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Cytoplasmic-Nuclear Localization of NFAT Responsiveness By The Journal of Immunology Active Protein Kinase B Regulates TCR Responsiveness by Modulating Cytoplasmic-Nuclear Localization of NFAT and NF-␬B Proteins1 Amiya K. Patra, Shin-Young Na, and Ursula Bommhardt2 T cell activation leads to the induction of the transcription factors of the NFAT and NF-␬B families, important regulators of T cell activation and function. In this study we demonstrate that TCR/CD3-stimulated T cells from mice expressing a constitutively active form of protein kinase B (myr PKB␣) lack significant nuclear accumulation/shuttling of NFATc1 and NFATp as well as NF-␬〉p65 and RelB proteins. Notably, despite this deficit in nuclear NFAT and NF-␬B proteins, myr PKB T cells show lower activation threshold for proliferation, enhanced cell cycle progression and increased production of Th1 and Th2 cytokines similar to signals provided by CD28 costimulation. The enhanced T cell response correlates with increased expression of cyclins D3 and B1 and cytokine-induced Src homology 2 protein, and inactivation of the forkhead transcription factor FKHR. In addition, coimmunoprecipitation studies indicate a direct regulation of NFATc1 by active PKB. Together, our results demonstrate that the positive regulatory role of myr PKB on TCR responsiveness, subsequent cell division, and effector function is linked to a negative regulatory mechanism on the nuclear accumulation/shuttling of NFAT and NF-␬〉 proteins. The Journal of Immunology, 2004, 172: 4812–4820. D4 T cell activation, expansion, and differentiation re- by the immunosuppressants cyclosporin A (CsA)3 or FK506 pre- quire recognition of specific Ag presented by MHC class vents activation and nuclear entry of NFAT. The shuttling of C II molecules on APCs in context with costimulatory sig- NFATs between nucleus and cytoplasm is finely balanced and pre- nals, such as those provided by CD28, inducible costimulator, cisely controlled. Kinases implicated in the rephosphorylation of CD40 ligand, or OX40 (CD134) (1). TCR-initiated signaling leads NFATs in the nucleus, thereby rendering them inactive and trig- to the induction of a complex array of kinases, phosphatases, and gering their nuclear export, include glycogen synthase kinase 3 downstream transcription factors, which regulate cellular functions (GSK3) and the mitogen-activated protein kinases (MAPKs), Ja- such as metabolism, cell cycle, survival, and cell death, thereby nus N-terminal kinase (JNK) and p38. ␬ controlling appropriate T cell responses. The members of the NF- B/Rel proteins can exist as homo- or heterodimers com- NFAT and NF-␬B/Rel families of transcription factors are criti- posed of almost any combination of the five mammalian family members, c-Rel, p65/RelA, RelB, p50/NF-␬B1, and p52/NF-␬B2 cally involved in many of these cellular processes, and their dys- (5, 6). Dimerization as well as DNA binding are mediated by the regulation is usually connected with the development of patho- conserved N-terminal Rel homology domain. Although most physiological states, including oncogenesis. NF-␬B dimers are transcriptional activators, the p50/p50 and p52/ Of the four NFAT family members that share significant se- p52 homodimers can repress target gene transcription. In resting quence and functional similarity, NFATc1, NFATc2/NFATp, and unstimulated cells, most NF-␬B dimers are complexed with an NFATc3 mainly function in the immune system, whereas NFATc4 inhibitory protein of the I␬B family, which masks one of the dual is involved in regulation of cardiac hypertrophy and hippocampal nuclear localization signals (NLS) on NF-␬B, thereby, in the sim- neuronal signaling (2–4). NFAT proteins in resting T cells are plistic model, retaining them in the cytoplasm. In response to a mainly located in the cytoplasm, in a highly phosphorylated form. variety of stimuli, including TCR and CD28 costimulation, I␬B␣ The calcium calmodulin-dependent phosphatase calcineurin is and -␤, the prototypes of the I␬B family, are phosphorylated by the known to dephosphorylate serine residues within the N terminus of I␬B kinase complex, followed by their ubiquitination and degra- NFAT proteins, thereby facilitating their entry into the nucleus and dation in the proteasome. The release of I␬Bs unmasks the NLS subsequent target gene expression. Inhibition of this phosphatase and allows NF-␬B to enter the nucleus (7). Protein kinase B (PKB) is a serine/threonine kinase that in lym- phocytes is activated by TCR and CD28 costimulation, insulin, cytokines, and chemokines, among others (8). By positively or Institute of Virology and Immunobiology, University of Wu¨rzburg, Wu¨rzburg, Ger- negatively regulating anti- and proapoptotic molecules such as many Bcl-xL or Bad, cell cycle regulators, kinases, and transcription fac- Received for publication October 24, 2003. Accepted for publication February tors such as GSK3 and NF-␬B, respectively, PKB exerts pleiotro- 5, 2004. pic effects on cell proliferation, survival, and cell death (9–11). 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 a grant to the Forschergruppe 303 TPA3 from the 3 Abbreviations used in this paper: CsA, cyclosporin A; CE, cytoplasmic extract; CIS, Deutsche Forschungsgemeinschaft. cytokine-induced Src homology 2 protein; GSK3, glycogen synthase kinase 3; JNK, 2 Address correspondence and reprint requests to Dr. Ursula Bommhardt, Institute of Janus N-terminal kinase; MAPK, mitogen-activated protein kinase; NE, nuclear ex- Virology and Immunobiology, Versbacher Strasse 7, D-97078 Wu¨rzburg, Germany. tract; NLS, nuclear localization signal; PI3K, phosphatidylinositol 3-kinase; PKB, E-mail address: [email protected] protein kinase B; pPKB, phospho-PKB; tg, transgenic; wt, wild type. Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 4813 Overexpression of active PKB is connected with induction of in- Western blot analysis and immunoprecipitation flammatory processes and development of tumors (12). For preparation of nuclear and cytoplasmic cell extracts (NE and CE, re- We have previously shown that the expression of a constitu- spectively), 2 ϫ 107 CD4ϩ T cells were stimulated with plate-bound anti- tively active form of PKB (myr PKB) in transgenic mice influ- CD3 mAb alone or in combination with anti-CD28 mAb in the absence or ences thymocyte selection, leads to an accumulation of CD4ϩ T the presence of CsA for the time periods indicated. Kinase inhibitors ␮ ␮ ␮ cells in peripheral lymphoid organs, and enhances their survival in PD98059 (100 M), LY294002 (20 M), SB202190 (40 M), and stau- rosporine (10 nM; all from Calbiochem) were added 1 h before stimulation the presence of various apoptosis-inducing reagents (13). In this of cells. Cells were harvested, washed twice in cold PBS, and suspended in study we examined the influence of PKB on T cell proliferation 200 ␮l of buffer A (10 mM KCl, 10 mM HEPES (pH 7.9), 0.1 mM EGTA and effector function and have identified a new role for PKB in (pH 7.9), 0.1 mM EDTA (pH 7.9), protease inhibitor mixture (Roche, regulating the nuclear translocation of NFAT and NF-␬B proteins. Basel, Switzerland), 1 mM DTT, 1 mM sodium orthovanadate, and 0.5% Nonidet P-40) for 3 min on ice. After immediate centrifugation at 14,000 Notably, the PKB-mediated deficit in nuclear accumulation of rpm, the supernatant was collected as CE. The pellet was washed twice in these transcription factors does not block T cell activation, but, buffer A and incubated with 100 ␮l of buffer C (420 mM NaCl, 20 mM rather, is connected with lower thresholds for proliferative re- HEPES (pH 7.9), 1 mM EGTA (pH 7.9), 1 mM EDTA (pH 7.9), protease sponses, enhanced cell cycle progression, and increased produc- inhibitor mixture, 1 mM DTT, and 1 mM sodium orthovanadate) for 2 h tion of Th1 and Th2 cytokines. The down-modulation of nuclear with constant shaking at 4°C. After incubation, NE was collected by cen- ␬ trifugation at 14,000 rpm for 20 min. The protein concentrations of CE and activities of NF- B and NFAT family members thus defines a NE were determined using Bradford’s reagent (Bio-Rad, Munich, Ger- novel regulatory mechanism by which PKB exerts positive effects many). Ten micrograms of protein for each sample for both CE and NE on T cell function, which, as discussed, may be one underlying was separated on 8–12% SDS-PAGE and electroblotted to nitrocellulose mechanism contributing to tumor development. membranes. Specific proteins were detected by Western blot analysis using the following primary Abs: anti-PKB, anti-phospho-PKB (Ser473), anti- phospho-GSK3␣␤ (Ser21/9), anti-phospho-FKHR (Ser256), anti-phospho- Materials and Methods JNK, and anti-phospho-p38 (all from Cell Signaling, Beverly, MA); anti- NFATc1 (Alexis, Carlsbad, CA); anti-NFATp (a gift from Dr. A. Rao, Mice Center for Blood Research, Harvard Medical School, Boston, MA); and Human CD2-myr PKB␣ (myr PKB) transgenic (tg) mice have been de- anti-NF-␬Bp65, anti-p50, anti-RelB, anti-cyclins B1 and D3, anti-CIS, and scribed previously (13). Myr PKB tg and wild-type (wt) mice used in this anti-I␬B␣ (all from Santa Cruz Biotechnology, Santa Cruz, CA). Primary study (backcrossed more than five generations to C57BL/6J mice) were Abs were detected by goat anti-rabbit (Santa Cruz Biotechnology), goat 6–8 wk of age. anti-mouse, or rabbit anti-goat Abs coupled with HRP (both from Jackson ImmunoResearch Laboratories, West Grove, PA) and ECL (Pierce, Rock- ford, IL). Blots were reprobed with anti-actin Ab (Santa Cruz Biotechnol- Isolation of T cells and flow cytometry ogy) to control protein loading in the case of CE. Reprobing NE with Peripheral and mesenteric lymph nodes were homogenized through nylon anti-actin Ab showed that NE were free from cytoplasmic protein contam- cell strainers to obtain single-cell suspensions.
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