Acetylation Modulates IL-2 Receptor Signaling in T Cells Taku Kuwabara, Hirotake Kasai and Motonari Kondo This information is current as J Immunol published online 31 October 2016 of September 26, 2021. http://www.jimmunol.org/content/early/2016/10/31/jimmun ol.1601174 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/10/31/jimmunol.160117 Material 4.DCSupplemental Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on September 26, 2021 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 © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 31, 2016, doi:10.4049/jimmunol.1601174 The Journal of Immunology Acetylation Modulates IL-2 Receptor Signaling in T Cells Taku Kuwabara,* Hirotake Kasai,†,1 and Motonari Kondo* Ligand binding to the cognate cytokine receptors activates intracellular signaling by recruiting protein tyrosine kinases and other protein modification enzymes. However, the roles of protein modifications other than phosphorylation remain unclear. In this study, we examine a novel regulatory mechanism of Stat5, based on its acetylation. As for phosphorylation, IL-2 induces the acetylation of signaling molecules, including Stat5, in the murine T cell line CTLL-2. Stat5 is acetylated in the cytoplasm by CREB-binding protein (CBP). Acetylated Lys696 and Lys700 on Stat5 are critical indicators for limited proteolysis, which leads to the generation of a truncated form of Stat5. In turn, the truncated form of Stat5 prevents transcription of the full-length form of Stat5. We also demonstrate that CBP physically associates with the IL-2 receptor b-chain. CBP, found in the nucleus in resting CTLL-2 cells, relocates to the cytoplasm after IL-2 stimulation in an MEK/ERK pathway–dependent manner. Thus, IL-2–mediated acetylation plays an important role in the modulation of cytokine signaling and T cell fate. The Journal of Immunology, 2016, 197: 000–000. Downloaded from nterleukin-2 and its receptor, IL-2R, constitute the prototypic into three subregions: the serine-rich (S) region (aa 267–322), the cytokine/cytokine receptor system and have been extensively acidic (A) region (aa 313–382), and the proline-rich (P) region (aa I investigated during several decades (1). IL-2 is a 15-kDa 378–525) (10). Among these three IL-2Rb-chain subdomains, the glycoprotein that is mainly produced by activated T cells (2). S region is indispensable for activating signal cascades via IL-2R Although IL-2 was initially identified as a T cell growth factor, it upon IL-2 binding, mainly because this region contains a Jak1- is now known that IL-2 is a cytokine with diverse actions. For binding site (7). http://www.jimmunol.org/ example, IL-2 is an essential factor for the development, main- The cytoplasmic tails of IL-2Rb-chains contain six tyrosine tenance, and proper suppressive function of regulatory T cells (3). residues that are immediately phosphorylated upon IL-2 binding The cognate receptor for IL-2, IL-2R, is a heterotrimeric complex to IL-2R (11–14). Of these, Y392 and Y510 are located in the P consisting of IL-2Ra (CD25), IL-2Rb (CD122), and common region; both can be included in Stat5 docking sites and are nec- g-chains (gc; CD132) (4, 5). Interaction of the IL-2Rb and gc essary for IL-2–mediated Stat5 activation (15). Of the four tyro- cytoplasmic domains upon IL-2 binding leads to the activation of sine residues in the A region, three (i.e., Y355, Y358, and Y361) the Janus family kinases, Jak1 and Jak3, which are associated with are dispensable for growth signal transduction (4). Phosphoryla- IL-2Rb and gc, respectively (6). In turn, the activated Jak kinases tion of the fourth, Y338, allows association of the Shc adaptor phosphorylate the cytoplasmic tails of the IL-2Rb-chain and gc protein (15, 16), which provides a platform for activation of the by guest on September 26, 2021 and generate docking sites for various signal molecules, which are Ras-Raf-MEK-ERK kinase pathway (noted as the MEK/ERK also phosphorylated by Jak1, Jak3, and other kinases, including an pathway in this study) and promotes cell growth (4). Addition- Src tyrosine kinase, Lck (7–9). ally, Lck binds to the A region and activates the PI3K pathway, The cytoplasmic regions of IL-2Rb-chain and gc contain unique leading to the induction of expression of antiapoptotic proteins subdomains that are composed of stretches of specific amino acid and maintenance of cell survival (17, 18). Although each of these residues. For example, the cytoplasmic tail of IL-2Rb is divided cascades is strictly regulated to maintain the balance of IL-2R signaling, the intensity of Stat5 signaling might influence the bi- ological function of IL-2, as a constitutively active form of Stat5 *Department of Molecular Immunology, Toho University School of Medicine, Tokyo has been shown to mimic the IL-2 effect by sustaining tran- 143-8540, Japan; and †Department of Immunology, Duke University Medical Center, Durham, NC 27710 scription of its target genes (19). Therefore, Stat5 plays an im- 1Current address: Department of Microbiology, Faculty of Medicine, Yamanashi portant role in IL-2R signaling. However, the molecular basis University, Chuo, Japan. underlying Stat5 regulation remains to be elucidated. ORCIDs: 0000-0001-5985-8245 (T.K.); 0000-0002-1565-8804 (M.K.). The Stat family proteins—Stat1, Stat2, Stat3, Stat4, Stat5a, Received for publication July 6, 2016. Accepted for publication October 5, 2016. Stat5b, and Stat6—are structurally and functionally related. All This work was supported by Japan Society for the Promotion of Science Grant-in-Aid Stat proteins contain N-terminal (NH2), coiled-coil, DNA-binding, for Scientific Research 25460600 (to T.K.), as well as by grants from the Takeda Src homology 2 (SH2), conserved tyrosine, and transactivation Science Foundation (to T.K.) and the Public Foundation of the Vaccination Research domains (TAD) (20). Stats are latent cytoplasmic factors that can Center (to M.K.). be activated by dimerization upon phosphorylation of the tyrosine Address correspondence and reprint requests to Dr. Taku Kuwabara, Department of Molecular Immunology, Toho University School of Medicine, 5-21-16 Omori-Nishi, on the SH2 domain by the members of the Jak family (21). Homo- Ota-ku, Tokyo 143-8540, Japan. E-mail address: [email protected] or heterodimers of Stat proteins translocate to the nucleus, The online version of this article contains supplemental material. resulting in the transcriptional activation of target genes. Two Abbreviations used in this article: A region, acidic region; gc, common g-chain; CBP, highly related Stat5 proteins, Stat5a and Stat5b, share 96% iden- CREB-binding protein; CIS, cytokine-inducible Src homology 2–containing protein; tity at the protein level and are encoded by two different genes. CIS-luc, CIS promoter–luciferase; CRM1, chromosome region maintenance 1; KAT, lysine acetyltransferase; 2KRmt, 2KR Stat5 mutant; MS/MS, tandem mass spectrom- Stat5 is activated in response to a variety of cytokines such as IL-2 etry; NPC, nuclear pore complex; P region, proline-rich region; RanBP3, Ran- and IL-3 family cytokines, growth hormone, and prolactin (22). binding protein 3; S region, serine-rich region; SH2, Src homology 2; siRNA, small Deletion of the Stat5a and Stat5b genes in mice revealed that Stat5 interfering RNA; TAD, transactivation domain; tStat5, truncated form of Stat5. plays important roles in erythropoiesis and lymphopoiesis (23). Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 Therefore, Stat5 activity needs to be strictly controlled. It has been www.jimmunol.org/cgi/doi/10.4049/jimmunol.1601174 2 ACETYLATION MODULATES Stat5 ACTIVITY reported that Stat5a is cleaved between aa 719 (tyrosine) and 720 Construction of expression vectors and site-directed (methionine) through the action of Stat proteases in myeloid mutagenesis cells (24). The truncated form of Stat5 (tStat5) lacks the TAD Mouse T cell cDNA was used as a template for PCR to amplify sequences and functions as the dominant negative form of Stat5 (24–26). encoding Stat5A. PCR was performed with ExTaq DNA polymerase Accordingly, tStat5 can dimerize with Stat5 to reduce its activ- (Takara Bio). The PCR products were ligated into the expression vector ities. However, it is not clear whether tStat5 is generated pCMV3 (Agilent Technologies). Site-directed mutagenesis was performed in lymphocytes in which IL-2 family cytokines such as IL-4 and using the QuickChange kit method (Stratagene). The following Stat5A residues were individually substituted with arginine: K582, K583, K600, IL-7 play a critical role in the development and function of T and K610, K632, K644, K675, K681, K696, and K700. All expression con- B lymphocytes. structs and mutations were verified by DNA sequencing. Although protein phosphorylation has a major regulatory role in Lysine acetyltransferase activity assay signal transduction via cell surface receptors, other posttransla- tional modifications of signaling proteins such as ubiquitination Cultured CTLL-2 cells were harvested at the indicated time points and the and SUMOylation also mediate protein signal transduction, sub- IL-2Rb-chain fraction was prepared. Lysine acetyltransferase (KAT) ac- tivity in the IL-2Rb fraction was measured using the EpiQuik HAT cellular localization, or commitment to degradation (27–31).