Effect of Potent and Selective Inhibitors of the Grb2 SH2 Domain on Cell Motility

Abstract

Cell motility has been correlated both with oncogenic invasiveness and metastatic potential. The development of selective inhibitors of motility has thus great potential importance. Grb2 is a SH2/SH3 domain-containing adaptor protein that links growth factor receptor tyrosine kinases to the Ras signaling pathway. We have developed specific small molecule inhibitors of the Grb2 SH2 domain as potential leads for drug discovery. Synthesis of the inhibitors and their effects on growth factor-induced growth in cells have been reported previously. In the current study, we establish that these inhibitors inhibit hepatocyte growth factor/scatter factor-induced A431 and Madin-Darby canine kidney cell motility and various cell motility-related events, including epidermal growth factor-induced ruffling of A431 cells and epidermal growth factor-induced translocation of the small GTPase Rac in these cells. We demonstrate for the first time a direct role for Grb2 in cell motility and indicate a new avenue for cancer therapeutics. Cell motility has been correlated both with oncogenic invasiveness and metastatic potential. The development of selective inhibitors of motility has thus great potential importance. Grb2 is a SH2/SH3 domain-containing adaptor protein that links growth factor receptor tyrosine kinases to the Ras signaling pathway. We have developed specific small molecule inhibitors of the Grb2 SH2 domain as potential leads for drug discovery. Synthesis of the inhibitors and their effects on growth factor-induced growth in cells have been reported previously. In the current study, we establish that these inhibitors inhibit hepatocyte growth factor/scatter factor-induced A431 and Madin-Darby canine kidney cell motility and various cell motility-related events, including epidermal growth factor-induced ruffling of A431 cells and epidermal growth factor-induced translocation of the small GTPase Rac in these cells. We demonstrate for the first time a direct role for Grb2 in cell motility and indicate a new avenue for cancer therapeutics. growth factor epidermal growth factor hepatocyte growth factor/scatter factor glutathione S -transferase Src homology 2 EGF receptor Ras-binding domain Madin-Darby canine kidney enzyme-linked immunosorbent assay Acquisition of cell motility is a prerequisite to biological processes taking place in tissue remodeling. It has been described as a major event in morphogenesis and in pathological situations such as invasion and metastasis of tumor cells (1Thiery J.P. Cell Differ. 1984; 15: 1-15Crossref PubMed Scopus (55) Google Scholar, 2Strauli P. Weiss L. Eur. J. Cancer. 1977; 13: 1-12Abstract Full Text PDF PubMed Scopus (88) Google Scholar). Most cell motility factors are also growth factors (GFs),1 a most intriguing duality of function that implies the possibility of common or related signaling transduction pathways (3Stocker M. Gherardi E. Biochim. Biophys. Acta. 1991; 1072: 81-102PubMed Google Scholar). Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a pleiotropic GF that, besides promoting cell survival and proliferation, has the ability to dissociate epithelial sheets and to stimulate cell motility (3Stocker M. Gherardi E. Biochim. Biophys. Acta. 1991; 1072: 81-102PubMed Google Scholar). HGF/SF predominantly acts as a paracrine factor. Nearly 90% of human malignant tumors arise from epithelial tissue, and most carcinoma cells express the c-Met/HGF receptor and are likely to use HGF/SF produced from stromal tissue (4Matsumoto K. Horikoshi M. Rikimaru K. Enomoto S. J. Oral Pathol. Med. 1989; 18: 498-501Crossref PubMed Scopus (76) Google Scholar, 5Tajima H. Matsumoto K. Nakamura T. Exp. Cell Res. 1992; 202: 423-431Crossref PubMed Scopus (171) Google Scholar). On the other hand, GFs such as the epidermal growth factor (EGF) can enhance motility in an autocrine manner in several tumor cell lines (6Liotta L.A. Mandler R. Murano G. Katz D.A. Gordon R.K. Hing P.K. Shiffmann E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3302-3306Crossref PubMed Scopus (473) Google Scholar). The EGF receptor (EGFR) has also been extensively studied. GF activation of EGFR influences a number of phenotypic properties in malignant cells in vitro , including mitogenesis and cell motility stimulation (7Lung-Johansen M. Bjerkvig R. Humphrey A.P. Bigner H.S. Bigner D.D. Laerum O. Cancer Res. 1990; 50: 6039-6044PubMed Google Scholar). Interestingly, overexpression of EGFR is correlated more strongly with metastasis and invasion than with tumor size (7Lung-Johansen M. Bjerkvig R. Humphrey A.P. Bigner H.S. Bigner D.D. Laerum O. Cancer Res. 1990; 50: 6039-6044PubMed Google Scholar).Growth/motility factors such as EGF and HGF/SF, initiate a response by binding to receptors and thereby activating a tyrosine kinase domain located in the cytoplasmic portion of the receptor (8Cross M. Dexter T.M. Cell. 1991; 64: 271-280Abstract Full Text PDF PubMed Scopus (608) Google Scholar). This event leads to the autophosphorylation of the receptors on tyrosine residues. The presence of phosphotyrosine at specific sites on receptors is crucial for downstream signaling: within a specific sequence context, phosphotyrosines act as binding sites for many signal-transducing molecules within the cells (9Cantley L.C. Songyang Z. J. Cell Sci. 1994; 18: 121-126Crossref Google Scholar, 10Panayotou G. Waterfield M.D. Bioessays. 1993; 15: 171-177Crossref PubMed Scopus (114) Google Scholar). The common element that confers the specific property of phosphotyrosine-containing sequence recognition to all these target molecules is the Src homology 2 (SH2) domain (11Pawson T. Schlessinger J. Curr. Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (574) Google Scholar).Grb2 is an adaptor protein made of one SH2 domain flanked by two SH3 domains. Grb2 interacts via its SH2 domain with activated receptors and via its SH3 domains to the nucleotide exchange factor Sos, which thus becomes activated as a positive regulator of Ras (12Schlessinger J. Curr. Opin. Genet. Dev. 1994; 4: 25-30Crossref PubMed Scopus (400) Google Scholar). Design of molecules that block the interaction between the phosphotyrosine-containing activated receptors and the SH2 domain of Grb2 should interrupt the Ras signaling pathway and may promise therapeutic leads for diseases like cancer, in which the Ras signaling pathway plays a major role (13Sastry L. Cao T. King R. Int. J. Cancer. 1997; 70: 208-213Crossref PubMed Scopus (26) Google Scholar). Grb2 may have other functions as well. Micro-injection studies with anti-Grb2 antibodies in rat kidney cells suggest that Grb2 may play a role in signaling from receptor tyrosine kinases to the small GTPase Rac, a protein involved in motility-related events (14Matuoka K. Shibasaki F. Shibata M. Takenawa T. EMBO J. 1993; 12: 3467-3473Crossref PubMed Scopus (100) Google Scholar).We have reported the rational drug design of CGP78850, a potent and selective inhibitor of Grb2 SH2 domain (15Furet P. Gay B. Caravatti G. Garcia-Echeverria C. Rahuel J. Schoepfer J. Fretz H. J. Med. Chem. 1998; 41: 3442-3449Crossref PubMed Scopus (85) Google Scholar). We have already demonstrated that CGP78850 inhibits Grb2 SH2 domain binding and Ras activation in live cells and prevents anchorage independence of growth (16Gay B. Suarez S. Caravatti G. Furet P. Meyer T. Schoepfer J. Int. J. Cancer. 1999; (in press)PubMed Google Scholar). CGP85793 is a prodrug derivative of CGP78850 with improved cell penetration (16Gay B. Suarez S. Caravatti G. Furet P. Meyer T. Schoepfer J. Int. J. Cancer. 1999; (in press)PubMed Google Scholar).In this study, using both compounds, we explored the possibility that Grb2 may also play a role in cell motility. Significantly, we demonstrate that inhibition of Grb2 SH2 domain prevents HGF/SF-induced A431 and MDCK cell motility and cytoskeletal rearrangements essential for this process. We also establish that EGF can no longer activate Rac in A431 cells.DISCUSSIONA parameter often associated with epithelial invasiveness is motility. Cell motility requires several distinct steps that must occur in a coordinated fashion for cellular translocation to occur. Following the establishment of adhesion to the underlying substratum, the cell must be able to form protrusions, establishing new adhesions, and be able to break older adhesions for translocation to occur (3Stocker M. Gherardi E. Biochim. Biophys. Acta. 1991; 1072: 81-102PubMed Google Scholar). The fact that so many GFs can, at least in some cells, stimulate movement as well as growth suggests that there may be some link in the early stage of signal transduction. Grb2 is a SH2/SH3 domain-containing adaptor protein that links receptor tyrosine kinases to the Ras signaling pathway and mitogenesis (18Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi D. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar). Earlier studies have shown that the injection of an antibody against Grb2 into normal rat kidney-derived fibroblasts had an effect on the cytoskeletal rearrangements induced by EGF and platelet-derived growth factor (14Matuoka K. Shibasaki F. Shibata M. Takenawa T. EMBO J. 1993; 12: 3467-3473Crossref PubMed Scopus (100) Google Scholar), suggesting the possibility that Grb2 functions in pathways involving cell motility.Scattering of MDCK and A431 cells is indeed prevented by CGP78850 or its prodrug derivative CGP85793. Because regulated changes in actin microfilament organization underlie cell motility, we have also monitored early changes in the cytoskeleton of A431 cells. Membrane ruffles and lamellipodia are found at the leading edges of motile cells and are believed to play a fundamental role in migration (28Bussolino F. Di Renzo M.F. Ziche M. Bochietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1195) Google Scholar, 29Small J.V. Electron Microsc. Rev. 1988; 1: 155-174Crossref PubMed Scopus (142) Google Scholar). Membrane ruffling formation is due to extensive actin polymerization. In this respect it is of interest to mention that EGF has been demonstrated as causing actin polymerization (30Rijken P.J. Hage W.J. Van Bergen en Henegowen P.M.P. Verkeij A.J. Boonstra J. J. Cell Sci. 1991; 100: 491-499PubMed Google Scholar). The strong colocalization of Grb2 to membrane ruffles and the observation that Grb2 SH2 inhibitors prevented membrane ruffle formation support a role for Grb2 in cell motility and provide evidence that Grb2 is essential for tyrosine kinase signaling to the cytoskeleton.The GTPase that appears most likely to control cell surface protrusive activity associated with translational locomotion is Rac (21Ridley A.J. Comoglio P.M. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Crossref PubMed Google Scholar). Overexpression of constitutively active Rac promotes the formation of ruffling lamellae in fibroblasts (26Nobes C. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar). In quiescent A431 cells, we observed Rac in the cytosol, correlating with the inactive state and its translocation to the cell periphery to regions of ruffling activity upon stimulation. CGP85793 completely blocked this translocation, indicating that Rac translocation is dependent on Grb2 binding to phosphoproteins. However, the different localization of Grb2 (mostly membranous) and Rac (cytosolic) before stimulation suggests that Grb2 effect on Rac translocation is indirect and may occur through Ras. Moreover, our demonstration that Rac and Grb2 are recruited and colocalize upon stimulation to the sites of ruffling activity suggests that Grb2 may be involved in the two aspects of GF regulation of Rac activity, i.e. not only in the recruitment of Rac from the cytosol to the membrane but also, as for Ras, in its guanine nucleotide exchange activity, possibly, as recently proposed, through homology domain Bar-Sagi D. 1998; PubMed Scopus Google Scholar).We establish for the first time that Grb2 is involved in the regulation of cell motility. The also that, at least in A431 cells, Grb2 plays a role in cell motility-related including Rac regulation and cytoskeletal rearrangements downstream of receptor tyrosine Grb2 SH2 inhibitors are for a new of therapeutic that may control the ability of tumor cells to and Acquisition of cell motility is a prerequisite to biological processes taking place in tissue remodeling. It has been described as a major event in morphogenesis and in pathological situations such as invasion and metastasis of tumor cells (1Thiery J.P. Cell Differ. 1984; 15: 1-15Crossref PubMed Scopus (55) Google Scholar, 2Strauli P. Weiss L. Eur. J. Cancer. 1977; 13: 1-12Abstract Full Text PDF PubMed Scopus (88) Google Scholar). Most cell motility factors are also growth factors (GFs),1 a most intriguing duality of function that implies the possibility of common or related signaling transduction pathways (3Stocker M. Gherardi E. Biochim. Biophys. Acta. 1991; 1072: 81-102PubMed Google Scholar). Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a pleiotropic GF that, besides promoting cell survival and proliferation, has the ability to dissociate epithelial sheets and to stimulate cell motility (3Stocker M. Gherardi E. Biochim. Biophys. Acta. 1991; 1072: 81-102PubMed Google Scholar). HGF/SF predominantly acts as a paracrine factor. Nearly 90% of human malignant tumors arise from epithelial tissue, and most carcinoma cells express the c-Met/HGF receptor and are likely to use HGF/SF produced from stromal tissue (4Matsumoto K. Horikoshi M. Rikimaru K. Enomoto S. J. Oral Pathol. Med. 1989; 18: 498-501Crossref PubMed Scopus (76) Google Scholar, 5Tajima H. Matsumoto K. Nakamura T. Exp. Cell Res. 1992; 202: 423-431Crossref PubMed Scopus (171) Google Scholar). On the other hand, GFs such as the epidermal growth factor (EGF) can enhance motility in an autocrine manner in several tumor cell lines (6Liotta L.A. Mandler R. Murano G. Katz D.A. Gordon R.K. Hing P.K. Shiffmann E. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3302-3306Crossref PubMed Scopus (473) Google Scholar). The EGF receptor (EGFR) has also been extensively studied. GF activation of EGFR influences a number of phenotypic properties in malignant cells in vitro , including mitogenesis and cell motility stimulation (7Lung-Johansen M. Bjerkvig R. Humphrey A.P. Bigner H.S. Bigner D.D. Laerum O. Cancer Res. 1990; 50: 6039-6044PubMed Google Scholar). Interestingly, overexpression of EGFR is correlated more strongly with metastasis and invasion than with tumor size (7Lung-Johansen M. Bjerkvig R. Humphrey A.P. Bigner H.S. Bigner D.D. Laerum O. Cancer Res. 1990; 50: 6039-6044PubMed Google Scholar). factors such as EGF and HGF/SF, initiate a response by binding to receptors and thereby activating a tyrosine kinase domain located in the cytoplasmic portion of the receptor (8Cross M. Dexter T.M. Cell. 1991; 64: 271-280Abstract Full Text PDF PubMed Scopus (608) Google Scholar). This event leads to the autophosphorylation of the receptors on tyrosine residues. The presence of phosphotyrosine at specific sites on receptors is crucial for downstream signaling: within a specific sequence context, phosphotyrosines act as binding sites for many signal-transducing molecules within the cells (9Cantley L.C. Songyang Z. J. Cell Sci. 1994; 18: 121-126Crossref Google Scholar, 10Panayotou G. Waterfield M.D. Bioessays. 1993; 15: 171-177Crossref PubMed Scopus (114) Google Scholar). The common element that confers the specific property of phosphotyrosine-containing sequence recognition to all these target molecules is the Src homology 2 (SH2) domain (11Pawson T. Schlessinger J. Curr. Biol. 1993; 3: 434-442Abstract Full Text PDF PubMed Scopus (574) Google Scholar). Grb2 is an adaptor protein made of one SH2 domain flanked by two SH3 domains. Grb2 interacts via its SH2 domain with activated receptors and via its SH3 domains to the nucleotide exchange factor Sos, which thus becomes activated as a positive regulator of Ras (12Schlessinger J. Curr. Opin. Genet. Dev. 1994; 4: 25-30Crossref PubMed Scopus (400) Google Scholar). Design of molecules that block the interaction between the phosphotyrosine-containing activated receptors and the SH2 domain of Grb2 should interrupt the Ras signaling pathway and may promise therapeutic leads for diseases like cancer, in which the Ras signaling pathway plays a major role (13Sastry L. Cao T. King R. Int. J. Cancer. 1997; 70: 208-213Crossref PubMed Scopus (26) Google Scholar). Grb2 may have other functions as well. Micro-injection studies with anti-Grb2 antibodies in rat kidney cells suggest that Grb2 may play a role in signaling from receptor tyrosine kinases to the small GTPase Rac, a protein involved in motility-related events (14Matuoka K. Shibasaki F. Shibata M. Takenawa T. EMBO J. 1993; 12: 3467-3473Crossref PubMed Scopus (100) Google Scholar). We have reported the rational drug design of CGP78850, a potent and selective inhibitor of Grb2 SH2 domain (15Furet P. Gay B. Caravatti G. Garcia-Echeverria C. Rahuel J. Schoepfer J. Fretz H. J. Med. Chem. 1998; 41: 3442-3449Crossref PubMed Scopus (85) Google Scholar). We have already demonstrated that CGP78850 inhibits Grb2 SH2 domain binding and Ras activation in live cells and prevents anchorage independence of growth (16Gay B. Suarez S. Caravatti G. Furet P. Meyer T. Schoepfer J. Int. J. Cancer. 1999; (in press)PubMed Google Scholar). CGP85793 is a prodrug derivative of CGP78850 with improved cell penetration (16Gay B. Suarez S. Caravatti G. Furet P. Meyer T. Schoepfer J. Int. J. Cancer. 1999; (in press)PubMed Google Scholar). In this study, using both compounds, we explored the possibility that Grb2 may also play a role in cell motility. Significantly, we demonstrate that inhibition of Grb2 SH2 domain prevents HGF/SF-induced A431 and MDCK cell motility and cytoskeletal rearrangements essential for this process. We also establish that EGF can no longer activate Rac in A431 cells. parameter often associated with epithelial invasiveness is motility. Cell motility requires several distinct steps that must occur in a coordinated fashion for cellular translocation to occur. Following the establishment of adhesion to the underlying substratum, the cell must be able to form protrusions, establishing new adhesions, and be able to break older adhesions for translocation to occur (3Stocker M. Gherardi E. Biochim. Biophys. Acta. 1991; 1072: 81-102PubMed Google Scholar). The fact that so many GFs can, at least in some cells, stimulate movement as well as growth suggests that there may be some link in the early stage of signal transduction. Grb2 is a SH2/SH3 domain-containing adaptor protein that links receptor tyrosine kinases to the Ras signaling pathway and mitogenesis (18Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi D. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar). Earlier studies have shown that the injection of an antibody against Grb2 into normal rat kidney-derived fibroblasts had an effect on the cytoskeletal rearrangements induced by EGF and platelet-derived growth factor (14Matuoka K. Shibasaki F. Shibata M. Takenawa T. EMBO J. 1993; 12: 3467-3473Crossref PubMed Scopus (100) Google Scholar), suggesting the possibility that Grb2 functions in pathways involving cell motility.Scattering of MDCK and A431 cells is indeed prevented by CGP78850 or its prodrug derivative CGP85793. Because regulated changes in actin microfilament organization underlie cell motility, we have also monitored early changes in the cytoskeleton of A431 cells. Membrane ruffles and lamellipodia are found at the leading edges of motile cells and are believed to play a fundamental role in migration (28Bussolino F. Di Renzo M.F. Ziche M. Bochietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1195) Google Scholar, 29Small J.V. Electron Microsc. Rev. 1988; 1: 155-174Crossref PubMed Scopus (142) Google Scholar). Membrane ruffling formation is due to extensive actin polymerization. In this respect it is of interest to mention that EGF has been demonstrated as causing actin polymerization (30Rijken P.J. Hage W.J. Van Bergen en Henegowen P.M.P. Verkeij A.J. Boonstra J. J. Cell Sci. 1991; 100: 491-499PubMed Google Scholar). The strong colocalization of Grb2 to membrane ruffles and the observation that Grb2 SH2 inhibitors prevented membrane ruffle formation support a role for Grb2 in cell motility and provide evidence that Grb2 is essential for tyrosine kinase signaling to the cytoskeleton.The GTPase that appears most likely to control cell surface protrusive activity associated with translational locomotion is Rac (21Ridley A.J. Comoglio P.M. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Crossref PubMed Google Scholar). Overexpression of constitutively active Rac promotes the formation of ruffling lamellae in fibroblasts (26Nobes C. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar). In quiescent A431 cells, we observed Rac in the cytosol, correlating with the inactive state and its translocation to the cell periphery to regions of ruffling activity upon stimulation. CGP85793 completely blocked this translocation, indicating that Rac translocation is dependent on Grb2 binding to phosphoproteins. However, the different localization of Grb2 (mostly membranous) and Rac (cytosolic) before stimulation suggests that Grb2 effect on Rac translocation is indirect and may occur through Ras. Moreover, our demonstration that Rac and Grb2 are recruited and colocalize upon stimulation to the sites of ruffling activity suggests that Grb2 may be involved in the two aspects of GF regulation of Rac activity, i.e. not only in the recruitment of Rac from the cytosol to the membrane but also, as for Ras, in its guanine nucleotide exchange activity, possibly, as recently proposed, through homology domain Bar-Sagi D. 1998; PubMed Scopus Google Scholar).We establish for the first time that Grb2 is involved in the regulation of cell motility. The also that, at least in A431 cells, Grb2 plays a role in cell motility-related including Rac regulation and cytoskeletal rearrangements downstream of receptor tyrosine Grb2 SH2 inhibitors are for a new of therapeutic that may control the ability of tumor cells to and parameter often associated with epithelial invasiveness is motility. Cell motility requires several distinct steps that must occur in a coordinated fashion for cellular translocation to occur. Following the establishment of adhesion to the underlying substratum, the cell must be able to form protrusions, establishing new adhesions, and be able to break older adhesions for translocation to occur (3Stocker M. Gherardi E. Biochim. Biophys. Acta. 1991; 1072: 81-102PubMed Google Scholar). The fact that so many GFs can, at least in some cells, stimulate movement as well as growth suggests that there may be some link in the early stage of signal transduction. Grb2 is a SH2/SH3 domain-containing adaptor protein that links receptor tyrosine kinases to the Ras signaling pathway and mitogenesis (18Lowenstein E.J. Daly R.J. Batzer A.G. Li W. Margolis B. Lammers R. Ullrich A. Skolnik E.Y. Bar-Sagi D. Schlessinger J. Cell. 1992; 70: 431-442Abstract Full Text PDF PubMed Scopus (1331) Google Scholar). Earlier studies have shown that the injection of an antibody against Grb2 into normal rat kidney-derived fibroblasts had an effect on the cytoskeletal rearrangements induced by EGF and platelet-derived growth factor (14Matuoka K. Shibasaki F. Shibata M. Takenawa T. EMBO J. 1993; 12: 3467-3473Crossref PubMed Scopus (100) Google Scholar), suggesting the possibility that Grb2 functions in pathways involving cell motility. of MDCK and A431 cells is indeed prevented by CGP78850 or its prodrug derivative CGP85793. Because regulated changes in actin microfilament organization underlie cell motility, we have also monitored early changes in the cytoskeleton of A431 cells. Membrane ruffles and lamellipodia are found at the leading edges of motile cells and are believed to play a fundamental role in migration (28Bussolino F. Di Renzo M.F. Ziche M. Bochietto E. Olivero M. Naldini L. Gaudino G. Tamagnone L. Coffer A. Comoglio P.M. J. Cell Biol. 1992; 119: 629-641Crossref PubMed Scopus (1195) Google Scholar, 29Small J.V. Electron Microsc. Rev. 1988; 1: 155-174Crossref PubMed Scopus (142) Google Scholar). Membrane ruffling formation is due to extensive actin polymerization. In this respect it is of interest to mention that EGF has been demonstrated as causing actin polymerization (30Rijken P.J. Hage W.J. Van Bergen en Henegowen P.M.P. Verkeij A.J. Boonstra J. J. Cell Sci. 1991; 100: 491-499PubMed Google Scholar). The strong colocalization of Grb2 to membrane ruffles and the observation that Grb2 SH2 inhibitors prevented membrane ruffle formation support a role for Grb2 in cell motility and provide evidence that Grb2 is essential for tyrosine kinase signaling to the The GTPase that appears most likely to control cell surface protrusive activity associated with translational locomotion is Rac (21Ridley A.J. Comoglio P.M. Hall A. Mol. Cell. Biol. 1995; 15: 1110-1122Crossref PubMed Google Scholar). Overexpression of constitutively active Rac promotes the formation of ruffling lamellae in fibroblasts (26Nobes C. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3700) Google Scholar). In quiescent A431 cells, we observed Rac in the cytosol, correlating with the inactive state and its translocation to the cell periphery to regions of ruffling activity upon stimulation. CGP85793 completely blocked this translocation, indicating that Rac translocation is dependent on Grb2 binding to phosphoproteins. However, the different localization of Grb2 (mostly membranous) and Rac (cytosolic) before stimulation suggests that Grb2 effect on Rac translocation is indirect and may occur through Ras. Moreover, our demonstration that Rac and Grb2 are recruited and colocalize upon stimulation to the sites of ruffling activity suggests that Grb2 may be involved in the two aspects of GF regulation of Rac activity, i.e. not only in the recruitment of Rac from the cytosol to the membrane but also, as for Ras, in its guanine nucleotide exchange activity, possibly, as recently proposed, through homology domain Bar-Sagi D. 1998; PubMed Scopus Google Scholar). We establish for the first time that Grb2 is involved in the regulation of cell motility. The also that, at least in A431 cells, Grb2 plays a role in cell motility-related including Rac regulation and cytoskeletal rearrangements downstream of receptor tyrosine Grb2 SH2 inhibitors are for a new of therapeutic that may control the ability of tumor cells to and