Abstract
The Src family kinase Lck is essential for T cell Ag receptor-mediated signaling. In this study, we report the effects of acute elimination of Lck in Jurkat TAg and primary T cells using RNA interference mediated by short-interfering RNAs. In cells with Lck knockdown (kd), proximal TCR signaling was strongly suppressed as indicated by reduced zeta-chain phosphorylation and intracellular calcium mobilization. However, we observed sustained and elevated phosphorylation of ERK1/2 in Lck kd cells 30 min to 2 h after stimulation. Downstream effects on immune function as determined by activation of a NFAT-AP-1 reporter, and TCR/CD28-stimulated IL-2 secretion were strongly augmented in Jurkat and primary T cells, respectively. As expected, overexpression of SHP-1 in Jurkat cells inhibited TCR-induced NFAT-AP-1 activation, but this effect could be overcome by simultaneous kd of Lck. Furthermore, acute elimination of Lck also suppressed TCR-mediated activation of SHP-1, suggesting the possible role of SHP-1 in a negative feedback loop originating from Lck. This report underscores Lck as an important mediator of proximal TCR signaling, but also indicates a suppressive role on downstream immune function.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Blotting, Northern
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CD28 Antigens / immunology
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CD28 Antigens / metabolism
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Calcium / metabolism
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Cell Line, Tumor
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Enzyme Activation / immunology
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Humans
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Immunoprecipitation
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Interleukin-2 / immunology
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Interleukin-2 / metabolism
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Intracellular Signaling Peptides and Proteins / immunology
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Intracellular Signaling Peptides and Proteins / metabolism
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Jurkat Cells
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Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / deficiency*
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Lymphocyte Specific Protein Tyrosine Kinase p56(lck) / immunology*
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Mitogen-Activated Protein Kinase 1 / immunology
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Mitogen-Activated Protein Kinase 1 / metabolism
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Mitogen-Activated Protein Kinase 3 / immunology
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Mitogen-Activated Protein Kinase 3 / metabolism
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NFATC Transcription Factors / immunology
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NFATC Transcription Factors / metabolism
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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Protein Tyrosine Phosphatases / immunology
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Protein Tyrosine Phosphatases / metabolism
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RNA, Small Interfering
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Receptors, Antigen, T-Cell / immunology*
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Signal Transduction / immunology*
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T-Lymphocytes / immunology*
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Transfection
Substances
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CD28 Antigens
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Interleukin-2
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Intracellular Signaling Peptides and Proteins
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NFATC Transcription Factors
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RNA, Small Interfering
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Receptors, Antigen, T-Cell
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Lymphocyte Specific Protein Tyrosine Kinase p56(lck)
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Mitogen-Activated Protein Kinase 1
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Mitogen-Activated Protein Kinase 3
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PTPN6 protein, human
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Protein Tyrosine Phosphatase, Non-Receptor Type 6
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Protein Tyrosine Phosphatases
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Calcium