Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells

T-cell-receptor (TCR)-mediated LAT (linker fdr activation of T cells) phosp horylation is critical for the membrane recruitment of signalling complexes required for T-cell activation. Although tyrosine phosphorylation of LAT i s required for recruitment and activation-of signalling proteins, the molec ular mechanism associated with this event is unclear. In the present study we reconstituted the LAT signalling pathway by demonstrating that a direct tyrosine phosphorylation of LAT with activated protein-tyrosine kinase Zap7 0 is necessary and sufficient for the association and activation of signall ing proteins. Zap-70 efficiently phosphorylates LAT on tyrosine residues at positions 226, 191, 171, 132 and 127, By,substituting these tyrosine resid ues in LAT with phenylalanine and by utilizing phosphorylated peptides deri ved from these-sites, we mapped the tyrosine residues in LAT. required for the direct interaction and activation of Vav, p85/p1-10 alpha. and phosphol ipase C gamma1 (PLC gamma1). Our results indicate that Tyr(226) and Tyr(101 ) are required for Vav binding, whereas Tyr(171) and Tyr(132) are necessary for association and activation of phosphoinositide 3-kinase activity and P LC gamma1 respectively. Furthermore, by expression of LAT mutants in LAT-de ficient T cells, we demonstrate that Tyr(101) and Tyr(171) are required for T-cell activation and Tyr132 is required for the activation of PLC gamma1 and Ras,signalling pathways.