The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the Tyr292 negative regulatory phosphorylation site of ZAP-70

Mark L. Lupher, Zhou Songyang, Steven E. Shoelson, Lewis C. Cantley, Hamid Band

Research output: Contribution to journalArticle

180 Citations (Scopus)

Abstract

The Cul protooncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases through currently undefined mechanisms. Therefore, determining how Cul 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 Cul (Cul-N), which mediated direct binding to ZAP-70 tyrosine kinase. Here, we have screened a degenerate phosphopeptide library and show that the CblPTB 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 Cul-N. A ZAP-70/Y292F mutant failed to bind to Cul-N, whereas a D290A mutant resulted in a 64% decrease in binding, confirming the importance of the Tyr(P) and Y-2 residues in Cul-PTB domain recognition. Finally the ZAP-70/Y292F mutant also failed to associate with Cul-N or full-length Cul in vivo. These results identify a potential Cul-PTB domain-dependent role for Cul in the negative regulation of ZAP-70 and predict potential CblPTB domain binding sites on other protein tyrosine kinases known to interact with Cul.

Original languageEnglish (US)
Pages (from-to)33140-33144
Number of pages5
JournalJournal of Biological Chemistry
Volume272
Issue number52
DOIs
StatePublished - Dec 26 1997

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Phosphotyrosine
Phosphorylation
Protein-Tyrosine Kinases
Phosphopeptides
ZAP-70 Protein-Tyrosine Kinase
Binding Sites

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the Tyr292 negative regulatory phosphorylation site of ZAP-70. / Lupher, Mark L.; Songyang, Zhou; Shoelson, Steven E.; Cantley, Lewis C.; Band, Hamid.

In: Journal of Biological Chemistry, Vol. 272, No. 52, 26.12.1997, p. 33140-33144.

Research output: Contribution to journalArticle

Lupher, Mark L. ; Songyang, Zhou ; Shoelson, Steven E. ; Cantley, Lewis C. ; Band, Hamid. / The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the Tyr292 negative regulatory phosphorylation site of ZAP-70. In: Journal of Biological Chemistry. 1997 ; Vol. 272, No. 52. pp. 33140-33144.
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abstract = "The Cul protooncogene product has emerged as a novel negative regulator of receptor and non-receptor tyrosine kinases through currently undefined mechanisms. Therefore, determining how Cul 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 Cul (Cul-N), which mediated direct binding to ZAP-70 tyrosine kinase. Here, we have screened a degenerate phosphopeptide library and show that the CblPTB 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 Cul-N. A ZAP-70/Y292F mutant failed to bind to Cul-N, whereas a D290A mutant resulted in a 64{\%} decrease in binding, confirming the importance of the Tyr(P) and Y-2 residues in Cul-PTB domain recognition. Finally the ZAP-70/Y292F mutant also failed to associate with Cul-N or full-length Cul in vivo. These results identify a potential Cul-PTB domain-dependent role for Cul in the negative regulation of ZAP-70 and predict potential CblPTB domain binding sites on other protein tyrosine kinases known to interact with Cul.",
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