Abstract
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.
Publication types
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Acylation
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Adaptor Proteins, Signal Transducing*
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Calcium / metabolism
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Carrier Proteins / metabolism
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Cell Line
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DNA-Binding Proteins / metabolism
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Electrophoresis, Polyacrylamide Gel
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Enzyme Activation
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Enzyme-Linked Immunosorbent Assay
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Gene Expression Regulation, Enzymologic
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Humans
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Immunoblotting
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Interleukin-2 / metabolism
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Isoenzymes / metabolism*
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Jurkat Cells
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Luciferases / metabolism
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Membrane Microdomains / metabolism*
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Membrane Proteins*
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Microscopy, Confocal
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Microscopy, Fluorescence
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NFATC Transcription Factors
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Nuclear Proteins*
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Phospholipase C gamma
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Phosphoproteins / metabolism
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Phosphorylation
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Plasmids / metabolism
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Precipitin Tests
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Protein Transport
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Protein-Tyrosine Kinases / metabolism
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Signal Transduction
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Subcellular Fractions
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T-Lymphocytes / enzymology*
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Transcription Factors / metabolism
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Transcription, Genetic
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Transcriptional Activation
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Transfection
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Type C Phospholipases / metabolism*
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Tyrosine / metabolism
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ZAP-70 Protein-Tyrosine Kinase
Substances
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Adaptor Proteins, Signal Transducing
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Carrier Proteins
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DNA-Binding Proteins
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Interleukin-2
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Isoenzymes
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LAT protein, human
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Membrane Proteins
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NFATC Transcription Factors
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Nuclear Proteins
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Phosphoproteins
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SLP-76 signal Transducing adaptor proteins
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Transcription Factors
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Tyrosine
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Luciferases
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Protein-Tyrosine Kinases
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ZAP-70 Protein-Tyrosine Kinase
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ZAP70 protein, human
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Type C Phospholipases
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Phospholipase C gamma
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Calcium