Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line

The adaptor molecule LAT (linker for activation of I cells) is a palmitoyla ted integral membrane protein that localizes to the glycolipid-enriched mic rodomains in the plasma membrane. Upon TCR engagement, LAT becomes phosphor ylated on multiple tyrosine residues and then binds several critical signal ing molecules. Here, we describe the generation and characterization of a L AT-deficient cell line, Using this cell line, we demonstrate that LAT is re quired for TCR-mediated Ca2+ mobilization and optimal tyrosine phosphorylat ion of phospholipase C-gamma 1,Vav and SLP-76, LAT is also required for Erk activation, CD69 up-regulation, and AP- and NFAT-mediated gene transcripti on. We also demonstrate, by reconstituting this cell line with LAT mutants, that the LAT transmembrane domain and palmitoylation at Cys26, but not Cys 29, are required for LAT function and TCR signaling. These studies provide further evidence for the crucial role of the LAT molecule, and demonstrate the use of a LAT-deficient cell line for the analysis of LAT structure and function.