNA fragment coding for amino acids 250–502. WASP full-length (WT and H246DΔSH), CRIB (131–309; WT and H246DΔSH) and VCA (414–502) were obtained as glutathione S-transferase (GST) fusion proteins or Histidine (His)-tagged proteins. GST-full length, -CRIB, and -VCA were constructed by inserting the cDNA fragments into pGEX-4T-1 (Amersham Pharmacia Biotech). The His-CRIB construction was obtained by subcloning the cDNA fragments into pQE 30 (Qiagen). Cdc42 and profilin I were also obtained as GST fusion proteins (14Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (559) Google Scholar, 15Suetsugu S. Miki H. Takenawa T. EMBO J. 1998; 17: 6516-6526Crossref PubMed Scopus (193) Google Scholar). GST fusion and His-tagged proteins were expressed and purified as described previously (14Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (559) Google Scholar, 16Miki H. Miura K. Matuoka K. Nakata T. Hirokawa N. Orita S. Kaibuchi K. Takai Y. Takenawa T. J. Biol. Chem. 1994; 269: 5489-5492Abstract Full Text PDF PubMed Google Scholar). WASP (WT and mutants) and Cdc42 (G12V and T17N) were subcloned into pEF-BOS mammalian expression vectors (14Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (559) Google Scholar). COS7 cells were transfected with these plasmids by electroporation and subjected to immunofluorescence microscopy as described previously (14Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (559) Google Scholar). About 70% of transfected cells indicated each phenotype. WASP (WT, H246DΔSH, or ΔPR)-expressing COS7 cell lysates were prepared as follows. Cells were harvested with lysis buffer (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 1 mm EDTA, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride) and sonicated. After centrifugation, the soluble fraction was collected as cell lysates. 150 μg of GST-Cdc42 was immobilized on 30 μl of glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech). The GST-Cdc42 beads were incubated in 50 μl of GTPγS or GDP-binding buffer (50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 10 mm GTPγS or GDP) at 30 °C for 10 min, and then MgCl2 was added to a final concentration of 10 mm. The GTPγS or GDP-loaded GST-Cdc42 beads were suspended and incubated in WASP-expressing COS7 lysates (WT or H246DΔSH) with 5 mm MgCl2 at 4 °C for 2 h with rotation. In the case of profilin I, 150 μg of GST-profilin I was immobilized on 30 μl of glutathione-Sepharose 4B beads. The GST-profilin I beads were suspended and incubated in WASP-expressing COS7 lysates (WT or ΔPR) at 4 °C for 2 h with rotation. After being washed with lysis buffer (without Triton X-100 and phenylmethylsulfonyl fluoride), the beads were suspended in SDS sample buffer, and Western blotting was performed. GST and GST-CRIB (WT or H246DΔSH) were subjected to SDS-polyacrylamide gel electrophoresis followed by blotting to a polyvinylidene difluoride membrane. After blocking and washing with binding buffer (50 mm Tris-HCl, pH 7.5, 5 mm MgCl2, 0.1% Triton X-100), the membranes were incubated with probes for 5 min and washed with binding buffer. The signal was visualized by autoradiography. The probes were prepared as follows. Cdc42 cleaved from GST fusion protein by thrombin digestion was dialyzed against 50 mm Tris-HCl, pH 7.5, 5 mm EDTA. Then the GTP-binding reaction was performed. 300 μl of reaction buffer (50 mm Tris-HCl, pH 7.5, 5 mm EDTA, 6 μg of Cdc42, 30 μCi of [γ-32P]GTP) was incubated at 30 °C for 10 min, and then 8μl, 40, and 200 μl of this Cdc42 added to 492, 460, and 300 μl of binding buffer and MgCl2 (final 10 mm), respectively. 30 μg of GST-VCA was immobilized on 15 μl of glutathione-Sepharose 4B beads. The beads were incubated with various concentrations of His-CRIB protein (WT or H246DΔSH) in buffer (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 1 mm EDTA) at 4 °C for 2 h with rotation. Then they were washed, and Western blotting was performed. 10 μg of GST-full length (WT or H246DΔSH) or -VCA was immobilized on 40 μl of glutathione-Sepharose 4B beads. The beads were incubated with 0.2 or 1 μmArp2/3 complex in the buffer (50 mm Tris-HCl, pH 7.5, and 200 mm KCl) at 4 °C for 2 h with rotation. Then they were washed, and Western blotting was performed. The Arp2/3 complex was purified from bovine brain by the method described previously (19Machesky L.M. Mullins R.D. 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 (601) Google Scholar). We first tried to determine which region of WASP is essential for the regulation of actin cytoskeleton. As described above, WASP is composed of many functional domains. In the case of N-WASP, another WASP family protein, both the VCA domain and the PR domain have been shown to be important for the formation of actin microspikes (14Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (559) Google Scholar, 15Suetsugu S. Miki H. Takenawa T. EMBO J. 1998; 17: 6516-6526Crossref PubMed Scopus (193) Google Scholar, 17Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (547) Google Scholar, 20Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Crossref PubMed Scopus (113) Google Scholar). The VCA domain and the PR domain of N-WASP directly bind to actin and profilin, respectively (15Suetsugu S. Miki H. Takenawa T. EMBO J. 1998; 17: 6516-6526Crossref PubMed Scopus (193) Google Scholar, 17Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (547) Google Scholar). In reference to these results, to examine which domain of WASP is important for the actin clustering, we prepared two deletion constructs ΔPRVCA (lacking both the PR and VCA domains) and ΔPR (lacking only the PR domain) schematically shown in Fig.1 A, and transfected COS7 cells with them. We checked the expression of each protein by Western blotting and confirmed that the two proteins are expressed at similar levels (Fig. 1 B). We next immunostained the cells with anti-WASP antibody and phalloidin to visualize the expressed WASP (including mutants) and actin filaments (F-actin), respectively. As a result, ΔPR was found to induce actin clustering as well as did the wild type (WT), whereas ΔPRVCA did not (Fig.2 A), suggesting that the VCA domain is essential for the formation of actin clustering. This conclusion is consistent with a previous report in which the deletion of the VCA domain was shown to abolish the actin clustering activity (3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar)Figure 2Verprolin-cofilin-acidic domain is essential for the actin clustering induced by WASP. A, immunofluorescence staining of cells untransfected and transfected with WT, ΔPRVCA, and ΔPR WASP. COS7 cells were transfected with WASP constructs (WT, ΔPRVCA, or ΔPR). WASP and F-actin were visualized with anti-WASP antibody and phalloidin, respectively. B, profilin-WASP binding assay. WASP (WT or ΔPR)-expressing COS7 cell lysates were incubated with GST or GST-profilin I-immobilized glutathione beads. The bound proteins were subjected to SDS-polyacrylamide gel electrophoresis followed by Western blotting with anti-WASP antibody. GST or GST-profilin was visualized by Coomassie Brilliant Blue staining of the gels.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The results presented above also suggest that direct binding to profilin is not required for the actin clustering, because the PR domain in N-WASP is the site for direct binding to profilin. To confirm this hypothesis, we examined the possible interaction between WASP and profilin. WASP (WT or ΔPR)-expressing COS7 cell lysate was incubated with GST or GST-profilin I immobilized on beads, and then the bound protein was examined by Western blotting with anti-WASP antibody. Fig.2 B shows that ΔPR failed to bind to profilin, whereas WT did bind. Taken together, these results indicate that WASP induces actin clustering in a manner independent of direct binding with profilin via the PR domain. It was shown that ΔPRVCA composed only of the N-terminal 310 residues of WASP clusters especially around the nucleus like the wild type WASP (Fig. 2 A). This indicates that the unidentified signal that determines the intracellular localization of WASP exists within the 310 residues. The N terminus of WASP contains many functional domains such as the PH domain, WASP insert (WI) domain, which is WASP-unique proline-rich domain compared with N-WASP, basic-rich region (BR), and GBD/CRIB motif (Fig. 1 A). To determine which domains are important for the clustering of WASP, we transfected COS7 cells with various N-terminal deletion constructs such as ΔPHWI, ΔWI, and ΔPH (Fig. 1 A). As shown in Fig.3 A, ΔWI was shown to cluster around the nuclei with F-actin as well as did the WT, indicating that the WI domain is not required for the clustering. In contrast, ΔPHWI and ΔPH did not cluster around the nuclei but instead induced marked retraction of the plasma membrane (Fig. 3 A). Most of the expressed ΔPHWI and ΔPH proteins seemed to accumulate at the plasma membrane. These results suggest that the PH domain is important for WASP clustering. We next examined whether the PH domain is sufficient for the clustering by expressing other N terminus-modified constructs such as PH, PHWI, and PHBR (Fig. 1 A). Although PH (data not shown) and PHWI were dispersed throughout the cytoplasm, PHBR clustered around the nuclei as did wild type WASP (Fig. 3 B). These results indicate that the region from the PH domain to the basic-rich region is essential for WASP clustering. A previous study by Symons et al. (3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar) showed that the expression of dominant-negative Cdc42 suppresses WASP-induced actin clustering. Indeed, we also confirmed that this inhibition occurs in COS7 cells (described later). To examine whether direct binding is required for actin cluster formation, we prepared a GBD/CRIB motif-modified construct (H246DΔSH; Fig. 1 A). We first checked whether this mutation really abolishes binding to Cdc42. First, we investigated the binding by using GST-Cdc42-immobilized beads. WASP (WT or H246DΔSH)-expressing COS7 cell lysates were incubated with GST or GST-Cdc42 (pre-loaded with GDP or GTPγS) immobilized on beads. Then Western blotting with anti-WASP antibody was performed. H246DΔSH failed to bind to GTPγS-loaded GST-Cdc42, whereas WT did bind (Fig.4 A). Second, we performed immunoprecipitation. WASP (WT or H246DΔSH) and c-Myc-tagged Cdc42G12V were co-expressed in COS7 cells, and the cell lysates were subjected to immunoprecipitation with anti-c-Myc antibody. Fig. 4 B shows that Cdc42 did not co-precipitate with H246DΔSH at all. Third, we examined the direct association between GBD/CRIB motif in WASP and Cdc42. GST and GST-CRIB (WT and H246DΔSH) blotted on membranes were incubated with Cdc42 (loaded with 32P-labeled GTP), and a positive signal was detected by autoradiography. As shown in Fig.4 C, Cdc42 bound to only wild type GST-CRIB. The results of these three experiments clearly indicate that H246DΔSH does not bind to Cdc42. We next examined the effect of H246DΔSH expression by immunofluorescence microscopy. Unexpectedly, this Cdc42-binding defective mutant also induced actin clusters like wild type WASP (Fig.5 A). In the case of N-WASP, the actin-regulating VCA domain is thought to be masked at the resting state by the intra- and/or intermolecular interaction with the region containing the GBD/CRIB motif (14Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (559) Google Scholar). Another effector of Cdc42, p21-activated kinase is also thought to be kept inactive at the resting state, and its GBD/CRIB motif-modified mutant that does not bind to Cdc42 becomes constitutively active (21Sells M.A. Knaus U.G Bagrodia S. Ambrose D.M. Bokoch G.M. Chernoff J. Curr. Biol. 1996; 7: 202-210Abstract Full Text Full Text PDF Scopus (575) Google Scholar, 22Bokoch G.M. Reilly A.M. Daniels R.H. King C.C. Olivera A. Spiegel S. Knaus U.G. J. Biol. Chem. 1997; 273: 8137-8144Abstract Full Text Full Text PDF Scopus (173) Google Scholar). Because there is a possibility that the GBD/CRIB motif-modified mutant, H246DΔSH, is also constitutively active, we checked for interaction between WASP (WT and H246DΔSH) and its downstream effector, the Arp2/3 complex (23Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). For this purpose, we first prepared anti-Arp3 antibody (Fig.5 B). Using this antibody, it was found that full-length WASP (WT and H246DΔSH) weakly binds to the Arp2/3 complex compared with the VCA protein and that there is no significant difference in binding to the Arp2/3 complex between the WT and H246DΔSH full-length WASPs (Fig. 5 C). This result strongly supports the idea that the H246DΔSH mutant still keeps the normal protein conformation. In support of this possibility, the GBD/CRIB motif-containing region of H246DΔSH associated with the VCA domain-containing region with the same affinity as the WT GBD/CRIB motif-containing region (Fig.5 D). Taken together, the results suggest that direct binding with Cdc42 is dispensable for actin cluster formation. Further, to examine whether H246DΔSH-induced actin cluster formation is affected by dominant-negative Cdc42, we co-transfected COS7 cells with H246DΔSH and Cdc42T17N. As shown in Fig.6, the co-expression of dominant-negative Cdc42 also suppressed the formation of actin clusters induced by H246DΔSH. Thus, we conclude that WASP has some functional interaction with Cdc42, but direct physical interaction between them is not required for actin cluster formation.Figure 6Cdc42T17N inhibits actin cluster formation induced by GBD/CRIB motif-mutated WASP, H246DΔSH. COS7 cells were transfected with both H246DΔSH and Cdc42T17N (c-Myc-tagged). The cells were stained with anti-WASP antibody, anti-c-Myc antibody, and phalloidin to visualize WASP, Cdc42, and F-actin, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It has been suggested that WASP plays a key role in regulating cytoskeletal reorganization. However, little is known about the mechanism by which it does so. WASP has many functional domains through which it interacts with various signaling and/or cytoskeletal proteins. Ectopic expression of WASP has been shown to induce the clustering of the expressed WASP itself (3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). In addition, actin filaments are also accumulated at the clusters of WASP. Although the physiological relevance of this WASP/actin cluster is unknown, the existence of similar vesicle-like structures is reported in phorbol ester-treated MEG-01 magakaryoblastic cell line (11Miki H. Nonoyama S. Zhu Q. Aruffo A. Ochs H.D. Takenawa T. Cell Growth Differ. 1997; 8: 195-202PubMed Google Scholar), suggesting the importance of the clusters in vivo. It has been reported that Cdc42 regulates the formation of these WASP/actin clusters (3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar), because activated Cdc42 directly binds to the GBD/CRIB motif of WASP and the expression of its dominant-negative mutant completely inhibits the cluster formation (3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). Thus, we started to investigate which domains are required for the actin clustering and the accumulation of WASP itself and the role of Cdc42 in the process. First, the VCA domain was shown to be essential for the accumulation of actin filaments. This result is consistent with a previous study in which the deletion of the C-terminal 59 amino acid residues resulted in the inability to induce the actin clustering (3Symons M. Derry J.M. Karlak B. Jiang S. Lemahieu V. Mccormick F. Francke U. Abo A. Cell. 1996; 84: 723-734Abstract Full Text Full Text PDF PubMed Scopus (743) Google Scholar). The VCA domain consists of a verprolin homology (VPH) domain, a cofilin homology domain, and a highly acidic region. We reported that direct interaction between the VPH domain and actin is essential for cytoskeletal reorganization in the case of N-WASP, suggesting that the VPH domain of WASP might also be required for actin clustering (20Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Crossref PubMed Scopus (113) Google Scholar). Recently, it has been reported that the Arp2/3 complex, which plays an important role in nucleating actin filaments, binds to the C terminus of WASP (23Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar). The binding region was narrowed down to the C-terminal 38 residues in the case of WAVE/Scar1, a WASP/N-WASP-related protein that also possess a similar VCA domain. Moreover, it was demonstrated that the region plays a critical role in Rac-induced reorganization of the actin cytoskeleton (23Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (739) Google Scholar, 24Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (567) Google Scholar). The C-terminal Arp2/3 binding region is corresponds to the cofilin homology domain and the acidic region. The deletion of eight residues in the cofilin homology domain (amino acids 473–480), as expected, resulted in the inhibition of the accumulation of actin filaments (data not shown). Thus, both the actin binding to the VPH domain and the Arp2/3-binding to the region from cofilin homology domain to the acidic region are important in actin clustering. This may trigger the actin nucleation by the Arp2/3 complex and induce the subsequent actin polymerization on newly formed actin filaments. The region important for the clustering of WASP itself was narrowed down to one covering from the N-terminal PH domain to the basic residue-rich region. The PH domain seemed to be essential, because the deletion mutants of the PH domain showed a different intracellular localization from the wild type WASP. It has been reported that the PH domain may be responsible for cellular localization via interactions with specific lipids and/or proteins (25Harlan J.E. Hajduk P.J. Yoon H.S. Fesik S.W. Nature. 1994; 371: 168-170Crossref PubMed Scopus (672) Google Scholar, 26Tsukada S. Simon M.I. Witte O.N. Katz A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11256-11260Crossref PubMed Scopus (238) Google Scholar, 27Yao L. Kawakami Y. Kawakami T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9175-9179Crossref PubMed Scopus (308) Google Scholar, 28Varnai P. Balla T. J. Cell Biol. 1998; 143: 501-510Crossref PubMed Scopus (665) Google Scholar, 29Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar, 30Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar, 31Inglese J. Luttrell L.M. Iniguez-Lluhi J.A. Touhara K. Koch W.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3637-3641Crossref PubMed Scopus (71) Google Scholar). In the case of phospholipase Cδ1, the PH domain specifically interacts with phosphatidylinositol 4,5-bisphosphate and localizes to plasma membrane (28Varnai P. Balla T. J. Cell Biol. 1998; 143: 501-510Crossref PubMed Scopus (665) Google Scholar). In contrast, the PH domain of β-adrenergic receptor kinase specifically recognizes the βγ subunit of the G-protein and, through the interaction with Gβγ, inactivates G-protein-coupled receptor (29Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar, 30Koch W.J. Hawes B.E. Inglese J. Luttrell L.M. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 6193-6197Abstract Full Text PDF PubMed Google Scholar, 31Inglese J. Luttrell L.M. Iniguez-Lluhi J.A. Touhara K. Koch W.J. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3637-3641Crossref PubMed Scopus (71) Google Scholar). Therefore, it is quite probable and reasonable that the PH domain of WASP also recognizes some specific lipids and/or proteins and locates WASP to the special structures seen in the photographs shown in the figures. Lastly, we found that WASP does not need to associate directly with Cdc42 to induce the actin cluster formation. The GBD/CRIB motif-containing region was shown to bind to the actin-regulating VCA domain (Fig. 5 D), suggesting that WASP also takes some folded inactive structure as N-WASP. Thus, some other proteins and/or specific lipids play a role as an activator of WASP in cells. However, the results shown in Fig. 6 indicate that Cdc42 affects the actin cluster formation induced by WASP, even in the case of Cdc42-binding defective WASP mutant. Some other factors link Cdc42 and WASP, signal transduction pathways of WASP and Cdc42 are both required for actin cluster formation, or Cdc42 functions downstream of WASP. Although we cannot answer here which possibility is true, this is a new concept and gives us a novel insight into the regulation and/or function of WASP. We thank Shiro Suetsugu in our laboratory for profilin constructs.

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