Selective interaction of LAT (linker of activated T cells) with the open-active form of Lck in lipid rafts reveals a new mechanism for the regulation of Lck in T cells.
In T cells, the lipid raft-associated Lck is strongly tyrosine phosphorylated and has reduced enzymic activity in contrast with the detergent-soluble pool, which has substantial activity. Lck tagged at the C-terminus (Lck/V5-His) was efficiently captured by epitope-specific reagents from the detergent-soluble fraction but not from lipid rafts. Binding was restored following urea denaturation, suggesting that Lck/V5-His is in a 'closed' conformation in these domains. In agreement with this hypothesis, the Tyr(505) --> Phe/V5-His and Arg(154) --> Lys/V5-His mutants, which disrupt the SH2-Tyr(505) intramolecular interaction, were efficiently precipitated from lipid rafts. In contrast to Lck, Fyn/V5-His was precipitated equally well from both fractions. In the LAT(linker of activated T cells)-deficient J.CaM2 cells, Tyr(505) phosphorylation of raft-associated Lck was reduced whereas its enzymic activity was elevated. This correlated with decreased levels of raft-localized Csk (C-terminal Src kinase) kinase. Increased tyrosine phosphorylation of Lck was restored in LAT-reconstituted J.CaM2 cells suggesting that LAT negatively regulates Lck activity in lipid rafts. Co-immunoprecipitation experiments from Tyr(505) --> Phe/V5-His-expressing cells revealed that LAT preferentially interacts with the 'open' form of Lck in T cell raft domains. These results demonstrate that, unlike the non-raft pool, Lck in lipid rafts has a 'closed'-inactive structure, and that LAT plays a role in maintaining this conformation, possibly by facilitating critical associations within lipid rafts via its capacity to interact with the 'open' form of the kinase.
|Title:||Selective interaction of LAT (linker of activated T cells) with the open-active form of Lck in lipid rafts reveals a new mechanism for the regulation of Lck in T cells.|
|Keywords:||Adaptor Proteins, Signal Transducing, Base Sequence, Blotting, Western, Carrier Proteins, DNA Primers, Humans, Jurkat Cells, Lipid Metabolism, Membrane Proteins, Phosphoproteins, Phosphorylation, Precipitin Tests, Protein Binding, Protein Conformation, T-Lymphocytes, Tyrosine|
|UCL classification:||UCL > School of Life and Medical Sciences
UCL > School of Life and Medical Sciences > Faculty of Medical Sciences
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