Abstract
cAMP negatively regulates T cell immune responses by activation of type I protein kinase A (PKA), which in turn phosphorylates and activates C-terminal Src kinase (Csk) in T cell lipid rafts. Using yeast two-hybrid screening, far-Western blot, immunoprecipitation and immunofluorescense analyses, and small interfering RNA-mediated knockdown, we identified Ezrin as the A-kinase anchoring protein that targets PKA type I to lipid rafts. Furthermore, Ezrin brings PKA in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of PKA/Ezrin/Ezrin-binding protein 50, Csk, and Csk-binding protein/phosphoprotein associated with glycosphingolipid-enriched microdomains. The complex is initially present in immunological synapses when T cells contact APCs and subsequently exits to the distal pole. Introduction of an anchoring disruptor peptide (Ht31) into T cells competes with Ezrin binding to PKA and thereby releases the cAMP/PKA type I-mediated inhibition of T cell proliferation. Finally, small interfering RNA-mediated knockdown of Ezrin abrogates cAMP regulation of IL-2. We propose that Ezrin is essential in the assembly of the cAMP-mediated regulatory pathway that modulates T cell immune responses.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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A Kinase Anchor Proteins / chemistry
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A Kinase Anchor Proteins / metabolism
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A Kinase Anchor Proteins / physiology*
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Actins / metabolism
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Cells, Cultured
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Cloning, Molecular
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Cyclic AMP / pharmacology*
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Cyclic AMP-Dependent Protein Kinase Type I / antagonists & inhibitors
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Cyclic AMP-Dependent Protein Kinase Type I / genetics
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Cyclic AMP-Dependent Protein Kinase Type I / metabolism*
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Cytoskeletal Proteins / antagonists & inhibitors
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Cytoskeletal Proteins / genetics
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Cytoskeletal Proteins / physiology*
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Cytoskeleton / immunology
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Cytoskeleton / metabolism
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Humans
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Immunosuppressive Agents / pharmacology*
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Jurkat Cells
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Membrane Microdomains / metabolism
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Membrane Microdomains / physiology*
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Peptide Mapping
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Phosphoproteins / physiology
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Protein Binding
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RNA, Small Interfering / pharmacology
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Receptor-CD3 Complex, Antigen, T-Cell / metabolism
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Signal Transduction / immunology
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Sodium-Hydrogen Exchangers / physiology
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T-Lymphocytes / enzymology
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T-Lymphocytes / immunology*
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T-Lymphocytes / metabolism*
Substances
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A Kinase Anchor Proteins
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Actins
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Cytoskeletal Proteins
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Immunosuppressive Agents
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Phosphoproteins
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RNA, Small Interfering
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Receptor-CD3 Complex, Antigen, T-Cell
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Sodium-Hydrogen Exchangers
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ezrin
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sodium-hydrogen exchanger regulatory factor
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Cyclic AMP
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Cyclic AMP-Dependent Protein Kinase Type I