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J. Biol. Chem., Vol. 279, Issue 34, 36132-36141, August 20, 2004

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Biochemical Basis for the Requirement of Kinase Activity for Cbl-dependent Ubiquitinylation and Degradation of a Target Tyrosine Kinase^*

Amiya K. Ghosh, Alagarsamy L. Reddi, Navin L. Rao¶, Lei Duan, Vimla Band, and Hamid Band**

From the Divisions of Molecular Oncology and ^||Cancer Biology, Department of Medicine Evanston Northwestern Healthcare Research Institute, Feinberg School of Medicine and Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Evanston, Illinois 60201 and the Division of Rheumatology, Immunology, and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Members of the Cbl family of ubiquitin ligases have emerged as crucial negative regulators of tyrosine kinase signaling. These proteins preferentially interact with and target activated tyrosine kinases for ubiquitinylation, thereby facilitating the lysosomal sorting of receptor tyrosine kinases or proteasomal degradation of nonreceptor tyrosine kinases. Recent work has indicated a crucial role of the target kinase activity in Cbl-dependent ubiquitinylation and degradation, but the biochemical basis for this requirement is not understood. Here, we have used the Src-family kinase Fyn, a well characterized Cbl target, to address this issue. Using defined Fyn mutants, we demonstrate that the kinase activity of Fyn is crucial for its Cbl-dependent ubiquitinylation and degradation, but a low level of ubiquitinylation and degradation of kinase-inactive Fyn mutants was consistently observed. Mutational induction of an open conformation enhanced the susceptibility of kinase-active Fyn to Cbl but was insufficient to promote the ubiquitinylation and degradation of kinase-inactive Fyn. Notably, the Cbl-dependent degradation of Fyn did not require the Fyn-mediated phosphorylation of Cbl. Finally, we show that the major determinant of the susceptibility of Fyn protein to Cbl-dependent ubiquitinylation and degradation is the extent to which it physically associates with Cbl; kinase activity of Fyn serves as a critical determinant to promote its association with Cbl, which we demonstrate is mediated by multiple protein-protein interactions. Our results strongly suggest that promotion of association with Cbl is the primary mechanism by which the kinase activity of the targets of Cbl contributes to their susceptibility to Cbl.

Received for publication, April 15, 2004 , and in revised form, June 18, 2004.

^* The work reported here was supported by National Institute of Health Grants CA 87986, CA 76118, CA 99900, and CA99163 (to H. B.) and CA 81076 and CA 70195 (to V. B.) and United States Army Breast Cancer Research Program trainee award DAMD17-99-1-9086 (to A. G. and L. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Present address: Johnson & Johnson Pharmaceutical R&D LLC, San Diego, CA.

^** To whom correspondence should be addressed: ENH Research Institute, 1001 University Pl., Evanston, IL 60201. Tel.: 224-364-7401/7424; Fax: 224-364-7402; E-mail: h-band{at}northwestern.edu.

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