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Possible v-Crk-induced transformation through activation of Src kinases
H Sabe1, S E Shoelson, H Hanafusa
1Laboratory of Molecular Oncology, Rockefeller University, New York, New York 10021-6399, USA.
Abstract:
p47gag-crk (v-Crk) encoded by avian sarcoma virus CT10, causes an elevation of tyrosine phosphorylation of several cellular proteins. The lack of a protein-tyrosine kinase domain in v-Crk suggests its co-operation with cellular protein-tyrosine kinase activity. We have shown that suppression of a certain fraction of c-Src activity by Csk may require the binding of Csk to tyrosine-phosphorylated paxillin. In this study, we detected co-immunoprecipitation of tyrosine-phosphorylated paxillin with v-Crk in CT10-transformed chicken embryo fibroblasts (CEF), and demonstrated that v-Crk binding to paxillin can inhibit Csk binding to paxillin. A phosphotyrosine peptide, which can inhibit v-Crk binding to paxillin, did not inhibit Csk binding to paxillin, suggesting that v-Crk and Csk bind to different tyrosine-phosphorylated sites in paxillin. We also found that the kinase activity of the endogenous c-Src in CEF is elevated severalfold after CT10-transformation. We therefore suggest that the competitive binding of overexpressed v-Crk affects an efficient interaction of Csk with tyrosine-phosphorylated paxillin in CT10-transformed CEF. This would result in a failure in the suppression of the kinase activities of a population of c-Src and other Src family protein-tyrosine kinases as well, and these kinases may then contribute to the phosphorylation of cellular proteins in CT10-transformed CEF.
Insights
Avian sarcoma virus CT10
Area of Science:
- Oncology
- Molecular Biology
- Cellular Biology
Background:
- The v-Crk oncoprotein, lacking kinase activity, interacts with cellular tyrosine kinases.
- Cellular Src (c-Src) kinase activity is regulated by C-terminal Src kinase (Csk).
- Paxillin, a focal adhesion protein, is a substrate for tyrosine phosphorylation and binds to Csk.
Purpose of the Study:
- To investigate the interaction between v-Crk, paxillin, and Csk in CT10-transformed cells.
- To determine how v-Crk affects the regulation of c-Src kinase activity.
Main Methods:
- Co-immunoprecipitation assays to detect protein interactions.
- Use of phosphotyrosine peptides to study binding specificities.
- Measurement of endogenous c-Src kinase activity in transformed cells.
Main Results:
- Tyrosine-phosphorylated paxillin co-immunoprecipitated with v-Crk in CT10-transformed chicken embryo fibroblasts (CEF).
- v-Crk binding to paxillin inhibited Csk binding to paxillin, suggesting distinct binding sites.
- Endogenous c-Src kinase activity was significantly elevated in CT10-transformed CEF.
Conclusions:
- Overexpressed v-Crk competitively inhibits Csk binding to tyrosine-phosphorylated paxillin.
- This inhibition leads to impaired suppression of c-Src and other Src family kinase activities.
- Unregulated kinase activity contributes to cellular protein phosphorylation in CT10-transformed CEF.