The transforming gene product of the S13 avian erythroblastosis virus, the env-sea protein, is a member of the hepatocyte growth factor receptor family of tyrosine kinases comprising Met, Ron, and Sea. Like all three members of this family, the env-sea protein has a socalled bidentate motif (Y 557 INMAVTY 564 VNL) composed of two tandemly arranged tyrosines in the carboxyl terminus. To investigate whether the tyrosine residues in this motif are essential for the env-sea-mediated transformation,

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... we generated tyrosine to phenylalanine mutations. Substitutions of both tyrosine residues resulted in complete loss of the transforming activity. In contrast, single mutations at either tyrosine did not inhibit transformation of Rat1 cells, and mutation of tyrosine 564 actually increased transformation of Rat 1 cells. To define signaling pathways activated by the env-sea protein, we looked for protein-protein interactions mediated by these tyrosine residues. We show that the bidentate motif is responsible for interaction with the adapter protein Grb2, phosphatidylinositol 3-kinase, and the tyrosine phosphatase SHP-2. Furthermore, we show that microinjected Src homology 2 domains from either Grb2 or SHP-2 blocked the transforming activity of the envsea protein. Together, these results suggest that the tyrosines within the bidentate motif are essential for the env-sea transformation. EXPERIMENTAL PROCEDURES Reagents, Cells, and Antibodies-All reagents, unless specified, were purchased from Sigma. All cell lines were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. S13 Rat1 cells are Rat1 fibroblasts expressing a temperature-sensitive env-sea protein (11). S13 Rat1 cells were grown at 35 and 39.5°C with 7% CO 2 . Rat1 and COS1 cells were grown at 37°C with 7% CO 2 .