A soluble LAT deletion mutant inhibits T-cell activation: reduced recruitment of signalling molecules to glycolipid-enriched microdomains.
The type III transmembrane adaptor protein linker for activation of T cells (LAT) is essential for membrane recruitment of signalling molecules following TCR activation. Here we show that although LAT deleted in the transmembrane domain is completely soluble, it can be tyrosine phosphorylated after anti-CD3 stimulation or pervanadate treatment. Overexpression of this deletion mutant in transiently transfected Jurkat TAg cells inhibits transcriptional activation of nuclear factor of activated T cells (NF-AT)/AP-1 reporter construct in a concentration-dependent manner. Furthermore, by selection of transiently transfected cells, a clear reduction of TCR-induced CD69 expression was observed in cells expressing the mutant. These dominant negative effects seemed to be dependent both on the ability of the membrane deletion mutant to reduce phosphorylation of endogenous LAT and to reduce interaction of endogenous LAT with PLC-gamma1 and Grb2. Consistent with this, the redistribution of PLC-gamma1 and Grb2 to glycolipid-enriched microdomains, called lipid rafts, after stimulation was inhibited when the soluble form of LAT was overexpressed. We suggest that the dominant negative effect is caused by the ability of the mutant to sequester signalling molecules in cytosol and thereby inhibit redistribution of signalling molecules to lipid rafts upon T-cell activation.
Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway.
K M
KM
Torgersen
J T
JT
Vaage
B
B
Rolstad
K
K
Taskén
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D018274:Electroporation; D017353:Gene Deletion; D006017:Glycolipids / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000378:metabolism; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism; D016296:Mutagenesis; D010750:Phosphoproteins / Q000235:genetics*; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D015398:Signal Transduction; D013601:T-Lymphocytes / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D006017:Glycolipids; D007527:Isoenzymes; C110810:LAT protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D014157:Transcription Factors; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases