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Phosphorylation of WAVE Downstream of Mitogen-activated Protein Kinase Signaling

1999; Elsevier BV; Volume: 274; Issue: 39 Linguagem: Inglês

10.1074/jbc.274.39.27605

1083-351X

Hiroaki Miki, Makoto Fukuda, Eisuke Nishida, Tadaomi Takenawa,

Protein Kinase Regulation and GTPase Signaling

WAVE is a Wiskott-Aldrich syndrome protein (WASP)-family protein that functions in membrane-ruffling formation induced by Rac, a Rho family small GTPase. Here we report that WAVE is a phosphoprotein whose phosphorylation increases in response to various external stimuli that activate mitogen-activated protein (MAP) kinase signaling. When Swiss 3T3 cells are stimulated with platelet-derived growth factor, electrophoretic mobility shift occurs to WAVE, which reflects hyperphosphorylation. This is perfectly inhibited by the addition of PD98059, a specific inhibitor of MAP kinase kinase. Indeed, the ectopic expression of an activated mutant of MAP kinase kinase induces WAVE mobility shift. When MAP kinase activation is suppressed by PD98059, the intensity of platelet-derived growth factor-induced membrane ruffling is greatly reduced. In various cancer cell lines, the amount of WAVE mobility shift was found to increase significantly, suggesting the importance of WAVE hyperphosphorylation in the formation of membrane ruffles and oncogenic transformation. WAVE is a Wiskott-Aldrich syndrome protein (WASP)-family protein that functions in membrane-ruffling formation induced by Rac, a Rho family small GTPase. Here we report that WAVE is a phosphoprotein whose phosphorylation increases in response to various external stimuli that activate mitogen-activated protein (MAP) kinase signaling. When Swiss 3T3 cells are stimulated with platelet-derived growth factor, electrophoretic mobility shift occurs to WAVE, which reflects hyperphosphorylation. This is perfectly inhibited by the addition of PD98059, a specific inhibitor of MAP kinase kinase. Indeed, the ectopic expression of an activated mutant of MAP kinase kinase induces WAVE mobility shift. When MAP kinase activation is suppressed by PD98059, the intensity of platelet-derived growth factor-induced membrane ruffling is greatly reduced. In various cancer cell lines, the amount of WAVE mobility shift was found to increase significantly, suggesting the importance of WAVE hyperphosphorylation in the formation of membrane ruffles and oncogenic transformation. Wiskott-Aldrich syndrome protein glutathioneS-transferase lysophosphatidic acid mitogen-activated protein MAP kinase kinase platelet-derived growth factor baby hamster kidney hemagglutinin Madin-Darby canine kidney Wiskott-Aldrich syndrome protein (WASP)1 has been identified as the gene product whose mutation causes a human hereditary disease, Wiskott-Aldrich syndrome (1Derry J.M. Ochs H.D. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (823) Google Scholar). Ectopic expression studies revealed that WASP induces the formation of clusters of actin filaments in a manner dependent on Cdc42, a Rho family small GTPase (2Symons 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 (742) Google Scholar). Soon after the discovery of WASP, we identified a WASP-related molecule in a search of Grb2/Ash-binding proteins (3Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (545) Google Scholar). This novel protein, named neural WASP (N-WASP), has been shown to function downstream of Cdc42 to induce the formation of actin microspikes (filopodia) (4Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (557) Google Scholar). At the carboxyl-terminal regions of both WASP and N-WASP, there exist verprolin homology domains that are homologous to one part of verprolin, a yeast protein that regulates the actin cytoskeleton (5Donnelly S.F. Pocklington M.J. Pallotta D. Orr E. Mol. Microbiol. 1993; 10: 585-596Crossref PubMed Scopus (86) Google Scholar,6Vaduva G. Martin N.C. Hopper A.K. J. Cell Biol. 1997; 139: 1821-1833Crossref PubMed Scopus (105) Google Scholar). We demonstrated that the verprolin homology domain in N-WASP directly binds to actin (7Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Crossref PubMed Scopus (113) Google Scholar).To identify a novel WASP-related molecule, we performed a data base search using the verprolin homology domain amino acid sequence and identified WAVE (WASP-family verprolin homologous protein), which was originally isolated in a random cDNA sequencing project as KIAA0269 (8Nagase T. Seki N. Ishikawa K. Ohira M. Kawarabayasi Y. Ohara O. Tanaka A. Kotani H. Miyajima N. Nomura N. DNA Res. 1996; 3: 321-329Crossref PubMed Scopus (205) Google Scholar, 9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar). The carboxyl-terminal half of WAVE was structurally similar to both WASP and N-WASP, in that WAVE also possesses the verprolin homology domain and a proline-rich region. Thus, WAVE was thought to be a new member of the WASP family of proteins.We then demonstrated that WAVE regulates the actin reorganization that is essential for the formation of membrane ruffles induced by Rac, another Rho family member (9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar). Recently, Machesky and Insall (10Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar) reported that the carboxyl-terminal fragment of WAVE (they call the same protein Hs-Scar1) binds to Arp2/3 protein complex and that the ectopic expression of the fragment suppresses the PDGF-induced formation of membrane ruffles. Because PDGF-induced membrane-ruffling formation occurs in a Rac-dependent manner (11Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3050) Google Scholar), this result also supports the possibility that WAVE functions downstream of Rac.Thus, all WASP family proteins have been shown to regulate the reorganization of the actin cytoskeleton downstream of Cdc42 or Rac, and Cdc42 has been shown to bind directly to WASP and N-WASP (2Symons 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 (742) Google Scholar, 4Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (557) Google Scholar,12Aspenstrom P. Lindberg U. Hall A. Curr. Biol. 1996; 6: 70-75Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). In contrast, we could not detect any direct interaction between WAVE and Rac by conventional far Western blot assay, and the regulation mechanism of WAVE remains unclear.To understand its regulation mechanism, we have examined here whether WAVE is modified in response to external stimuli that cause membrane ruffling. As a result, we found that WAVE is hyperphosphorylated by various signals that activate a MAP kinase signaling cascade.DISCUSSIONMAP kinase is a protein kinase that is activated by various external stimuli and regulates many fundamental processes such as cell growth and differentiation. The best known function of MAP kinase is to receive signals from Ras and transmit the signal to the nucleus, regulating transcription of specific genes that affect the fate of cells. However, there has been accumulating evidence that MAP kinase not only transmits the signal to the nucleus but also regulates cytoplasmic events such as cell motility (25Klemke R.L. Cai S. Giannini A.L. Gallagher P.J. de Lanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1099) Google Scholar).We found in this study that suppression of MAP kinase activation resulted in significant, though not perfect, reduction of membrane ruffling. WAVE is a strong candidate for a downstream target of MAP kinase for proper formation of membrane ruffles, because WAVE is hyperphosphorylated downstream of MAP kinase signaling and has been shown to be a critical regulator of membrane ruffling downstream of Rac (9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar). The WAVE hyperphosphorylation seems to inhibit specifically the association with Grb2/Ash, although we do not know yet the physiological relevance of this inhibition. It is quite probable that Grb2/Ash recruits WAVE to the activated PDGF receptor through the association between the SH3 domains and the proline-rich region as in the case of Sos (26Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell D. Nature. 1993; 363: 83-85Crossref PubMed Scopus (835) Google Scholar, 27Li N. Batzer A. Daly R. Yajnik V. Skolnik E. Chardin P. Bar-Sagi D. Margolis B. Schlessinger J. Nature. 1993; 363: 85-88Crossref PubMed Scopus (797) Google Scholar), a well known Ras activator. The hyperphosphorylation that follows by MAP kinase pathway may free WAVE from Grb2/Ash, and then WAVE may become fully active in inducing the membrane-ruffling formation.However, it should be noted that the hyperphosphorylation of WAVE is not required for membrane-ruffling formation. As described above, PD98059 treatment could only partially suppress the membrane-ruffling formation. Furthermore, the WAVE hyperphosphorylation is not sufficient for membrane ruffling. Indeed, stimulation of cells with LPA or serum, both of which induce significant WAVE hyperphosphorylation, does not induce membrane ruffling. More directly, we confirmed that expression of active MEK alone did not induce membrane ruffling (data not shown). Thus, we conclude that the Rac pathway is the main route to induction of the membrane-ruffling formation and that the MAP kinase pathway modulates the signaling cascade at some points including WAVE. Now we do not know how WAVE is regulated by Rac. Because WAVE and Rac can form protein complexes when co-expressed in COS 7 cells (9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar), some adaptor molecule may link between Rac and WAVE. We have performed two-hybrid screening using various parts of WAVE as bait and identified several WAVE-specific binding proteins including known and unknown ones (data not shown), among which we hope the “linking protein” exists.The important question is what kinase “directly” hyperphosphorylates WAVE. We performed an in vitro kinase assay using activated MAP kinase purified from Xenopusoocyte extracts and found that it indeed phosphorylates the carboxyl-terminal GST fusion fragment of WAVE (data not shown). However, the phosphorylation efficiency was very weak (< 150the phosphorylation efficiency of myelin basic protein). More importantly, the full-length WAVE protein immunoprecipitated from Swiss 3T3 cells not treated with PDGF was not phosphorylated at all (data not shown). Thus, it is questionable that MAP kinase itself directly hyperphosphorylates WAVE. Several kinases have been found to be activated downstream of MAP kinases (28Stokoe D. Campbell D.G. Nakielny S. Hidaka H. Leevers S.J. Marshall C. Cohen P. EMBO J. 1992; 11: 3985-3994Crossref PubMed Scopus (391) Google Scholar), and we think that such a kinase(s) phosphorylates WAVE.WAVE mobility shift occurs not only in cells stimulated with growth factors but also in oncogenically transformed cells. It has been shown that a MAP kinase cascade is essential for oncogenic transformation by various oncogenes (29Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar). In addition, LA-SDSE mutant MEK, which is constitutively active and can remain in the nucleus, can transform NIH3T3 fibroblasts by itself (19Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar). Rac has also been demonstrated to be involved in transformation. For example, expression of the dominant negative form of Rac can suppress transformation by oncogenic Ras (30Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (812) Google Scholar). Rac also participates in invasion of carcinoma cells into normal tissues (31Shaw L.M. Rabinovitz I. Wang H.H. Toker A. Mercurio A.M. Cell. 1997; 91: 949-960Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). Thus, taken together, this information suggests that both MAP kinase and Rac produce critical signals inducing oncogenic transformation. Considering this, we conclude that WAVE might be a critical regulator of tumorigenesis by integrating two important signals for transformation, MAP kinase and Rac. Wiskott-Aldrich syndrome protein (WASP)1 has been identified as the gene product whose mutation causes a human hereditary disease, Wiskott-Aldrich syndrome (1Derry J.M. Ochs H.D. Francke U. Cell. 1994; 78: 635-644Abstract Full Text PDF PubMed Scopus (823) Google Scholar). Ectopic expression studies revealed that WASP induces the formation of clusters of actin filaments in a manner dependent on Cdc42, a Rho family small GTPase (2Symons 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 (742) Google Scholar). Soon after the discovery of WASP, we identified a WASP-related molecule in a search of Grb2/Ash-binding proteins (3Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (545) Google Scholar). This novel protein, named neural WASP (N-WASP), has been shown to function downstream of Cdc42 to induce the formation of actin microspikes (filopodia) (4Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (557) Google Scholar). At the carboxyl-terminal regions of both WASP and N-WASP, there exist verprolin homology domains that are homologous to one part of verprolin, a yeast protein that regulates the actin cytoskeleton (5Donnelly S.F. Pocklington M.J. Pallotta D. Orr E. Mol. Microbiol. 1993; 10: 585-596Crossref PubMed Scopus (86) Google Scholar,6Vaduva G. Martin N.C. Hopper A.K. J. Cell Biol. 1997; 139: 1821-1833Crossref PubMed Scopus (105) Google Scholar). We demonstrated that the verprolin homology domain in N-WASP directly binds to actin (7Miki H. Takenawa T. Biochem. Biophys. Res. Commun. 1998; 243: 73-78Crossref PubMed Scopus (113) Google Scholar). To identify a novel WASP-related molecule, we performed a data base search using the verprolin homology domain amino acid sequence and identified WAVE (WASP-family verprolin homologous protein), which was originally isolated in a random cDNA sequencing project as KIAA0269 (8Nagase T. Seki N. Ishikawa K. Ohira M. Kawarabayasi Y. Ohara O. Tanaka A. Kotani H. Miyajima N. Nomura N. DNA Res. 1996; 3: 321-329Crossref PubMed Scopus (205) Google Scholar, 9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar). The carboxyl-terminal half of WAVE was structurally similar to both WASP and N-WASP, in that WAVE also possesses the verprolin homology domain and a proline-rich region. Thus, WAVE was thought to be a new member of the WASP family of proteins. We then demonstrated that WAVE regulates the actin reorganization that is essential for the formation of membrane ruffles induced by Rac, another Rho family member (9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar). Recently, Machesky and Insall (10Machesky L.M. Insall R.H. Curr. Biol. 1998; 8: 1347-1356Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar) reported that the carboxyl-terminal fragment of WAVE (they call the same protein Hs-Scar1) binds to Arp2/3 protein complex and that the ectopic expression of the fragment suppresses the PDGF-induced formation of membrane ruffles. Because PDGF-induced membrane-ruffling formation occurs in a Rac-dependent manner (11Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3050) Google Scholar), this result also supports the possibility that WAVE functions downstream of Rac. Thus, all WASP family proteins have been shown to regulate the reorganization of the actin cytoskeleton downstream of Cdc42 or Rac, and Cdc42 has been shown to bind directly to WASP and N-WASP (2Symons 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 (742) Google Scholar, 4Miki H. Sasaki T. Takai Y. Takenawa T. Nature. 1998; 391: 93-96Crossref PubMed Scopus (557) Google Scholar,12Aspenstrom P. Lindberg U. Hall A. Curr. Biol. 1996; 6: 70-75Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). In contrast, we could not detect any direct interaction between WAVE and Rac by conventional far Western blot assay, and the regulation mechanism of WAVE remains unclear. To understand its regulation mechanism, we have examined here whether WAVE is modified in response to external stimuli that cause membrane ruffling. As a result, we found that WAVE is hyperphosphorylated by various signals that activate a MAP kinase signaling cascade. DISCUSSIONMAP kinase is a protein kinase that is activated by various external stimuli and regulates many fundamental processes such as cell growth and differentiation. The best known function of MAP kinase is to receive signals from Ras and transmit the signal to the nucleus, regulating transcription of specific genes that affect the fate of cells. However, there has been accumulating evidence that MAP kinase not only transmits the signal to the nucleus but also regulates cytoplasmic events such as cell motility (25Klemke R.L. Cai S. Giannini A.L. Gallagher P.J. de Lanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1099) Google Scholar).We found in this study that suppression of MAP kinase activation resulted in significant, though not perfect, reduction of membrane ruffling. WAVE is a strong candidate for a downstream target of MAP kinase for proper formation of membrane ruffles, because WAVE is hyperphosphorylated downstream of MAP kinase signaling and has been shown to be a critical regulator of membrane ruffling downstream of Rac (9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar). The WAVE hyperphosphorylation seems to inhibit specifically the association with Grb2/Ash, although we do not know yet the physiological relevance of this inhibition. It is quite probable that Grb2/Ash recruits WAVE to the activated PDGF receptor through the association between the SH3 domains and the proline-rich region as in the case of Sos (26Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell D. Nature. 1993; 363: 83-85Crossref PubMed Scopus (835) Google Scholar, 27Li N. Batzer A. Daly R. Yajnik V. Skolnik E. Chardin P. Bar-Sagi D. Margolis B. Schlessinger J. Nature. 1993; 363: 85-88Crossref PubMed Scopus (797) Google Scholar), a well known Ras activator. The hyperphosphorylation that follows by MAP kinase pathway may free WAVE from Grb2/Ash, and then WAVE may become fully active in inducing the membrane-ruffling formation.However, it should be noted that the hyperphosphorylation of WAVE is not required for membrane-ruffling formation. As described above, PD98059 treatment could only partially suppress the membrane-ruffling formation. Furthermore, the WAVE hyperphosphorylation is not sufficient for membrane ruffling. Indeed, stimulation of cells with LPA or serum, both of which induce significant WAVE hyperphosphorylation, does not induce membrane ruffling. More directly, we confirmed that expression of active MEK alone did not induce membrane ruffling (data not shown). Thus, we conclude that the Rac pathway is the main route to induction of the membrane-ruffling formation and that the MAP kinase pathway modulates the signaling cascade at some points including WAVE. Now we do not know how WAVE is regulated by Rac. Because WAVE and Rac can form protein complexes when co-expressed in COS 7 cells (9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar), some adaptor molecule may link between Rac and WAVE. We have performed two-hybrid screening using various parts of WAVE as bait and identified several WAVE-specific binding proteins including known and unknown ones (data not shown), among which we hope the “linking protein” exists.The important question is what kinase “directly” hyperphosphorylates WAVE. We performed an in vitro kinase assay using activated MAP kinase purified from Xenopusoocyte extracts and found that it indeed phosphorylates the carboxyl-terminal GST fusion fragment of WAVE (data not shown). However, the phosphorylation efficiency was very weak (< 150the phosphorylation efficiency of myelin basic protein). More importantly, the full-length WAVE protein immunoprecipitated from Swiss 3T3 cells not treated with PDGF was not phosphorylated at all (data not shown). Thus, it is questionable that MAP kinase itself directly hyperphosphorylates WAVE. Several kinases have been found to be activated downstream of MAP kinases (28Stokoe D. Campbell D.G. Nakielny S. Hidaka H. Leevers S.J. Marshall C. Cohen P. EMBO J. 1992; 11: 3985-3994Crossref PubMed Scopus (391) Google Scholar), and we think that such a kinase(s) phosphorylates WAVE.WAVE mobility shift occurs not only in cells stimulated with growth factors but also in oncogenically transformed cells. It has been shown that a MAP kinase cascade is essential for oncogenic transformation by various oncogenes (29Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar). In addition, LA-SDSE mutant MEK, which is constitutively active and can remain in the nucleus, can transform NIH3T3 fibroblasts by itself (19Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar). Rac has also been demonstrated to be involved in transformation. For example, expression of the dominant negative form of Rac can suppress transformation by oncogenic Ras (30Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (812) Google Scholar). Rac also participates in invasion of carcinoma cells into normal tissues (31Shaw L.M. Rabinovitz I. Wang H.H. Toker A. Mercurio A.M. Cell. 1997; 91: 949-960Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). Thus, taken together, this information suggests that both MAP kinase and Rac produce critical signals inducing oncogenic transformation. Considering this, we conclude that WAVE might be a critical regulator of tumorigenesis by integrating two important signals for transformation, MAP kinase and Rac. MAP kinase is a protein kinase that is activated by various external stimuli and regulates many fundamental processes such as cell growth and differentiation. The best known function of MAP kinase is to receive signals from Ras and transmit the signal to the nucleus, regulating transcription of specific genes that affect the fate of cells. However, there has been accumulating evidence that MAP kinase not only transmits the signal to the nucleus but also regulates cytoplasmic events such as cell motility (25Klemke R.L. Cai S. Giannini A.L. Gallagher P.J. de Lanerolle P. Cheresh D.A. J. Cell Biol. 1997; 137: 481-492Crossref PubMed Scopus (1099) Google Scholar). We found in this study that suppression of MAP kinase activation resulted in significant, though not perfect, reduction of membrane ruffling. WAVE is a strong candidate for a downstream target of MAP kinase for proper formation of membrane ruffles, because WAVE is hyperphosphorylated downstream of MAP kinase signaling and has been shown to be a critical regulator of membrane ruffling downstream of Rac (9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar). The WAVE hyperphosphorylation seems to inhibit specifically the association with Grb2/Ash, although we do not know yet the physiological relevance of this inhibition. It is quite probable that Grb2/Ash recruits WAVE to the activated PDGF receptor through the association between the SH3 domains and the proline-rich region as in the case of Sos (26Rozakis-Adcock M. Fernley R. Wade J. Pawson T. Bowtell D. Nature. 1993; 363: 83-85Crossref PubMed Scopus (835) Google Scholar, 27Li N. Batzer A. Daly R. Yajnik V. Skolnik E. Chardin P. Bar-Sagi D. Margolis B. Schlessinger J. Nature. 1993; 363: 85-88Crossref PubMed Scopus (797) Google Scholar), a well known Ras activator. The hyperphosphorylation that follows by MAP kinase pathway may free WAVE from Grb2/Ash, and then WAVE may become fully active in inducing the membrane-ruffling formation. However, it should be noted that the hyperphosphorylation of WAVE is not required for membrane-ruffling formation. As described above, PD98059 treatment could only partially suppress the membrane-ruffling formation. Furthermore, the WAVE hyperphosphorylation is not sufficient for membrane ruffling. Indeed, stimulation of cells with LPA or serum, both of which induce significant WAVE hyperphosphorylation, does not induce membrane ruffling. More directly, we confirmed that expression of active MEK alone did not induce membrane ruffling (data not shown). Thus, we conclude that the Rac pathway is the main route to induction of the membrane-ruffling formation and that the MAP kinase pathway modulates the signaling cascade at some points including WAVE. Now we do not know how WAVE is regulated by Rac. Because WAVE and Rac can form protein complexes when co-expressed in COS 7 cells (9Miki H. Suetsugu S. Takenawa T. EMBO J. 1998; 17: 6932-6941Crossref PubMed Scopus (565) Google Scholar), some adaptor molecule may link between Rac and WAVE. We have performed two-hybrid screening using various parts of WAVE as bait and identified several WAVE-specific binding proteins including known and unknown ones (data not shown), among which we hope the “linking protein” exists. The important question is what kinase “directly” hyperphosphorylates WAVE. We performed an in vitro kinase assay using activated MAP kinase purified from Xenopusoocyte extracts and found that it indeed phosphorylates the carboxyl-terminal GST fusion fragment of WAVE (data not shown). However, the phosphorylation efficiency was very weak (< 150the phosphorylation efficiency of myelin basic protein). More importantly, the full-length WAVE protein immunoprecipitated from Swiss 3T3 cells not treated with PDGF was not phosphorylated at all (data not shown). Thus, it is questionable that MAP kinase itself directly hyperphosphorylates WAVE. Several kinases have been found to be activated downstream of MAP kinases (28Stokoe D. Campbell D.G. Nakielny S. Hidaka H. Leevers S.J. Marshall C. Cohen P. EMBO J. 1992; 11: 3985-3994Crossref PubMed Scopus (391) Google Scholar), and we think that such a kinase(s) phosphorylates WAVE. WAVE mobility shift occurs not only in cells stimulated with growth factors but also in oncogenically transformed cells. It has been shown that a MAP kinase cascade is essential for oncogenic transformation by various oncogenes (29Cowley S. Paterson H. Kemp P. Marshall C.J. Cell. 1994; 77: 841-852Abstract Full Text PDF PubMed Scopus (1845) Google Scholar). In addition, LA-SDSE mutant MEK, which is constitutively active and can remain in the nucleus, can transform NIH3T3 fibroblasts by itself (19Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (880) Google Scholar). Rac has also been demonstrated to be involved in transformation. For example, expression of the dominant negative form of Rac can suppress transformation by oncogenic Ras (30Qiu R.G. Chen J. Kirn D. McCormick F. Symons M. Nature. 1995; 374: 457-459Crossref PubMed Scopus (812) Google Scholar). Rac also participates in invasion of carcinoma cells into normal tissues (31Shaw L.M. Rabinovitz I. Wang H.H. Toker A. Mercurio A.M. Cell. 1997; 91: 949-960Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). Thus, taken together, this information suggests that both MAP kinase and Rac produce critical signals inducing oncogenic transformation. Considering this, we conclude that WAVE might be a critical regulator of tumorigenesis by integrating two important signals for transformation, MAP kinase and Rac. We thank Kyoko Miki for skillful technical assistance. We are also grateful to Dr. W. Birchmeier (Max-Delbruck Center for Molecular Medicine, Berlin, Germany) and Dr. Yoshimi Takai and Dr. Masato Umikawa (University of Osaka, Osaka, Japan) for giving us MDCK cells, to Dr. Motoharu Seiki (University of Tokyo, Tokyo, Japan) for HT1080 cells, to Dr. Mikako Shirouzu and Dr. Shigeyuki Yokoyama (Institute of Physical and Chemical Research (RIKEN), Saitama, Japan) for RasG12V cDNA, and to Dr. Tadashi Yamamoto (University of Tokyo, Tokyo, Japan) for the GST-Fyn SH3 construct.