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.

cAMP negatively regulates T cell immune responses by activation of type I protein kinase A 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 type I to lipid rafts. Furthermore, Ezrin brings duanyu1529 in proximity to its downstream substrate Csk in lipid rafts by forming a multiprotein complex consisting of 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 duanyu1529 and thereby releases the 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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