Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck

J Biol Chem. 1999 May 14;274(20):14229-37. doi: 10.1074/jbc.274.20.14229.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution
  • Binding Sites
  • Catalysis
  • Humans
  • Jurkat Cells
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phenotype
  • Phosphorylation
  • Protein-Tyrosine Kinases / genetics
  • Protein-Tyrosine Kinases / metabolism*
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / metabolism*
  • Structure-Activity Relationship
  • Tyrosine / metabolism*
  • Up-Regulation
  • ZAP-70 Protein-Tyrosine Kinase
  • src Homology Domains*

Substances

  • Receptors, Antigen, T-Cell
  • Tyrosine
  • Protein-Tyrosine Kinases
  • Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
  • ZAP-70 Protein-Tyrosine Kinase
  • ZAP70 protein, human