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.
Kabouridis
Panagiotis S
PS
Bone and Joint Research Unit, Barts and the London School of Medicine and Dentistry, Queen Mary, University of London, Charterhouse Square, London EC1M 6BQ, UK. p.s.kabouridis@qmul.ac.uk
D048868:Adaptor Proteins, Signal Transducing; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D019169:Jurkat Cells; D050356:Lipid Metabolism; D008565:Membrane Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D013601:T-Lymphocytes / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D017931:DNA Primers; C110810:LAT protein, human; C491734:Lat protein, mouse; D008565:Membrane Proteins; D010750:Phosphoproteins; C402726:Sh2d2a protein, mouse; D014443:Tyrosine