The Cbl Proto-Oncogene Product Negatively Regulates the Src-Family Tyrosine Kinase Fyn by Enhancing Its Degradation

Abstract

Fyn is a prototype Src-family tyrosine kinase that plays specific roles in neural development, keratinocyte differentiation, and lymphocyte activation, as well as roles redundant with other Src-family kinases. Similar to other Src-family kinases, efficient regulation of Fyn is achieved through intramolecular binding of its SH3 and SH2 domains to conserved regulatory regions. We have investigated the possibility that the tyrosine kinase regulatory protein Cbl provides a complementary mechanism of Fyn regulation. We show that Cbl overexpression in 293T embryonic kidney and Jurkat T-lymphocyte cells led to a dramatic reduction in the active pool of Fyn; this was seen as a reduction in Fyn autophosphorylation, reduced phosphorylation of in vivo substrates, and inhibition of transcription from a Src-family kinase response element linked to a luciferase reporter. Importantly, a Fyn mutant (FynY528F) relieved of intramolecular repression was still negatively regulated by Cbl. The Cbl-dependent negative regulation of Fyn did not appear to be mediated by inhibition of Fyn kinase activity but was correlated with enhanced protein turnover. Consistent with such a mechanism, elevated levels of Fyn protein were observed in cell lines derived from Cbl^−/− mice compared to those in wild-type controls. The effects of Cbl on Fyn were not observed when the 70ZCbl mutant protein was analyzed. Taken together, these observations implicate Cbl as a component in the negative regulation of Fyn and potentially other Src-family kinases, especially following kinase activation. These results also suggest that protein degradation may be a general mechanism for Cbl-mediated negative regulation of activated tyrosine kinases.

ACKNOWLEDGMENTS

This work was supported in part by grants to H.B. from the National Institutes of Health (CA64503, CA75075, and CA76118) and the American Cancer Society (CIM-89513). C.E.A. is a U.S. Department of Defense Breast Cancer Research Program Postdoctoral Fellow, N.L.L. is a recipient of a U.S. Department of Defense Breast Cancer Research Program Career Development Award, and M. L. L. is a Breast Cancer Research Fellow of the Massachusetts Department of Public Health.

We thank R. Perlmutter (Howard Hughes Medical Institute, University of Washington, Seattle) and A. Shaw (Washington University School of Medicine, St. Louis, Mo.) for Fyn constructs, K. Alexandropoulos (Department of Pharmacology, Columbia University, New York, N.Y.) for the SRE-luciferase construct, and B. Druker (Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oreg.) for the 4G10 antibody.