T-cell-receptor (TCR)-mediated LAT (linker for activation of T cells) phosphorylation is critical for the membrane recruitment of signalling complexes required for T-cell activation. Although tyrosine phosphorylation of LAT is required for recruitment and activation of signalling proteins, the molecular 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 Zap70 is necessary and sufficient for the association and activation of signalling proteins. Zap-70 efficiently phosphorylates LAT on tyrosine residues at positions 226, 191, 171, 132 and 127. By substituting these tyrosine residues in LAT with phenylalanine and by utilizing phosphorylated peptides derived from these sites, we mapped the tyrosine residues in LAT required for the direct interaction and activation of Vav, p85/p110α and phospholipase Cγ1 (PLCγ1). Our results indicate that Tyr226 and Tyr191 are required for Vav binding, whereas Tyr171 and Tyr132 are necessary for association and activation of phosphoinositide 3-kinase activity and PLCγ1 respectively. Furthermore, by expression of LAT mutants in LAT-deficient T cells, we demonstrate that Tyr191 and Tyr171 are required for T-cell activation and Tyr132 is required for the activation of PLCγ1 and Ras signalling pathways.
Abbreviations used: AP-1, activator protein-1 (a transcription factor); TCR, T-cell receptor; PTK, protein tyrosine kinase; ITAM, immunoreceptor tyrosine-based activation motif; MAP kinase, mitogen-activated protein kinase; PLCγ1, phospholipase Cγ1; LAT, linker for activation of T cells; NFAT, nuclear factor in activated T cells; SH2, Src homology 2; GEM, glycolipid-enriched domains; GST, glutathione S-transferase; PTyr or pY, phosphotyrosine; PI 3-kinase, phosphoinositol 3-kinase; PDGF, platelet-derived growth factor; ERK, extracellular-signal-regulated kinase; [Y132F]LAT (etc.), LAT mutant in which Tyr132 has been mutated to Phe (etc.); FLAG, the epitope DYKDDDDK; Sf9 cells, Spodoptera frugiperda (fall armyworm) cells; PIP2, phosphatidylinositol 4,5-bisphosphate; PE, phosphatidylethanolamine; NP40, Nonidet P40; GEF, guanine nucleotide exchange factor; WT, wild-type; Vav, a signalling protein with guanine-nucleotide-exchange activity; p85/p110α, the α-isoform of PI 3-kinase; Grb2, growth-factor receptor bound-2, an adapter protein; Ras, a small GTPase; Cbl-b and Slp76, adapter proteins; Sos, a guanine nucleotide exchange factor; p42 and p44, ERK kinases; GAP, GTPase activating protein for Ras; Lck/Fyn and Zap-70/Syk, protein-tyrosine kinases; OKT3, an anti-CD3 monoclonal antibody.
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