BCR PHOSPHORYLATED ON TYROSINE-177 BINDS GRB2

We and others have shown that the Bcr-Abl oncoprotein binds activators of the Ras pathway such as Grb2 and She, Grb2 binding is mediated thr ough a phosphorylated tyrosine residue (Y177) located within a consens us Grb2 binding site encoded by the first exon of the BCR gene, Our re sults indicate that P160 BCR is tyrosine phosphorylated at the same si te by Bcr-Abl in kinase assays (Pull ef al., 1994), We performed exper iments to determine whether Bcr, which was tyrosine phosphorylated wit hin cells by activated c-Abl, could also bind Grb2, and whether phosph otyrosine 177 was the major binding site. Complexes between Bcr and Ab l were detected in a hemopoietic cell line lacking Bcr-Abl and in COS1 cells coexpressing both Bcr and Abl proteins, P160 BCR was tyrosine p hosphorylated in COS1 cells coexpressing Abl and Bcr proteins, Similar ly, various deletion mutants of Bcr including BCRN553, BCRN413 and BCR N221 were tyrosine phosphorylated by activated c-Abl whereas BCRN159 w as not, Wild-type Bcr and Bcr Y177F were examined under these conditio ns for their ability to co-precipitate with Grb2. The results showed t hat while wild-type tyrosine phosphorylated Bcr efficiently bound Grb2 , tyrosine phosphorylated Bcr Y177F had greatly reduced Grb2-binding a bility, Studies with GST-SH2 (Grb2) revealed that tyrosine phosphoryla ted Bcr was able to bind to GST SH2 (Grb2) but tyrosine phosphorylated Bcr Y177F was deficient in binding, These results indicate that the B cr protein when phosphorylated at tyrosine 177 binds Grb2, thereby imp licating Bcr as a potantial activator of the Ras pathway.