Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck.
T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases. One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) ... and, more recently, that Tyr319 of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr319 mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr319, YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr319 to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence Y319SDP into Y319EEI. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr319-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.
Mesh Terms:
Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Catalysis, Humans, Jurkat Cells, Lymphocyte Specific Protein Tyrosine Kinase p56(lck), Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Phenotype, Phosphorylation, Protein-Tyrosine Kinases, Receptors, Antigen, T-Cell, Structure-Activity Relationship, Tyrosine, Up-Regulation, ZAP-70 Protein-Tyrosine Kinase, src Homology Domains
Amino Acid Sequence, Amino Acid Substitution, Binding Sites, Catalysis, Humans, Jurkat Cells, Lymphocyte Specific Protein Tyrosine Kinase p56(lck), Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, Phenotype, Phosphorylation, Protein-Tyrosine Kinases, Receptors, Antigen, T-Cell, Structure-Activity Relationship, Tyrosine, Up-Regulation, ZAP-70 Protein-Tyrosine Kinase, src Homology Domains
J. Biol. Chem.
Date: May. 14, 1999
PubMed ID: 10318843
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