The Cbl Phosphotyrosine-binding Domain Selects a D(N/D)XpY Motif and Binds to the Tyr292Negative Regulatory Phosphorylation Site of ZAP-70*
- From the ‡Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital, the ¶Division of Signal Transduction, Beth Israel Deaconess Hospital Medical Center, and the ‖Research Division, Joslin Diabetes Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Abstract
The Cbl protooncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases through currently undefined mechanisms. Therefore, determining how Cbl physically interacts with tyrosine kinases is of substantial interest. We recently identified a phosphotyrosine binding (PTB) domain residing within the N-terminal transforming region of Cbl (Cbl-N), which mediated direct binding to ZAP-70 tyrosine kinase. Here, we have screened a degenerate phosphopeptide library and show that the Cbl-PTB domain selects a D(N/D)XpY motif, reminiscent of but distinct from the NPXpY motif recognized by the PTB domains of Shc and IRS-1/2. A phosphopeptide predicted by this motif and corresponding to the in vivo negative regulatory phosphorylation site of ZAP-70 (Tyr(P)292) specifically inhibited binding of ZAP-70 to Cbl-N. A ZAP-70/Y292F mutant failed to bind to Cbl-N, whereas a D290A mutant resulted in a 64% decrease in binding, confirming the importance of the Tyr(P) and Y-2 residues in Cbl-PTB domain recognition. Finally the ZAP-70/Y292F mutant also failed to associate with Cbl-N or full-length Cbl in vivo. These results identify a potential Cbl-PTB domain-dependent role for Cbl in the negative regulation of ZAP-70 and predict potential Cbl-PTB domain binding sites on other protein tyrosine kinases known to interact with Cbl.
Footnotes
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↵* This work was supported by Grants AR36308 and CA64503 from the National Institutes of Health and Grant IM-770 from the American Cancer Society (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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↵§ Breast Cancer Research Fellow of the Massachusetts Dept. of Public Health.
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↵** To whom correspondence should be addressed: Smith Bldg., Rm. 538C, 75 Francis St., Boston, MA 02115. Tel.: 617-525-1101; Fax: 617-525-1102; E-Mail: hband{at}rics.bwh.harvard.edu.
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↵1 The abbreviations used are: TCR, T cell receptor; CD, cluster of differentiation; GST, glutathioneS-transferase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; EGF, epidermal growth factor; EGFR, EGF receptor; PTB, phosphotyrosine-binding domain; Cbl-N, N-terminal transforming region of Cbl; HA, hemagglutinin; PDGFRα, platelet-derived growth factor α receptor.
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↵2 M. L. Lupher, Jr. and H. Band, unpublished data.
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↵3 Z. Songyang, unpublished data.
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↵4 M. Lupher and H. Band, unpublished data.
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- Received June 20, 1997.
- Revision received November 6, 1997.
