Fyn, Yes, and Syk Phosphorylation Sites in c-Cbl Map to the Same Tyrosine Residues That Become Phosphorylated in Activated T Cells*
- From the ‡Department of Microbiology and Immunology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 and the §Department of Pathology, University of Western Australia, Nedlands 6009, Australia
Abstract
Protooncogenic protein c-Cbl undergoes tyrosine phosphorylation in response to stimulation through the receptors for antigens, immunoglobulins, cytokines, and growth factors as well as through the integrins. Tyrosine phosphorylation of c-Cbl may play a functional role in signal transduction, since c-Cbl interacts with many crucial signaling molecules including protein-tyrosine kinases, adaptor proteins, and phosphatidylinositol 3′-kinase. Therefore, it is essential for our understanding of the functions of c-Cbl in signal transduction to identify its tyrosine phosphorylation sites, to determine the protein-tyrosine kinases that phosphorylate these sites, and to elucidate the role of these sites in the interactions of c-Cbl with other signaling proteins. In this report, we demonstrate that tyrosines 700, 731, and 774 are the major tyrosine phosphorylation sites of c-Cbl in T cells in response to pervanadate treatment, as well as in response to TcR/CD3 ligation. Coexpression experiments in COS cells demonstrate that among T cell-expressed Src- and Syk-related protein-tyrosine kinases, Fyn, Yes, and Syk appear to play a major role in phosphorylation of c-Cbl, whereas Lck and Zap phosphorylate c-Cbl ineffectively. Fyn, Yes, and Syk phosphorylate the same sites of c-Cbl that become phosphorylated in stimulated T cells. Among these kinases, Fyn and Yes demonstrate strong binding to c-Cbl, which involves both phosphotyrosine-dependent and phosphotyrosine-independent mechanisms.
Footnotes
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↵* This work was funded by Council for Tobacco Research Award SA048 (to A. Y. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Kresge Bldg., Room 506, Dept. of Microbiology and Immunology, Temple University School of Medicine, 3400 Broad St., Philadelphia, PA 19140. Tel.: 215-707-1745; E-mail: tsygan{at}astro.ocis.temple.edu.
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↵1 The abbreviations used are: PTK, protein-tyrosine kinase; HA, hemagglutinin tag; mAb, monoclonal antibody; Tyr(P), phosphotyrosine; PAGE, polyacrylamide gel electrophoresis; TcR, T cell antigen receptor; SH2, Src homology 2; MOPS, 4-morpholinepropanesulfonic acid.
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↵2 Denoted in the figures as 5Y→F and in the text as Tyr5→Phe.
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- Received October 22, 1997.
- Revision received December 22, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
