Association of Grb2 Gads, and phospholipase C-gamma 1 with phosphorylated LAT tyrosine residues - Effect of LAT tyrosine mutations on T cell antigen receptor-mediated signaling

The linker for activation of T cells (LAT) is a critical adaptor molecule r equired for T cell antigen receptor (TCR)-mediated signaling and thymocyte development. Upon T cell activation, LAT becomes highly phosphorylated on t yrosine residues, and Grb2, Gads, and phospholipase C (PLC)-gamma 1 bind LA T via Src homology-2 domains. In LAT-deficient mutant Jurkat cells, TCR eng agement fails to induce ERK activation, Ca2+ flux, and activation of AP-1 a nd NF-AT. We mapped the tyrosine residues in LAT responsible for interactio n with these specific signaling molecules by expressing LAT mutants with ty rosine to phenylalanine mutations in LAT-deficient cells. Our results showe d that three distal tyrosines, Tyr(171), Tyr(191), and Tyr(226), are respon sible for Grb2-binding; Tyr171, and Tyr(191), but not Tyr(226) necessary fo r Gads binding. Mutation of Tyr(132) alone abolished PLC-gamma 1 binding. M utation of all three distal tyrosines also abolished PLC-gamma 1 binding, s uggesting there might be multiple binding sites for PLC-gamma 1. Mutation o f Tyr(132) affected calcium flux and blocked Erk and NF-AT activation. Sinc e Grb2 binding is not affected by this mutation, these results strongly sug gest that PLC-gamma activation regulates Ras activation in these cells. Mut ation of individual Grb2 binding sites had no functional effect, but mutati on of two or three of these sites, in combination, also affected Erk. and N F-AT activation.