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: 4812Ð4820.

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 (2Ð4). 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 (9Ð11). 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.

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 staurosporine (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 8Ð12% 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 6Ð8 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. For isolation of CD4ϩ or ination. Controls shown in the figures as n.s. for NE and CE are nonspecific ϩ ␬ ␣ CD8 T cells, total lymph node cells were treated with a mixture of rat ones used as protein loading controls. For I B analysis, cells were pre- ␮ anti-mouse CD4 (GK1.5) or anti-CD8 (53-6.73) and anti-CD19 (1D3), treated with cycloheximide (50 g/ml; Sigma-Aldrich, St. Louis, MO) for ␮ anti-MHC class II (2G9), and anti-NK1.1 (4D11) hybridoma supernatants 15 min before stimulation of cells with soluble anti-CD3 mAb (1 g/ml) in ␮ for 25 min on ice and then washed twice in cold PBS. BioMag anti-rat IgG combination with anti-CD28 mAb (5 g/ml) for the indicated time periods. coupled to magnetic beads (Qiagen, Hilden, Germany) was added for 25 For long term stimulation, cells were activated with plate-bound anti-CD3 ϩ ϩ ␮ ϫ 6 min at 4¡C, and CD4 or CD8 T cells were collected by negative selec- mAb (5 g/ml). Total protein extracts were prepared from 3 10 cells tion using magnetic cell separation. The purity of isolated CD4ϩ or CD8ϩ and were analyzed by Western blot as described previously (13). ϫ 7 ϩ T cells was analyzed for each experiment by flow cytometry and routinely For immunoprecipitation experiments, 1 10 CD4 T cells from myr was 90Ð95%. Abs for flow cytometry were purchased from BD PharMin- PKB tg or wt mice were lysed in buffer A, and cytoplasmic protein extracts 473 gen (San Diego, CA) as FITC-, PE-, or biotin-labeled conjugates; the latter were incubated with anti-PKB, anti-phospho-PKB (pPKB) (Ser ), anti- were revealed with streptavidin-CyChrome (BD PharMingen). Cells were Lamin A (all from Cell Signaling), or anti-NFATc1 Ab (Alexis) overnight ␮ stained using standard procedure and were analyzed on a FACSCalibur 4¡C with shaking. After addition of 25 l of 50% protein G-Sepharose using CellQuest software (BD Biosciences, Mountain View, CA). (Amersham Pharmacia Biotech, Uppsala, Sweden) for1hat4¡C, extracts were centrifuged at 10,000 rpm for 1 min, and immunoprecipitates were washed four times with buffer A. After the final wash, pellets were boiled Proliferation and cytokine assays. in loading buffer, and supernatants were resolved on 8% SDS-PAGE. Co- For proliferation assays, 2 ϫ 105 purified CD4ϩ or CD8ϩ T cells were precipitation was analyzed for association of NFATc1 with PKB or pPKB. cultured in triplicate in complete RPMI 1640 medium supplemented with 5% FCS in 96-well plates coated with anti-CD3 mAb (145.2C11; BD PharMingen) or anti-CD3 plus anti-CD28 mAbs (37.51; BD PharMingen). Results CsA (Calbiochem, Schwalbach, Germany) was added at the beginning of Active PKB lowers the activation threshold of CD4ϩ and CD8ϩ culture at the concentrations indicated. After 48 h, cells were pulsed with T cells and enables proliferation in the presence of CsA 1 ␮Ci of [3H]thymidine/well (ICN Pharmaceuticals, Asse-Relegem, Bel- gium) for 12 h. Measurement of cytokine production was made in super- To examine the effects of constitutively active PKB (myr PKB) on natants collected 24 h after stimulation of CD4ϩ T cells using ELISA or activation and proliferation of peripheral T cells, CD4ϩ and CD8ϩ cytokine bead array (BD PharMingen) according to the manufacturer’s T cells from wt and myr PKB tg mice were activated with either instructions. high (10 ␮g/ml) or limiting (1 ␮g/ml) concentrations of anti-TCR/ CD3 mAb alone or in the presence of CD28 costimulation. As Cell cycle analysis shown in Fig. 1, strong TCR/CD3 stimuli led to comparable pro- For analysis of cell division, purified T cells (1 ϫ 107/ml) were washed liferation, whereas weak TCR/CD3 signals induced significant twice with PBS and labeled with CFSE (Molecular Probes Europe, Leiden, proliferation only in myr PKB CD4ϩ and CD8ϩ T cells. CD28 The Netherlands) at a final concentration of 2 ␮M in PBS for 5 min at room temperature. Cells were washed twice with RPMI 1640 medium containing costimulation had only small enhancing effects in tg T cells, 10% FCS and thereafter cultured in RPMI 1640/5% FCS medium at a whereas in wt T cells, proliferation was increased 10- to 20-fold. density of 2 ϫ 106 cells/ml in 96-well plates. Cells were activated by In addition, in wt cells the response to TCR engagement alone or plate-bound anti-CD3 mAb (10 ␮g/ml) alone or anti-CD3 (1 ␮g/ml) plus to CD28 costimulation in combination with weak TCR engage- anti-CD28 mAb (5 ␮g/ml) for 2 or 3 days and analyzed by FACS. For short term activation, CFSE-labeled cells were stimulated with plate-bound anti- ment was totally blocked in the presence of CsA, whereas myr CD3 mAb (5 ␮g/ml) for 12 or 18 h, harvested, and recultured in medium PKB T cells showed significant proliferation under these condi- without further stimulus until 24 or 48 h. tions. Thus, myr PKB signaling lowers the threshold for activation 4814 MYR PKB REGULATES NUCLEAR LOCALIZATION OF NFAT/NF␬B PROTEINS

CD4ϩ T cells. These data attribute an augmentative role to PKB in cell cycle progression of CD4ϩ and CD8ϩ T cells by providing CD28-like costimulatory signals. To further dissect the role of PKB in cell cycle progression, CD4ϩ T cells from tg and wt mice were activated with anti-CD3 mAb for 12 or 18 h and were recultured without further stimulus until 24 or 48 h. As shown in Fig. 2C, a 12-h stimulation pulse was sufﬁcient for 16% of myr PKB CD4ϩ T cells to enter the cell cycle and divide once within 24 h; on day 2, 54% of tg cells had undergone one to three cell divisions. In stark contrast, stimulation of wt CD4ϩ T cells for 12 h induced only 5% of cells to divide once within 24 h, and ϳ3-fold fewer cells had divided twice or three times on day 2 compared with tg cells. When the activating stimulus was extended to 18 h, 19% of wt cells showed one round of cell division on day 2, similar to what was observed for tg cells on day 1. Together, these data clearly show that myr PKB strongly enhances cell cycle entry and progression and also sustains cell division. To deﬁne molecular mechanisms underlying the positive effect of myr PKB on the cell cycle, we analyzed the expression of cy-

clins D3 and B1, proteins regulating cell cycle entry and G2 phase transition, respectively. As shown in Fig. 2D, myr PKB accelerated and enhanced the induction of cyclin D3 and also increased cyclin B1 expression in activated T cells, thus providing a link FIGURE 1. Active PKB lowers the threshold for T cell activation and between active PKB and the observed enhanced cell division. We confers significant CsA resistance in proliferation. Purified CD4ϩ (A) and also studied inactivation of the forkhead transcription factor CD8ϩ (B) T cells from wt and tg mice were cultured in medium only or FKHR, which, like other forkhead family members, is located in stimulated with plate-bound anti-CD3 mAb (CD3) at a concentration of 10 the nucleus in resting cells and is critically involved in cell cycle or 1 ␮g/ml or with anti-CD3 (1 ␮g/ml) plus anti-CD28 (CD28; 5 ␮g/ml) regulation. In various cell lines phosphorylation of these factors by 3 mAbs in the presence or the absence of CsA (100 ng/ml). [ H]thymidine PKB leads to their nuclear exclusion and thereby facilitates cell incorporation was measured 48 h after initiation of cultures. Data show cycle entry and progression into M phase (14). In nuclear and averages from triplicate cultures of two wt and tg mice each and are rep- cytoplasmic extracts of unstimulated wt cells (Fig. 2E, upper left resentative for three experiments. panels), phosphorylated forms of FKHR could hardly be detected, whereas after TCR/CD3 engagement, phospho-FKHR was strongly present in both fractions. Interestingly, cytoplasmic phos- by providing costimulatory signals similar to those induced by phorylated FKHR was already prominent in unstimulated myr CD28 and confers partial resistance to CsA treatment with regard PKB CD4ϩ T cells, and only low levels of nuclear FKHR ap- to proliferation. peared in stimulated cells (Fig. 2E, upper right panels), suggesting Myr PKB enhances cell cycle progression similar to CD28 that most FKHR proteins had been phosphorylated and shuttled costimulation out of the nucleus. This correlates with tg PKB also being located in the nucleus (Fig. 2E, lower panels). Thus, increased or consti- Next we analyzed the effect of myr PKB on cell cycle by labeling tutive inactivation and nuclear export of FKHR is one component T cells with CFSE, which allows tracing the number of cell divi- that contributes to the enhanced cell cycle progression promoted sions undergone at any particular time point. As shown in Fig. 2A, ϩ by myr PKB. 2 days after anti-CD3 activation, ϳ30% of wt and myr PKB CD4 T cells had divided once; however, 3-fold more (26%) myr PKB ϩ CD4 cells had progressed through a second cell division com- ϩ Enhanced Th1 and Th2 cytokine production in myr PKB CD4 pared with wt cells (9%). After activation with anti-CD3 plus anti- T cells CD28 mAbs, cell cycle in wt cells resembled that of myr PKB CD4ϩ T cells stimulated with anti-CD3 mAb only. In contrast, T cell growth and apoptosis are highly dependent on various cy- costimulation had only small enhancing effects on cell cycle pro- tokines. To better understand why myr PKB T cells could prolif- gression in myr PKB CD4ϩ T cells. Comparable results were ob- erate after addition of CsA, we first surveyed the production of served for day 3 cultures, in which the majority of myr PKB CD4ϩ IL-2, which is an important growth factor in T cell activation and T cells activated with either anti-CD3 mAb alone or in combina- in an autocrine fashion regulates the expression of the high affinity tion with anti-CD28 mAb had undergone four to six cell cycles, IL-2R (15, 16). We found that surface expression of the IL-2R corresponding to the same pattern from CD28-costimulated wt ␣-chain (CD25) in CD3-stimulated wt cells was down-regulated to cells. The wt cells stimulated with anti-CD3 mAb only showed a levels found in unstimulated cells when CsA was added to the ϩ less synchronous cell division profile, with ϳ20% of cells each cultures. In contrast, a higher percentage of myr PKB CD4 T having divided one to three times and with only 14% of cells cells still expressed high levels of CD25 in the presence of CsA having completed five or six cell divisions compared with 54% of (data not shown). This suggested that myr PKB signaling allows myr PKB CD4ϩ T cells. As evident from Fig. 2B, myr PKB shows sufficient production of IL-2 in the presence of CsA, thus main- the same enhancing effects on cell cycle progression in CD8ϩ T taining proliferation. Indeed, as shown in Fig. 3A, myr PKB CD4ϩ cells; the observed advantage in the progression of the cell cycle in T cells produced significant amounts of IL-2 when only low TCR the case of CD8ϩ T cells is even more prominent than that in signals were provided and when CsA was administered. The The Journal of Immunology 4815

FIGURE 2. Active PKB enhances cell cycle progression. A and B, Wt and myr PKB CD4ϩ (A) and CD8ϩ (B) T cells labeled with CFSE were stimulated with anti-CD3 mAb only or with anti-CD3 plus anti-CD28 mAbs for 2 or 3 days as indicated. Cell division was analyzed by FACS gating on viable cells on the basis of forward and side scatter characteristics. Numbers at the top of histograms indicate the number of cell divisions undergone. Panels on the right give the percentage of wt and tg cells that have undergone the indicated numbers of cell divisions. C, Faster cell cycle entry of tg CD4ϩ T cells. CFSE-labeled CD4ϩ T cells from wt and tg mice were activated with plate-bound anti-CD3 mAb for 12 or 18 h and then cultured without further stimulus until 24 or 48 h. Cell division was determined as described above. D, Myr PKB positively regulates the expression of cell cycle regulators. Wt and tg CD4ϩ T cells were stimulated by TCR/CD3 ligation for the time periods indicated, and the expression of cyclin D3 and B1 proteins in total cell extracts was analyzed by Western blot. E, Enhanced phosphorylation of FKHR in tg T cells. Western blot analysis of pFKHR levels in CE and NE from wt and tg CD4ϩ T cells 8 h after stimulation with anti-CD3 mAb (5 ␮g/ml; upper panels). n.s., Nonspeciﬁc loading control. The lower panels show expression of endogenous (end) and tg PKB in CE and NE from unstimulated wt and myr PKB tg CD4ϩ T cells.

amounts of IL-2 produced under these conditions, therefore, would only obvious when CD28 costimulatory signal was provided in be sufﬁcient for myr PKB T cells to induce high afﬁnity IL-2R and addition to low TCR engagement, and under these conditions IL-2 to sustain expansion. In contrast, IL-2 production of wt cells was production was completely ablated by CsA treatment. 4816 MYR PKB REGULATES NUCLEAR LOCALIZATION OF NFAT/NF␬B PROTEINS

(Fig. 3B). Positive regulation of CIS by active PKB thus may contribute to the enhanced functional responses of myr PKB T cells.

Myr PKB impairs nuclear accumulation of NFAT proteins T cell activation involves induction of the transcription factors NFATc1 and NFATp, which play important roles in cytokine gene induction, Th cell differentiation, and apoptosis (2Ð4, 21, 22). Sus- tained mobilization of intracellular Ca2ϩ ion triggered by TCR engagement activates calcineurin, which, in turn, dephosphorylates the phosphoserine residues in the NFAT homology region of NFATs and unmasks the NLS, resulting in their entry into the nucleus. In view of the effects of myr PKB on partial resistance to CsA treatment in proliferation and cytokine production, we examined whether this phenotype could be linked to increased or con- stitutive activation of NFAT proteins. As shown in Fig. 4A,NE and CE of CD4ϩ T cells from myr PKB and wt mice stimulated with anti-CD3 mAb alone or anti-CD3 plus anti-CD28 mAbs in the absence or the presence of CsA were analyzed by Western blot

FIGURE 3. A, Myr PKB promotes the production of Th1 and Th2 cytokines. CD4ϩ T cells from wt and tg mice were left unstimulated or were treated with anti-CD3 mAb (1 ␮g/ml) without or with CD28 costimulation (5 ␮g/ml) in the presence or the absence of CsA (100 ng/ml). Secretion of the indicated cytokines was determined after 24 h of culture. B, Enhanced expression of CIS protein in myr PKB T cells. Wt and tg CD4ϩ T cells were stimulated with anti-CD3 mAb (5 ␮g/ml) for the indicated time points, and CIS expression in total cellular extracts was determined by Western blot. Actin expression is given as a control for protein loading.

Furthermore, under the same stimulatory conditions we determined the production of other cytokines. We found that besides IL-2, other Th1 cytokines, such as IFN-␥ and TNF-␣, and notably also Th2 cytokines such as IL-4 and IL-5 were increased in cultures from myr PKB CD4ϩ T cells, even in the presence of CsA (Fig. 3A). As RNase protection assays revealed that mRNAs for other Th2 cytokines, including IL-10 and IL-13, were also enhanced after TCR/CD3 stimulation (data not shown), myr PKB has an overall positive regulatory effect on Th1 and Th2 cytokine production. Recently, a number of reports have emphasized that members of the suppressor of cytokine signaling family regulate cytokine signal transduction, thereby regulating immune responses and homeostasis (17Ð19). In this context, the expression of the family member cytokine-induced Src homology 2 protein (CIS) is in- FIGURE 4. Myr PKB impairs nuclear accumulation of NFAT proteins. duced in T cells by TCR stimulation, and overexpression of CIS in ϩ A, Wt and tg CD4 T cells were stimulated with anti-CD3 mAbs alone or the CD4 T cell lineage enhances proliferative responses and sur- in combination with anti-CD28 mAbs with or without CsA (100 ng/ml) for vival of T cells (20). In view of the positive regulatory effects of 8h(upper panel)or16h(lower panel). NE and CE were analyzed for myr PKB on proliferation and cytokine production, we studied the nuclear translocation of NFATc1 in Western blot. B, CE and NE from wt expression of CIS and detected accelerated and enhanced expres- and tg CD4ϩ T cells stimulated for 4 h, as described in A, were probed for ϩ sion of CIS in myr PKB CD4 T cells compared with wt cells NFATp activation and nuclear localization. The Journal of Immunology 4817 for nuclear translocation of NFATc1. Eight hours after activation, proteins. In particular, overexpression of GSK3 has been shown to most induced NFATc1 protein in wt CD4ϩ T cells was translo- signiﬁcantly reduce nuclear import, whereas inhibition of GSK3 cated to the nucleus, and nuclear entry was clearly abolished in the slows nuclear export of NFATc1 (23). As shown in Fig. 5A,8h presence of CsA. Strikingly, whereas in cytoplasmic extracts of after TCR/CD3 activation, phosphorylated GSK3␤ is clearly de- myr PKB CD4ϩ T cells, NFATc1 induction after anti-CD3 and tectable in cytoplasmic and nuclear extracts of myr PKB T cells, anti-CD3 plus anti-CD28 costimulation was obvious, nuclear but is detected only very weakly or not at all in case of cytoplasmic translocation of NFATc1 was hardly detectable (Fig. 4A, upper and nuclear extracts of wt cells. This correlates with myr PKB panel). At earlier time points of activation, e.g., at 4 h (data not being located in the nucleus (see Fig. 2E) and published data that shown), nuclear translocation of NFATc1 could also not be de- active PKB can phosphorylate GSK3␤, thereby leading to its in- tected and was drastically diminished even 16 h after activation activation (24). However, although inactivation of GSK3␤ by (Fig. 4A, lower panel). phosphorylation through PKB should foster nuclear retention of To test whether this intriguing effect of myr PKB on nuclear NFAT, the opposite, namely, grossly diminished nuclear NFATc1 shuttling was restricted to NFATc1, we examined NFATp activa- or NFATp, was detected in myr PKB T cells. We therefore ana- tion (Fig. 4B). Similarly, although induction of NFATp in the cy- lyzed cytoplasmic and nuclear levels of active JNK and p38, which toplasm was evident, nuclear translocation of NFATp was hardly by rephosphorylating NFAT would induce its nuclear extrusion. detectable in myr PKB CD4ϩ T cells, similar to the blockade seen As shown in Fig. 5A, myr PKB T cells elicit elevated levels of in CsA-treated wt cells. Thus, myr PKB promotes proliferation and phosphorylated p38 and JNK proteins compared with wt cells cytokine production in T cells despite immensely diminished nu- upon stimulation. These data suggest that rephosphorylation of clear levels of NFATc1 and NFATp. NFAT by these kinases could be involved in the altered cytoplasmic-nuclear shuttling of NFAT in myr PKB T cells. Enhanced nuclear levels of p-p38, pJNK, and pGSK3 in myr PKB T cells Inhibition of MAPK/SAPK and phosphatidylinositol 3-kinase As phosphorylation and dephosphorylation are the basis of NFAT (PI3K) pathways fails to repair the altered nuclear shuttling of translocation and subsequent gene expression, we examined the NFATc1 activities of JNK, p38, and GSK3␤, kinases that have been impli- In view of the reported involvement of JNK, p38, and PI3K (25) cated in the regulation of cytoplasmic-nuclear shuttling of NFAT in opposing nuclear translocation of NFAT and our observation

FIGURE 5. A, Active PKB enhances phosphorylation of MAPKs, p38 and JNK, and of GSK3␤. Wt and tg CD4ϩ T cells were cultured in medium only or were activated with anti-CD3 mAb (5 ␮g/ml) for 8 h. The level of phosphorylation of the indicated kinases in CE (left panel) and NE (right panel) was analyzed by Western blot using phospho-speciﬁc Abs. B, Inhibition of MAPKs and PI3K does not lead to higher nuclear localization of NFATc1. Wt and tg CD4ϩ T cells were stimulated with anti-CD3 mAb for4hintheabsence or the presence of the kinase inhibitors, PD98059 (PD), SB202190 (SB), LY294002 (LY), or staurosporine (stau), and NE was analyzed for NFATc1 nuclear translocation. C and D, PKB coprecipitates with NFATc1. C, Cytoplasmic protein extracts from unstimulated myr PKB CD4ϩ T cells were immunoprecipitated with either anti-phospho-PKB or anti-NFATc1 Abs. Immune complexes were resolved by Western blot and probed for interaction with NFATc1 and pPKB, respectively. D, Interaction of NFATc1 with endogenous PKB in unstimulated and stimulated (5 ␮g/ml anti-CD3 mAb for 4 h) wt CD4ϩ T cells was analyzed in immunoprecipitation assays. Anti-Lamin A Ab was used to control the speciﬁcity of the interaction. 4818 MYR PKB REGULATES NUCLEAR LOCALIZATION OF NFAT/NF␬B PROTEINS

regarding higher pJNK and p-p38 levels in myr PKB T cells, we sulted in pull-down of NFATc1. Together these data show that studied whether inhibition of these signaling pathways could repair transgenic PKB as well as endogenous PKB interact with the alteration. For this purpose we treated cells with the pharma- NFATc1. cological reagents PD98059, SB202190, and LY294002, inhibitors of MAPKs and PI3K, respectively, as well as with staurospor- Myr PKB impairs TCR-induced nuclear translocation of RelB ␬ ␬ ine, a broad spectrum inhibitor of kinases. The concentrations of and NF- Bp65, but not NF- Bp50, proteins inhibitors used in these experiments clearly blocked induction of Next we investigated whether the striking effect on nuclear trans- CD25 expression at4hofstimulation, the time point when protein location is confined to NFAT proteins or also occurs for members extracts were prepared, and also inhibited proliferation when mea- of the NF-␬B family, which in various cell systems and with dif- sured by [3H]thymidine incorporation at 24 h, indicating that they ferent stimuli have been shown to become activated via PKB (26Ð effectively blocked the respective kinases (data not shown). As 28). Similar to NFAT analysis, CE and NE from wt and myr PKB none of these inhibitors led to higher nuclear accumulation of CD4ϩ T cells stimulated in the presence or the absence of CsA NFATc1 (Fig. 5B), the deficiency in nuclear NFATc1 in myr PKB were tested for nuclear translocation of NF-␬Bp50 and p65 pro- CD4ϩ T cells is regulated by mechanisms not involving PI3K or teins. Induction of cytoplasmic NF-␬Bp50, nuclear translocation, the MAPKs, extracellular signal-regulated kinase, p38, and JNK. and inhibitory effects of CsA were similar for wt and myr PKB We therefore hypothesized whether PKB itself could act as an CD4ϩ T cells (Fig. 6A). However, in strong contrast to NF-␬Bp50, NFAT kinase and investigated its direct interaction with NFATc1. nuclear translocation of NF-␬Bp65 in the case of myr PKB CD4ϩ Immunoprecipitation experiments, as shown in Fig. 5C, clearly T cells at 16 h of activation was markedly diminished, with nuclear detected in vivo association of pPKB with NFATc1 in cytoplasmic NF-␬Bp65 levels similar to those in unstimulated or CsA-treated extracts from tg CD4ϩ T cells. Whether this interaction also occurs wt cells. Similar to NFAT proteins, nuclear NF-␬Bp65 was also in wt T cells was analyzed by immunoprecipitation studies with absent or strongly diminished at earlier time points after activation anti-pPKB and anti-PKB Abs in unstimulated and stimulated wt T (data not shown). Because of this differential effect of myr PKB on cells (Fig. 5D). In unstimulated wt T cells, significant coprecipi- NF-␬B subunits, we investigated translocation of RelB, another tation of NFATc1 with PKB could only be detected with anti-PKB, member of the NF-␬B family. As shown in Fig. 6B, in resting cells but not with anti-pPKB, Abs. This probably reflects the minute and in response to both stimulation with CD3 alone and with CD3 amounts of active PKB present in unstimulated T cells, as in stim- plus CD28, no nuclear RelB could be detected in myr PKB CD4ϩ ulated wt cells immunoprecipitation with anti-pPKB Abs also re- T cells. Again, this is in stark contrast to wt cells, which showed

FIGURE 6. A and B, Myr PKB diminishes nuclear translocation of NF␬B subunits p65 and RelB. Western blot analysis of NF␬Bp50, p65, and RelB in CE and NE from wt and tg CD4ϩ T cells stimulated as described in Fig. 4. NF␬Bp50 and p65 expression is shown for cells stimulated for8h(A), and RelB expression is shown for cells 4 h after activation (B). n.s., Nonspecific loading control. C, Similar degradation of I␬B␣ after TCR/CD28 stimulation. Wt and tg CD4ϩ T cells, pretreated with cycloheximide for 15 min, were stimulated with anti-CD3 plus anti-CD28 mAbs for the time periods indicated. Whole cell extracts were analyzed for I␬B␣ degradation. Protein loading was controlled by actin expression. The Journal of Immunology 4819 the expected induction of RelB protein after TCR engagement. In down-modulation of immune responses when cells return to a qui- repeated experiments nuclear levels of NF-␬Bp65, RelB, and escent state (43). Furthermore, NFAT has been indicated to be NFAT proteins were always drastically diminished, but not com- involved in the expression of stage-specific cyclins, unveiling a pletely absent, indicating that nuclear shuttling is not completely role for NFAT in the regulation of the cell cycle (44). We therefore shut off, but is down-regulated to a major extent. propose that by direct interaction, active PKB regulates NFAT As activation of NF-␬Bp65 and RelB, but not of NF-␬Bp50, pro- localization and thereby partially exerts its positive regulatory role teins is regulated via I␬B proteins, we compared the degradation of on T cell activation and effector function. The modified regulation I␬B␣ in wt and tg T cells. As shown in Fig. 6C, degradation of I␬B␣ of subcellular localization of NFAT and other to date unknown after CD3/CD28 stimulation in myr PKB CD4ϩ T cells was only proteins in cells with constitutively active PKB may contribute to slightly reduced. This suggests that myr PKB-mediated mechanisms the development of T cell lymphomas, as observed in our myr other than targeting I␬B␣ degradation are the major factors in the PKB homozygous mice (data not shown) and other reports where substantial loss of nuclear NF-␬Bp65 and RelB proteins. overexpression of active PKB is associated with tumor development (45Ð47). Discussion We also have to consider that myr PKB down-modulates the In this report we demonstrate a strong positive regulatory role of nuclear localization of NF-␬Bp65 and RelB proteins in TCR/CD3- constitutively active PKB (myr PKB) on T cell activation and pro- activated CD4ϩ T cells. Our observations are in contrast with stud- liferation as well as production of Th1 and Th2 cytokines, with ies in which PI3K/PKB signaling in transient transfection systems myr PKB obviating signals that in vivo can be provided by CD28 and using PI3K inhibitors was found to enhance NF-␬B activation costimulation (29). The most intriguing finding is that the positive (26, 28, 48Ð51). Some of these studies point to a possible role for effects on T cell responses are coupled with a negative regulatory PI3K/PKB signaling in activating the I␬B kinase ␣␤ complex and role of myr PKB on the nuclear localization of NFAT and NF-␬B NF-␬B trans-activational activities (34, 52). However, a number of proteins. As coprecipitation experiments reveal an interaction be- other research groups found no evidence for PI3K signaling in tweem PKB and NFATc1 in vivo, active PKB could modify this NF-␬B activation (53Ð55). The opposing results probably reflect transcription factor either directly or via some substrate associated the use of different experimental systems, constructs, and stimuli. within a multiprotein complex. To date we do not know whether In T cells expressing a transgenic gag-PKB construct, I␬B␣ deg- the functional loss of nuclear accumulation of NFAT proteins is radation and DNA binding of NF-␬B were enhanced compared the result of inhibition of nuclear import or enhancement of nu- with those in wt cells (27). We assume that the differences between clear export. Mutations in the NLS sequences of NFATs have been the two PKB transgenic systems result from differences in tempo- reported to reduce their nuclear localization (30, 31). Thus, in the ral and spatial expression levels of active PKB. In our transgenic simplest scenario, sustained activation of PKB could interfere with system the role of active PKB seems to entail the down-modula- unmasking of the NLSs and thereby retaining NFAT in the tion of certain NF-␬B and also NFAT responses, rather than en- cytoplasm. hancing TCR-mediated activation of these proteins. Hence, our Recently, analysis of the regulation of nuclear shuttling of findings may reflect differences in the overall activation/differen- NFAT proteins has identified a number of NFAT kinases that op- tiation status of myr PKB T cells compared with wt cells or cell pose NFAT nuclear translocation, including GSK3␤ and the lines transiently transfected with active PKB. Our observations MAPKs, JNK and p38 (32Ð35). However, the exact mechanisms thus would reveal the function of PKB in down-modulation of of the regulation of NFAT activation and its subcellular localiza- transcriptional activities such as might occur when activated T tion by these kinases are not completely understood. GSK3␤ itself cells return to a resting state or during specific differentiation is a known target of PKB signaling, and phosphorylation leads to processes. its inactivation (24). Although we detected higher levels of phos- To date we do not know how myr PKB affects NF-␬Bp65/RelB phorylated GSK3␤ in the nucleus of myr PKB T cells compared nuclear translocation, and a variety of different mechanisms can be with wt cells, accumulation of nuclear NFAT was reduced, rather envisaged. In addition to the regulation via NF-␬B:I␬B complexes, than increased. Considering the data on stronger activation of p38 it is now evident that NF-␬B/Rel proteins are regulated post-trans- and JNK in myr PKB T cells, one possibility could be that phos- lationally via phosphorylation (56) or acetylation events (57). phorylation of NFAT by these kinases, which would foster nuclear Thus, as the overall I␬B␣ degradation in myr PKB T cells was extrusion, dominates over nuclear retention signals expected to be normal, it is likely that myr PKB or one of its substrates acts provided by inactive GSK3␤. However, in experiments in which downstream of I␬B␣ degradation to inhibit nuclear translocation activation of MAPKs and PI3K was inhibited by pharmacological of NF-␬B. In view of altered regulation of NFAT as well as NF-␬〉 inhibitors, we did not observe any reversal of the block in nuclear family members, it may also be possible that myr PKB acts by a translocation of NFATc1 in tg T cells. Thus, the enhanced JNK similar mechanism on the Rel homology domain, which is com- and p38 activities in myr PKB CD4ϩ T cells could contribute to mon in both classes of transcription factors. NF-␬Bp50 may not be the T cell hyper-responsiveness without being involved in the reg- affected, because its activation depends on processing of a larger ulation of NFATs. precursor protein and also lacks the C-terminal trans-activation NFAT proteins are normally thought to be positive regulators of domain(s) that might be critical. transcription, thereby regulating lymphoid homeostasis (36, 37). From a host of studies we know that a delicate balance between However, studies from NFAT knockout mice also document a neg- positive and negative regulatory mechanisms determines the spec- ative role of NFAT proteins in T cell function (38Ð41). In this ificity and magnitude of immune responses. Despite all the above context, in mice doubly deficient in NFATp and NFATc3 Th cells considerations, the novel finding that active PKB is involved in the show hyper-responsiveness independent of CD28 coengagement negative regulation of nuclear transcription factors highlights the (42), similar to our PKB transgenic system in which hyper-respon- role of PKB in processes related to the inactivation of gene ex- siveness of T cells and enhanced production of Th1 and Th2 cy- pression programs through the subcellular localization of tran- tokines are coupled with reduced nuclear NFAT activity. Recently, scription factors. PKB has been implicated in various forms of a direct association of NFATp with the histone deacetylase 1 in cancer. The mechanism of retaining transcription factors in the regulation of the CDK4 promoter has implicated NFAT in the cytoplasm or inducing their nuclear extrusion, thereby preventing 4820 MYR PKB REGULATES NUCLEAR LOCALIZATION OF NFAT/NF␬B PROTEINS

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