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The Journal of Immunology, 2004, 172: 4812-4820.
Copyright © 2004 by The American Association of Immunologists

Active Protein Kinase B Regulates TCR Responsiveness by Modulating Cytoplasmic-Nuclear Localization of NFAT and NF-{kappa}B Proteins1

Amiya K. Patra, Shin-Young Na and Ursula Bommhardt2

Institute of Virology and Immunobiology, University of Würzburg, Würzburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
T cell activation leads to the induction of the transcription factors of the NFAT and NF-{kappa}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{alpha}) lack significant nuclear accumulation/shuttling of NFATc1 and NFATp as well as NF-{kappa}Bp65 and RelB proteins. Notably, despite this deficit in nuclear NFAT and NF-{kappa}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-{kappa}B proteins.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4 T cell activation, expansion, and differentiation require recognition of specific Ag presented by MHC class II molecules on APCs in context with costimulatory signals, such as those provided by CD28, inducible costimulator, CD40 ligand, or OX40 (CD134) (1). TCR-initiated signaling leads to the induction of a complex array of kinases, phosphatases, and downstream transcription factors, which regulate cellular functions such as metabolism, cell cycle, survival, and cell death, thereby controlling appropriate T cell responses. The members of the NFAT and NF-{kappa}B/Rel families of transcription factors are critically involved in many of these cellular processes, and their dysregulation is usually connected with the development of pathophysiological states, including oncogenesis.

Of the four NFAT family members that share significant sequence and functional similarity, NFATc1, NFATc2/NFATp, and NFATc3 mainly function in the immune system, whereas NFATc4 is involved in regulation of cardiac hypertrophy and hippocampal neuronal signaling (2, 3, 4). NFAT proteins in resting T cells are mainly located in the cytoplasm, in a highly phosphorylated form. The calcium calmodulin-dependent phosphatase calcineurin is known to dephosphorylate serine residues within the N terminus of NFAT proteins, thereby facilitating their entry into the nucleus and subsequent target gene expression. Inhibition of this phosphatase by the immunosuppressants cyclosporin A (CsA)3 or FK506 prevents activation and nuclear entry of NFAT. The shuttling of NFATs between nucleus and cytoplasm is finely balanced and precisely controlled. Kinases implicated in the rephosphorylation of NFATs in the nucleus, thereby rendering them inactive and triggering their nuclear export, include glycogen synthase kinase 3 (GSK3) and the mitogen-activated protein kinases (MAPKs), Janus N-terminal kinase (JNK) and p38.

NF-{kappa}B/Rel proteins can exist as homo- or heterodimers composed of almost any combination of the five mammalian family members, c-Rel, p65/RelA, RelB, p50/NF-{kappa}B1, and p52/NF-{kappa}B2 (5, 6). Dimerization as well as DNA binding are mediated by the conserved N-terminal Rel homology domain. Although most NF-{kappa}B dimers are transcriptional activators, the p50/p50 and p52/p52 homodimers can repress target gene transcription. In resting unstimulated cells, most NF-{kappa}B dimers are complexed with an inhibitory protein of the I{kappa}B family, which masks one of the dual nuclear localization signals (NLS) on NF-{kappa}B, thereby, in the simplistic model, retaining them in the cytoplasm. In response to a variety of stimuli, including TCR and CD28 costimulation, I{kappa}B{alpha} and -{beta}, the prototypes of the I{kappa}B family, are phosphorylated by the I{kappa}B kinase complex, followed by their ubiquitination and degradation in the proteasome. The release of I{kappa}Bs unmasks the NLS and allows NF-{kappa}B to enter the nucleus (7).

Protein kinase B (PKB) is a serine/threonine kinase that in lymphocytes is activated by TCR and CD28 costimulation, insulin, cytokines, and chemokines, among others (8). By positively or negatively regulating anti- and proapoptotic molecules such as Bcl-xL or Bad, cell cycle regulators, kinases, and transcription factors such as GSK3 and NF-{kappa}B, respectively, PKB exerts pleiotropic effects on cell proliferation, survival, and cell death (9, 10, 11). Overexpression of active PKB is connected with induction of inflammatory processes and development of tumors (12).

We have previously shown that the expression of a constitutively active form of PKB (myr PKB) in transgenic mice influences thymocyte selection, leads to an accumulation of CD4+ T cells in peripheral lymphoid organs, and enhances their survival in the presence of various apoptosis-inducing reagents (13). In this study we examined the influence of PKB on T cell proliferation and effector function and have identified a new role for PKB in regulating the nuclear translocation of NFAT and NF-{kappa}B proteins. Notably, the PKB-mediated deficit in nuclear accumulation of these transcription factors does not block T cell activation, but, rather, is connected with lower thresholds for proliferative responses, enhanced cell cycle progression, and increased production of Th1 and Th2 cytokines. The down-modulation of nuclear activities of NF-{kappa}B and NFAT family members thus defines a novel regulatory mechanism by which PKB exerts positive effects on T cell function, which, as discussed, may be one underlying mechanism contributing to tumor development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Human CD2-myr PKB{alpha} (myr PKB) transgenic (tg) mice have been described previously (13). Myr PKB tg and wild-type (wt) mice used in this study (backcrossed more than five generations to C57BL/6J mice) were 6–8 wk of age.

Isolation of T cells and flow cytometry

Peripheral and mesenteric lymph nodes were homogenized through nylon cell strainers to obtain single-cell suspensions. For isolation of CD4+ or CD8+ T cells, total lymph node cells were treated with a mixture of rat anti-mouse CD4 (GK1.5) or anti-CD8 (53-6.73) and anti-CD19 (1D3), anti-MHC class II (2G9), and anti-NK1.1 (4D11) hybridoma supernatants for 25 min on ice and then washed twice in cold PBS. BioMag anti-rat IgG coupled to magnetic beads (Qiagen, Hilden, Germany) was added for 25 min at 4°C, and CD4+ or CD8+ T cells were collected by negative selection using magnetic cell separation. The purity of isolated CD4+ or CD8+ T cells was analyzed for each experiment by flow cytometry and routinely was 90–95%. Abs for flow cytometry were purchased from BD PharMingen (San Diego, CA) as FITC-, PE-, or biotin-labeled conjugates; the latter were revealed with streptavidin-CyChrome (BD PharMingen). Cells were stained using standard procedure and were analyzed on a FACSCalibur using CellQuest software (BD Biosciences, Mountain View, CA).

Proliferation and cytokine assays.

For proliferation assays, 2 x 105 purified CD4+ or CD8+ T cells were 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). CsA (Calbiochem, Schwalbach, Germany) was added at the beginning of culture at the concentrations indicated. After 48 h, cells were pulsed with 1 µCi of [3H]thymidine/well (ICN Pharmaceuticals, Asse-Relegem, Belgium) for 12 h. Measurement of cytokine production was made in supernatants collected 24 h after stimulation of CD4+ T cells using ELISA or cytokine bead array (BD PharMingen) according to the manufacturer’s instructions.

Cell cycle analysis

For analysis of cell division, purified T cells (1 x 107/ml) were washed twice with PBS and labeled with CFSE (Molecular Probes Europe, Leiden, The Netherlands) at a final concentration of 2 µM in PBS for 5 min at room temperature. Cells were washed twice with RPMI 1640 medium containing 10% FCS and thereafter cultured in RPMI 1640/5% FCS medium at a density of 2 x 106 cells/ml in 96-well plates. Cells were activated by plate-bound anti-CD3 mAb (10 µg/ml) alone or anti-CD3 (1 µg/ml) plus anti-CD28 mAb (5 µg/ml) for 2 or 3 days and analyzed by FACS. For short term activation, CFSE-labeled cells were stimulated with plate-bound anti-CD3 mAb (5 µg/ml) for 12 or 18 h, harvested, and recultured in medium without further stimulus until 24 or 48 h.

Western blot analysis and immunoprecipitation

For preparation of nuclear and cytoplasmic cell extracts (NE and CE, respectively), 2 x 107 CD4+ T cells were stimulated with plate-bound anti-CD3 mAb alone or in combination with anti-CD28 mAb in the absence or the presence of CsA for the time periods indicated. Kinase inhibitors PD98059 (100 µM), LY294002 (20 µM), SB202190 (40 µM), and staurosporine (10 nM; all from Calbiochem) were added 1 h before stimulation of cells. Cells were harvested, washed twice in cold PBS, and suspended in 200 µl of buffer A (10 mM KCl, 10 mM HEPES (pH 7.9), 0.1 mM EGTA (pH 7.9), 0.1 mM EDTA (pH 7.9), protease inhibitor mixture (Roche, 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 rpm, the supernatant was collected as CE. The pellet was washed twice in buffer A and incubated with 100 µl of buffer C (420 mM NaCl, 20 mM HEPES (pH 7.9), 1 mM EGTA (pH 7.9), 1 mM EDTA (pH 7.9), protease inhibitor mixture, 1 mM DTT, and 1 mM sodium orthovanadate) for 2 h with constant shaking at 4°C. After incubation, NE was collected by centrifugation at 14,000 rpm for 20 min. The protein concentrations of CE and NE were determined using Bradford’s reagent (Bio-Rad, Munich, Germany). Ten micrograms of protein for each sample for both CE and NE was separated on 8–12% SDS-PAGE and electroblotted to nitrocellulose membranes. Specific proteins were detected by Western blot analysis using the following primary Abs: anti-PKB, anti-phospho-PKB (Ser473), anti-phospho-GSK3{alpha}{beta} (Ser21/9), anti-phospho-FKHR (Ser256), anti-phospho-JNK, and anti-phospho-p38 (all from Cell Signaling, Beverly, MA); anti-NFATc1 (Alexis, Carlsbad, CA); anti-NFATp (a gift from Dr. A. Rao, Center for Blood Research, Harvard Medical School, Boston, MA); and anti-NF-{kappa}Bp65, anti-p50, anti-RelB, anti-cyclins B1 and D3, anti-CIS, and anti-I{kappa}B{alpha} (all from Santa Cruz Biotechnology, Santa Cruz, CA). Primary Abs were detected by goat anti-rabbit (Santa Cruz Biotechnology), goat anti-mouse, or rabbit anti-goat Abs coupled with HRP (both from Jackson ImmunoResearch Laboratories, West Grove, PA) and ECL (Pierce, Rockford, IL). Blots were reprobed with anti-actin Ab (Santa Cruz Biotechnology) to control protein loading in the case of CE. Reprobing NE with anti-actin Ab showed that NE were free from cytoplasmic protein contamination. Controls shown in the figures as n.s. for NE and CE are nonspecific ones used as protein loading controls. For I{kappa}B{alpha} analysis, cells were pretreated with cycloheximide (50 µg/ml; Sigma-Aldrich, St. Louis, MO) for 15 min before stimulation of cells with soluble anti-CD3 mAb (1 µg/ml) in combination with anti-CD28 mAb (5 µg/ml) for the indicated time periods. For long term stimulation, cells were activated with plate-bound anti-CD3 mAb (5 µg/ml). Total protein extracts were prepared from 3 x 106 cells and were analyzed by Western blot as described previously (13).

For immunoprecipitation experiments, 1 x 107 CD4+ T cells from myr PKB tg or wt mice were lysed in buffer A, and cytoplasmic protein extracts were incubated with anti-PKB, anti-phospho-PKB (pPKB) (Ser473), anti-Lamin A (all from Cell Signaling), or anti-NFATc1 Ab (Alexis) overnight 4°C with shaking. After addition of 25 µl of 50% protein G-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 h at 4°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 in loading buffer, and supernatants were resolved on 8% SDS-PAGE. Coprecipitation was analyzed for association of NFATc1 with PKB or pPKB.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Active PKB lowers the activation threshold of CD4+ and CD8+ T cells and enables proliferation in the presence of CsA

To examine the effects of constitutively active PKB (myr PKB) on activation and proliferation of peripheral T cells, CD4+ and CD8+ T cells from wt and myr PKB tg mice were activated with either high (10 µg/ml) or limiting (1 µg/ml) concentrations of anti-TCR/CD3 mAb alone or in the presence of CD28 costimulation. As shown in Fig. 1, strong TCR/CD3 stimuli led to comparable proliferation, whereas weak TCR/CD3 signals induced significant proliferation only in myr PKB CD4+ and CD8+ T cells. CD28 costimulation had only small enhancing effects in tg T cells, whereas in wt T cells, proliferation was increased 10- to 20-fold. In addition, in wt cells the response to TCR engagement alone or to CD28 costimulation in combination with weak TCR engagement was totally blocked in the presence of CsA, whereas myr PKB T cells showed significant proliferation under these conditions. Thus, myr PKB signaling lowers the threshold for activation by providing costimulatory signals similar to those induced by CD28 and confers partial resistance to CsA treatment with regard to proliferation.



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  		FIGURE 1. Active PKB lowers the threshold for T cell activation and confers significant CsA resistance in proliferation. Purified CD4+ (A) and CD8+ (B) T cells from wt and tg mice were cultured in medium only or stimulated with plate-bound anti-CD3 mAb (CD3) at a concentration of 10 or 1 µg/ml or with anti-CD3 (1 µg/ml) plus anti-CD28 (CD28; 5 µg/ml) mAbs in the presence or the absence of CsA (100 ng/ml). [3H]thymidine incorporation was measured 48 h after initiation of cultures. Data show averages from triplicate cultures of two wt and tg mice each and are representative for three experiments.

 
Myr PKB enhances cell cycle progression similar to CD28 costimulation

Next we analyzed the effect of myr PKB on cell cycle by labeling T cells with CFSE, which allows tracing the number of cell divisions undergone at any particular time point. As shown in Fig. 2A, 2 days after anti-CD3 activation, ~30% of wt and myr PKB CD4+ T cells had divided once; however, 3-fold more (26%) myr PKB CD4+ cells had progressed through a second cell division compared with wt cells (9%). After activation with anti-CD3 plus anti-CD28 mAbs, cell cycle in wt cells resembled that of myr PKB CD4+ T cells stimulated with anti-CD3 mAb only. In contrast, costimulation had only small enhancing effects on cell cycle progression in myr PKB CD4+ T cells. Comparable results were observed for day 3 cultures, in which the majority of myr PKB CD4+ T cells activated with either anti-CD3 mAb alone or in combination with anti-CD28 mAb had undergone four to six cell cycles, corresponding to the same pattern from CD28-costimulated wt cells. The wt cells stimulated with anti-CD3 mAb only showed a less synchronous cell division profile, with ~20% of cells each having divided one to three times and with only 14% of cells having completed five or six cell divisions compared with 54% of myr PKB CD4+ T cells. As evident from Fig. 2B, myr PKB shows the same enhancing effects on cell cycle progression in CD8+ T cells; the observed advantage in the progression of the cell cycle in the case of CD8+ T cells is even more prominent than that in CD4+ T cells. These data attribute an augmentative role to PKB in cell cycle progression of CD4+ and CD8+ T cells by providing CD28-like costimulatory signals.



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  		FIGURE 2. Active PKB enhances cell cycle progression. A and B, Wt and myr PKB CD4+ (A) and CD8+ (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 CD4+ T cells. CFSE-labeled CD4+ 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 CD4+ 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 CD4+ T cells 8 h after stimulation with anti-CD3 mAb (5 µg/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 CD4+ T cells.

 
To further dissect the role of PKB in cell cycle progression, CD4+ 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 CD4+ 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 CD4+ T cells for 12 h induced only 5% of cells to divide once within 24 h, and ~3-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 cyclins 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 between active PKB and the observed enhanced cell division. We also studied inactivation of the forkhead transcription factor FKHR, which, like other forkhead family members, is located in the nucleus in resting cells and is critically involved in cell cycle regulation. In various cell lines phosphorylation of these factors by PKB leads to their nuclear exclusion and thereby facilitates cell cycle entry and progression into M phase (14). In nuclear and cytoplasmic extracts of unstimulated wt cells (Fig. 2E, upper left panels), phosphorylated forms of FKHR could hardly be detected, whereas after TCR/CD3 engagement, phospho-FKHR was strongly present in both fractions. Interestingly, cytoplasmic phosphorylated FKHR was already prominent in unstimulated myr PKB CD4+ T cells, and only low levels of nuclear FKHR appeared in stimulated cells (Fig. 2E, upper right panels), suggesting that most FKHR proteins had been phosphorylated and shuttled out of the nucleus. This correlates with tg PKB also being located in the nucleus (Fig. 2E, lower panels). Thus, increased or constitutive inactivation and nuclear export of FKHR is one component that contributes to the enhanced cell cycle progression promoted by myr PKB.

Enhanced Th1 and Th2 cytokine production in myr PKB CD4+ T cells

T cell growth and apoptosis are highly dependent on various cytokines. To better understand why myr PKB T cells could proliferate after addition of CsA, we first surveyed the production of IL-2, which is an important growth factor in T cell activation and in an autocrine fashion regulates the expression of the high affinity IL-2R (15, 16). We found that surface expression of the IL-2R {alpha}-chain (CD25) in CD3-stimulated wt cells was down-regulated to levels found in unstimulated cells when CsA was added to the cultures. In contrast, a higher percentage of myr PKB CD4+ T cells still expressed high levels of CD25 in the presence of CsA (data not shown). This suggested that myr PKB signaling allows sufficient production of IL-2 in the presence of CsA, thus maintaining proliferation. Indeed, as shown in Fig. 3A, myr PKB CD4+ T cells produced significant amounts of IL-2 when only low TCR signals were provided and when CsA was administered. The amounts of IL-2 produced under these conditions, therefore, would be sufficient for myr PKB T cells to induce high affinity IL-2R and to sustain expansion. In contrast, IL-2 production of wt cells was only obvious when CD28 costimulatory signal was provided in addition to low TCR engagement, and under these conditions IL-2 production was completely ablated by CsA treatment.



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  		FIGURE 3. A, Myr PKB promotes the production of Th1 and Th2 cytokines. CD4+ T cells from wt and tg mice were left unstimulated or were treated with anti-CD3 mAb (1 µg/ml) without or with CD28 costimulation (5 µg/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 CD4+ T cells were stimulated with anti-CD3 mAb (5 µg/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-{gamma} and TNF-{alpha}, and notably also Th2 cytokines such as IL-4 and IL-5 were increased in cultures from myr PKB CD4+ 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 (17, 18, 19). In this context, the expression of the family member cytokine-induced Src homology 2 protein (CIS) is induced in T cells by TCR stimulation, and overexpression of CIS in the CD4 T cell lineage enhances proliferative responses and survival of T cells (20). In view of the positive regulatory effects of myr PKB on proliferation and cytokine production, we studied the expression of CIS and detected accelerated and enhanced expression of CIS in myr PKB CD4+ T cells compared with wt cells (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 (2, 3, 4, 21, 22). Sustained mobilization of intracellular Ca2+ 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 constitutive activation of NFAT proteins. As shown in Fig. 4A, NE and CE of CD4+ 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 for nuclear translocation of NFATc1. Eight hours after activation, most induced NFATc1 protein in wt CD4+ T cells was translocated to the nucleus, and nuclear entry was clearly abolished in the presence of CsA. Strikingly, whereas in cytoplasmic extracts of myr PKB CD4+ T cells, NFATc1 induction after anti-CD3 and anti-CD3 plus anti-CD28 costimulation was obvious, nuclear translocation of NFATc1 was hardly detectable (Fig. 4A, upper panel). At earlier time points of activation, e.g., at 4 h (data not shown), nuclear translocation of NFATc1 could also not be detected and was drastically diminished even 16 h after activation (Fig. 4A, lower panel).



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  		FIGURE 4. Myr PKB impairs nuclear accumulation of NFAT proteins. A, Wt and tg CD4+ T cells were stimulated with anti-CD3 mAbs alone or in combination with anti-CD28 mAbs with or without CsA (100 ng/ml) for 8 h (upper panel) or 16 h (lower panel). NE and CE were analyzed for nuclear translocation of NFATc1 in Western blot. B, CE and NE from wt and tg CD4+ T cells stimulated for 4 h, as described in A, were probed for NFATp activation and nuclear localization.

 
To test whether this intriguing effect of myr PKB on nuclear shuttling was restricted to NFATc1, we examined NFATp activation (Fig. 4B). Similarly, although induction of NFATp in the cytoplasm was evident, nuclear translocation of NFATp was hardly detectable in myr PKB CD4+ T cells, similar to the blockade seen in CsA-treated wt cells. Thus, myr PKB promotes proliferation and cytokine production in T cells despite immensely diminished nuclear levels of NFATc1 and NFATp.

Enhanced nuclear levels of p-p38, pJNK, and pGSK3 in myr PKB T cells

As phosphorylation and dephosphorylation are the basis of NFAT translocation and subsequent gene expression, we examined the activities of JNK, p38, and GSK3{beta}, kinases that have been implicated in the regulation of cytoplasmic-nuclear shuttling of NFAT proteins. In particular, overexpression of GSK3 has been shown to significantly reduce nuclear import, whereas inhibition of GSK3 slows nuclear export of NFATc1 (23). As shown in Fig. 5A, 8 h after TCR/CD3 activation, phosphorylated GSK3{beta} is clearly detectable in cytoplasmic and nuclear extracts of myr PKB T cells, but is detected only very weakly or not at all in case of cytoplasmic and nuclear extracts of wt cells. This correlates with myr PKB being located in the nucleus (see Fig. 2E) and published data that active PKB can phosphorylate GSK3{beta}, thereby leading to its inactivation (24). However, although inactivation of GSK3{beta} by phosphorylation through PKB should foster nuclear retention of NFAT, the opposite, namely, grossly diminished nuclear NFATc1 or NFATp, was detected in myr PKB T cells. We therefore analyzed cytoplasmic and nuclear levels of active JNK and p38, which by rephosphorylating NFAT would induce its nuclear extrusion. As shown in Fig. 5A, myr PKB T cells elicit elevated levels of phosphorylated p38 and JNK proteins compared with wt cells upon stimulation. These data suggest that rephosphorylation of NFAT by these kinases could be involved in the altered cytoplasmic-nuclear shuttling of NFAT in myr PKB T cells.



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  		FIGURE 5. A, Active PKB enhances phosphorylation of MAPKs, p38 and JNK, and of GSK3{beta}. Wt and tg CD4+ T cells were cultured in medium only or were activated with anti-CD3 mAb (5 µg/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 CD4+ T cells were stimulated with anti-CD3 mAb for 4 h in the absence 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 CD4+ 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 µg/ml anti-CD3 mAb for 4 h) wt CD4+ T cells was analyzed in immunoprecipitation assays. Anti-Lamin A Ab was used to control the specificity of the interaction.

 
Inhibition of MAPK/SAPK and phosphatidylinositol 3-kinase (PI3K) pathways fails to repair the altered nuclear shuttling of NFATc1

In view of the reported involvement of JNK, p38, and PI3K (25) in opposing nuclear translocation of NFAT and our observation regarding higher pJNK and p-p38 levels in myr PKB T cells, we studied whether inhibition of these signaling pathways could repair the alteration. For this purpose we treated cells with the pharmacological reagents PD98059, SB202190, and LY294002, inhibitors of MAPKs and PI3K, respectively, as well as with staurosporine, a broad spectrum inhibitor of kinases. The concentrations of inhibitors used in these experiments clearly blocked induction of CD25 expression at 4 h of stimulation, the time point when protein extracts were prepared, and also inhibited proliferation when measured by [3H]thymidine incorporation at 24 h, indicating that they effectively blocked the respective kinases (data not shown). As none of these inhibitors led to higher nuclear accumulation of NFATc1 (Fig. 5B), the deficiency in nuclear NFATc1 in myr PKB CD4+ T cells is regulated by mechanisms not involving PI3K or the MAPKs, extracellular signal-regulated kinase, p38, and JNK.

We therefore hypothesized whether PKB itself could act as an NFAT kinase and investigated its direct interaction with NFATc1. Immunoprecipitation experiments, as shown in Fig. 5C, clearly detected in vivo association of pPKB with NFATc1 in cytoplasmic extracts from tg CD4+ T cells. Whether this interaction also occurs in wt T cells was analyzed by immunoprecipitation studies with anti-pPKB and anti-PKB Abs in unstimulated and stimulated wt T cells (Fig. 5D). In unstimulated wt T cells, significant coprecipitation of NFATc1 with PKB could only be detected with anti-PKB, but not with anti-pPKB, Abs. This probably reflects the minute amounts of active PKB present in unstimulated T cells, as in stimulated wt cells immunoprecipitation with anti-pPKB Abs also resulted in pull-down of NFATc1. Together these data show that transgenic PKB as well as endogenous PKB interact with NFATc1.

Myr PKB impairs TCR-induced nuclear translocation of RelB and NF-{kappa}Bp65, but not NF-{kappa}Bp50, proteins

Next we investigated whether the striking effect on nuclear translocation is confined to NFAT proteins or also occurs for members of the NF-{kappa}B family, which in various cell systems and with different stimuli have been shown to become activated via PKB (26, 27, 28). Similar to NFAT analysis, CE and NE from wt and myr PKB CD4+ T cells stimulated in the presence or the absence of CsA were tested for nuclear translocation of NF-{kappa}Bp50 and p65 proteins. Induction of cytoplasmic NF-{kappa}Bp50, nuclear translocation, and inhibitory effects of CsA were similar for wt and myr PKB CD4+ T cells (Fig. 6A). However, in strong contrast to NF-{kappa}Bp50, nuclear translocation of NF-{kappa}Bp65 in the case of myr PKB CD4+ T cells at 16 h of activation was markedly diminished, with nuclear NF-{kappa}Bp65 levels similar to those in unstimulated or CsA-treated wt cells. Similar to NFAT proteins, nuclear NF-{kappa}Bp65 was also absent or strongly diminished at earlier time points after activation (data not shown). Because of this differential effect of myr PKB on NF-{kappa}B subunits, we investigated translocation of RelB, another member of the NF-{kappa}B family. As shown in Fig. 6B, in resting cells and in response to both stimulation with CD3 alone and with CD3 plus CD28, no nuclear RelB could be detected in myr PKB CD4+ T cells. Again, this is in stark contrast to wt cells, which showed the expected induction of RelB protein after TCR engagement. In repeated experiments nuclear levels of NF-{kappa}Bp65, RelB, and NFAT proteins were always drastically diminished, but not completely absent, indicating that nuclear shuttling is not completely shut off, but is down-regulated to a major extent.



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  		FIGURE 6. A and B, Myr PKB diminishes nuclear translocation of NF{kappa}B subunits p65 and RelB. Western blot analysis of NF{kappa}Bp50, p65, and RelB in CE and NE from wt and tg CD4+ T cells stimulated as described in Fig. 4. NF{kappa}Bp50 and p65 expression is shown for cells stimulated for 8 h (A), and RelB expression is shown for cells 4 h after activation (B). n.s., Nonspecific loading control. C, Similar degradation of I{kappa}B{alpha} after TCR/CD28 stimulation. Wt and tg CD4+ 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{kappa}B{alpha} degradation. Protein loading was controlled by actin expression.

 
As activation of NF-{kappa}Bp65 and RelB, but not of NF-{kappa}Bp50, proteins is regulated via I{kappa}B proteins, we compared the degradation of I{kappa}B{alpha} in wt and tg T cells. As shown in Fig. 6C, degradation of I{kappa}B{alpha} after CD3/CD28 stimulation in myr PKB CD4+ T cells was only slightly reduced. This suggests that myr PKB-mediated mechanisms other than targeting I{kappa}B{alpha} degradation are the major factors in the substantial loss of nuclear NF-{kappa}Bp65 and RelB proteins.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this report we demonstrate a strong positive regulatory role of constitutively active PKB (myr PKB) on T cell activation and proliferation as well as production of Th1 and Th2 cytokines, with myr PKB obviating signals that in vivo can be provided by CD28 costimulation (29). The most intriguing finding is that the positive effects on T cell responses are coupled with a negative regulatory role of myr PKB on the nuclear localization of NFAT and NF-{kappa}B proteins. As coprecipitation experiments reveal an interaction betweem PKB and NFATc1 in vivo, active PKB could modify this transcription factor either directly or via some substrate associated within a multiprotein complex. To date we do not know whether the functional loss of nuclear accumulation of NFAT proteins is the result of inhibition of nuclear import or enhancement of nuclear export. Mutations in the NLS sequences of NFATs have been reported to reduce their nuclear localization (30, 31). Thus, in the simplest scenario, sustained activation of PKB could interfere with unmasking of the NLSs and thereby retaining NFAT in the cytoplasm.

Recently, analysis of the regulation of nuclear shuttling of NFAT proteins has identified a number of NFAT kinases that oppose NFAT nuclear translocation, including GSK3{beta} and the MAPKs, JNK and p38 (32, 33, 34, 35). However, the exact mechanisms of the regulation of NFAT activation and its subcellular localization by these kinases are not completely understood. GSK3{beta} itself is a known target of PKB signaling, and phosphorylation leads to its inactivation (24). Although we detected higher levels of phosphorylated GSK3{beta} in the nucleus of myr PKB T cells compared with wt cells, accumulation of nuclear NFAT was reduced, rather than increased. Considering the data on stronger activation of p38 and JNK in myr PKB T cells, one possibility could be that phosphorylation of NFAT by these kinases, which would foster nuclear extrusion, dominates over nuclear retention signals expected to be provided by inactive GSK3{beta}. However, in experiments in which activation of MAPKs and PI3K was inhibited by pharmacological inhibitors, we did not observe any reversal of the block in nuclear translocation of NFATc1 in tg T cells. Thus, the enhanced JNK and p38 activities in myr PKB CD4+ T cells could contribute to the T cell hyper-responsiveness without being involved in the regulation of NFATs.

NFAT proteins are normally thought to be positive regulators of transcription, thereby regulating lymphoid homeostasis (36, 37). However, studies from NFAT knockout mice also document a negative role of NFAT proteins in T cell function (38, 39, 40, 41). In this context, in mice doubly deficient in NFATp and NFATc3 Th cells show hyper-responsiveness independent of CD28 coengagement (42), similar to our PKB transgenic system in which hyper-responsiveness of T cells and enhanced production of Th1 and Th2 cytokines are coupled with reduced nuclear NFAT activity. Recently, a direct association of NFATp with the histone deacetylase 1 in regulation of the CDK4 promoter has implicated NFAT in the down-modulation of immune responses when cells return to a quiescent state (43). Furthermore, NFAT has been indicated to be involved in the expression of stage-specific cyclins, unveiling a role for NFAT in the regulation of the cell cycle (44). We therefore propose that by direct interaction, active PKB regulates NFAT localization and thereby partially exerts its positive regulatory role on T cell activation and effector function. The modified regulation of subcellular localization of NFAT and other to date unknown proteins in cells with constitutively active PKB may contribute to the development of T cell lymphomas, as observed in our myr PKB homozygous mice (data not shown) and other reports where overexpression of active PKB is associated with tumor development (45, 46, 47).

We also have to consider that myr PKB down-modulates the nuclear localization of NF-{kappa}Bp65 and RelB proteins in TCR/CD3-activated CD4+ T cells. Our observations are in contrast with studies in which PI3K/PKB signaling in transient transfection systems and using PI3K inhibitors was found to enhance NF-{kappa}B activation (26, 28, 48, 49, 50, 51). Some of these studies point to a possible role for PI3K/PKB signaling in activating the I{kappa}B kinase {alpha}{beta} complex and NF-{kappa}B trans-activational activities (34, 52). However, a number of other research groups found no evidence for PI3K signaling in NF-{kappa}B activation (53, 54, 55). The opposing results probably reflect the use of different experimental systems, constructs, and stimuli. In T cells expressing a transgenic gag-PKB construct, I{kappa}B{alpha} degradation and DNA binding of NF-{kappa}B were enhanced compared with those in wt cells (27). We assume that the differences between the two PKB transgenic systems result from differences in temporal and spatial expression levels of active PKB. In our transgenic system the role of active PKB seems to entail the down-modulation of certain NF-{kappa}B and also NFAT responses, rather than enhancing TCR-mediated activation of these proteins. Hence, our findings may reflect differences in the overall activation/differentiation status of myr PKB T cells compared with wt cells or cell lines transiently transfected with active PKB. Our observations thus would reveal the function of PKB in down-modulation of transcriptional activities such as might occur when activated T cells return to a resting state or during specific differentiation processes.

To date we do not know how myr PKB affects NF-{kappa}Bp65/RelB nuclear translocation, and a variety of different mechanisms can be envisaged. In addition to the regulation via NF-{kappa}B:I{kappa}B complexes, it is now evident that NF-{kappa}B/Rel proteins are regulated post-translationally via phosphorylation (56) or acetylation events (57). Thus, as the overall I{kappa}B{alpha} degradation in myr PKB T cells was normal, it is likely that myr PKB or one of its substrates acts downstream of I{kappa}B{alpha} degradation to inhibit nuclear translocation of NF-{kappa}B. In view of altered regulation of NFAT as well as NF-{kappa}B family members, it may also be possible that myr PKB acts by a similar mechanism on the Rel homology domain, which is common in both classes of transcription factors. NF-{kappa}Bp50 may not be affected, because its activation depends on processing of a larger precursor protein and also lacks the C-terminal trans-activation domain(s) that might be critical.

From a host of studies we know that a delicate balance between positive and negative regulatory mechanisms determines the specificity and magnitude of immune responses. Despite all the above considerations, the novel finding that active PKB is involved in the negative regulation of nuclear transcription factors highlights the role of PKB in processes related to the inactivation of gene expression programs through the subcellular localization of transcription factors. PKB has been implicated in various forms of cancer. The mechanism of retaining transcription factors in the cytoplasm or inducing their nuclear extrusion, thereby preventing them from exerting control over selective cellular activation, proliferation, and death-inducing processes, may be an important step in PKB-mediated initiation of transformation or progression of malignancy.


   Acknowledgments
 
We thank Drs. T. Hünig, A. Schimpl, E. Serfling, and I. Berberich for reagents and discussion, and Dr. A. Rao for generously providing anti-NFATp Ab.


   Footnotes
 
1 This work was supported by a grant to the Forschergruppe 303 TPA3 from the Deutsche Forschungsgemeinschaft. Back

2 Address correspondence and reprint requests to Dr. Ursula Bommhardt, Institute of Virology and Immunobiology, Versbacher Strasse 7, D-97078 Würzburg, Germany. E-mail address: bommhardt{at}vim.uni-wuerzburg.de Back

3 Abbreviations used in this paper: CsA, cyclosporin A; CE, cytoplasmic extract; CIS, cytokine-induced Src homology 2 protein; GSK3, glycogen synthase kinase 3; JNK, Janus N-terminal kinase; MAPK, mitogen-activated protein kinase; NE, nuclear extract; NLS, nuclear localization signal; PI3K, phosphatidylinositol 3-kinase; PKB, protein kinase B; pPKB, phospho-PKB; tg, transgenic; wt, wild type. Back

Received for publication October 24, 2003. Accepted for publication February 5, 2004.


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 Materials and Methods
 Results
 Discussion
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Differential expression of SHIP1 in CD56bright and CD56dim NK cells provides a molecular basis for distinct functional responses to monokine costimulation
Blood, April 15, 2005; 105(8): 3011 - 3018.
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P. S. Hammerman, C. J. Fox, R. M. Cinalli, A. Xu, J. D. Wagner, T. Lindsten, and C. B. Thompson
Lymphocyte Transformation by Pim-2 Is Dependent on Nuclear Factor-{kappa}B Activation
Cancer Res., November 15, 2004; 64(22): 8341 - 8348.
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U. Bommhardt, K. C. Chang, P. E. Swanson, T. H. Wagner, K. W. Tinsley, I. E. Karl, and R. S. Hotchkiss
Akt Decreases Lymphocyte Apoptosis and Improves Survival in Sepsis
J. Immunol., June 15, 2004; 172(12): 7583 - 7591.
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