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 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/p110alpha and phospholipase Cgamma1 (PLCgamma1). Our results indicate that Tyr(226) and Tyr(191) are required for Vav binding, whereas Tyr(171) and Tyr(132) are necessary for association and activation of phosphoinositide 3-kinase activity and PLCgamma1 respectively. Furthermore, by expression of LAT mutants in LAT-deficient T cells, we demonstrate that Tyr(191) and Tyr(171) are required for T-cell activation and Tyr(132) is required for the activation of PLCgamma1 and Ras signalling pathways.
Mesh Terms:
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Carrier Proteins, DNA Primers, Enzyme Precursors, Humans, Intracellular Signaling Peptides and Proteins, Isoenzymes, Jurkat Cells, Lymphocyte Activation, Lymphocyte Specific Protein Tyrosine Kinase p56(lck), Membrane Proteins, Molecular Sequence Data, Peptide Mapping, Phospholipase C gamma, Phosphoproteins, Phosphorylation, Protein-Tyrosine Kinases, Recombinant Fusion Proteins, T-Lymphocytes, Type C Phospholipases, Tyrosine, ZAP-70 Protein-Tyrosine Kinase, ras Proteins, src Homology Domains
Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Base Sequence, Binding Sites, Carrier Proteins, DNA Primers, Enzyme Precursors, Humans, Intracellular Signaling Peptides and Proteins, Isoenzymes, Jurkat Cells, Lymphocyte Activation, Lymphocyte Specific Protein Tyrosine Kinase p56(lck), Membrane Proteins, Molecular Sequence Data, Peptide Mapping, Phospholipase C gamma, Phosphoproteins, Phosphorylation, Protein-Tyrosine Kinases, Recombinant Fusion Proteins, T-Lymphocytes, Type C Phospholipases, Tyrosine, ZAP-70 Protein-Tyrosine Kinase, ras Proteins, src Homology Domains
Biochem. J.
Date: Jun. 01, 2001
PubMed ID: 11368773
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