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

Abstract

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, 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.A large number of oncogenes encode altered forms of growth factor receptor-tyrosine kinases (1, 2). Oncogenes derived from cellular receptor-tyrosine kinases (RTKs) 1 are constitutively activated by structural changes, such as point mutation, truncation, and rearrangement (3-6). The activation of RTKs triggers autophosphorylation of tyrosine residues, which then serve as specific binding sites for cellular signaling proteins containing Src-homology 2 (SH2) or phosphotyrosine binding domains (7-9). Individual SH2 domains recognize specific phosphotyrosine residues flanked by distinct sequences (10). Signal transduction by RTKs involves activation of multiple pathways following association with different cellular target proteins.The env-sea oncogene is derived from a cellular proteintyrosine kinase, c-sea, the extracellular and transmembrane domains of which have been replaced by viral envelope sequences (11). Due to the fusion with viral envelope domain, the env-sea protein has a structure similar to RTKs (12). Previous studies showed that the env-sea tyrosine kinase activity is necessary for transformation by the env-sea protein (13) and that cell surface localization, oligomerization by viral envelope domains, and autophosphorylation are all necessary for the transforming activity of the env-sea protein (14 -16). Using a temperature-sensitive mutant, previous work demonstrated that the phosphorylation of the Shc proteins correlated with the activation of the tyrosine kinase activity of the env-sea protein (17). However, Shc did not form a stable complex with the env-sea protein. In contrast, the Grb2 protein did associate with the env-sea pro...

GRB2-mediated recruitment of GAB2, but not GAB1, to SF-STK supports the expansion of Friend virus-infected erythroid progenitor cells

“…All Gab family members contain multiple p85-binding sites that become phosphorylated upon receptor activation (Liu and Rohrschneider, 2002), and we demonstrate here that the p85-binding sites in Gab2 are required for efficient cytokine-independent growth of Friend virus-infected erythroblasts. The ability of Gab proteins to amplify Sf-Stk signaling through the activation of PI3K is consistent with previous findings from our laboratory that demonstrate a requirement for PI3K activation in the transformation of cells by FV (Park and Hayman, 1999;Finkelstein et al, 2002). Our data also extend previous studies demonstrating constitutive tyrosine phosphorylation of Gab2 and its association with p85 in FV-infected cells grown in the absence of Epo (Lecoq-Lafon et al, 1999;Wickrema et al, 1999;Nishigaki et al, 2000).…”

“…In these cells, PI3K is constitutively active, and PI3K activity, but not EpoR phosphorylation, is required for the proliferation of these cells in the absence of Epo (Nishigaki et al, 2000). Furthermore, v-sea, which induces erythroleukemia in chickens, preferentially interacts with Gab2 and is essential for cellular transformation induced by v-sea (Ischenko et al, 2003), and mutagenesis studies revealed a critical role in v-sea-induced transformation for PI3K activation (Park and Hayman, 1999).…”

The C Terminus of RON Tyrosine Kinase Plays an Autoinhibitory Role

“…Mutation of these two tyrosines (Y1353F/Y1360F) suppressed the transforming ability of activated forms of RON (15). A similar mutation caused a complete loss of transforming ability of the related SEA kinase (22). These results could be the result of the inability of the double mutant to engage SH2 domain-containing downstream signaling proteins.…”

The Tyrosine Phosphatase SHP-2 Is Required for Sustained Activation of Extracellular Signal-Regulated Kinase and Epithelial Morphogenesis Downstream from the Met Receptor Tyrosine Kinase

“…Two tyrosine residues within the carboxyl terminus of Met (Y1349 and Y1356), which are highly conserved between the other members of the Met receptor tyrosine kinase gene family, Sea and Ron, are crucial for cell scatter and epithelial morphogenesis in Madin-Darby canine kidney (MDCK) epithelial cells (29,48,58,74,83). Y1356 forms a multisubstrate-binding site, coupling the Met receptor with the Grb2 and Shc adapter proteins, as well as the Cbl proto-oncogene and the Grb2-associated binder 1 (Gab1) (10-12, 44, 51, 73).…”