Src Phosphorylation of the Epidermal Growth Factor Receptor at Novel Sites Mediates Receptor Interaction with Src and P85α

Abstract

Following ligand binding, the epidermal growth factor receptor (EGF-R) autophosphorylates itself on tyrosine residues located in its carboxyl terminus; in vitro, three sites are highly phosphorylated, while two other sites are phosphorylated to lesser extents. In the presence of the Src protein-tyrosine kinase, in vitro phosphorylation of the minor autophosphorylation sites was increased, and four additional residues were phosphorylated. Following EGF stimulation, two (Tyr-891 and Tyr-920) were found to be phosphorylated in a colorectal cell line (DLD-1) and in a breast tumor cell line (MCF7). The remaining in vitro sites were not found to be highly phosphorylated in vivo. The sequences surrounding Tyr-891 and Tyr-920 match the reported consensus binding sequences for the SH2 domains of Src and the regulatory domain of phosphatidylinositol 3-kinase (p85α), respectively. In vitro, both of these proteins were found to bind to Src-phosphorylated EGF-R with ∼100-fold greater affinity than to autophosphorylated EGF-R, demonstrating that Src creates new sites for SH2 binding. Furthermore, Csk-inactivated Src was activated by interaction with Src-phosphorylated EGF-R but not by autophosphorylated EGF-R. Upon EGF treatment of MCF7 or three colorectal carcinoma cell lines (WiDr, DLD-1, and LS174T), the EGF-R coimmunoprecipitated with both p85α and Src. Evidence is also presented that suggests that an EGF-R-related protein, ErbB2, may be involved in similar Src-mediated interactions. These data demonstrate that EGF-R is phosphorylated in vivo at non-autophosphorylation sites and that these novel sites can act as docking sites for Src, P85α, and potentially other SH2-containing proteins. In addition, the data suggest a tyrosine phosphatase-independent mechanism for the elevation of Src activity in cells exposed to growth factors. Overexpression of Src, EGF-R, and/or ErbB2 in breast and colorectal tumor cells suggests the potential that such interactions may contribute to the transformed phenotype of these carcinomas. Following ligand binding, the epidermal growth factor receptor (EGF-R) autophosphorylates itself on tyrosine residues located in its carboxyl terminus; in vitro, three sites are highly phosphorylated, while two other sites are phosphorylated to lesser extents. In the presence of the Src protein-tyrosine kinase, in vitro phosphorylation of the minor autophosphorylation sites was increased, and four additional residues were phosphorylated. Following EGF stimulation, two (Tyr-891 and Tyr-920) were found to be phosphorylated in a colorectal cell line (DLD-1) and in a breast tumor cell line (MCF7). The remaining in vitro sites were not found to be highly phosphorylated in vivo. The sequences surrounding Tyr-891 and Tyr-920 match the reported consensus binding sequences for the SH2 domains of Src and the regulatory domain of phosphatidylinositol 3-kinase (p85α), respectively. In vitro, both of these proteins were found to bind to Src-phosphorylated EGF-R with ∼100-fold greater affinity than to autophosphorylated EGF-R, demonstrating that Src creates new sites for SH2 binding. Furthermore, Csk-inactivated Src was activated by interaction with Src-phosphorylated EGF-R but not by autophosphorylated EGF-R. Upon EGF treatment of MCF7 or three colorectal carcinoma cell lines (WiDr, DLD-1, and LS174T), the EGF-R coimmunoprecipitated with both p85α and Src. Evidence is also presented that suggests that an EGF-R-related protein, ErbB2, may be involved in similar Src-mediated interactions. These data demonstrate that EGF-R is phosphorylated in vivo at non-autophosphorylation sites and that these novel sites can act as docking sites for Src, P85α, and potentially other SH2-containing proteins. In addition, the data suggest a tyrosine phosphatase-independent mechanism for the elevation of Src activity in cells exposed to growth factors. Overexpression of Src, EGF-R, and/or ErbB2 in breast and colorectal tumor cells suggests the potential that such interactions may contribute to the transformed phenotype of these carcinomas. INTRODUCTIONThe cellular events that result from extracellular signaling by growth factors are initiated primarily by members of the transmembrane protein-tyrosine kinase family (for review, see Ullrich and Schlessinger, 1990Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4583) Google Scholar). Typically, these receptor kinases dimerize upon binding ligand, which results in enzyme activation and the subsequent inter- and/or intra-molecular autophosphorylation on tyrosine residues. These receptor autophosphorylation sites act as docking sites for a wide range of regulatory molecules, each of which contain at least one Src homology 2 (SH2) domain that mediates interaction with the receptor (for reviews, see Koch et al., 1991Koch A.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1429) Google Scholar and Cantley et al., 1991Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2177) Google Scholar). Facilitation of substrate phosphorylation, regulation of enzymatic activity, and alterations in subcellular localization are three documented effects of SH2-mediated docking (Sugimoto et al., 1994Sugimoto S. Wandless T.J. Shoelson S.E. Neel B.G. Walsh C.T. J. Biol. Chem. 1994; 269: 13614-13622Abstract Full Text PDF PubMed Google Scholar; Uchida et al., 1994Uchida T. Matozaki T. Noguchi T. Yamao T. Horita K. Suzuki T. Fujioka Y. Sakamoto C. Kasuga M. J. Biol. Chem. 1994; 269: 12220-12228Abstract Full Text PDF PubMed Google Scholar; Moran et al., 1991Moran M.F. Polakis P. McCormick F. Pawson T. Ellis C. Mol. Cell. Biol. 1991; 11: 1804-1812Crossref PubMed Scopus (209) Google Scholar; Vega et al., 1992Vega Q.C. Cochet C. Filhol O. Chang C. Rhee S.G. Gill G.N. Mol. Cell. Biol. 1992; 12: 128-135Crossref PubMed Scopus (79) Google Scholar; and Pronk et al., 1993Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar).Five sites of autophosphorylation have been identified in the EGF-R,1( 1The abbreviations used are: EGF-Repidermal growth factor receptorRPTKreceptor protein-tyrosine kinasePDGF-Rplatelet-derived growth factor receptorpTyrphosphotyrosine. )three major (Tyr-1068, Tyr-1148, and Tyr-1173) and two minor (Tyr-992 and Tyr-1086) (Downward et al., 1984Downward J. Parker P. Waterfield M.D. Nature. 1984; 311: 283-285Crossref Scopus (465) Google Scholar; Hsuan et al., 1989Hsuan J.J. Totty N. Waterfield M.D. Biochem. J. 1989; 262: 659-663Crossref PubMed Scopus (49) Google Scholar; Margolis et al., 1989Margolis B.L. Lax I. Kris R. Dombalagain M. Honegger A.M. Howk R. Givol D. Ullrich A. Schlessinger J. J. Biol. Chem. 1989; 264: 10667-10671Abstract Full Text PDF PubMed Google Scholar; and Walton et al., 1990Walton G.M. Chen W.S. Rosenfeld M.G. Gill G.N. J Biol. Chem. 1990; 265: 1750-1754Abstract Full Text PDF PubMed Google Scholar). Several SH2-containing proteins have been demonstrated to bind to one or more of these sites both in vitro and in vivo (Grb2, phospholipase C-γ, SHC, Syp, and GTPase-activating protein) (Batzer et al., 1994Batzer A.G. Rotin D. Urena J.M. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar; Margolis et al., 1990Margolis B. Li N. Koch A. Mohammadi M. Hurwitz D.R. Zilberstein A. Ullrich A. Pawson T. Schlessinger J. EMBO J. 1990; 9: 4375-4380Crossref PubMed Scopus (215) Google Scholar; Rotin et al., 1992Rotin D. Margolis B. Mohammadi M. Daly R.J. Daum G. Li N. Fischer E.H. Burgess W.H. Ullrich A. Schlessinger J. EMBO J. 1992; 11: 559-567Crossref PubMed Scopus (250) Google Scholar; Feng et al., 1994Feng G.S. Shen R. Heng H.H. Tsui L.C. Kazlauskas A. Pawson T. Oncogene. 1994; 9: 1545-1550PubMed Google Scholar; Xiao et al., 1994Xiao S. Roses D.W. Sasaoka T. Maegawa H. Burke Jr., T.R. Roller P.P. Shoelson S.E. Olefsky J.M. J. Biol. Chem. 1994; 269: 21244-21248Abstract Full Text PDF PubMed Google Scholar). Recently, an unprecedented collaboration of laboratories has undertaken a thorough survey of the consensus binding sequences for many of the SH2 domains (Songyang et al., 1993Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar, Songyang et al., 1994Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar). They have found that the SH2 domains can generally be placed into families of related binding specificities, with the three residues on the carboxy side of the phosphotyrosine having the greatest influence on affinity. However, binding of SH2-containing proteins to whole proteins appears to be less predictable than the interactions between isolated SH2 domains and simple peptides (Soler et al., 1994Soler C. Beguinot L. Carpenter G. J. Biol. Chem. 1994; 269: 12320-12324Abstract Full Text PDF PubMed Google Scholar).Other proteins have been reported to bind to the EGF-R, but the site or sites of binding have not been identified. Recently, the prototype SH2-containing protein, pp60c-src (Src) was reported to associate with the EGF-R (also known as ErbB1) and a related protein, ErbB2, in a breast tumor cell line (Luttrell et al., 1994Luttrell D.K. Lee A. Lansing T.J. Crosby R.M. Jung K.D. Willard D. Luther M. Rodriguez M. Berman J. Gilmer T.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 83-87Crossref PubMed Scopus (264) Google Scholar). This correlates with evidence that overexpression of Src enhances the mitogenic response of cells to EGF (Chang et al., 1993Chang J.H. Wilson L.K. Moyers J.S. Zhang K. Parson S.J. Oncogene. 1993; 8: 959-967PubMed Google Scholar). However, none of the autophosphorylation sites in the EGF-R are similar to the reported consensus binding sequence for Src-SH2 (pTyr-acidic-acidic-hydrophobic) (Songyang et al., 1993Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar). Similarly, the regulatory domain of phosphatidylinositol 3-kinase (P85α) has also been reported to bind EGF-R, but none of the autophosphorylated sites match its consensus sequence either (pYXXM) (Songyang et al., 1993Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar; Hu et al., 1992Hu P. Margolis B. Skolnik E.Y. Lammers R. Ullrich A. Schlessinger J. Mol. Cell. Biol. 1992; 12: 981-990Crossref PubMed Scopus (246) Google Scholar; McGlade et al., 1992McGlade C.J. Ellis C. Reedijk M. Anderson D. Mbamalu G. Reith A.D. Panayotou G. End P. Bernstein A. Kazlauskas A. Waterfield M.D. Pawson T. Mol. Cell. Biol. 1992; 12: 991-997Crossref PubMed Google Scholar). There would seem to be three possible explanations for these findings: 1) these observed bindings are artifacts and are not physiologically important, 2) consensus sequences based on peptides are not reliable predictors of protein binding or, 3) there may be other tyrosine residues within the EGF-R that are phosphorylated and can bind SH2-containing not the that the two explanations may be for of the in the in evidence that Src can the EGF-R on novel sites in vitro, that Src and associate with the EGF-R has been phosphorylated by Src, that these Src-phosphorylated sites consensus binding sites for Src and are phosphorylated in cells in response to that and Src bind to the EGF-R in these cells in an and that Csk-inactivated Src is upon binding to the Src phosphorylated EGF-R. In addition, ErbB2, a related receptor kinase, is also phosphorylated by Src in The and Src consensus binding sites found in the EGF-R are highly in ErbB2, similar SH2 interactions may with the EGF-R growth factor receptor This evidence suggests the possible of these interactions in breast and colorectal EGF receptor and ErbB2 have each been demonstrated to sites in vitro to these sites in response to ligand stimulation, that autophosphorylation also in vivo. In demonstrate that a tyrosine kinase, Src, is of each of these to a In vitro, Src of the EGF-R autophosphorylation sites an additional tyrosine residues. of these additional sites (Tyr-891 and Tyr-920) were observed to be phosphorylated in a colorectal carcinoma cell line (DLD-1) and in a breast tumor cell line In cells that EGF-R autophosphorylation may these additional sites have been in the that have in vivo phosphotyrosine EGF-R is in of the carcinoma cell lines used in Src is also highly et al., I. K. A. H. H. 1990; Scopus Google Scholar). The of the overexpression of Src with phosphorylation of these sites that Src may be the kinase for This is by the that phosphorylation of these new sites is observed Src is found to associate with the et al., Mol. Cell. Biol. 1991; 11: PubMed Scopus Google Scholar have demonstrated that in cells transformed by the EGF-R is phosphorylated at Furthermore, phospholipase is also phosphorylated on tyrosine in these phospholipase is an in vivo substrate of the EGF-R kinase, the that the activity of EGF-R been activated by However, in phosphorylation of EGF-R by Src has on EGF-R activity in vitro not suggest that the phosphorylation of phospholipase in cells may be to phosphorylation by Src or phospholipase may be of binding to one of the sites of phosphorylation by a substrate for EGF-R in the of and has been to bind to a minor autophosphorylated In site was a minor on an However, Src phosphorylation, site was as highly phosphorylated as the major sites of with of EGF-R phosphorylation in vivo EGF These data suggest that phospholipase may to a site is phosphorylated by Src or kinase in vivo is not Src and have been found to bind to the EGF-R in and results evidence that is the in the cell in However, none of the autophosphorylation sites of EGF-R the consensus binding sites for either Src or the SH2 and binding of these proteins to the autophosphorylated EGF-R. would that is more than an of the three sites observed to be phosphorylated in one is a of a binding site while the binding of Src-SH2 Furthermore, the sequences surrounding these two sites are more between EGF-R and ErbB2 than are of the major autophosphorylation these a for a for Src phosphorylation of these tyrosine has been reported that the a EGF-R has its known autophosphorylation sites can and However, each of the autophosphorylation sites are to the EGF-R is to a is a was that more of its may suggest an for the three tyrosine residues phosphorylated by Src in the carboxyl of the kinase domain are to these effects of However, Src may not be to these sites the EGF-R has been autophosphorylated the may and these a for which there is evidence et al., G.N. Chen W.S. Rosenfeld M.G. Biol. PubMed Google the autophosphorylation sites are the be the remaining sites are from phosphorylated. the other would the these sites for phosphorylation by Src, and signaling from SH2 proteins that with these sites et al., Proc. Natl. Sci. U. S. A. 1994; 91: PubMed Scopus (49) Google Scholar have reported that a kinase of EGF-R the to upon EGF This kinase was phosphorylated on tyrosine in response to EGF by the as an tyrosine there is evidence that additional tyrosine kinases in the response to growth factors such as signaling has been demonstrated to be by overexpression of Src and may the activation of Src (Luttrell et al., D.K. S.J. Mol. Biol. 8: PubMed Scopus Google Scholar). that Src creates additional SH2 docking sites on EGF-R and that one or more of these is a docking site for Src suggests an of these effects may of Src has been in but has also been observed that Src phosphorylated in response to EGF The of Src in EGF signaling is not to the of additional SH2 docking sites on the receptor and is involved in the phosphorylation of regulatory is also possible that of the proteins that have been to be of EGF-R may be phosphorylated by Src to the EGF-R. Src has been demonstrated to associate with EGF-R, ErbB2, and the growth factor receptor et al., M. Honegger A.M. Rotin D. Fischer R. F. Li M. M. Schlessinger J. Mol. Biol. 1991; 11: PubMed Google Scholar; et al., 1994Luttrell D.K. Lee A. Lansing T.J. Crosby R.M. Jung K.D. Willard D. Luther M. Rodriguez M. Berman J. Gilmer T.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 83-87Crossref PubMed Scopus (264) Google Scholar; et al., R. D. M. Waterfield M.D. M.F. EMBO J. 1993; 12: PubMed Scopus Google Scholar; et al., C. Hu R. T. J. Chem. 1994; 269: Full Text PDF PubMed Google Scholar). These interactions the that Src is involved with many growth factor signaling Furthermore, with of the within the et al., P. C. R. A. Cell. 1991; 64: Full Text PDF PubMed Scopus Google Scholar). not have in the or in However, are in Src is for the of is not for cell that its is or that that would cell in the of EGF is not the growth factor that to Src in its of cells results in activation and phosphorylation of Src, and the with Src a autophosphorylated ErbB2 has been reported to associate with Src in tumor cells (Luttrell et al., 1994Luttrell D.K. Lee A. Lansing T.J. Crosby R.M. Jung K.D. Willard D. Luther M. Rodriguez M. Berman J. Gilmer T.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 83-87Crossref PubMed Scopus (264) Google Scholar). that the EGF-R and ErbB2 are also phosphorylated by Src on non-autophosphorylation and is these additional sites that are for Src binding. Src may be an of many growth factor signaling This is with the of Src in cell evidence that these in at least tumor cell is contribute in to or are involved in EGF in with EGF-R the sites phosphorylated by Src, to the of these sites in EGF INTRODUCTIONThe cellular events that result from extracellular signaling by growth factors are initiated primarily by members of the transmembrane protein-tyrosine kinase family (for review, see Ullrich and Schlessinger, 1990Ullrich A. Schlessinger J. Cell. 1990; 61: 203-212Abstract Full Text PDF PubMed Scopus (4583) Google Scholar). Typically, these receptor kinases dimerize upon binding ligand, which results in enzyme activation and the subsequent inter- and/or intra-molecular autophosphorylation on tyrosine residues. These receptor autophosphorylation sites act as docking sites for a wide range of regulatory molecules, each of which contain at least one Src homology 2 (SH2) domain that mediates interaction with the receptor (for reviews, see Koch et al., 1991Koch A.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1429) Google Scholar and Cantley et al., 1991Cantley L.C. Auger K.R. Carpenter C. Duckworth B. Graziani A. Kapeller R. Soltoff S. Cell. 1991; 64: 281-302Abstract Full Text PDF PubMed Scopus (2177) Google Scholar). Facilitation of substrate phosphorylation, regulation of enzymatic activity, and alterations in subcellular localization are three documented effects of SH2-mediated docking (Sugimoto et al., 1994Sugimoto S. Wandless T.J. Shoelson S.E. Neel B.G. Walsh C.T. J. Biol. Chem. 1994; 269: 13614-13622Abstract Full Text PDF PubMed Google Scholar; Uchida et al., 1994Uchida T. Matozaki T. Noguchi T. Yamao T. Horita K. Suzuki T. Fujioka Y. Sakamoto C. Kasuga M. J. Biol. Chem. 1994; 269: 12220-12228Abstract Full Text PDF PubMed Google Scholar; Moran et al., 1991Moran M.F. Polakis P. McCormick F. Pawson T. Ellis C. Mol. Cell. Biol. 1991; 11: 1804-1812Crossref PubMed Scopus (209) Google Scholar; Vega et al., 1992Vega Q.C. Cochet C. Filhol O. Chang C. Rhee S.G. Gill G.N. Mol. Cell. Biol. 1992; 12: 128-135Crossref PubMed Scopus (79) Google Scholar; and Pronk et al., 1993Pronk G.J. McGlade J. Pelicci G. Pawson T. Bos J.L. J. Biol. Chem. 1993; 268: 5748-5753Abstract Full Text PDF PubMed Google Scholar).Five sites of autophosphorylation have been identified in the EGF-R,1( 1The abbreviations used are: EGF-Repidermal growth factor receptorRPTKreceptor protein-tyrosine kinasePDGF-Rplatelet-derived growth factor receptorpTyrphosphotyrosine. )three major (Tyr-1068, Tyr-1148, and Tyr-1173) and two minor (Tyr-992 and Tyr-1086) (Downward et al., 1984Downward J. Parker P. Waterfield M.D. Nature. 1984; 311: 283-285Crossref Scopus (465) Google Scholar; Hsuan et al., 1989Hsuan J.J. Totty N. Waterfield M.D. Biochem. J. 1989; 262: 659-663Crossref PubMed Scopus (49) Google Scholar; Margolis et al., 1989Margolis B.L. Lax I. Kris R. Dombalagain M. Honegger A.M. Howk R. Givol D. Ullrich A. Schlessinger J. J. Biol. Chem. 1989; 264: 10667-10671Abstract Full Text PDF PubMed Google Scholar; and Walton et al., 1990Walton G.M. Chen W.S. Rosenfeld M.G. Gill G.N. J Biol. Chem. 1990; 265: 1750-1754Abstract Full Text PDF PubMed Google Scholar). Several SH2-containing proteins have been demonstrated to bind to one or more of these sites both in vitro and in vivo (Grb2, phospholipase C-γ, SHC, Syp, and GTPase-activating protein) (Batzer et al., 1994Batzer A.G. Rotin D. Urena J.M. Skolnik E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar; Margolis et al., 1990Margolis B. Li N. Koch A. Mohammadi M. Hurwitz D.R. Zilberstein A. Ullrich A. Pawson T. Schlessinger J. EMBO J. 1990; 9: 4375-4380Crossref PubMed Scopus (215) Google Scholar; Rotin et al., 1992Rotin D. Margolis B. Mohammadi M. Daly R.J. Daum G. Li N. Fischer E.H. Burgess W.H. Ullrich A. Schlessinger J. EMBO J. 1992; 11: 559-567Crossref PubMed Scopus (250) Google Scholar; Feng et al., 1994Feng G.S. Shen R. Heng H.H. Tsui L.C. Kazlauskas A. Pawson T. Oncogene. 1994; 9: 1545-1550PubMed Google Scholar; Xiao et al., 1994Xiao S. Roses D.W. Sasaoka T. Maegawa H. Burke Jr., T.R. Roller P.P. Shoelson S.E. Olefsky J.M. J. Biol. Chem. 1994; 269: 21244-21248Abstract Full Text PDF PubMed Google Scholar). Recently, an unprecedented collaboration of laboratories has undertaken a thorough survey of the consensus binding sequences for many of the SH2 domains (Songyang et al., 1993Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar, Songyang et al., 1994Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky S. Feldman R.A. Cantley L.C. Mol Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar). They have found that the SH2 domains can generally be placed into families of related binding specificities, with the three residues on the carboxy side of the phosphotyrosine having the greatest influence on affinity. However, binding of SH2-containing proteins to whole proteins appears to be less predictable than the interactions between isolated SH2 domains and simple peptides (Soler et al., 1994Soler C. Beguinot L. Carpenter G. J. Biol. Chem. 1994; 269: 12320-12324Abstract Full Text PDF PubMed Google Scholar).Other proteins have been reported to bind to the EGF-R, but the site or sites of binding have not been identified. Recently, the prototype SH2-containing protein, pp60c-src (Src) was reported to associate with the EGF-R (also known as ErbB1) and a related protein, ErbB2, in a breast tumor cell line (Luttrell et al., 1994Luttrell D.K. Lee A. Lansing T.J. Crosby R.M. Jung K.D. Willard D. Luther M. Rodriguez M. Berman J. Gilmer T.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 83-87Crossref PubMed Scopus (264) Google Scholar). This correlates with evidence that overexpression of Src enhances the mitogenic response of cells to EGF (Chang et al., 1993Chang J.H. Wilson L.K. Moyers J.S. Zhang K. Parson S.J. Oncogene. 1993; 8: 959-967PubMed Google Scholar). However, none of the autophosphorylation sites in the EGF-R are similar to the reported consensus binding sequence for Src-SH2 (pTyr-acidic-acidic-hydrophobic) (Songyang et al., 1993Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar). Similarly, the regulatory domain of phosphatidylinositol 3-kinase (P85α) has also been reported to bind EGF-R, but none of the autophosphorylated sites match its consensus sequence either (pYXXM) (Songyang et al., 1993Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W.G. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2373) Google Scholar; Hu et al., 1992Hu P. Margolis B. Skolnik E.Y. Lammers R. Ullrich A. Schlessinger J. Mol. Cell. Biol. 1992; 12: 981-990Crossref PubMed Scopus (246) Google Scholar; McGlade et al., 1992McGlade C.J. Ellis C. Reedijk M. Anderson D. Mbamalu G. Reith A.D. Panayotou G. End P. Bernstein A. Kazlauskas A. Waterfield M.D. Pawson T. Mol. Cell. Biol. 1992; 12: 991-997Crossref PubMed Google Scholar). There would seem to be three possible explanations for these findings: 1) these observed bindings are artifacts and are not physiologically important, 2) consensus sequences based on peptides are not reliable predictors of protein binding or, 3) there may be other tyrosine residues within the EGF-R that are phosphorylated and can bind SH2-containing not the that the two explanations may be for of the in the in evidence that Src can the EGF-R on novel sites in vitro, that Src and associate with the EGF-R has been phosphorylated by Src, that these Src-phosphorylated sites consensus binding sites for Src and are phosphorylated in cells in response to that and Src bind to the EGF-R in these cells in an and that Csk-inactivated Src is upon binding to the Src phosphorylated EGF-R. In addition, ErbB2, a related receptor kinase, is also phosphorylated by Src in The and Src consensus binding sites found in the EGF-R are highly in ErbB2, similar SH2 interactions may with the EGF-R growth factor receptor This evidence suggests the possible of these interactions in breast and colorectal carcinomas.