Abstract
The Tpr-Met oncoprotein consists of the catalytic kinase domain of the hepatocyte growth factor/scatter factor receptor tyrosine kinase (Met) fused downstream from sequences encoded by the tpr gene. Tpr-Met is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement and has constitutive kinase activity. We have previously demonstrated that a single carboxyl-terminal tyrosine residue, Tyr489, is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and forms a multisubstrate binding site for Grb2, phosphatidylinositol 3' kinase, phospholipase Cgamma, SHP2, and an unknown protein of 110 kDa. A mutant Tpr-Met protein that selectively fails to bind Grb2 has reduced transforming activity, implicating pathways downstream of Grb2 in Tpr-Met mediated cell transformation. We show here that the 110-kDa Tpr-Met substrate corresponds to the recently identified Grb2-associated protein, Gab1. Moreover, we show that tyrosine phosphorylation of the Cbl protooncogene product as well as Gab1 required Tyr489 and correlated with the ability of Tpr-Met to associate with Grb2 and to transform cells, providing evidence that pathways downstream of Gab1 and/or Cbl may play a role in Tpr-Met-mediated cell transformation.
Highlights
The receptor for hepatocyte growth factor/scatter factor (HGF/SF),1 the Met receptor tyrosine kinase (RTK), is expressed primarily in epithelial and endothelial cells in vivo and in vitro where it mediates the pleiotropic biological responses of HGF/SF [1,2,3,4,5]
Utilizing carboxyl-terminal tyrosine mutant Tpr-Met proteins, Y482F, Y489F, and Y482F/Y489F, as well as a mutant that has selectively lost the ability to associate with Grb2, N491H, we have demonstrated that pathways downstream of Grb2 and Shc are required for transformation of Fischer rat 3T3 (Fr3T3) cells by Tpr-Met [20]
We have shown here that tyrosine phosphorylation of the Cbl protooncogene product and the multisubstrate docking protein, Gab1, correlates with the ability of the Tpr-Met oncoprotein to associate with the Grb2 adaptor protein and to transform cells
Summary
The receptor for hepatocyte growth factor/scatter factor (HGF/SF), the Met receptor tyrosine kinase (RTK), is expressed primarily in epithelial and endothelial cells in vivo and in vitro where it mediates the pleiotropic biological responses of HGF/SF [1,2,3,4,5]. To define signal transduction pathways required for transformation of fibroblasts by Tpr-Met, we have shown that a single carboxyl-terminal tyrosine residue, Tyr489, is phosphorylated [18] and essential for the association of Tpr-Met with the Grb adaptor protein, phospholipase Cg, and the tyrosine phosphatase, SHP2 [19, 20]. This tyrosine is required for the activation of phosphatidylinositol 39-kinase and the tyrosine phosphorylation of and/or association with an unknown protein of 110 kDa [19]. In the present study we demonstrate that association of Grb with Tpr-Met is essential for the efficient tyrosine phosphorylation of the Cbl protooncogene product, and a previously characterized 110-kDa protein which we show corresponds to the Grb2associated docking protein, Gab
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