Cbl-mediated Negative Regulation of Platelet-derived Growth Factor Receptor-dependent Cell Proliferation
A CRITICAL ROLE FOR Cbl TYROSINE KINASE-BINDING DOMAIN*
- 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
Abstract
The Cbl proto-oncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases. Our previous observations that Cbl overexpression in NIH3T3 cells enhanced the ubiquitination and degradation of the platelet-derived growth factor receptor-α (PDGFRα) and that the expression of oncogenic Cbl mutants up-regulated the PDGFRα signaling machinery strongly suggested that Cbl negatively regulates PDGFRα signaling. Here, we show that, similar to PDGFRα, selective stimulation of PDGFRβ induces Cbl phosphorylation, and its physical association with the receptor. Overexpression of wild type Cbl in NIH3T3 cells led to an enhancement of the ligand-dependent ubiquitination and subsequent degradation of the PDGFRβ, as observed with PDGFRα. We show that Cbl-dependent negative regulation of PDGFRα and β results in a reduction of PDGF-induced cell proliferation and protection against apoptosis. A point mutation (G306E) that inactivates the tyrosine kinase binding domain in the N-terminal transforming region of Cbl compromised the PDGF-inducible tyrosine phosphorylation of Cbl although this mutant could still associate with the PDGFR. More importantly, the G306E mutation abrogated the ability of Cbl to enhance the ligand-induced ubiquitination and degradation of the PDGFR and to inhibit the PDGF-dependent cell proliferation and protection from apoptosis. These results demonstrate that Cbl can negatively regulate PDGFR-dependent biological responses and that this function requires the conserved tyrosine kinase binding domain of Cbl.
Footnotes
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↵* This work was supported in part by National Institutes of Health Grants CA76118 and CA75075 and American Cancer Society Grant CIM-89513 (to H. B.).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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↵‡ Fellow of the Uehara Memorial Foundation, Japan.
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↵§ Scholar of the Massachusetts Dept. of Public Health Breast Cancer Program.
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↵¶ Recipient of the U. S. Dept. of Defense Breast Cancer Research Program Career Development Award DAMD 17-98-1-8038.
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↵‖ Fellow of Association pour la Recherche Contre le Cancer and is a scholar of the Massachusetts Dept. of Public Health Breast Cancer Program.
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↵** To whom correspondence should be addressed: Smith Bldg., Room 538C, One Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1101, Fax: 617-525-1102 or 525-1010; E-mail: hband{at}rics.bwh.harvard.edu.
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↵2 The region referred to as the TKB domain was previously referred to as a phosphotyrosine-binding (PTB) domain, but structural studies show that this region is an integrated phosphopeptide-binding platform composed of a four-helical domain, an EF hand, and an SH2 domain (40).
- Abbreviations:
- PTK
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protein tyrosine kinase
- ECL
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enhanced chemiluminescence
- EGFR
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epidermal growth factor receptor
- GST
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glutathione S-transferase
- TKB
-
tyrosine kinase-binding
- HRPO
-
horseradish peroxidase
- PDGFR
-
platelet-derived growth factor receptor
- PVDF
-
polyvinylidene difluoride
- PAGE
-
polyacrylamide gel electrophoresis
- Tyr(P)
-
phosphotyrosine
- α-MEM
-
α-minimal essential medium
- FCS
-
fetal calf serum
- HA
-
hemagglutinin
- aa
-
amino acid(s)
- TUNEL
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terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling
- PI 3-kinase
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phosphatidylinositol 3-kinase
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- Received December 14, 1998.
- Revision received April 2, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
