Protein kinase Cß1, a major regulator of TCR–CD28-activated signal transduction leading to IL-2 gene transcription and secretion

Uschi E. Dreikhausen1, Katrin Gorf1, Klaus Resch1 and Marta Szamel1

1 Institute of Pharmacology, Medical School Hannover, 30623 Hannover, Germany

The first two authors contributed equally to this work
Correspondence to: M. Szamel; E-mail: Szamel.Marta{at}MH-Hannover.de
Transmitting editor: T. Hünig


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The aim of this study was to investigate the influence of protein kinase C (PKC) {alpha} and ß on the TCR–CD28-stimulated protein kinase cascades participating in regulation of IL-2 gene transcription and secretion. Inhibition of the synthesis of PKC{alpha} and ß by specific phosphorothioate-modified antisense oligonucleotides (ODN) resulted in suppression of phosphorylation and activation of Raf-1, mitogen-activated extracellular-regulated kinase kinases and extracellular-regulated kinases in stimulated Jurkat T cells. Furthermore, a marked reduction of I{kappa}B kinase-{alpha}-catalyzed I{kappa}B{alpha} phosphorylation was observed in both PKC{alpha}- and ß-specific antisense oligonucleotide-treated cells. In sharp contrast, TCR–CD28-stimulated phosphorylation and activation of the Jun-N-terminal kinase (JNK) cascade was specifically suppressed upon treatment with PKCß-specific antisense ODN, suggesting that PKCß was a specific upstream regulator of the JNK protein kinase cascade. Significant inhibition of high-affinity NF-AT binding and transactivation, IL-2 gene expression, reduction of IL-2 mRNA synthesis, and, most impressively, a complete suppression of IL-2 secretion were observed in PKCß antisense ODN-treated cells. The data indicate a highly specific function of PKCß for regulation of TCR–CD28 induced-signaling, IL-2 gene expression and secretion in Jurkat T cells.

Keywords: cytokine, protein kinase/phosphatase, signal transduction, T lymphocyte, transcription factor


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Stimulation of T lymphocytes involves several signal transduction cascades leading to the activation of transcription factors which subsequently regulate a set of pleiotropic cellular responses that induce proliferation and cytokine secretion. T cell activation via the TCR–CD3 complex is associated with the rapid generation of inositol phosphates and diacylglycerols that regulate intracellular calcium and protein kinase C (PKC) respectively. PKC consists of a family of isoforms divided into three groups based on enzymatic properties. These are the classical ({alpha}, ß and {gamma}), novel ({delta}, {epsilon}, {eta}, {theta} and µ) and atypical ({zeta} and {lambda}/{iota}) subfamilies (1). T lymphocytes express the PKC isoforms {alpha}, ß1, {delta}, {epsilon}, {eta}, {theta} and {zeta}. PKC has been shown to play a key role in T cell activation (2). Various studies have been attempted to elucidate specific roles of individual isoforms in T lymphocyte activation. Using constitutive active PKC isoforms it has been shown that PKC{alpha} regulates generation of the transcription factors AP-1 and NF-AT (3). PKC{theta}, which associates with the immunological synapse, appears to be an upstream activator of c-Jun-N-terminal kinases (JNK)/stress-activated protein kinases and of the IL-2 promoter activation in vivo and in vitro (48). More recently, it has been shown that PKC{theta} synergized with Vav in the activation of the transcription factor NF-{kappa}B in T cells (9).

Introducing neutralizing antibodies by electroporation, we have previously demonstrated that different PKC isoenzymes regulated either IL-2 synthesis or IL-2 receptor expression in human peripheral blood lymphocytes. These results suggested PKCß to be the major regulator of IL-2 synthesis (10,11). However, the molecular mechanisms whereby PKCß affected IL-2 gene expression could not be investigated in detail by this approach.

The IL-2 gene is activated by the concerted action of several transcription factors (12,13). Generation, activation and nuclear translocation of these transcription factors is the ultimate function of extracellular-regulated kinase (ERK), JNK and p38 mitogen-activated proteinkinase (MAPK) signal transduction cascades activated via TCR and CD28 co-receptor (14). As a consequence of TCR-induced elevation of intracellular calcium, the calcium–calmodulin-dependent phosphatase calcineurin additionally activates NF-AT (12,13). Furthermore, stimulation via TCR–CD28 activates specific protein kinase cascade(s) leading to activation and nuclear translocation of NF-{kappa}B (15).

The aim of this study was to investigate the influence of PKC{alpha} and ß on the protein kinase cascades participating in regulation of the IL-2 gene in Jurkat T cells. PKC isoenzyme-specific phosphorothioate antisense oligonucleotides (ODN) were introduced into these cells by electroporation. Antisense technology provides a highly specific approach for functional studies of individual protein isoforms in signal transduction processes (16,17). To do so, the antisense oligonucleotides should be designed to hybridize with high affinity to mRNA sequences unique for a given protein, thereby inhibiting RNA translation and expression of this protein. Due to the long half-lives of some proteins requiring several days to significantly lower protein levels, it is advantageous to decrease sensitivity of antisense ODN to endogenous nucleases by chemical modifications, e.g. use of phosphorothioate analogs (16,17).

The results show that phosphorothioate-modified antisense ODN effectively inhibited new synthesis of PKC{alpha} and ß protein. TCR–CD28-induced JNK activation was specifically impaired in PKCß-depleted cells, whereas other signaling cascades, like Raf/mitogen-activated ERK kinase (MEK)/ERK or I{kappa}B kinase (IKK) catalyzed I{kappa}B phosphorylation were dependent on both PKC{alpha} and ß. A significant decrease in IL-2 gene transcription was observed and IL-2 secretion was completely abolished in PKCß-specific antisense ODN-treated cells. The results substantiate the decisive role of PKCß in regulation of IL-2 synthesis and secretion in the course of T cell activation.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cells and cell culture
Jurkat T cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 1 mM pyruvate, 2 mM glutamine, 100 U/ml penicillin and 0.1 mg/ml streptomycin at 37°C/5% CO2. Cells were seeded in fresh medium with 0.1% FCS at 1 x 106 cells/ml overnight before addition of 2.5 µg/ml OKT3 antibody and 1.25 µg/ml mouse anti-human CD28 antibody (a kind gift from Professor B. Schraven, Magdeburg, Germany) for the times indicated in the experiments.

Human peripheral blood lymphocytes were separated by Ficoll gradient centrifugation from heparinized blood of healthy volunteers. Cells were washed with RPMI and stimulated for 15 min with 2.5 µg/ml OKT3 antibody and 1.25 µg/ml mouse anti-human CD28 antibody in serum-free RPMI medium for activation of the JNK protein kinase cascade or in RPMI supplemented with 5% FCS for 16 h for determination of IL-2 synthesis.

Introduction of PKC-specific antisense ODN by electroporation
Jurkat T cells were adjusted to a cell density of 2 x 107/ml in RPMI 1640 medium with 1% FCS (heat-inactivated at 65°C for 30 min). An aliquot of 8 x 106 cells was electroporated for 5 ms at 400 V (rectangle impulse) in the presence and absence of 10 nmol of a 20mer PKC{alpha} (5'-GTTCTCGCTGGTGAGTTTCA-3')- and 13 nmol of a 15mer PKCß (5'-CGCAGCCGGGTC AGC-3')-specific phosphorothioate-modified antisense ODN (MWG Biotech, Ebersberg, Germany). After electroporation, cells were transferred into RPMI 1640 medium with 10% FCS (65°C heat inactivated) and adjusted to 2.5 x 105 cells/ml. After culturing for 48 h, cells were taken for stimulation in serum-free medium.

With minor modifications, Jurkat cells stably transfected with a plasmid containing the luciferase reporter gene together with either a single copy of the minimal IL-2 promoter (3) or four copies of the distal NF-AT-binding domain of the IL-2 promoter were also used for electroporation. Thereby, 8 x 106 cells at a cell density of 1 x 107/ml were electroporated with specific PKC{alpha} (10 nmol) and PKCß (13 nmol) antisense ODN for 5 ms at 350 V, and cultured for 34 (IL-2 promoter activity) or 42 h (NF-AT-binding domain activity).

Flow cytometry
Jurkat cells were electroporated as described in the previous section with or without 10 nmol PKC{alpha} or 13 nmol PKCß FITC-labeled antisense ODN or 50 µg/ml FITC–dextran (Sigma, Deisenhofen, Germany). Immediately after electroporation, and 24 and 48 h later an aliquot of cells was washed twice in PBS, pH 7.4 with 0.1% BSA. From these samples, 105 events were acquired using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) and analyzed with the Lysis II software.

Preparation of subcellular fractions
For preparation of subcellular fractions, cells were resuspended in a buffer consisting of 20 mM HEPES, 1 mM EDTA, 1 mM EGTA, 2 mM MgCl2, 1 mM DTT, 50 µg/ml leupeptin, 400 nM ocadaic acid and 1 mM sodium vanadate, pH 7.4. Cells were sonicated (50 W, 5 impulses) and centrifuged (10 min at 2500 g). For preparation of cytoplasmic fractions, the 2500 g supernatant was further centrifuged at 100,000 g for 30 min. The pellet (crude membrane fraction) was washed with the buffer described above.

Alternatively, nuclear-free lysates were obtained by incubating cells for 30 min on ice in a buffer containing 10 mM Tris, 30 mM sodium pyrophosphate, 50 mM NaCl, 5 mM NaF, 20 mM ß-glycerophosphate, 1 mM Na3VO4, 400 nM okadaic acid, 0.5 mM PMSF, 10 µg/ml leupeptin, 0.5 µg/ml pepstatin, 10 mM p-nitrophenylphosphate, 1 mM DTT and 1% Triton X-100, pH 7.4. Nuclei were removed by centrifugation for 15 min at 6000 g and 4°C.

Protein concentration of the subcellular fractions was determined using the bicincholinic acid assay and BSA as a standard.

Detection of phosphorylated protein kinases by immunoblot analysis
Cellular proteins were separated by SDS–PAGE. Proteins were transferred onto nitrocellulose. For the detection of protein kinases, membranes were blocked in TBST (20 mM Tris, pH 7.6, 137 mM NaCl and 0.1% Tween 20) supplemented with 5% non-fat milk for 1 h at 20°C, thoroughly rinsed in TBST followed by incubation with the primary antibodies raised against PKC{alpha} and ß, Raf-1, MEK-1 (Transduction Laboratories, Lexington, UK), phospho-Raf-1, phospho-MEK-1/2, pan-ERK, phospho-ERK-1/2, phospho-MAPK kinase (MKK)-4, phospho-JNK, p38 MAPK, phospho-p38 MAPK and phospho-I{kappa}B (NEB, Beverly, MA) in TBST over night at 4°C. The membranes were washed again and incubated for 1 h at room temperature with appropriate secondary antibodies conjugated to horseradish peroxidase. Finally, membranes were rinsed and developed using the enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, IL). In some cases blots were stripped and reprobed.

Determination of protein kinase activities
For in vitro determination of JNK activity, 50 µg of cytosolic protein in 2 x kinase buffer (100 mM Tris and 20 mM MgCl2, pH 7.6) was incubated with 1.5 µg human recombinant GST-c-jun (NEB, Beverly, MA) and 5 µM ATP as substrates for 20 min at 37°C. Phosphorylated GST-c-jun was obtained by GSH beads and separated by SDS–PAGE. Phosphorylated c-jun was detected in immunoblots by specific antibodies (NEB).

Determination of luciferase activity
NF-AT-binding domain activity and IL-2 reporter gene activity were detected using 1 x 106 cells/ml of stably transfected Jurkat cells seeded in serum-free RPMI 1640 medium and stimulated for 6 (NF-AT-binding domain) and 14 h (IL-2 promoter activity) with or without OKT3 and anti-CD28 antibody in PKC{alpha} and ß ODN-treated cells. Incubation was stopped by washing three times with ice-cold PBS, pH 7.4. Lysis of cells resulted from incubation in a buffer containing 91.5 mM K2HPO4, 8.5 mM KH2PO4, 0.5 mM PMSF, 2.5 µg/ml leupeptin, 0.7 µg/ml pepstatin and 0.2% Triton X-100 for 15 min at 4°C. Luciferase activity was determined in the supernatants gained after centrifugation for 15 min at 13,000 r.p.m. and 4°C. Finally, the resulting relative light units were normalized for protein concentration.

RNA isolation and RT-PCR
Jurkat T cells (5 x 106) were stimulated with OKT3 and anti-CD28 antibody for the times indicated in the experiments. Cells were then harvested and RNA isolated with a Qiagen RNA isolation kit according to the manufacturer’s instructions (Qiagen, Hilden, Germany).

For RT-PCR, 3 µg RNA was incubated with 500 ng poly(dT) (Amersham Pharmacia Biotech, Freiburg, Germany) as a primer, and dATP, dCTP, dGTP and dTTP, 10 nmol of each (Q-Biogene, Heidelberg, Germany) at 65°C for 5 min. Samples were chilled on ice and incubated for 2 min in the presence of 40 U RNase inhibitor (RNaseOut; Gibco, Karlsruhe, Germany) for 2 min at 37°C. Synthesis of cDNA was started by addition of 200 U DNA polymerase (M-MLV-RT; Gibco) in a buffer containing 50 mM Tris, 75 mM KCl, 3 mM MgCl2 and 10 mM DTT. Following incubation at 37°C for 50 min, the reaction was terminated at 95°C for 10 min.

The primers used were from MWG Biotech and from Stratagene (Amsterdam, Netherlands). IL-2 sense: 5'-ATG TAC AGG ATG CAA CTC CTG TCT T-3'; antisense: 5'-GTC AGT GTT GAG ATG ATG CTT TGA C-3' (457 kb PCR product). GAPDH sense: 5'-CCA CCC ATG GCA AAT TCC ATG GCA-3'; antisense: 5'-TCT AGA CGG CAG GTC AGG TCC ACC-3' (598 kb PCR product).

PCR was carried out in a buffer containing 10 mM Tris–HCl, 50 mM KCl, 2 mM MgCl2 and 0.8% Nonidet P-40 with 2 µl cDNA in the presence of the specific primers (10 pmol of each), dATP, dCTP, dGTP and dTTP (10 nmol of each), and 1 U Taq polymerase (Q-Biogene) with the further specifications: denaturation at 94°C for 5 min, annealing at 60°C for 1 min, elongation at 72°C for 1 min and 30 cycles (within the linear range).

Agarose gel electrophoresis
PCR products were separated in a 1.7% agarose gel in 40 mM Tris–CH3COOH, pH 8.0, 1 mM EDTA and 0.5 µg/ml ethidium bromide, and documented with the geldocumentation system from Bio-Rad (Hercules, CA).

Determination of IL-2 synthesis
IL-2-specific ELISA was from R & D Systems (Minneapolis, MN). Jurkat T cells (1 x 106/ml) were incubated in RPMI with 2% FCS, and stimulated with or without OKT3 and anti-CD28 antibody for 16 h. IL-2 concentration of the supernatants was determined by a specific ELISA according to the manufacturer’s instructions.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Phosphorothioate-modified antisense ODN effectively inhibit new synthesis of PKC{alpha} and ß protein
Figure 1(A) depicts that the amount of PKC{alpha} or ß protein was significantly reduced upon treatment with 10 or 13 nmol of PKC{alpha}- and ß-specific antisense ODN. Introduction of higher amounts of the antisense ODN into the cells inhibited PKC levels even more effectively. However, these concentrations resulted in apoptosis of the cells (data not shown). Based on these results, all further experiments were carried out with the concentration of ODN as described above. Due to the long half-life of PKC, cells were incubated with the antisense ODN for 48 h to achieve a significant reduction in the amount of PKC{alpha} and ß.




View larger version (50K):
[in this window]
[in a new window]
 
Fig. 1. (A) PKC isotype-specific phosphorothioate antisense ODN effectively inhibit synthesis of PKC{alpha} and ß protein. Jurkat T cells (8 x 106) were electroporated for 5 ms at 400 V in the presence and absence of 10 nmol of PKC{alpha}- or 13 nmol of PKCß-specific phosphorothioate-modified antisense ODN, and transferred into RPMI 1640 medium containing 10% FCS for 48 h. Nuclear-free extracts were obtained, subjected to SDS–PAGE, and immunoblotted with PKC{alpha}- and ß-specific antibodies. For details, see Methods. Results are representative of 17 independent experiments. (B) Jurkat cells were electroporated in the presence of 10 nmol PKC{alpha} or 13 nmol PKCß FITC-labeled antisense ODN, or 50 µg/ml FITC–dextran, as described in (A). Immediately after electroporation, and 24 and 48 h later an aliquot of cells was washed twice in PBS, pH 7.4 with 0.1% BSA and analyzed by FACScan. Results are representative of three individual experiments.

 
Effective loading of cells with PKC{alpha}- and ß-specific ODN was ascertained by introduction of FITC-labeled antisense ODN, and analysis by FACScan immediately, and 24 and 48 h after electropermeabilization. As shown in Fig. 1(B), 95% of the cells proved to be positive for FITC-labeled antisense ODN under the experimental conditions used. As expected, the intensity of FITC-labeling was decreased after 24 and 48 h; however, the vast majority (i.e. 90%) of the cells remained FITC+ over this time period. To rule out the possibility that reduction of fluorescence intensity was due to degradation of FITC-labeled PKC-specific antisense ODN by cellular endonucleases, FITC-labeled dextran (18) was introduced into the cells, and analyzed after 0, 24 and 48 h after electropermeabilization. The data depicted in Fig. 1(B) show that the fluorescence intensity in FITC–dextran-labeled cells also decreased during the incubation period. These results suggested that the decrease in the fluorescence intensity of FITC-labeled antisense ODN-loaded cells was either due to bleaching or loss of fluorescent stain caused by the multidrug resistance pumps of the plasma membrane rather than to degradation of the ODN during incubation for 48 h.

Phosphorylation and activation of protein kinases of the TCR-stimulated Raf/MEK/ERK cascade is effectively inhibited in PKC{alpha}- and ß-specific antisense ODN-treated cells
Stimulation of cells with a combination of anti-CD3/CD28 antibodies resulted in enhanced phosphorylation and thus activation of Raf-1, MEK-1/2 as well as ERK-1/2. As shown in Fig. 2(A), electroporation itself had no influence on the amount of phosphorylated Raf-1; neither was hyperphosphorylation and subsequent activation of this protein kinase influenced. In sharp contrast, the amount of phosphorylated Raf-1 was reduced by >50% in PKC{alpha}- and to 80% in PKCß-specific antisense ODN-treated cells compared to control, as determined by densitometric analysis.



View larger version (41K):
[in this window]
[in a new window]
 
Fig. 2. Phosphorylation and subsequent activation of protein kinases of the TCR-stimulated Raf/MEK/ERK cascade is inhibited in PKC{alpha}- and ß-specific antisense ODN-treated cells. Cells (8 x 106) were electroporated in the presence and absence of PKC{alpha}- or ß-specific phosphorothioate-modified antisense ODN. Cells were then cultured at a cell density of 2.5 x 105 cells/ml for 48 h. Cells were stimulated with anti-CD3/CD28 antibodies for 20 min, sonicated, centrifuged and supernatants subjected to SDS–PAGE. Specific antibodies raised against the phosphorylated forms of Raf-1 (A), MEK-1/2 (B) and ERK-1/2 (C) were used for immunoblotting. Finally, equal protein loading was confirmed by treating the blots with antibodies raised against the non-phosphorylated form of ERK-2 proteins (D). Results are representative of three experiments.

 
As shown in Fig. 2(B), stimulation of cells with anti-CD3/anti-CD28 antibodies resulted in enhanced phosphorylation of MEK-1/2 in control and electropermeabilized cells. Cells pretreated with PKC{alpha}-specific antisense ODN showed a 90% reduction of MEK phosphorylation and activation. MEK phosphorylation and activation was completely inhibited in cells treated with PKCß-specific antisense ODN. As a consequence of the inhibition of MEK, the activation of the downstream kinases p42/44 MAPK (ERK-1/2) were under the detection limit in both PKC{alpha}- and -ß-specific antisense ODN-treated cells (Fig. 2C).

Both PKC{alpha} and ß regulate IKK-catalyzed I{kappa}B phosphorylation
NF-{kappa}B is one of the most important transcription factors regulating the IL-2 gene (8,9,12,15). In resting cells I{kappa}B is associated with and retards NF-{kappa}B in the cytoplasm in its inactive form. Upon activation via TCR–CD28, I{kappa}B is phosphorylated by IKK, dissociates and NF-{kappa}B translocates to the nucleus (15,19). Figure 3 shows that stimulation of cells with anti-CD3/CD28 resulted in an enhanced phosphorylation of I{kappa}B{alpha}. Introduction of both PKC{alpha}- and ß-specific antisense ODN resulted in a marked reduction of IKK-catalyzed I{kappa}B{alpha} phosphorylation. In cells pretreated with the proteosome inhibitor Z-Leu-Leu-Phe-CHO, a more pronounced phosphorylation of I{kappa}B{alpha} was observed that was significantly inhibited in PKC{alpha} and ß ODN-treated cells.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3. IKK-catalyzed I{kappa}B phosphorylation is inhibited in both PKC{alpha}- and ß-specific antisense ODN-treated cells. PKC{alpha}- and ß-specific antisense ODN were introduced as described in Methods. Cells were preincubated with the proteosome inhibitor Z-Leu-Leu-Phe-CHO (10 µM) for 1 h and stimulated with CD3/CD28 for 15 min. After resolving cellular proteins by SDS–PAGE, the phosphorylated form of I{kappa}B{alpha} was detected in immunoblots. Equal protein loading was proved by immunoblotting against non-phosphorylated ERK-2. Results are representative of three experiments.

 
Lack of effect of PKC-specific antisense ODN on TCR–CD28-stimulated p38 MAPK
It should be emphasized that neither PKC{alpha} nor ß antisense ODN treatment had any influence on phosphorylation and thus activation of TCR–CD28-stimulated p38 MAPK (Fig. 4).



View larger version (28K):
[in this window]
[in a new window]
 
Fig. 4. PKC{alpha} and ß have no influence on activation of p38 MAPK. Jurkat T cells were pretreated with the PKC{alpha}- and ß-specific antisense ODN as described in Methods. Cells were then stimulated with CD3/CD28 for 15 min. Nuclear-free lysates were obtained as described in Methods. Proteins were separated by SDS–PAGE. The phosphorylated form of p38 MAPK was detected in immunoblots. Equal protein loading was proved by immunoblotting against p38 MAPK. Results are representative of four experiments.

 
Protein kinases of the TCR–CD28-activated JNK cascade are specifically inhibited in PKCß-specific antisense ODN-treated cells
Co-stimulation of Jurkat T cells via TCR–CD28 resulted in activation of the JNK cascade. JNK are critically involved in phosphorylation of important transcription factors (ATF-2, c-jun) and in subsequent regulation of the IL-2 gene (20,21). As shown in Fig. 5(A), JNK themselves and their upstream activator, MKK-4, were effectively phosphorylated, and thus activated upon anti-CD3/CD28 stimulation. Treatment of cells with PKCß-specific antisense ODN effectively suppressed MKK-4 as well as JNK phosphorylation. In sharp contrast, no significant changes could be detected in cells treated with PKC{alpha}-specific antisense ODN. Accordingly, activation of JNK-catalyzed c-jun phosphorylation was effectively and specifically suppressed in cells treated with PKCß-specific antisense ODN, whereas introduction of PKC{alpha}-specific antisense ODN treatment was without effect (Fig. 5A).





View larger version (100K):
[in this window]
[in a new window]
 
Fig. 5. (A) Protein kinases of the TCR–CD28-activated JNK cascade are specifically inhibited in PKCß-specific antisense ODN-treated cells. Jurkat T cells were electroporated in the presence and absence of PKC{alpha}- or ß-specific phosphorothioate-modified antisense ODN. Cells were cultured for 48 h and then stimulated with anti-CD3/CD28 antibodies for 15 min. Cells were sonicated and post-nuclear supernatants were centrifuged at 100,000 g for 1 h. Cytoplasmic proteins were subjected to SDS–PAGE. Specific antibodies raised against the phosphorylated forms of MKK-4 (A) and JNK-1 and -2 (B) were used for immunoblotting. The same blots were treated with antibodies raised against the non-phosphorylated form of ERK-2 protein (D). JNK activity was determined with 50 µg of cytosolic protein, 1.5 µg GST-c-jun and 5 µM ATP as substrates as described in Methods. Phosphorylated GST-c-jun was obtained by GSH beads and detected in immunoblots by use of specific antibodies raised against phosphorylated c-jun (C). Results are representative of seven independent experiments. (B) Protein kinases of the TCR–CD28-activated JNK cascade are inhibited by the PKC-specific inhibitor Gö 6976. Jurkat T cells were preincubated with Gö 6976 at the concentrations indicated, and then stimulated with or without OKT3 and anti-CD28 antibodies for 15 min. Cytoplasmic proteins were separated by SDS–PAGE. The phosphorylated forms of MKK-4 and JNK were detected in immunoblots. Equal protein loading was proved by immunoblotting against ERK-2. Results are representative of four experiments. (C) Phosphorylation and activation of the TCR-activated JNK cascade are inhibited by the PKC-specific inhibitor Gö 6976 in human peripheral blood lymphocytes. Human peripheral blood lymphocytes were preincubated with Gö 6976 at the concentrations indicated, and then stimulated OKT3 and anti-CD28 antibodies for 15 min. Cytoplasmic proteins were separated by SDS–PAGE. The phosphorylated forms of MKK-4 and JNK were detected in immunoblots. Equal protein loading was proved by immunoblotting against ERK-2.

 
These results were further assured by a different approach using PKC isoform-specific inhibitors (22). Different inhibitors exerted differential effects on phosphorylation and activation of the JNK protein kinase cascade. As shown in Fig. 5(B), phosphorylation and activation of MKK-4 and JNK was inhibited in a dose-dependent manner by Gö 6976. This substance exhibits high(er) affinity to the PKCß isoform (22). Phosphorylation and activation of MKK-4 and JNK was suppressed at 100 nM Gö 6976. Higher concentrations of this inhibitor completely abolished activation of the JNK protein kinase cascade (Fig. 5B). Ro 32-0432 (showing a higher selectivity to PKC{alpha}) or Gö 6850 exerted no significant effects on phosphorylation and activation of JNK (23).

Strikingly similar effects of the PKC isotype-specific inhibitors were observed on peripheral blood lymphocytes. Gö 6976 exerted a marked inhibitory effect on activation of the JNK protein kinase cascade (Fig. 5C). TCR-induced enhanced phosphorylation of MKK-4 and p46 JNK was inhibited in a concentration-dependent manner; 100 nM of the inhibitor has been effective in significant inhibition of phosphorylation and activation of both enzymes. Ro 32-0432 and Gö 6850 exerted only marginal effects on activation of JNK (data not shown).

Activation of NF-AT and IL-2 gene expression requires PKCß in stimulated Jurkat T cells
As depicted in Table 1, different PKC-specific inhibitors exerted differential effects on activation of the IL-2 reporter gene expression. Ro 32-0432 exerted no significant effect on IL-2 gene expression. Gö 6976 exerted a marked inhibitory effect on IL-2 reporter gene activation. At the concentration used, Gö 6976 proved to be as effective as cyclosporin A in inhibiting IL-2 gene expression, suggesting that proper functioning of PKCß was necessary for onset of IL-2 gene expression (Table 1).


View this table:
[in this window]
[in a new window]
 
Table 1. Gö 6976 efficiently inhibits reporter gene activity of the minimal IL-2 promoter and of the NF-AT-binding domain of the IL-2 promoter
 
The effect of Gö 6976 was investigated in human peripheral blood lymphocytes to show the ‘physiological’ role of PKCß in regulation of IL-2 synthesis. As shown in Table 2, at a concentration of 100 nM Gö 6976 effectively inhibited TCR-induced IL-2 secretion. In contrast, Ro 32-0432 and Gö 6850 did not exert any significant effect on IL-2 synthesis of activated human T cells (data not shown).


View this table:
[in this window]
[in a new window]
 
Table 2.
 
Activation and high-affinity DNA binding of NF-AT is a necessary step of IL-2 gene expression in activated T cells (12,13,24). Upon stimulation with anti-CD3/CD28, a nearly 70-fold elevation of NF-AT activation was detected, as measured by reporter gene activation of firefly luciferase in stably transfected cells. Introduction of PKCß antisense ODN resulted in a significant, i.e. ~50%, inhibition of NF-AT activation (Fig. 6A). Even a 2-fold elevation of NF-AT luciferase activity was measured in cells treated with PKC{alpha}-specific antisense ODN.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 6. Activation of IL-2 gene expression and synthesis is specifically suppressed in PKCß-specific antisense ODN-treated cells. (A) Activation of NF-AT binding and IL-2 gene expression is suppressed in PKCß-, but not {alpha}-, specific antisense ODN-treated cells. Jurkat cells stably transfected with a plasmid containing the luciferase reporter gene together with either a single copy of the minimal IL-2 promoter or four copies of the distal NF-AT-binding domain were electropermeabilized in the presence of PKC{alpha}- and ß-specific antisense ODN under conditions as described in Methods. Cells were stimulated with anti-CD3/CD28 for 6 h for NF-AT binding and for 14 h for IL-2 gene expression. Cellular extracts were prepared and luciferase activity determined as described in Methods. Results are representative of five to seven experiments. (B) IL-2 mRNA synthesis is specifically and significantly inhibited in PKCß-specific antisense ODN-treated cells. Cells were pretreated with PKC{alpha}- or ß-specific antisense ODN as described in Methods. Cells were stimulated with OKT3 and anti-CD28 antibody for 2 h. RNA isolation and RT-PCR were carried out as described in Methods. Results are representative of three experiments. (C) IL-2 synthesis and secretion are specifically and completely inhibited in PKCß-specific antisense ODN-treated cells. Jurkat T cells were seeded in 24-well-flat bottom tissues plates in RPMI with 2% FCS, and stimulated with or without OKT3 and anti-CD28 antibodies for 16 h. IL-2 concentration of the supernatants was determined by a specific ELISA according to the manufacturer’s instructions. IL-2 secreted by non-stimulated cells was under the detection limit. TCR–CD28-stimulated cells synthesized 500–600 pg/ml IL-2 (set to 100%). Results are representative of five experiments.

 
Figure 6(A) shows that TCR–CD28 co-stimulation resulted in a nearly 20-fold elevation of IL-2 reporter gene activation. Electroporation of cells had no influence on IL-2 gene activation; it was moderately elevated in PKC{alpha} antisense ODN-treated cells. Inhibition of PKCß protein synthesis by antisense ODN resulted in ~40% inhibition of IL-2 gene expression. These results were in agreement with determination of endogenous IL-2 mRNA synthesis by means of RT-PCR. A significant enhancement of IL-2-specific mRNA was detected in control and PKC{alpha}-specific antisense ODN-treated cells upon activation via TCR–CD28 (Fig. 6B). In sharp contrast, inhibition of PKCß protein synthesis by specific antisense ODN resulted in a nearly complete inhibition of IL-2 mRNA synthesis. Most interestingly, treatment of cells with PKCß-specific antisense ODN resulted in complete inhibition of secreted IL-2 in the supernatants of TCR–CD28-stimulated cells (Fig. 6C). This effect is highly specific. Introduction of PKC{alpha} antisense ODN into the cells resulted in a 2-fold elevation of IL-2 synthesis and secretion, as compared to the TCR–CD28-stimulated control.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The results of this paper establish a specific function of PKCß for regulation of TCR–CD28-induced signaling in Jurkat T cells. Depletion of cells for PKCß protein by means of specific antisense ODN results in the significant inhibition of several protein kinase cascades, and finally in inhibition of IL-2 gene expression, synthesis and export.

In several in vitro model systems, including transfected Jurkat T cells, PKC{alpha} was considered to be the ‘prototype’ for classical PKC and its influence on TCR–CD28-induced signaling was investigated (3,5,6). Our data confirm the recent observations showing that regulation of the Raf/MEK/ERK protein kinase cascade, as well as activation of the transcription factor NF-{kappa}B, is a function of classical PKC (Figs 2 and 3), illustrating overlapping biological activities of PKC{alpha} and ß for these protein kinase cascades (25,26). Furthermore, both PKC did not influence activation of p38 MAPK, additionally proving the specificity of PKC antisense ODN used in these experiments (Fig. 4).

Regulation by PKC isoenzymes of TCR-induced NF-{kappa}B signaling is well documented (2729). Our data indicate that besides PKC{theta}, PKC{alpha} and ß also participate in activation of IKK-induced I{kappa}B phosphorylation. The decisive function of PKCß controlling I{kappa}B kinase activation has been shown very recently in response to BCR signaling (30).

The molecular mechanisms involved in the activation of the Raf/MEK/ERK protein kinase cascade are well established (31,32). Phosphorylation and thus activation of the Raf/MEK/ERK cascade was impaired in TCR–CD28-stimulated Jurkat T cells depleted of PKC{alpha} or ß. Both PKC{alpha} and ß have been suggested to regulate the Raf/MEK/ERK cascade in epidermal growth factor-stimulated Cos cells, although in a different manner (33). To our knowledge, there are no published data on the participation of PKCß in regulation of TCR-induced Raf-1 activation.

Several studies have provided evidence that the Raf/MEK/ERK protein kinase cascade is a necessary (but not sufficient) effector pathway for NF-AT activation. These conclusions, which mainly result from expression of dominant-negative or constitutive active p21Ras, Raf-1 or MEK-1 mutants, have created a paradigm that the Raf-1/MEK-1/ERK-2 pathway was one of the major routes for activation of IL-2 gene transcription in TCR–CD3-stimulated cells (3436). Our results show that both PKC{alpha} and ß interfered with this protein kinase cascade in a similar way. However, only the PKCß isoform seems to be specifically involved in activation of NF-AT binding, IL-2 gene expression and IL-2 secretion. This implies the contribution of other TCR–CD28-regulated signal transduction pathways for regulation of IL-2 synthesis and secretion.

Regulation of TCR–CD28 induced activation of the JNK cascade seemed to be a specific function of PKCß in our experimental system. Phosphorylation of MKK-4 and subsequent activation of JNK was significantly reduced in PKCß-depleted cells (Fig. 5A).

The function of JNK and regulation of their activity by PKC isoenzymes is a matter of discussion. While enhanced activity of JNK was an absolute necessity for regulation of IL-2 gene expression in T cell lines (5,6,21), a defect in JNK signaling was claimed to be involved in T cell differentiation, but not in T cell activation in vivo, i.e. in JNK-deficient animals (37,38). It has been demonstrated that activation of NFAT and subsequent regulation of cytokine gene expression was impaired in JNK-deficient mice (39). The importance of JNK in IL-2 gene expression has been further emphasized by showing that calcineurin and JNK synergized in induction of IL-2 synthesis (5,6,20 21). Furthermore, JNK-catalyzed phosphorylation of c-jun, as well as JNK-regulated elevation of c-jun synthesis, was a prerequisite of high-affinity NF-AT binding and subsequent cytokine synthesis in T lymphocytes (20,21,38,39). It is well documented that JNK participating in the re-phosphorylation of NF-AT in the nucleus regulates the duration (length) of NF-AT–DNA interactions involved in cytokine gene expression (39). Furthermore, it was suggested that JNK might be responsible for stabilization of IL-2-specific mRNA in T cells (40). Since activation and functioning of JNK is critically involved in regulation of the NF-AT-binding site of the IL-2 promoter, our data indicate that PKCß-regulated JNK activation is required for efficient, high-affinity NF-AT binding and IL-2 gene expression in Jurkat T cells. Our data imply that PKCß operates upstream of MKK-4, leading to enhanced phosphorylation, and thus activation of MKK-4-catalyzed JNK and subsequent phosphorylation of c-jun, as well as high-affinity NF-AT binding (Figs 5 and 6).

Numerous reports emphasized the importance of the calcium-independent PKC isoform, PKC{theta}, in the regulation of JNK activation as well as in the generation of transcription factors participating in enhancing the activation of the IL-2 promoter in vivo and in vitro (68,41). The hypothesis that PKC{theta} played a significant role in T lymphocyte activation, as indicated by numerous studies in cell lines, was recently confirmed in mice deficient in the expression of this enzyme (8). Peripheral T cells lacking PKC{theta} failed to activate NF-{kappa}B and AP-1, and failed to express IL-2, in response to TCR stimulation. This revealed a critical function for this PKC family member in linking membrane-proximal activation cascades to transcriptional responses governing T cell activation (8).

The molecular interactions involved in the engagement of PKC{theta} have not been fully delineated. In T cell lines, PKC{theta}-regulated activation of JNK was claimed to participate in IL-2 gene expression (57). In contrast, no change in JNK activation has been observed in T cells of PKC{theta}-deficient animals, although the amount of IL-2 was significantly decreased (8). The results indicated that PKC{theta} was an essential component activating the NF-{kappa}B signaling cascade and thus leading to expression of the IL-2 gene during T cell activation. However, stimulation with phorbol ester plus ionomycin resulted in restoration of IL-2 gene synthesis and cellular proliferation. These findings suggest the involvement of a conventional PKC which might compensate for the absence of PKC{theta}.

If PKCß can fit into this function, one would expect enhanced amounts (activity) of PKCß in T cells of PKC{theta}-deficient animals. In fact, elevated expression of cPKC has been observed in those cells (42).

Participation of cPKC, after all that of PKCß, in regulation of T cell activation was also controversially discussed, depending on the experimental system used. On the one hand, TCR-induced cellular proliferation was not influenced in PKCß-deficient mice (43); on the other hand, several reports dealt with regulation by cPKC, including PKCß of signal transduction and cytokine gene expression in T cells and T cell lines (3,6,11,44).

At first glance our results contradict those of Long et al. (44) showing that PKCß was specifically implicated in secretion (but not transcription) of IL-2 in HUT 78 cells. IL-2 gene expression was inhibited by 50% in Jurkat cells; however, IL-2 secretion was completely abolished upon PKCß depletion, implying that PKCß must be specifically involved in both processes. Since T cells and T cell lines showed marked functional differences, it is not surprising that this heterogeneity was also reflected in differential expression of PKC genes and protein. Gene expression and function of PKC was claimed to be strictly cell-type specific (42,45,46).

While Jurkat T cells express high amounts of both PKC{alpha} and ß, PKC{alpha} is the dominant PKC isoform in the HUT 78 cell line. It is obvious that IL-2 synthesis is differentially regulated in Jurkat and HUT 78 cells. Resembling TCR–CD3 signaling, the phenotypically non-activated Jurkat T cell line requires phorbol myristate acetate plus ionomycin for IL-2 synthesis. In contrast, HUT 78, a phenotypically activated T cell line, secreted IL-2 in response to phorbol myristate acetate alone, suggesting that a calcium-independent PKC isoform might be involved in regulation of IL-2 synthesis (47).

Both depletion of Jurkat cells for PKCß and repression of its activity by an isotype-specific inhibitor resulted in a significant reduction of IL-2 gene expression in our experimental system. In accordance with our recent data and those of other groups, the PKCß-specific inhibitor Gö 6976 was effective in suppression of JNK activation and IL-2 synthesis in human peripheral blood lymphocytes (11,47,48). These results underline the importance of PKCß in regulation of the IL-2 gene (Figs 5 and 6, and Tables 1 and 2) (23). Our more recent results showing that overexpression of PKCß or its catalytic subunit leads to a remarkable elevation of IL-2 synthesis support the assumption of the involvement of PKCß in regulation of IL-2 gene expression (manuscript in preparation).

In summary, data presented in this paper confirm and extend our hypothesis on the decisive role of PKCß in the regulation of IL-2 synthesis in T lymphocytes. The results suggest that interference of PKCß with activation of the JNK cascade might be one of the important mechanisms in the regulation of high-affinity NF-AT binding, IL-2 synthesis and secretion. The fact that a complete inhibition of IL-2 synthesis and secretion was achieved in PKCß-specific antisense ODN-treated cells indicates a highly specific (and most important) function of the PKCß isoform in the gene expression and processing of this cytokine.


    Acknowledgements
 
This work was supported by the Deutsche Forschungs-Gemeinschaft, from grants of SFB 265-A9.


    Abbreviations
 
ERK—extracellular-regulated kinase

ERK-1—p44 MAPK

ERK-2—p42 MAPK

IKK—I{kappa}B kinase

JNK—Jun-N-terminal kinase

MAPK—mitogen-activated protein kinase

MEK—mitogen-activated ERK kinase

MKK—MAPK kinase

ODN—oligodesoxynucleotide

PKC—protein kinase C


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Nishizuka, Y. 1992. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258:607.[ISI][Medline]
  2. Berry, N. and Nishizuka., Y. 1990. Protein kinase C and T cell activation. Eur. J. Biochem. 189:205.[Abstract]
  3. Genot, E. M., Parker, P. J. and Cantrell, D. A. 1995. Analysis of the role of protein kinase C-{alpha}, -{epsilon}, and {zeta} in T cell activation. J. Biol. Chem. 270:9833.[Abstract/Free Full Text]
  4. Monks, C. R., Kupfer, H., Tamir, I., Barlow, A. and Kupfer, A. 1997. Selective modulation of protein kinase C-theta during T cell activation. Nature 385:83.[CrossRef][ISI][Medline]
  5. Werlen, G., Jacinto, E., Xia, Y. and Karin, M. 1998. Calcineurin preferentially synergizes with PKC{theta} to activate JNK and IL-2 promoter in T lymphocytes. EMBO J. 17:3101.[Abstract/Free Full Text]
  6. Ghaffari-Tabrizi, N., Bauer, B., Villunger, A., Baier-Bitterlich, G., Altman, A., Utermann, G., Überall, F. and Baier, G. 1999. Protein kinase C{theta}, a selective upstream regulator of JNK/SAPK and IL-2 promoter activation in Jurkat T cells. Eur. J. Immunol. 28:132.[CrossRef]
  7. Altman, A., Isakov, N. and Baier, G. 2000. Protein kinase C{theta}: a new essential superstar on the T-cell stage. Immunol. Today 21:567.[CrossRef][ISI][Medline]
  8. Sun, Z., Arendt, C. W., Ellmeier, W., Schaeffer, E. M., Sunshine, M. J., Gandhi, L., Annes, J., Petrzilka, D., Kupfer, A., Schwartzberg, P. L. and Littman, D. R. 2000. PKC{theta} is required for TCR-induced NF-{kappa}B activation in mature but not immature T lymphocytes. Nature 404:402.[CrossRef][ISI][Medline]
  9. Dienz, O., Hehner, S. P., Dröge, W. and Schmitz, M. L. 2000. Synergistic activation of NF-{kappa}B by functional cooperation between Vav and PKC{theta} in T lymphocytes. J. Biol. Chem. 275:24547.[Abstract/Free Full Text]
  10. Szamel, M., Bartels, F. and Resch, K. 1993. Cyclosporin A inhibits T cell receptor-induced interleukin-2 synthesis of human T lymphocytes by selectively preventing a transmembrane signal transduction pathway leading to sustained activation of a protein kinase C isoenzyme, protein kinase C-beta. Eur. J. Immunol. 23:3072.[ISI][Medline]
  11. Szamel, M., Appel, A., Schwinzer, R. and Resch, K. 1998. Different protein kinase C isoenzymes regulate IL-2 receptor expression or IL-2 synthesis in human lymphocytes stimulated via the TCR. J. Immunol. 160:2207.[Abstract/Free Full Text]
  12. Serfling, E., Avots, A. and Neumann, M. 1995. The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation. Biochim. Biophys. Acta 263:181.
  13. Serfling, E., Berberich-Siebelt, F., Chuvpilo, S., Jankevics, E., Klein-Hessling, S., Twardzik, T. and Avots, A. 2000. The role of NF-AT transcription factors in T cell activation and differentiation. Biochim. Biophys. Acta 1498:1.[ISI][Medline]
  14. Rincón, M., Flavell, R. A. and Davis, R. 2001. Signal transduction by MAP kinases in T lymphocytes. Oncogene 20:2490.[CrossRef][ISI][Medline]
  15. Li, N. and Karin, M. 2000. Signaling pathways leading to nuclear factor-kappa B activation. Methods Enzymol. 319:273.[ISI][Medline]
  16. Koller, E., Gaarde, W. A. and Monia, B. R. 2000. Elucidating cell signaling mechanisms using antisense technology. Trends Pharmacol. Sci. 21:142.[CrossRef][ISI][Medline]
  17. Toulmé, J. J., Cazenave, C. and Moreau, S. 1997. Antisense Technology, p. 39, IRL Press/Oxford University Press, Oxford, UK.
  18. Grayson, M. H, Chaplin, D. D., Karl, E. I. and Hotchkiss, R. S. 2001. Confocal fluorescent intravital microscopy of the murine spleen. J. Immunol. Methods 256:56.
  19. Karin, M. and Ben-Neriah, Y. 2000. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu. Rev. Immunol. 18:621.[CrossRef][ISI][Medline]
  20. Kaminuma, O., Deckert, M., Elly, C., Liu, Y.-C. and Altman, A. 2001. Vav–Rac1-mediated activation of the c-Jun N-terminal kinase/c-Jun/AP-1 pathway plays a major role in stimulation of the distal NFAT site in the interleukin-2 gene promoter. Mol. Cell. Biol. 21:3126.[Abstract/Free Full Text]
  21. Avraham, A., Jung, S., Samuels, Y., Seger, R. and Ben-Neriah, Y. 1998. Co-stimulation-dependent activation of a JNK-kinase in T lymphocytes. Eur. J. Immunol. 28:2320.[CrossRef][ISI][Medline]
  22. Way, K. J., Chou, E. and King, G. I. 2000. Identification of PKC isoform-specific biological actions using pharmacological approaches. Trends Pharmacol. Sci. 21:181.[CrossRef][ISI][Medline]
  23. Dreikhausen, U. E., Gorf, K., Schmidt, M., Golombek, M., Resch, K. and Szamel, M. 2000. Molecular mechanisms of T lymphocyte activation: involvement of protein kinase C isoenzymes in regulation of signal transduction cascades controlling interleukin-2 gene expression. Naunyn-Schmiedebergs Arch. Pharmacol. 361:R67.[CrossRef]
  24. Crabtree, G. R. 2001. Calcium, calcineurin, and the control of transcription. J. Biol. Chem. 276:2313.[Free Full Text]
  25. Dhillon, A. S. and Kolch W. 2002. Untying the regulation of the Raf-1 kinase. Arch. Biochem. Biophys. 404:3.[CrossRef][ISI][Medline]
  26. Trushin, S. A., Pennington, K. N., Ageciras-Schimnich, A. and Paya, C. V. 1999. Protein kinase C and calcineurin synergize to activate I{kappa}B kinase and NF-{kappa}B in T lymphocytes. J. Biol. Chem. 274:22923.[Abstract/Free Full Text]
  27. Lin, X., O‘Mahony, A., Mu, Y., Geleziunas, R. and Greene, W. C. 2000. Protein kinase C-participates in NF-B activation induced by CD3–CD28 costimulation through selective activation of I{kappa}B kinase. Mol. Cell. Biol. 20:2933.[Abstract/Free Full Text]
  28. Coudronniere, N., Villalba, M., Englund, N. and Altman, A. 2000. NF-{kappa}B activation induced by T cell receptor/CD28 costimulation is mediated by protein kinase C-{theta}. Proc. Natl Acad. Sci. USA 97:3394.[Abstract/Free Full Text]
  29. Khoshnan, A., Bae, D., Tindell, C. A. and Nel, A. E. 2000. The physical association of protein kinase C theta with a lipid raft-associated inhibitor of kappa B factor kinase (IKK) complex plays a role in the activation of the NF-kappa B cascade by TCR and CD28. J. Immunol. 165:6933.[Abstract/Free Full Text]
  30. Su, T. T., Guo, B., Kawakami, Y., Sommer, K., Chae, K., Humphries, L. A., Kato, R. M., Kang, S., Patrone, L., Wall, R., Teitell, M., Leitges, M., Kawakami, T. and Rawlings, D. J. 2002. PKC{theta} controls I{kappa}B kinase lipid raft recruitment and activation in response to BCR signaling. Nat. Immunol. 3:780.[ISI][Medline]
  31. Rincon, M. 2002. MAP kinase signalling pathways in T cells. Curr. Opin. Immunol. 13:339.[CrossRef][ISI]
  32. Kolch, W. 2000. Meaningful relationships: the regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem. J. 351:289.[CrossRef][ISI][Medline]
  33. Schönwasser, D. C., Marais, R. M., Marshall, C. J. and Parker, P. J. 1998. Activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway by conventional, novel, and atypical protein kinase C isotypes. Mol. Cell. Biol. 18:790.[Abstract/Free Full Text]
  34. Genot, E., Cleverley, S., Henning, S. and Cantrell, D. 1996. Multiple p21ras effector pathways regulate nuclear factor of activated T cells. EMBO J. 15:3923.[Abstract]
  35. Izquierdo, M., Bowden, S. and Cantrell, D. 1994. The role of Raf-1 in the regulation of extracellular signal-regulated kinase 2 by the T cell antigen. J. Exp. Med. 180:401.[Abstract]
  36. Whitehurst, C. E. and Geppert, T. D. 1996. MEK1 and the extracellular signal-regulated kinases are required for the stimulation of IL-2 gene transcription in T cells. J. Immunol. 156:1020.[Abstract]
  37. Dong, C., Yang, D. D., Wysk, M., Whitmarsh, A. J., Davis, R. J. and Flavell, R. A. 1998. Defective T cell differentiation in the absence of Jnk1. Science 282:2092.[Abstract/Free Full Text]
  38. Dong, C., Yang, D. D., Tournier, C., Whitmarsh, A. J., Xu, J., Davis, R. J. and Flavell, R. A. 2000. JNK is required for effector T-cell function but not for T-cell activation. Nature 405:91.[CrossRef][ISI][Medline]
  39. Chow, C.-W., Dong, C., Flavell, R. A. and Davis, R. J. 2000. c-Jun NH2-terminal kinase inhibits targeting of the protein phosphatase calcineurin to NFATc1. Mol. Cell. Biol. 20:5227.[Abstract/Free Full Text]
  40. Chen, C. Y., Del Gatto-Konczak, F., Wu, Z. and Karin, M. 1998. Stabilization of interleukin-2 mRNA by the c-Jun NH2-terminal kinase pathway. Science 280:1945.[Abstract/Free Full Text]
  41. Kempiak, S. J., Hiura, T. S. and Nel, A. E. 1999. The Jun kinase cascade is responsible for activating the CD28 response element of the IL-2 promoter: proof of cross-talk with the I{kappa}B kinase cascade. J. Immunol. 162:3176.[Abstract/Free Full Text]
  42. Baier, G. 2003. The PKC gene module: molecular biosystematics to resolve its T cell functions. Immunol. Rev. 192:64.[ISI][Medline]
  43. Leitges, M., Schmedt, C., Guinamard, R., Davoust, J., Schaal, S., Stabel, S. and Tarakhovsky, A. 1996. Immunodeficiency in protein kinase Cbeta-deficient mice. Science 9:788.
  44. Long, A., Kelleher, D., Lynch, S. and Volkov, Y. 2001. Protein kinase Cß expression is critical for export of IL-2 from T cells. J. Immunol. 167:636.[Abstract/Free Full Text]
  45. Bauer, B., Krumbock, N., Ghaffari-Tabrizi, N., Kampfer, S., Villunger, A., Wilda, M., Hameister, H., Utermann, G., Leitges, M., Uberall, F. and Baier, G. 2000. T cell expressed PKCtheta demonstrates cell-type selective function. Eur. J. Immunol. 12:3645.[CrossRef]
  46. Wilda, M., Ghaffari-Tabrizi, N., Reisert, I., Utermann, G., Baier, G. and Hameister, H. 2001. Protein kinase C isoenzyme: selective expression pattern of protein kinase C-theta; during mouse development. Mech. Dev. 103:197.[CrossRef][ISI][Medline]
  47. Keenan, C., Long, A. and Kelleher, D. 1997. Protein kinase C and T cell function. Biochim. Biophys. Acta 1358:113.[CrossRef][ISI][Medline]
  48. Mochly-Rosen, D. and Kauvar, L. M. 2000. Pharmacological regulation of network kinetics by protein kinase C localization. Semin. Immunol. 12:55.[CrossRef][ISI][Medline]




This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (2)
Request Permissions
Google Scholar
Articles by Dreikhausen, U. E.
Articles by Szamel, M.
PubMed
PubMed Citation
Articles by Dreikhausen, U. E.
Articles by Szamel, M.