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* The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway;
Biomedicum Helsinki, Department of Pathology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland;
Institut für Biochemie, Universität Kassel, Kassel, Germany; and
Department of Pathology, University of Turku and Turku University Central Hospital, Turku, Finland
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.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by grants from The National Programme for Research in Functional Genomics in Norway, The Research Council of Norway, The Norwegian Cancer Society, Novo Nordic Foundation Committee, the European Union (Grants QLK3-CT-2002-02149 and 037189, thera-cAMP), and the Deutsche Forschungsgemeinschaft (He1818/3).
2 Current address: Diagenic ASA, N-0663 Oslo, Norway.
3 Current address: Institute of Experimental Medical Research, Ullevål University Hospital, N-0407 Oslo, Norway.
4 Current address: Department of Pharmacology, University of California San Diego, La Jolla, CA 92093.
5 Address correspondence and reprint requests to Dr. Kjetil Taskén, The Biotechnology Centre, University of Oslo, P.O. Box 1125, Blindern, N-0317 Oslo, Norway. E-mail address: kjetil.tasken{at}biotek.uio.no
6 Abbreviations used in this paper: PKA, protein kinase A; AKAP, A-kinase anchoring protein; Cbp, Csk-binding protein; CFTR, cystic fibrosis transmembrane conductance regulator; 8-CPT, 8-(4-chlorophenylthio)-cAMP; Csk, C-terminal Src kinase; EBP50, Ezrin-Radixin-Moesin-binding phosphoprotein 50; FERM, 4.1/ezrin/radixin/moesin; IPTG, isopropyl 
-D-thiogalactoside; LAT, linker for activation of T cells; PAG, phosphoprotein associated with glycosphingolipid-enriched microdomain; PVDF, polyvinylidene difluoride; siRNA, small interfering RNA.
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