Cbl-mediated Negative Regulation of the Syk Tyrosine Kinase

doi:10.1074/jbc.273.52.35273

Cbl-mediated Negative Regulation of the Syk Tyrosine Kinase

A CRITICAL ROLE FOR Cbl PHOSPHOTYROSINE-BINDING DOMAIN BINDING TO Syk PHOSPHOTYROSINE 323*

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^‡Department of Immunology, University of Washington, Seattle, Washington 98195, and the ^§§Division of Hematology and Medical Oncology, Oregon Health Sciences University, Portland, Oregon, 97201

Abstract

The proto-oncogene product Cbl has emerged as a potential negative regulator of the Syk tyrosine kinase; however, the nature of physical interactions between Cbl and Syk that are critical for this negative regulation remains unclear. Here we show that the phosphotyrosine-binding (PTB) domain within the N-terminal transforming region of Cbl (Cbl-N) binds to phosphorylated Tyr³²³in the linker region between the Src homology 2 and kinase domains of Syk, confirming recent results by another laboratory using the yeast two-hybrid approach (Deckert, M., Elly, C., Altman, A., and Liu, Y. C. (1998) J. Biol. Chem. 273, 8867–8874). A PTB domain-inactivating point mutation (G306E), corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue SLI-1, severely compromised Cbl-N/Syk binding in vitro and Cbl/Syk association in transfected COS-7 cells. Using heterologous expression in COS-7 cells, we investigated the role of Cbl PTB domain binding to Syk Tyr³²³ in the negative regulation of Syk. Co-expression of Cbl with Syk in COS-7 cells led to a dose-dependent decrease in the autophosphorylated pool of Syk and in phosphorylation of an in vivo substrate, CD8-ζ. Unexpectedly, these effects were largely due to the loss of Syk protein. Both the decrease in Syk and CD8-ζ phosphorylation and reduction in Syk protein levels were blocked by either G306E mutation in Cbl or by Y323F mutation in Syk. These results demonstrate a critical role for the Cbl PTB domain in the recruitment of Cbl to Syk and in Cbl-mediated negative regulation of Syk.

Footnotes

↵* This work was supported in part by National Institutes of Health Grant CA76118 and American Cancer Society Grants RPG-95-066-CIM and RPG-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.
↵‡ A Breast Cancer Research Scholar of the Massachusetts Department of Public Health. Present address: ICOS Corp., Bothell, Washington 98021.
↵§ A predoctoral fellow of the Howard Hughes Medical Institute.
↵¶ Recipient of U.S. Department of Defense Breast Cancer Research Program Career Development Award DAMD17-98-1-8038.
↵‖ An Athelstan and Amy Saw Medical Research Fellow (Australia) and recipient of a postdoctoral fellowship from the U.S. Department of Defense Breast Cancer Research Program (BC972746).
↵** A fellow of The Uehara Memorial Foundation, Japan.
↵¶¶ To whom correspondence should be addressed: Smith Bldg., Rm. 538C, 1 Jimmy Fund Way, Boston, MA 02115. Tel.: 617-525-1101; Fax: 617-525-1102/1010; E-mail:hband{at}rics.bwh.harvard.edu.
Abbreviations:

SH2 and -3

Src homology 2 and 3, respectively

Tyr(P) or pY

phosphotyrosine

CD

cluster of differentiation

mAb

monoclonal antibody

GST

glutathioneS-transferase

PAGE

polyacrylamide gel electrophoresis

PVDF

polyvinylidene difluoride

EGFR

epidermal growth factor receptor

PDGFRα

platelet-derived growth factor receptor α

HA

hemagglutinin

PTB

phosphotyrosine-binding

mAb

monoclonal antibody.
- Received August 18, 1998.
- Revision received September 29, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.