Membrane raft-dependent regulation of phospholipase Cgamma-1 activation in T lymphocytes.
Numerous signaling molecules associate with lipid rafts, either constitutively or after engagement of surface receptors. One such molecule, phospholipase Cgamma-1 (PLCgamma1), translocates from the cytosol to lipid rafts during T-cell receptor (TCR) signaling. To investigate the role played by lipid rafts in the activation of this molecule in T cells, an influenza virus hemagglutinin A (HA)-tagged PLCgamma1 was ectopically expressed in Jurkat T cells and targeted to these microdomains by the addition of a dual-acylation signal. Raft-targeted PLCgamma1 was constitutively tyrosine phosphorylated and induced constitutive NF-AT-dependent transcription and interleukin-2 secretion in Jurkat cells. Tyrosine phosphorylation of raft-targeted PLCgamma1 did not require Zap-70 or the interaction with the adapters Lat and Slp-76, molecules that are necessary for TCR signaling. In contrast, the Src family kinase Lck was required. Coexpression in HEK 293T cells of PLCgamma1-HA with Lck or the Tec family kinase Rlk resulted in preferential phosphorylation of raft-targeted PLCgamma1 over wild-type PLCgamma1. These data show that localization of PLCgamma1 in lipid rafts is sufficient for its activation and demonstrate a role for lipid rafts as microdomains that dynamically segregate and integrate PLCgamma1 with other signaling components.
Verí
M C
MC
Laboratory of Immunobiology, Division of Monoclonal Antibodies, Center for Biologics Evaluation & Research, National Institute o f Health, Bethesda, Maryland 20892, USA.
DeBell
K E
KE
Seminario
M C
MC
DiBaldassarre
A
A
Reischl
I
I
Rawat
R
R
Graham
L
L
Noviello
C
C
Rellahan
B L
BL
Miscia
S
S
Wange
R L
RL
Bonvini
E
E
D000215:Acylation; D048868:Adaptor Proteins, Signal Transducing; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D015151:Immunoblotting; D007376:Interleukin-2 / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D019169:Jurkat Cells; D008156:Luciferases / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D008565:Membrane Proteins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D051966:Phospholipase C gamma; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions; D013601:T-Lymphocytes / Q000201:enzymology*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D051746:ZAP-70 Protein-Tyrosine Kinase
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D007376:Interleukin-2; D007527:Isoenzymes; C110810:LAT protein, human; D008565:Membrane Proteins; D050778:NFATC Transcription Factors; D009687:Nuclear Proteins; D010750:Phosphoproteins; C092947:SLP-76 signal Transducing adaptor proteins; D014157:Transcription Factors; D014443:Tyrosine; D008156:Luciferases; D011505:Protein-Tyrosine Kinases; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium