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 Shc. Grb2 binding is mediated through a phosphorylated tyrosine residue (Y177) located within a consensus Grb2 binding site encoded by the first exon of the BCR gene. Our results indicate that P160 BCR is tyrosine phosphorylated at the same site by Bcr-Abl in kinase assays (Puil et al., 1994). We performed experiments to determine whether Bcr, which was tyrosine phosphorylated within cells by activated c-Abl, could also bind Grb2, and whether phosphotyrosine 177 was the major binding site. Complexes between Bcr and Abl were detected in a hemopoietic cell line lacking Bcr-Abl and in COS1 cells coexpressing both Bcr and Abl proteins. P160 BCR was tyrosine phosphorylated in COS1 cells coexpressing Abl and Bcr proteins. Similarly, various deletion mutants of Bcr including BCRN553, BCRN413 and BCRN221 were tyrosine phosphorylated by activated c-Abl whereas BCRN159 was not. Wild-type Bcr and Bcr Y177F were examined under these conditions for their ability to co-precipitate with Grb2. The results showed that while wild-type tyrosine phosphorylated Bcr efficiently bound Grb2, tyrosine phosphorylated Bcr Y177F had greatly reduced Grb2-binding ability. Studies with GST-SH2 (Grb2) revealed that tyrosine phosphorylated Bcr was able to bind to GST SH2 (Grb2) but tyrosine phosphorylated Bcr Y177F was deficient in binding. These results indicate that the Bcr protein when phosphorylated at tyrosine 177 binds Grb2, thereby implicating Bcr as a potantial activator of the Ras pathway.
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Department of Molecular Pathology, The University of Texas, M.D. Anderson Cancer Centre, Houston 77030, USA.
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Wu
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Liu
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Arlinghaus
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RB
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D019556:COS Cells; D051380:GRB2 Adaptor Protein; D005982:Glutathione Transferase / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases; D011506:Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D051562:Proto-Oncogene Proteins c-bcr; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D051380:GRB2 Adaptor Protein; D011506:Proteins; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D051562:Proto-Oncogene Proteins c-bcr