INTERACTIONS OF P59(FYN) AND ZAP-70 WITH T-CELL RECEPTOR ACTIVATION MOTIFS - DEFINING THE NATURE OF A SIGNALING MOTIF

The tyrosine-based activation motif is a 20- to 25-amino-acid sequence contained in the cytoplasmic domains of many hematopoietic receptors which is sufficient by itself to reconstitute signalling. This motif i s characterized by two YXXL/I sequences separated by approximately 10 residues. The molecular basis of signalling by this motif is unknown. Here we demonstrate that the tyrosine-based activation motif is requir ed and sufficient for association with the tyrosine kinases p59(fyn) a nd ZAP-70, suggesting that association with these kinases is a general feature of this motif. Focusing on the single activation motif presen t in epsilon, we analyzed which residues of the motif were critical fo r binding of p59(fyn) and ZAP-70. Surprisingly, we found that no singl e mutation of any residue of epsilon resulted in the loss of p59(fyn) association. In contrast, single mutations at five residues of the eps ilon activating motif abrogated ZAP-70 binding. Both of the tyrosines and the leucine or isoleucine residues that follow them were critical. The spacing between the tyrosines was also important, as deletion of two residues disrupted binding of ZAP-70, although p59(fyn) binding wa s not disrupted. Most of the defined features of the tyrosine activati on motif are therefore requirements for ZAP-70 binding. Interestingly, the interaction of ZAP-70 with the motif was dependent on the presenc e of both ZAP-70 SH2 domains and both of the tyrosine residues in the moth, suggesting that ZAP-70 interacts with two phosphotyrosine residu es and that the binding of the two SH2 domains is cooperative. In addi tion, we demonstrate that the interaction between the tyrosine activat ion moth is direct and requires prior tyrosine phosphorylation of the motif. We propose that the activation of cells by the tyrosine activat ing motif occurs in four discrete steps: binding of p59(fyn), phosphor ylation of the motif, binding of ZAP-70, and activation of ZAP-70 kina se activity.