The Cbl phosphotyrosine-binding domain selects a D(N/D)XpY motif and binds to the Tyr292 negative regulatory phosphorylation site of ZAP-70.
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.
Lupher
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ML
Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Songyang
Z
Z
Shoelson
S E
SE
Cantley
L C
LC
Band
H
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D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D050722:Oncogene Protein v-cbl; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015682:Retroviridae Proteins, Oncogenic / Q000378:metabolism*; D051746:ZAP-70 Protein-Tyrosine Kinase; D018909:src Homology Domains
D050722:Oncogene Protein v-cbl; D015682:Retroviridae Proteins, Oncogenic; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; C497262:Zap70 protein, mouse