Pleiotropic contributions of phospholipase C-gamma1 (PLC-gamma1) to T-cell antigen receptor-mediated signaling: reconstitution studies of a PLC-gamma1-deficient Jurkat T-cell line.
Phospholipase C-gamma1 (PLC-gamma1) plays a crucial role in the coupling of T-cell antigen receptor (TCR) ligation to interleukin-2 (IL-2) gene expression in activated T lymphocytes. In this study, we have isolated and characterized two novel, PLC-gamma1-deficient sublines derived from the Jurkat T-leukemic cell line. The P98 subline displays a >90% reduction in PLC-gamma1 expression, while the J.gamma1 subline contains no detectable PLC-gamma1 protein. The lack of PLC-gamma1 expression in J.gamma1 cells caused profound defects in TCR-dependent Ca(2+) mobilization and NFAT activation. In contrast, both of these responses occurred at normal levels in PLC-gamma1-deficient P98 cells. Unexpectedly, the P98 cells displayed significant and selective defects in the activation of both the composite CD28 response element (RE/AP) and the full-length IL-2 promoter following costimulation with anti-TCR antibodies and phorbol ester. These transcriptional defects were reversed by transfection of P98 cells with a wild-type PLC-gamma1 expression vector but not by expression of mutated PLC-gamma1 constructs that lacked a functional, carboxyl-terminal SH2 [SH2(C)] domain or the major Tyr(783) phosphorylation site. On the other hand, the amino-terminal SH2 [SH2(N)] domain was not essential for reconstitution of RE/AP- or IL-2 promoter-dependent transcription but was required for the association of PLC-gamma1 with LAT, as well as the tyrosine phosphorylation of PLC-gamma1 itself, in activated P98 cells. These studies demonstrate that the PLC-gamma1 SH2(N) and SH2(C) domains play functionally distinct roles during TCR-mediated signaling and identify a non-Ca(2+)-related signaling function linked to the SH2(C) domain, which couples TCR plus phorbol ester-CD28 costimulation to the activation of the IL-2 promoter in T lymphocytes.
Irvin
B J
BJ
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Williams
B L
BL
Nilson
A E
AE
Maynor
H O
HO
Abraham
R T
RT
D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007376:Interleukin-2 / Q000235:genetics*; D007527:Isoenzymes / Q000378:metabolism*; D019169:Jurkat Cells; D008213:Lymphocyte Activation / Q000235:genetics; D008565:Membrane Proteins; D016853:Muromonab-CD3 / Q000494:pharmacology; D016296:Mutagenesis / Q000235:genetics*; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D051966:Phospholipase C gamma; D010750:Phosphoproteins; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics*
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D007166:Immunosuppressive Agents; D007376:Interleukin-2; D007527:Isoenzymes; C110810:LAT protein, human; D008565:Membrane Proteins; D016853:Muromonab-CD3; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011948:Receptors, Antigen, T-Cell; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium