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 This information is current as of October 1, 2021. Anja Ruppelt, Randi Mosenden, Mikaela Grönholm, Einar M. Aandahl, Derek Tobin, Cathrine R. Carlson, Hilde Abrahamsen, Friedrich W. Herberg, Olli Carpén and Kjetil Taskén J Immunol 2007; 179:5159-5168; ; Downloaded from doi: 10.4049/jimmunol.179.8.5159 http://www.jimmunol.org/content/179/8/5159 References This article cites 63 articles, 39 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/179/8/5159.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology 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 Ezrin1 Anja Ruppelt,* Randi Mosenden,* Mikaela Gro¨nholm,† Einar M. Aandahl,* Derek Tobin,2* Cathrine R. Carlson,3* Hilde Abrahamsen,4* Friedrich W. Herberg,‡ Olli Carpe´n,†§ and Kjetil Taske´n5* 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, immunopre- cipitation 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 Downloaded from 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 immuno- logical 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 http://www.jimmunol.org/ responses. The Journal of Immunology, 2007, 179: 5159–5168. yclic AMP and protein kinase A (PKA)6 mediate intra- expression of PKA isoforms with different biochemical properties. cellular signals from a wide variety of hormones, neuro- In addition, A-kinase anchoring proteins (AKAPs) target PKA iso- C transmitters, growth factors, and cytokines (1). cAMP forms to defined subcellular sites in close proximity to relevant and PKA signaling affect a range of cellular processes, strongly substrates (2). AKAPs may also tune the sensitivity of the signal suggesting that this signaling pathway has the capacity to provide pathway by recruiting PKA into multiprotein complexes that may rapid and precise signaling with the required sensitivity and spec- include phosphodiesterases and protein phosphatases as well as ificity. Specificity can be achieved by tissue and cell type-specific other signal proteins (3). by guest on October 1, 2021 There are two major isoforms of PKA in eukaryotes, PKA type I and II. These isoforms can be distinguished by their regulatory *The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway; †Biomedicum Helsinki, Department of Pathology, University of Helsinki and Helsinki University subunits labeled RI and RII. Activation occurs upon binding of Central Hospital, Helsinki, Finland; ‡Institut fu¨r Biochemie, Universita¨t Kassel, Kas- cAMP to the R subunits, followed by the release of the active sel, Germany; and §Department of Pathology, University of Turku and Turku Uni- catalytic subunit. PKA type II is mainly particulate and associated versity Central Hospital, Turku, Finland with AKAPs, whereas PKA type I is both soluble and particulate. Received for publication March 19, 2007. Accepted for publication August 7, 2007. During T cell activation, PKA type I is redistributed during for- 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 mation of the immunological synapse and colocalizes with the with 18 U.S.C. Section 1734 solely to indicate this fact. TCR-CD3 complex (4). PKA type I inhibits T cell activation by 1 This work was supported by grants from The National Programme for Research in activation of C-terminal Src kinase (Csk) through phosphory- Functional Genomics in Norway, The Research Council of Norway, The Norwegian lation of serine 364 in Csk (5). Active Csk subsequently phos- Cancer Society, Novo Nordic Foundation Committee, the European Union (Grants QLK3-CT-2002-02149 and 037189, thera-cAMP), and the Deutsche Forschungsge- phorylates the C-terminal inhibitory tyrosine residue of Lck and meinschaft (He1818/3). thereby acts as a negative regulator of TCR signaling. Although 2 Current address: Diagenic ASA, N-0663 Oslo, Norway. the molecular details of this process are well understood, it is 3 Current address: Institute of Experimental Medical Research, Ullevål University not known how PKA type I is localized to lipid rafts during T Hospital, N-0407 Oslo, Norway. cell activation. 4 Current address: Department of Pharmacology, University of California San Diego, Ezrin is a 78-kDa protein belonging to the Ezrin-Radixin-Moe- La Jolla, CA 92093. sin (ERM) family of proteins that play structural and regulatory 5 Address correspondence and reprint requests to Dr. Kjetil Taske´n, The Biotechnol- roles in the assembly and stabilization of specialized plasma mem- ogy Centre, University of Oslo, P.O. Box 1125, Blindern, N-0317 Oslo, Norway. E-mail address: [email protected] brane domains by linking microfilaments to the membrane. ERM 6 Abbreviations used in this paper: PKA, protein kinase A; AKAP, A-kinase anchor- proteins have a highly homologous N-terminal 4.1/ezrin/radixin/ ing protein; Cbp, Csk-binding protein; CFTR, cystic fibrosis transmembrane conduc- moesin (FERM) domain and bind directly to a number of trans- tance regulator; 8-CPT, 8-(4-chlorophenylthio)-cAMP; Csk, C-terminal Src kinase; membrane proteins, including CD44, the transmembrane protein EBP50, Ezrin-Radixin-Moesin-binding phosphoprotein 50; FERM, 4.1/ezrin/radixin/ moesin; IPTG, isopropyl ␤-D-thiogalactoside; LAT, linker for activation of T cells; Na/H exchanger NEH1, CD43, and ICAMs (6–10), in addition to PAG, phosphoprotein associated with glycosphingolipid-enriched microdomain; indirect binding to other membrane proteins via the scaffolding PVDF, polyvinylidene difluoride; siRNA, small interfering RNA. proteins ERM-binding phosphoprotein 50 (EBP50) and sodium- Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 hydrogen exchanger type 3 kinase A regulatory protein. The www.jimmunol.org 5160 EZRIN TARGETS PKA TYPE I TO T CELL LIPID RAFTS FERM domain also binds to signaling molecules in the Rho path- Purification, culture, and transfection of human peripheral way, including Rho guanine dinucleotide dissociation domain and blood T cells and Jurkat T cells Dbl (11, 12). Most of Ezrin’s interactions are dependent on con- The human leukemic T cell line Jurkat Tag was cultured and transfected, formational activation of the molecule. In a dormant state, binding as described elsewhere (5). Purification of peripheral blood CD3ϩ T cells sites for interaction partners are masked due to an intramolecular by negative selection from buffycoats of normal healthy donors (Ullevaal interaction between the FERM domain and the C terminus. Upon University Hospital Blood Center) and T cell proliferation assays was con- ducted, as described in detail elsewhere (20). For transfections of periph- phosphorylation of a C-terminal threonine (T567) by protein ki- ϫ 6 ␳ eral blood T cells, cells (5 10 ) in 0.1 ml of Nucleofection solution nase C or kinase, the intramolecular bond is released and other (Amaxa) were mixed with 1200 nM siRNA and subjected to electropora- interactions can occur (13). tion in a Nucleofector (Amaxa) following the manufacturer’s protocol. The Previous reports have suggested that Ezrin recruits type II PKA cells were expanded in complete medium and incubated for 20 h at 37°C. 5 to the secretory canaliculi in gastric parietal cells when stimulated A total of 4 ϫ 10 T cells was treated with 10 ␮M, 50 ␮M, or without cAMP 15 min (37°C) and subsequently activated by addition of 8 ␮lof by gastrin (14). Ezrin has