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J. Biol. Chem. 275 (1): 367-377

© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

J Biol Chem, Vol. 275, Issue 1, 367-377, January 7, 2000

The Evolutionarily Conserved N-terminal Region of Cbl Is Sufficient to Enhance Down-regulation of the Epidermal Growth Factor Receptor*

Nancy L. LillDagger , Patrice Douillard§, Rana A. Awwad, Satoshi Ota, Mark L. Lupher Jr., Sachiko Miyakepar , Nichole Meissner-Lula, Victor W. Hsu**, and Hamid BandDagger Dagger

From the Lymphocyte Biology Section, Division of Rheumatology, Immunology, and Allergy, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115

The mammalian proto-oncoprotein Cbl and its homologues in Caenorhabditis elegans and Drosophila are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). Overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. We report that the Cbl tyrosine kinase-binding (TKB) domain is essential for this activity. Whereas wild-type Cbl enhanced ligand-dependent EGF-R ubiquitination, down-regulation from the cell surface, accumulation in intracellular vesicles, and degradation, a Cbl TKB domain-inactivated mutant (G306E) did not. Furthermore, the transforming truncation mutant Cbl-N (residues 1-357), comprising only the Cbl TKB domain, functioned as a dominant negative protein. It colocalized with EGF-R in intracellular vesicular structures, yet it suppressed down-regulation of EGF-R from the surface of cells expressing endogenous wild-type Cbl. Therefore, Cbl-mediated down-regulation of EGF-R requires the integrity of both the N-terminal TKB domain and additional C-terminal sequences. A Cbl truncation mutant comprising amino acids 1-440 functioned like wild-type Cbl in down-regulation assays. This mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. We conclude that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface.

* This work was supported in part by Grants CA75075 and CA76118 (to H. B.) from the National Institutes of Health and by Grant CIM-89513 from the American Cancer Society.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.

Dagger Recipient of a United States Department of the Army Breast Cancer Research Program Career Development Award DAMD17-98-1-8038.

§ Fellow of the Association pour la Recherche Contre le Cancer and a Breast Cancer Research Fellow of the Massachusetts Department of Public Health.

Breast Cancer Research Scholar of the Massachusetts Department of Public Health. Present address: ICOS Corp., Bothell, WA 98021.

par Fellow of the Uehara Memorial Foundation, Japan.

** Established investigator of the American Heart Association.

Dagger Dagger To whom correspondence should be addressed: Brigham and Women's Hospital, Smith Bldg., Rm. 538C, One Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1101; Fax: 617-525-1010; E-mail: hband@rics.bwh.harvard.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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