Functional analysis of LAT in TCR-mediated signaling pathways using a LAT-deficient Jurkat cell line.
The adaptor molecule LAT (linker for activation of T cells) is a palmitoylated integral membrane protein that localizes to the glycolipid-enriched microdomains in the plasma membrane. Upon TCR engagement, LAT becomes phosphorylated on multiple tyrosine residues and then binds several critical signaling molecules. Here, we describe the generation and characterization of a LAT-deficient cell line. Using this cell line, we demonstrate that LAT is required for TCR-mediated Ca2+ mobilization and optimal tyrosine phosphorylation of phospholipase C-gamma1, Vav and SLP-76. LAT is also required for Erk activation, CD69 up-regulation, and AP- and NFAT-mediated gene transcription. We also demonstrate, by reconstituting this cell line with LAT mutants, that the LAT transmembrane domain and palmitoylation at Cys26, but not Cys29, 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.
Zhang
W
W
Section on Lymphocyte Signaling, Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-5430, USA.
Irvin
B J
BJ
Trible
R P
RP
Abraham
R T
RT
Samelson
L E
LE
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD / Q000096:biosynthesis; D000945:Antigens, Differentiation, T-Lymphocyte / Q000096:biosynthesis; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D002469:Cell Separation; D002999:Clone Cells; D004789:Enzyme Activation / Q000276:immunology; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D019169:Jurkat Cells / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D037181:Lectins, C-Type; D008565:Membrane Proteins; D009154:Mutation; D015513:Oncogene Proteins / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D051567:Proto-Oncogene Proteins c-vav; D011948:Receptors, Antigen, T-Cell / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D015533:Transcriptional Activation / Q000276:immunology; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D015854:Up-Regulation / Q000276:immunology; D018909:src Homology Domains / Q000276:immunology; D000099192:CD69 Antigens
D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D000945:Antigens, Differentiation, T-Lymphocyte; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002352:Carrier Proteins; D007527:Isoenzymes; D037181:Lectins, C-Type; D008565:Membrane Proteins; D015513:Oncogene Proteins; D019308:Palmitic Acid; D051966:Phospholipase C gamma; D010750:Phosphoproteins; D051567:Proto-Oncogene Proteins c-vav; D011948:Receptors, Antigen, T-Cell; D010738:Type C Phospholipases; D014443:Tyrosine; D000099192:CD69 Antigens; C110810:LAT protein, human; C092947:SLP-76 signal Transducing adaptor proteins; C496618:VAV1 protein, human