The Tyrosines in the Bidentate Motif of the env-sea Oncoprotein Are Essential for Cell Transformation and Are Binding Sites for Grb2 and the Tyrosine Phosphatase SHP-2

• Published in: The Journal of biological chemistry 1999-03, Vol.274 (11), p.7583-7590
• Main Authors: Park, Chong Yon, Hayman, Michael J.
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing; Animals; Base Sequence; Binding Sites; Calcium-Calmodulin-Dependent Protein Kinases - metabolism; Cell Line; Cell Transformation, Neoplastic; DNA Primers; Enzyme Activation; GRB2 Adaptor Protein; Intracellular Signaling Peptides and Proteins; Microinjections; Mutagenesis, Site-Directed; Oncogene Proteins, Viral - chemistry; Oncogene Proteins, Viral - genetics; Oncogene Proteins, Viral - metabolism; Phosphatidylinositol 3-Kinases - metabolism; Phosphorylation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases - metabolism; Proteins - metabolism; Rats; Signal Transduction; Tyrosine - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.11.7583

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 so-called bidentate motif (Y557INMAVTY564VNL) 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, 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 env-sea protein. Together, these results suggest that the tyrosines within the bidentate motif are essential for the env-sea transformation.