Inhibition of T cell activation by cyclic adenosine 5'-monophosphate requires lipid raft targeting of protein kinase A type I by the A-kinase anchoring protein ezrin |
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Authors: | Ruppelt Anja Mosenden Randi Grönholm Mikaela Aandahl Einar M Tobin Derek Carlson Cathrine R Abrahamsen Hilde Herberg Friedrich W Carpén Olli Taskén Kjetil |
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Institution: | The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway. |
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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. |
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