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.

Highlights

  • Lck and ZAP-70, members of the Src and Syk families of nonreceptor PTKs,1 respectively, control in a sequential man

  • YSDP Motif from the Interdomain B of ZAP-70 Is Able to Bind LckSH2—The similar phenotype displayed by Y319F and Y493F ZAP-70 mutants [7, 16] together with the critical role of the Src homology 2 (SH2)-mediated interaction between ZAP-70 and Lck in T-cell antigen receptor (TCR) signaling [11, 18], suggested that Tyr319 could be a binding site for the LckSH2. This residue is located in the motif YSDP (Fig. 1), which considerably diverges from YEEI, an optimal binding sequence for the SH2 domain of Src PTKs [19]

  • This model was built with no other information than the structure of the LckSH2 domain, with the pY residue lodged in its binding pocket

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Summary

Introduction

Lck and ZAP-70, members of the Src and Syk families of nonreceptor PTKs,1 respectively, control in a sequential man-. When overexpressed, ZAP-70 generates its own binding sites on the ITAMs. In agreement with the initial hypothesis, expression of ZAP-YEEI at levels similar to ZAP-WT induced a much higher NFAT activity in unstimulated Jurkat cells (Fig. 4A).

Results
Conclusion
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