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Pathways downstream of Shc and Grb2 are required for cell transformation by the tpr-Met oncoprotein
E D Fixman1, T M Fournier, D M Kamikura
1Molecular Oncology Group, Royal Victoria Hospital, Department of Medicine, McGill University, Montreal, Quebec, H3A 1A1 Canada.
Abstract:
The Tpr-Met oncoprotein, which is a member of a family of tyrosine kinase oncoproteins generated following genomic rearrangement, 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. We have previously demonstrated that a single tyrosine residue in the carboxyl terminus, Tyr489, is highly phosphorylated and is essential for efficient transformation of Fr3T3 fibroblasts by Tpr-Met and for the association of Tpr-Met with the Grb2 adaptor protein and phosphatidylinositol 3'-kinase. We show here that Tyr489 is also required for association of Tpr-Met with phospholipase Cgamma and the tyrosine phosphatase, SHPTP2/Syp. To distinguish which of these substrates are required for cell transformation by the Tpr-Met oncoprotein, we generated a novel Tpr-Met mutant that selectively fails to associate with the Grb2 adaptor protein. Utilizing this mutant, together with additional Tpr-Met mutants containing Tyr to Phe substitutions, we have demonstrated that transformation of Fr3T3 fibroblasts by the Tpr-Met oncoprotein is dependent upon pathways downstream of Shc and Grb2 and that pathways downstream of phosphatidylinositol 3'-kinase, phospholipase Cgamma, and SHPTP2/Syp are insufficient for transformation.
Insights
Transformation by the Tpr-Met oncoprotein depends on the Shc and Grb2 adaptor proteins. Other downstream pathways, including phosphatidylinositol 3'-kinase, are insufficient for Tpr-Met-driven cell transformation.
Area of Science:
- Oncology
- Molecular Biology
- Cell Biology
Background:
- The Tpr-Met oncoprotein is a fusion protein involving the Met receptor tyrosine kinase.
- A key tyrosine residue, Tyr489, is crucial for Tpr-Met's association with signaling molecules and cell transformation.
- Previous studies highlighted Tyr489's role in Tpr-Met's interaction with Grb2 and phosphatidylinositol 3'-kinase.
Purpose of the Study:
- To determine which downstream pathways activated by Tpr-Met are essential for fibroblast transformation.
- To investigate the specific roles of various Tpr-Met substrates, including Grb2, phospholipase Cgamma, and SHPTP2/Syp, in cell transformation.
Main Methods:
- Generation of a novel Tpr-Met mutant unable to associate with Grb2.
- Utilizing Tpr-Met mutants with tyrosine-to-phenylalanine substitutions at key sites.
- Assessing the transformation of Fr3T3 fibroblasts by different Tpr-Met mutants.
Main Results:
- The Tpr-Met oncoprotein requires Tyr489 for association with phospholipase Cgamma and SHPTP2/Syp, in addition to Grb2 and phosphatidylinositol 3'-kinase.
- A Tpr-Met mutant selectively impaired in Grb2 association demonstrated that transformation is dependent on pathways downstream of Shc and Grb2.
- Pathways involving phosphatidylinositol 3'-kinase, phospholipase Cgamma, and SHPTP2/Syp were found to be insufficient for Tpr-Met-mediated cell transformation.
Conclusions:
- Cell transformation induced by the Tpr-Met oncoprotein critically relies on signaling cascades initiated by Shc and Grb2.
- Downstream effectors such as phosphatidylinositol 3'-kinase, phospholipase Cgamma, and SHPTP2/Syp are not sufficient to drive Tpr-Met-mediated transformation.
- These findings elucidate the specific signaling requirements for Tpr-Met oncoprotein-induced cell transformation.