A Novel Phosphotyrosine-binding Domain in the N-terminal Transforming Region of Cbl Interacts Directly and Selectively with ZAP-70 in T Cells

doi:10.1074/jbc.271.39.24063

A Novel Phosphotyrosine-binding Domain in the N-terminal Transforming Region of Cbl Interacts Directly and Selectively with ZAP-70 in T Cells*

From the ^‡ Lymphocyte Biology Section, Division of Rheumatology and Immunology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115 and the
^¶Department of Pathology, Queen Elizabeth II Medical Center, University of Western Australia, Nedlands, Australia 6009

^∥To whom correspondence should be addressed at:
Seeley G. Mudd Bldg., Rm. 514, 250 Longwood Ave., Boston, MA 02115.
Tel.: 617-432-1557; Fax: 617-432-2799; E-mail: band{at}mbcrr.harvard.edu

Abstract

The protooncogene product Cbl has emerged as a novel signal transduction protein downstream of a number of cell surface receptors coupled to tyrosine kinases. Recently, we and others have reported the activation-dependent association of Cbl with the Syk and ZAP-70 tyrosine kinases through presently undefined mechanisms. Potential Src homology 2 and 3 domain binding sites within the C-terminal half of Cbl mediate in vivo interactions with several signaling proteins; however, the N-terminal transforming region (Cbl-N) lacks recognizable catalytic or protein interaction motifs. Here, we show that in vitro Cbl-N (amino acids 1-357) but not Cbl-C (amino acids 358-906) binds to ZAP-70 in a T cell-activation-dependent manner. A point mutation in Cbl-N, G306E, corresponding to a loss-of-function mutation in the Caenorhabditis elegans Cbl homologue, SLI-1, severely compromised Cbl-N/ZAP-70 binding. Cbl-N/ZAP-70 binding was direct and phosphotyrosine-dependent, thus identifying a phosphotyrosine-binding domain within the transforming region of Cbl. In vivo, Cbl-N expressed in T cells selectively associated with the ZAP-70/ζ complex. These results identify a novel mechanism for the direct participation of the N-terminal region of Cbl in ZAP-70 signal transduction, and suggest a biochemical mechanism for the leukemogenicity of the oncogene v-cbl through potential interaction with proliferation-related phosphotyrosyl proteins.

Footnotes

↵^§ Fellow of The Uehara Memorial Foundation, Japan.
↵* This work was supported in part by American Cancer Society Grant IM-770 (to H. B.) and National Institutes of Health Grant AR36308 (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.
↵¹ The abbreviations used are:

TCR

T cell receptor

CD

cluster of differentiation

SH2

Src homology 2

SH3

Src homology 3

mAb

monoclonal antibody

GST

glutathione S-transferase

PAGE

polyacrylamide gel electrophoresis

PVDF

polyvinylidene difluoride

PA-HRPO

protein A-horseradish peroxidase

ECL

enhanced chemiluminescence

IP

immunoprecipitation

EGF

epidermal growth factor

PTB

phosphotyrosine-binding domain

HA

hemagglutinin.
↵²M. L. Lupher, Jr. and H. Band, unpublished data.
↵³W. Y. Langdon, unpublished data.
- Received June 25, 1996.
- Revision received July 26, 1996.