Stimulation of purified human PBL with mAbs raised against the T cell receptor resulted in an immediate and transient activation of protein kinase C-α (PKC-α) and PKC-θ, peaking at 10 min, whereas PKC-β, -δ, and -ε were translocated with a delay of >90 min and remained activated for up to 2 h. To characterize specific functions of distinct PKC isoenzymes, Abs against different PKC isoenzymes were introduced by means of electropermeabilization. Neutralization of PKC-α and -θ resulted in the complete inhibition of IL-2R expression, whereas anti-PKC-β, -δ, and -ε Abs inhibited IL-2 synthesis. Extensive control experiments have shown that neither electropermeabilization nor control Ig influenced PKC activity and cellular functions. Our data thus clearly show that specific PKC isoenzymes regulate different cellular functions in stimulated human lymphocytes.

Activation of lymphocytes via the TCR/CD3 complex leads to the increased hydrolysis of inositol phospholipids and to subsequent production of inositol polyphosphates and diacylglycerols (DAG)3 that result in elevation of intracellular calcium concentration and activation of protein kinase(s) C (PKC), respectively (1, 2). PKC represents a family of serine/threonine-specific protein kinases; at present, 11 different PKC isoenzymes are known, which were classified according to their structure and cofactor requirements for activation. Although all PKC isoforms are activated by phospholipids, with notable exceptions (DAG), particular isoenzymes differ markedly in their sensitivity toward calcium. PKC-α, -β1, -β2, and -γ are dependent on calcium for activation, whereas PKC-δ, -ε, -η, and -θ are not. A third group of PKC isoenzymes (PKC-λ, -ι, and -ζ) structurally belongs to the PKC family, but, atypically, is not activated by phorbol esters or DAGs (3, 4). A fourth group of enzymes has been described recently that binds DAG/phorbol esters and has structural homologies with the PKC family, but has unusual catalytic domains (5). Particular PKC isoenzymes differ in their substrate specificity in vitro, suggesting that different PKC isoforms may have distinct cellular functions, reflecting their substrate preferences in vivo.

Since the late seventies, the involvement PKCs in the course of T cell activation has been well established (6, 7). T lymphocyte activation represents a highly pleiotropic set of cellular responses that includes cell cycle entry and release of cytokines. Stimulation of T cells results in the transcription of several genes and expression of a variety of molecules, including cytokines and their specific receptors, such as IL-2 and its receptor.

PKCs regulate T cell activation genes via control of several transcription factors (1, 2, 8). T cells express multiple isotypes of PKC, including PKC-α, -β1, -δ, -ε, -η, -θ, and -ζ; however, the role of different PKC isotypes in the regulation of cellular signaling and gene transcription is not definitely established. Several conclusions regarding the role of PKC in T cell activation were based on experiments that examined the effects of phorbol esters or synthetic DAGs, which simultaneously activated multiple isotypes of PKC (8, 9, 10).

A significant approach to define the specific functions of PKC isoenzymes is to investigate functional changes in cells transfected with mutated constitutively active or inactive PKCs, respectively (11, 12, 13, 14, 15, 16, 17, 18). Such PKC mutants have been used to examine directly the functional effects of individual PKC isotypes in various cell lines (19, 20, 21, 22). However, recent data have shown significant differences in molecular mechanisms of signal transduction between T cell lines and normal resting T lymphocytes (23, 24).

Accordingly, to examine the role of PKC in T cell activation we have analyzed the consequences of neutralization of PKC-α, -β, -δ, -ε, -θ, and -ζ, as representatives for the three major subdivisions of the PKC family, by introduction of specific Abs into PBL. Of the many diverse PKC responses that could be explored, regulation of PKC-responsive IL-2 synthesis and surface expression of IL-2R were investigated. The results show that stimulation of T lymphocytes via the TCR/CD3 complex leads to activation and translocation of PKC isoenzymes with differential kinetics. The data show that PKC-α and -θ are the main regulators of IL-2R expression, while PKC-β, PKC-δ, and, to a minor extent, PKC-ε participate in the regulation of IL-2 synthesis.

All chemicals if not otherwise indicated were obtained from Sigma Chemical Co. (St. Louis, MO).

Mouse mAb, BMA 031 (IgG2b), raised against monomorphic determinants of the TCR was a gift from Dr. R. Kurrle (Behringwerke, Germany). OKT3 raised against the ε-chain of the TCR/CD3 complex was purified from CD3 hybridoma (American Type Culture Collection, Rockville, MD) supernatants by means of affinity chromatography on protein A-agarose. The Abs against protein kinases C were from different sources: anti-PKC-α, raised against the epitope corresponding to amino acids 651 to 672 mapping at the carboxyl terminus of cPKC-α, anti-PKC-β raised against an epitope corresponding to amino acids 656 to 671 mapping at the carboxyl terminus of cPKC-β, anti-PKC-δ against an epitope corresponding to amino acids 657 to 673 at the carboxyl terminus of nPKC-δ, anti-PKC-ε against an epitope corresponding to amino acids 722 to 736 mapping at the carboxyl terminus of nPKC-ε, and anti-PKC-ζ against an epitope corresponding to amino acids 573 to 592 mapping at the carboxyl terminus of nPKC-ζ were purchased from Dianova, (Hamburg, Germany). Anti-PKC-θ raised against residues 21 to 217 of the regulatory domain of nPKC-θ was purchased from Transduction Laboratories (Lexington, Kentucky). Anti-pan-PKC Abs were from Seikagaku Corp (Kyoto, Japan). For immunoblotting, mAbs against PKC isoenzymes from Transduction Laboratories were used.

PBL were isolated by Ficoll gradient centrifugation. Mononuclear cells were washed with RPMI 1640 and taken up in RPMI 1640 supplemented with 20% FCS. After adherence depletion overnight in plastic dishes, the cell suspension contained <1% of monocytes as assessed with an FITC-conjugated Ab against CD14 (Dianova) by flow cytometric analysis. Cells were cultured in RPMI 1640 supplemented with 10% FCS, 100 U of penicillin, 100 μg/ml streptomycin, and 2 mM glutamine in flat-bottom microtiter plates with 4 × 105 cells/well.

Lymphocytes were stimulated with BMA 031 (5 μg/ml), OKT3 (5 μg/ml), or PMA (10 ng/ml), respectively, as indicated in the experiments. Cells were incubated for 68 h (if not otherwise stated), then 0.5 μCi of [3H]TdR (20 Ci/mmol; Amersham, Arlington Heights, IL) was added for an additional 4 h. Cells were harvested with an automatic cell harvester, and incorporation of [3H]TdR into DNA was determined by liquid scintillation counting.

Electropermeabilization was performed by the capacitor discharge method, as described previously (25), in a Bio-Rad Gene Pulser (Bio-Rad, Richmond, CA) at 20°C with slight modifications. Briefly, human lymphocytes (2 × 106/ml) in permeabilization buffer containing 10 mM PIPES (pH 7.4), 0.5 mM MgCl2, 120 mM NaCl, 10 mM EGTA, and 8.2 mM CaCl2 were processed to electroporation in the presence of Abs raised against different PKCs at a concentration of 2 μg/ml. As control Ab, 2 μg/ml rabbit Ig (IgG1; Dianova) was used. Except when indicated otherwise, permeabilization was achieved by using a capacitance of 500 μF at 300 V current, which decreased to 1/e (= τ) in 8.5 ms (see also Table I). After electropermeabilization, lymphocytes were allowed to recover in RPMI/50% FCS for 1 h at 37°C. Cells were then washed, and viability was routinely tested by trypan blue exclusion test. Alternatively, propidium iodide incorporation was determined by incubation of 105 cells with 50 ng/ml propidium iodide for 20 min at 4°C. Propidium iodide-positive cells were analyzed by flow cytometry. Under the conditions used, >90% of cells proved to be viable following electropermeabilization.

Table I.

Influence of electropermeabilization conditions on viability of human T lymphocytesa

Capacitance (μF)Voltage (V)τ (ms)% of Living Cells
960 300 31.2 20 ± 6 
 250 28.9 50 ± 11 
500 300 8.7 89 ± 4 
 250 8.4 85 ± 10 
250 300 3.9 90 ± 7 
 250 4.1 84 ± 14 
125 300 2.0 96 ± 8 
 200 2.0 99 ± 11 
Capacitance (μF)Voltage (V)τ (ms)% of Living Cells
960 300 31.2 20 ± 6 
 250 28.9 50 ± 11 
500 300 8.7 89 ± 4 
 250 8.4 85 ± 10 
250 300 3.9 90 ± 7 
 250 4.1 84 ± 14 
125 300 2.0 96 ± 8 
 200 2.0 99 ± 11 
a

Lymphocytes 2 × 106 in 800 μl of “intracellular buffer” were electropermeabilized as described in Materials and Methods. After incubation in RPMI, 50% FCS, cell viability was determined by propidium iodide staining by means of flow cytometry. Results are means ± SD of two independent experiments.

Lymphocytes (5 × 106) were electropermeabilized in the presence of 2 μg/ml phycoerythrin (PE)-conjugated IgG2a (Dianova) as described above. Cells were then incubated in RPMI/50% FCS for 1 h. After washing with PBS containing 0.5% BSA, lymphocytes were incubated with 5 μg/ml BMA 031 for 30 min. Cells were then washed and incubated with dichlorotriazinylaminofluorescein (DTAF)-conjugated goat anti-mouse Ig (F(ab′)2) fragment (Dianova) for an additional 30 min and extensively washed with PBS/0.1% BSA. Flow cytometric analysis was conducted with a FACS (Becton Dickinson, Mountain View, CA).

Human lymphocytes were electropermeabilized, then stimulated with the mAb BMA 031 (5 μg/ml) or OKT3 (5 μg/ml), respectively, as indicated in the experiments, at a cell density of 2 × 106/ml in RPMI medium supplemented with 2% FCS for 20 h. Supernatants were harvested, and the concentration of IL-2 was quantitated by means of a specific ELISA with a sensitivity of 15 pg/ml. The Abs against human IL-2 were obtained from Genzyme (Cambridge, MA).

Lymphocytes (5 × 106) were stimulated with BMA 031 for 48 h in 2 ml of medium RPMI supplemented with 2% FCS. Cells were washed with PBS containing 0.5% BSA, then taken up in 20 μl of intraglobin (10 mg/ml). After incubation with 2 μg of PE-labeled anti-CD25 Abs (Dianova) for 30 min at 4°C, cells were washed three times in PBS containing 0.1% BSA. CD25-positive cells were determined by flow cytometric analysis.

Human lymphocytes (2 × 107) were stimulated with 5 μg/ml BMA 031 for the times indicated in the experiments. Total RNA was then isolated on RNeasy spin columns (Qiagen, Hilden, Germany) and transcribed into cDNA using reverse transcriptase from Moloney murine leukemia virus (Stratagene, La Jolla, CA). Amplification of cDNA was conducted with 2.5 U of Taq polymerase (Stratagene) in a Gene Amp PCR System 9600 (Perkin-Elmer/Cetus, Norwalk, CT) by 30 cycles. Three temperature cycles consisted of denaturation at 94°C (55 s), annealing at 55°C (60 s), and elongation at 72°C (60 s). The following primers were used: IL-2, 5′-GAATGGAATTAATAATTACAAGAATCCC-3′; antisense primer, 5′-TGTTTCAGATCCCTTTAGTTCCAG-3′; tubulin, 5′-TTCCCTGGCCAGCTGCAAAGCTGCAGCTGACCTAGCTCGCAAG-3′; and antisense primer, 5′-CATGCCCTCGCCAGCTGTGTACCAGTGAGCTAAGCTGAAGGC-3′.

PCR products were separated in 2% agarose gels and visualized by staining with ethidium bromide.

IL-2 binding was conducted as described previously (26). Briefly, lymphocytes were stimulated with OKT3 for 24 h. Cells were then washed and incubated with 0.1 μCi 125I-labeled IL-2 (sp. act., 600 Ci/mmol; Amersham) and different concentrations of human recombinant IL-2 ranging from 20 pmol to 200 nmol at 4°C for 1 h. Free and bound radioactivity was separated by centrifugation through silicon oil as described previously (26). The number of high affinity receptors was calculated by Scatchard analysis with a computer program.

Determination of PKC activity in permeabilized cells.

PKC activity was determined in permeabilized cells with the PKC-specific substrate, peptide GS (Bachem, Torrance, CA), as described previously (27). An essential component of this assay is the translocation of PKC to cellular membranes, since without translocation, PKC is rapidly lost from streptolysin O-permeabilized cells (27). Cells were washed before permeabilization with intracellular buffer, containing 120 mM KCl, 5 mM MgCl2, 5 mM PIPES (pH 7.4), 12.5 mM EGTA, and 8.17 mM CaCl2, resulting in final concentrations of 3.5 mM free Mg2+ and 150 nM free Ca2+, respectively. Cells were permeabilized with streptolysin-O (0.05 U/ml), and phosphorylation with 50 μM [32P]ATP (500 cpm/pmol) was conducted in a total volume of 250 μl at 37°C 5 min. The reaction was stopped by the addition of 100-μl portions of 25% (w/v) TCA in 2 M acetic acid. After being left for at least 10 min on ice, samples were centrifuged, and aliquots were spotted on P81 ion exchange chromatography paper (Whatman, Clifton, NJ), which was then washed three times in 30% (w/v) acetic acid containing 1% H3PO4. Assays were performed in duplicate, and a single tube containing no peptide was included for each pair of duplicate assays to estimate background phosphorylation of basic cell components that were not precipitated by TCA and that adhered to P81 paper. The values of such blanks were <10% of the values determined in the presence of peptide GS. Specific PKC activity is expressed as the difference between the values measured in the presence and those measured in the absence of the PKC-specific inhibitor, bis-indolylmaleimide (100 nM), in nanomoles of 32P transferred per minute per 2 × 106 cells.

Determination of PKC activity in subcellular fractions.

Cytoplasmic PKC was measured in aliquots of the cytosolic fraction immediately after isolation, without further purification, in a reaction mixture containing 40 mM HEPES, 10 mM MgCl2, 0.4 mM EGTA, 400 μM peptide GS, and 60 μM [32P]ATP (500 cpm/pmol) with or without 2 mM CaCl2, 200 μg/ml phosphatidylserine, and 20 μg/ml 1,2-diolein. Lipids were dispersed by sonification. The reaction was started by mixing 100 μl of reaction medium with 10 μg of sample protein in isolation buffer. After an incubation for 3 min at 37°C, the reaction was stopped by the addition of 100-μl portions of 25% (w/v) TCA in 2 M acetic acid. Aliquots were spotted on P81 ion exchange chromatography paper, which was then washed three times in 30% (w/v) acetic acid containing 1% H3PO4. The enzyme activity was calculated as the difference between the values measured in the presence and those measured in the absence of phospholipids and calcium in nanomoles of 32P transferred per minute per mg protein of the sample. As peptide substrates do not reflect the calcium dependency shown for polypeptide substrates (27), the specific activity of both classical and calcium-independent PKC isoforms could be determined by this assay.

Lymphocytes (5 × 106 cells/ml) were stimulated in serum-free medium RPMI with BMA 031 (5 μg/ml) and/or PMA (100 ng/ml) for different lengths of time as indicated in the experiments. Cells were then washed and resuspended in lysis buffer (20 mM Tricine (pH 7.4), 1 mM EGTA, 1 mM EDTA, 5 mM dithioerythritol, and 50 μg/ml leupeptin) and disrupted by nitrogen cavitation. Cytosolic and plasma membrane fractions were separated by removing nuclei and large granules (mitochondria, lysosomes) by centrifugation at 12,000 × g for 20 min, followed by centrifugation at 100,000 × g for 1 h. The membrane pellet was resuspended in lysis buffer as previously described (28). As resting lymphocytes contain a relatively small amount of endoplasmic reticulum, our membrane preparation can be considered a crude plasma membrane fraction containing <10% contamination by endoplasmic reticulum (29). Protein was determined by a method described previously (30).

Plasma membranes of stimulated lymphocytes were solubilized with 2 mg of 3-[(3-cholaminopropyl)dimethylammonio]-1 propanesulfonate/mg protein for 30 min at 4°C. Solubilized proteins were separated from nonsoluble material by centrifugation at 150,000 × g for 1 h as previously described (31).

Membrane protein (40 μg) was processed by SDS-PAGE on 10% polyacrylamide gels. Electroblotting of proteins to Immobilon membranes (Millipore Corp., Bedford, MA) was conducted at a constant current of 200 mA for 2 h. Blots were then incubated with mAbs against different PKC isoforms for 4 h. To visualize PKC subspecies, goat anti-mouse IgG and enhanced chemiluminescence reagent (Amersham) were used according to the manufacturer’s instructions.

It should be mentioned that all Abs used for electropermeabilization and neutralization of different PKCs were tested for their cross-reactivity by immunoblotting. While anti-PKC-α and -θ did not show any cross-reactivity, some, but not significant, cross-reactivity was detectable between PKC-β and -δ, but not PKC-ε, suggesting that anti-PKC Abs recognized their corresponding Ags, i.e., distinct PKC isoenzymes, with high specificity.

As shown in Figure 1, stimulation of human PBL with BMA 031 via the TCR/CD3 complex led to a bimodal activation of PKC activity. PKC activity was enhanced in the plasma membrane between 5 and 10 min following stimulation, then it declined to control levels after 30 min. After 90 to 120 min of stimulation, a second wave of PKC activation was detected.

FIGURE 1.

Bimodal activation of PKC in BMA 031-stimulated PBL. Lymphocytes (2 × 106 cells/ml) were stimulated with 5 μg/ml BMA 031 for the times indicated, and PKC activity was measured in streptolysin O-permeabilized cells with peptide GS as substrate, as described in Materials and Methods. Specific PKC activity varied between 21.3 and 33.3 pmol/2 × 106 cells/min in resting lymphocytes (dotted line), depending on the individual donors. Results are the means of duplicates from five independent experiments. ▪ indicates the control; • indicates stimulated.

FIGURE 1.

Bimodal activation of PKC in BMA 031-stimulated PBL. Lymphocytes (2 × 106 cells/ml) were stimulated with 5 μg/ml BMA 031 for the times indicated, and PKC activity was measured in streptolysin O-permeabilized cells with peptide GS as substrate, as described in Materials and Methods. Specific PKC activity varied between 21.3 and 33.3 pmol/2 × 106 cells/min in resting lymphocytes (dotted line), depending on the individual donors. Results are the means of duplicates from five independent experiments. ▪ indicates the control; • indicates stimulated.

Close modal

As shown in Figure 2, different PKC isoenzymes were translocated to the plasma membrane upon BMA 031 treatment in the early and late phases of T cell stimulation, corresponding to the first and second waves of PKC activation (see Fig. 1). PKC-α and PKC-θ isoenzymes were translocated rapidly, i.e., within 10 min of stimulation. PKC-β, -δ, and -ε isoenzymes were activated and translocated only after 90 min of stimulation. PKC-ζ was not activated via the TCR/CD3 complex (data not shown). It should be emphasized that upon activation with PMA, all conventional and calcium-independent PKC isoenzymes were activated and translocated (Fig. 2). As expected, PKC-ζ was not activated by PMA either (data not shown).

FIGURE 2.

BMA 031-induced translocation of different PKC isoenzymes in human PBL. Lymphocytes (2 × 106 cells/ml) were stimulated with BMA 031 (5 μg/ml) or with the combination of BMA 031 and PMA (10 ng/ml), respectively, for the times indicated. Plasma membranes were isolated and subjected to SDS-PAGE and immunoblotting as described in Materials and Methods. Results are representative of three independent experiments. In some experiments (see also Fig. 5) trace amounts of PKC were detectable in the plasma membranes of resting cells.

FIGURE 2.

BMA 031-induced translocation of different PKC isoenzymes in human PBL. Lymphocytes (2 × 106 cells/ml) were stimulated with BMA 031 (5 μg/ml) or with the combination of BMA 031 and PMA (10 ng/ml), respectively, for the times indicated. Plasma membranes were isolated and subjected to SDS-PAGE and immunoblotting as described in Materials and Methods. Results are representative of three independent experiments. In some experiments (see also Fig. 5) trace amounts of PKC were detectable in the plasma membranes of resting cells.

Close modal

To investigate regulation of activation by different PKC isoenzymes, Abs against different PKC isoforms were introduced by means of electropermeabilization into human T lymphocytes. To test optimal experimental conditions, cells were electropermeabilized, and cell viability was determined under different experimental conditions. As shown in Table I, at a capacitance of 960 μF, the majority of cells were dead, as measured by propidium iodide staining. At 500 μF, a significant decrease in 1/e = τ (i.e., decay time of current) was observed; consequently, cell viability proved to be about 90%. Upon decreasing voltage and capacitance, cell viability reached >90%. Preliminary experiments, however, have shown that only trace amounts of macromolecules could be introduced into the cells below 500 μF (28). Thus, in the additional experiments cells were electroporated at 500 μF and 300 V at 20°C.

As shown in Figure 3, under these experimental conditions the majority of cells carrying TCR (i.e., T lymphocytes) were loaded with PE-conjugated Ig (lower panels). Nonspecific adsorption of IgG to the cell surface was controlled by incubating lymphocytes with DTAF- or PE-labeled Igs (upper panels).

FIGURE 3.

Efficiency of introduction of PE-labeled Ig by means of electroporation into human PBL. Human lymphocytes were electropermeabilized in the presence or the absence of a PE-labeled Ig (2 μg/ml) under conditions described in Materials and Methods. For detection of the TCR by indirect immunofluorescence, lymphocytes were incubated with BMA 031- and DTAF-conjugated goat anti-mouse IgG F(ab′)2 as described in Materials and Methods. Dot plots show: top left, control, cells stained with DTAF-conjugated F(ab′)2 of goat anti-mouse IgG; top right,control, cells stained with PE-conjugated IgG; bottom left,TCR/CD3-positive cells, electropermeabilized lymphocytes stained with BMA 031- and DTAF-conjugated F(ab′)2 of goat anti-mouse IgG; and bottom right, PE-conjugated IgG containing electropermeabilized T lymphocytes (TCR/CD3 stained with BMA031- and DTAF-conjugated goat anti-mouse IgG F(ab′)2). Results are representative of four independent experiments.

FIGURE 3.

Efficiency of introduction of PE-labeled Ig by means of electroporation into human PBL. Human lymphocytes were electropermeabilized in the presence or the absence of a PE-labeled Ig (2 μg/ml) under conditions described in Materials and Methods. For detection of the TCR by indirect immunofluorescence, lymphocytes were incubated with BMA 031- and DTAF-conjugated goat anti-mouse IgG F(ab′)2 as described in Materials and Methods. Dot plots show: top left, control, cells stained with DTAF-conjugated F(ab′)2 of goat anti-mouse IgG; top right,control, cells stained with PE-conjugated IgG; bottom left,TCR/CD3-positive cells, electropermeabilized lymphocytes stained with BMA 031- and DTAF-conjugated F(ab′)2 of goat anti-mouse IgG; and bottom right, PE-conjugated IgG containing electropermeabilized T lymphocytes (TCR/CD3 stained with BMA031- and DTAF-conjugated goat anti-mouse IgG F(ab′)2). Results are representative of four independent experiments.

Close modal

Table II depicts the effects of different anti-PKC Abs on the specific PKC activity. In isolated cytoplasmic fractions, a pan-PKC Ab at a concentration of 2 μg/ml completely suppressed PKC activity in the cytosol.

Table II.

Influence of anti-PKC Abs on PKC activity of human lymphocytes

TreatmentPKC Activity (pmol/mg protein/min)
Ab introduced into cellsaAb added to cytosolb
None 900 ± 108  
Electroporation 890 ± 79 ND 
Control IgG 801 ± 88 810 ± 12 
Anti (pan) PKC Ab 89 ± 11 93 ± 5 
Anti PKC-α Ab 529 ± 77 416 ± 11 
Anti PKC-β Ab 494 ± 15 444 ± 62 
Anti PKC-δ Ab 764 ± 15 687 ± 56 
Anti PKC-ε Ab 404 ± 55 363 ± 60 
Anti PKC-θ Ab 710 ± 21 639 ± 66 
TreatmentPKC Activity (pmol/mg protein/min)
Ab introduced into cellsaAb added to cytosolb
None 900 ± 108  
Electroporation 890 ± 79 ND 
Control IgG 801 ± 88 810 ± 12 
Anti (pan) PKC Ab 89 ± 11 93 ± 5 
Anti PKC-α Ab 529 ± 77 416 ± 11 
Anti PKC-β Ab 494 ± 15 444 ± 62 
Anti PKC-δ Ab 764 ± 15 687 ± 56 
Anti PKC-ε Ab 404 ± 55 363 ± 60 
Anti PKC-θ Ab 710 ± 21 639 ± 66 
a

Lymphocytes 5 × 106 were electropermeabilized in the presence of different anti-PKC Abs as described in Materials and Methods. After recovery, cells were washed extensively and then homogenized, cytosol was prepared, and PKC activity was determined with peptide GS as substrate as described in Materials and Methods.

b

Cytosol was prepared and PKC activity was determined after direct addition of different anti-PKC Abs. Results are means ± SD of duplicates from two independent experiments.

Abs against different isoforms inhibited PKC activity partially and to varying extents. No significant inhibition was detectable upon addition of control Ig (Table II).

After introducing specific Abs raised against different PKC isoenzymes into intact lymphocytes, the sp. act. of the cytoplasmic PKC was decreased to a similar extent as in the cytosol upon direct addition of the respective Abs. It should be noticed that neither electropermeabilization itself nor the introduction of a control Ig influenced PKC activity significantly, as measured by phosphorylation of peptide GS. These results indicated that sufficient amounts of anti-PKC Abs have been introduced into the cells to neutralize and thus to inhibit selective PKC activities.

Introduction of PKC-specific Abs into human lymphocytes resulted in a significant inhibition of TCR/CD3-mediated cellular proliferation (Table III). Extensive control experiments have shown that neither electroporation nor introduction of control Ig influenced cellular proliferation. BMA 031 or OKT3-stimulated TdR incorporation was influenced by different Abs in a different way. While the inhibitory effects of anti-PKC-β, -δ, and -ε Abs were reversible upon the addition of 200 pg/ml human IL-2, inhibition of BMA 031- or OKT3-stimulated TdR incorporation by Abs against PKC-α and -θ could not be reversed by exogenous human IL-2. These results suggested that PKC-β and -δ might be the major PKC isoenzymes involved in the regulation of IL-2 synthesis, while PKC-α and -θ might be responsible for the regulation of high affinity IL-2R in stimulated human lymphocytes.

Table III.

Inhibition by different anti-PKC Abs of [3H]TdR incorporation in TCR/CD3-stimulated human lymphocytesa

Treatment[3H]TdR Incorporated/4 × 105 Cells (cpm)IL-2 Secreted (pg/ml)
−hr-IL-2+hr-IL-2
None 1.703 ± 53 1.477 ± 59 <15 
Electroporation 1.308 ± 78 1.743 ± 87 <15 
OKT3 13.774 ± 826 17.509 ± 875 133 ± 5 
+ electroporation 15.609 ± 788 14.758 ± 811 141 ± 7 
OKT3    
+ anti-PKC-α Ab 4.985 ± 224 5.536 ± 310 129 ± 9 
+ anti-PKC-β Ab 5.991 ± 335 14.480 ± 812 34 ± 2 
+ anti-PKC-δ Ab 8.983 ± 503 15.150 ± 848 53 ± 3 
+ anti-PKC-εAb 7.915 ± 443 17.672 ± 989 79 ± 6 
+ anti-PKC-θ Ab 8.543 ± 478 7.953 ± 445 132 ± 8 
+ anti-PKC-ζ Ab 15.459 ± 564 15.547 ± 432 ND 
+ anti-PKC-β + -δ Abs ND ND 31 ± 11 
BMA 031 18.742 ± 1049 18.755 ± 1.050 139 ± 10 
+ electroporation 19.609 ± 1098 17.471 ± 978 137 ± 8 
BMA 031    
+ anti-PKC- α Ab 5.498 ± 307 5.536 ± 312 131 ± 11 
+ anti-PKC-β Ab 6.599 ± 369 17.443 ± 976 34 ± 3 
+ anti-PKC-δ Ab 9.983 ± 559 18.151 ± 1.016 47 ± 7 
+ anti-PKC-ε Ab 8.795 ± 492 19.674 ± 1.101 69 ± 12 
+ anti-PKC-θ Ab 9.543 ± 534 7.953 ± 445 132 ± 6 
+ anti-PKC-ζ Ab 18.155 ± 668 18.549 ± 744 ND 
+ anti-PKC-β + -δ Abs ND ND 31 ± 11 
+ control IgG 17.666 ± 989 17.876 ± 962 127 ± 7 
Treatment[3H]TdR Incorporated/4 × 105 Cells (cpm)IL-2 Secreted (pg/ml)
−hr-IL-2+hr-IL-2
None 1.703 ± 53 1.477 ± 59 <15 
Electroporation 1.308 ± 78 1.743 ± 87 <15 
OKT3 13.774 ± 826 17.509 ± 875 133 ± 5 
+ electroporation 15.609 ± 788 14.758 ± 811 141 ± 7 
OKT3    
+ anti-PKC-α Ab 4.985 ± 224 5.536 ± 310 129 ± 9 
+ anti-PKC-β Ab 5.991 ± 335 14.480 ± 812 34 ± 2 
+ anti-PKC-δ Ab 8.983 ± 503 15.150 ± 848 53 ± 3 
+ anti-PKC-εAb 7.915 ± 443 17.672 ± 989 79 ± 6 
+ anti-PKC-θ Ab 8.543 ± 478 7.953 ± 445 132 ± 8 
+ anti-PKC-ζ Ab 15.459 ± 564 15.547 ± 432 ND 
+ anti-PKC-β + -δ Abs ND ND 31 ± 11 
BMA 031 18.742 ± 1049 18.755 ± 1.050 139 ± 10 
+ electroporation 19.609 ± 1098 17.471 ± 978 137 ± 8 
BMA 031    
+ anti-PKC- α Ab 5.498 ± 307 5.536 ± 312 131 ± 11 
+ anti-PKC-β Ab 6.599 ± 369 17.443 ± 976 34 ± 3 
+ anti-PKC-δ Ab 9.983 ± 559 18.151 ± 1.016 47 ± 7 
+ anti-PKC-ε Ab 8.795 ± 492 19.674 ± 1.101 69 ± 12 
+ anti-PKC-θ Ab 9.543 ± 534 7.953 ± 445 132 ± 6 
+ anti-PKC-ζ Ab 18.155 ± 668 18.549 ± 744 ND 
+ anti-PKC-β + -δ Abs ND ND 31 ± 11 
+ control IgG 17.666 ± 989 17.876 ± 962 127 ± 7 
a

Human lymphocytes were electropermeabilized in the presence of antibodies raised against different PKC isoenzymes as described in Materials and Methods. Cells were allowed to recover in RPMI-supplemented 50% FCS for 1 h, then washed resuspended in RPMI containing 10% FCS, then stimulated with BMA 031 or OKT3, respectively, and cultured in the presence or absence of 200 pg/ml hr-IL-2. [3H]TdR incorporation was measured after 68 h. IL-2 synthesis was determined from culture supernatants after 20 h of stimulation. Results are means ± SD of triplicates from three independent experiments.

In fact, introduction of anti-PKC-α and -θ Abs into human lymphocytes completely suppressed BMA 031-induced elevated expression of IL-2R as determined by two independent methods. BMA 031-induced up-regulation of CD25 was detected with PE-labeled anti-CD25 Abs by flow cytometry. While neutralization of PKC-α and -θ suppressed CD25 expression, no change in the number of IL-2R (CD25) was detectable in cells treated with Abs against other PKC isoenzymes (i.e., PKC-β, -δ, and -ε) or with control IgG (Fig. 4).

FIGURE 4.

Inhibition of cell surface expression of IL-2R (CD25) by anti-PKC Ab in BMA 031-stimulated human lymphocytes. Lymphocytes were electropermeabilized in the presence of Abs raised against different PKC isoenzymes as described in Materials and Methods. Cells were then stimulated with 5 μg/ml BMA 031 for 24 h, washed, and stained for CD25 expression as described in Materials and Methods. Histogram analysis of CD25 on the cell surface is shown. Results are representative of three independent experiments. 1, Control; 2, BMA 031; 3, control, permeabilized; 4, BMA 031, permeabilized; 5, BMA 031 and anti-PKC-α; 6, BMA 031 and anti-PKC-β; 7, BMA 031 and anti-PKC-δ; 8, BMA 031 and anti-PKC-ε; 9, BMA 031 and anti-PKC-θ; and 10, BMA 031 and PMA (10 ng/ml).

FIGURE 4.

Inhibition of cell surface expression of IL-2R (CD25) by anti-PKC Ab in BMA 031-stimulated human lymphocytes. Lymphocytes were electropermeabilized in the presence of Abs raised against different PKC isoenzymes as described in Materials and Methods. Cells were then stimulated with 5 μg/ml BMA 031 for 24 h, washed, and stained for CD25 expression as described in Materials and Methods. Histogram analysis of CD25 on the cell surface is shown. Results are representative of three independent experiments. 1, Control; 2, BMA 031; 3, control, permeabilized; 4, BMA 031, permeabilized; 5, BMA 031 and anti-PKC-α; 6, BMA 031 and anti-PKC-β; 7, BMA 031 and anti-PKC-δ; 8, BMA 031 and anti-PKC-ε; 9, BMA 031 and anti-PKC-θ; and 10, BMA 031 and PMA (10 ng/ml).

Close modal

Similar results were obtained when binding of [125I]IL-2 was measured in BMA 031-stimulated human lymphocytes. While introduction of anti-PKC-α and -θ Abs significantly suppressed specific IL-2 binding, anti-PKC-β, -δ, and -ε had no effect on the number and affinity of IL-2R (Table IV).

Table IV.

Inhibition by anti-PKC Abs of high affinity IL-2 receptor expression in stimulated human lymphocytesa

TreatmentKd (×10−12 M)Sites/Cell
None − <50 
Electroporation − <50 
BMA 031 4.15 1432 ± 111 
BMA 031+ electroporation 4.11 1394 ± 95 
BMA 031   
+ anti-PKC-α Ab − <50 
+ anti-PKC-β Ab 4.60 1566 ± 99 
+ anti-PKC-δ Ab 4.51 1743 ± 144 
+ anti-PKC-ε Ab 4.72 1712 ± 78 
+ anti-PKC-θ Ab 4.51 220 ± 14 
+ anti-PKC-ζ Ab 4.55 1558 ± 66 
+ control Ab 4.30 1443 ± 72 
TreatmentKd (×10−12 M)Sites/Cell
None − <50 
Electroporation − <50 
BMA 031 4.15 1432 ± 111 
BMA 031+ electroporation 4.11 1394 ± 95 
BMA 031   
+ anti-PKC-α Ab − <50 
+ anti-PKC-β Ab 4.60 1566 ± 99 
+ anti-PKC-δ Ab 4.51 1743 ± 144 
+ anti-PKC-ε Ab 4.72 1712 ± 78 
+ anti-PKC-θ Ab 4.51 220 ± 14 
+ anti-PKC-ζ Ab 4.55 1558 ± 66 
+ control Ab 4.30 1443 ± 72 
a

Lymphocytes (2 × 106) were stimulated with BMA 031 for 20 h. Cells were then washed and 125I-IL-2 binding was performed as described in Materials and Methods. Results are means ± SD of triplicates.

As shown in Table III, TCR/CD3-stimulated IL-2 synthesis was significantly inhibited upon introduction of anti-PKC-β, -δ, and, to a lesser extent, -ε Abs. Neither introduction of Abs against other PKC isoenzymes (i.e., PKC-α and -θ) nor control IgG had any influence on IL-2 synthesis of OKT3-stimulated human lymphocytes. Electropermeabilization itself was also without effect on the amount of IL-2 secreted by stimulated lymphocytes.

To determine whether PKC isoenzymes involved in the regulation of IL-2 synthesis exerted their effects via different signaling mechanisms, anti-PKC-β and -δ Abs were introduced simultaneously into the cells, and IL-2 synthesis was determined following TCR stimulation. As shown in Table III, combination of the Abs did not result in additional suppression of IL-2 synthesis, suggesting that at least PKC-β and -δ might interfere with cellular signaling at an identical site.

As PKC-β proved to be the major PKC isoform involved in the regulation of IL-2 gene expression, kinetics of PKC-β activation and translocation were correlated with IL-2 gene expression. As shown in Figure 5, activation of PKC-β preceded expression of IL-2 mRNA, as detected by RT-PCR. While an enhanced amount of PKC-β protein was translocated to the plasma membrane between 90 min and 2 h of stimulation, only trace amounts of IL-2 mRNA were detectable after 2 h of stimulation, while IL-2 gene expression was markedly enhanced after 4 h of activation.

FIGURE 5.

Correlation of PKC-β translocation and IL-2 gene expression in BMA 031-stimulated human lymphocytes. Human lymphocytes were stimulated with BMA 031 (5 μg/ml) for the times indicated. Plasma membranes were isolated, and translocation of PKC-β was detected by immunoblotting with an Ab against this PKC isoenzyme as described in Materials and Methods. RNA was isolated from the same cells, and reverse transcription-PCR for IL-2 mRNA was conducted as described in Materials and Methods. A, Translocation of PKC-β to the plasma membrane. B, IL-2 mRNA expression (0, 10, 30, 90, 120, and 240 min). Lanes 1, 3, 5, 7, 9, and 11, Tubulin; lanes 2, 4, 6, 8, 10, and 12, IL-2. Results are representative of two independent experiments.

FIGURE 5.

Correlation of PKC-β translocation and IL-2 gene expression in BMA 031-stimulated human lymphocytes. Human lymphocytes were stimulated with BMA 031 (5 μg/ml) for the times indicated. Plasma membranes were isolated, and translocation of PKC-β was detected by immunoblotting with an Ab against this PKC isoenzyme as described in Materials and Methods. RNA was isolated from the same cells, and reverse transcription-PCR for IL-2 mRNA was conducted as described in Materials and Methods. A, Translocation of PKC-β to the plasma membrane. B, IL-2 mRNA expression (0, 10, 30, 90, 120, and 240 min). Lanes 1, 3, 5, 7, 9, and 11, Tubulin; lanes 2, 4, 6, 8, 10, and 12, IL-2. Results are representative of two independent experiments.

Close modal

Since their classification as a family of serine/threonine-specific protein kinases, at least 11 different PKC isoenzymes have been identified and cloned (3, 4). Their different properties have prompted the idea that specific isoenzymes might be involved in different signal transduction pathways (8, 10, 19). Evidence has indeed been found in several cell lines using molecular biologic approaches such as overexpression of isoenzymes or deletion mutants (11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 32, 33). No data, however, are available for normal physiologic cells.

Here we show for the first time that in easily obtainable physiologic cells, i.e., in resting human PBL, the expressions of two different genes, namely IL-2 and IL-2R, are regulated by different PKC isoenzymes.

As in resting, nonproliferating cells, the majority of molecular biologic methods are hardly applicable, new methods had to be developed. Reversible permeabilization of cells by means of electroporation proved to be the most reliable method to introduce specific Abs into human lymphocytes. The conditions used allowed uptake of Abs without significant loss of cell viability. At least 80% of T lymphocytes contained Abs that neutralized their respective PKC isoenzymes, allowing functional analysis of PKCs. Thus, human T lymphocytes can effectively be loaded with anti-PKC Abs by means of electropermeabilization under the conditions used (Table I). Control experiments indicated that τ (= 1/e, decay time of current) was of special importance for reversible electropermeabilization and for viability of cells (Table I).

Pan-PKC Abs inhibited PKC activity nearly completely when added to isolated cytosol. PKC activity in the cytosol of electropermeabilized cells after introduction of pan-PKC Abs was inhibited in a similar extent (Table II). These results together with the data showing that the majority of T cells were stained with PE-labeled control Ig showed that the amounts of Abs entering T lymphocytes were sufficient to neutralize their respective PKC activities. These results were further supported by the finding that elevation of the Ab concentration did not result in further inhibition of cytoplasmic PKC activity either by direct addition or by electropermeabilization (data not shown). It should be emphasized that >95% of cells labeled with PE-coupled Ig were, in fact, T lymphocytes (Fig. 3).

IL-2 synthesis and IL-2R expression were regulated by different PKC isoenzymes upon stimulation of normal human lymphocytes. Introduction of anti-PKC-α and -θ Abs resulted in a nearly complete suppression of IL-2R expression as measured by three different, independent methods (Fig. 4 and Tables III and IV). The fact that neutralization of both PKC-α and -θ resulted in complete inhibition of TCR/CD3-induced up-regulation of CD25 suggested that these PKC isoforms might regulate differential and independent signal transduction pathways that converge at the level of the IL-2R gene. This finding was supported recently by very elegant experiments by Monks et al. (34) showing immediate activation and specific involvement of PKC-θ in Ag-stimulated T lymphocytes.

Neutralization by specific Abs of PKC-β, -δ, and -ε led to inhibition of IL-2 synthesis, although to different extents. As Abs raised against PKC-β inhibited IL-2 synthesis and secretion up to 70%, the results clearly show that PKC-β was the most predominant isoform involved in the regulation of IL-2 synthesis and secretion (Table III). Detailed analysis of the kinetics of PKC-β translocation to the plasma membrane and the onset of IL-2 gene expression, as shown in Figure 4, clearly demonstrates that under the experimental conditions used, activation of PKC-β preceded enhancement of transcription of IL-2 mRNA. It should be emphasized that there are marked differences in the activation of PKCs and IL-2 synthesis in T cell lines and resting T cells. While in Jurkat T cells PKC-β (and other PKC isoenzymes) were activated via TCR/CD3 within 5 to 10 min, PKC-β (-δ and -ε) in PBL were activated with a significant delay, i.e., between 90 and 120 min of stimulation (our manuscript in preparation) (Figs. 1 and 4). Thus, activation kinetics of PKC-β, the putative main regulator of IL-2 synthesis, correlated well with that of IL-2 gene expression, as TCR/CD3-stimulated IL-2 mRNA expression in PBL was not observed before 2 h, reaching maximal levels as late as 4 h (Fig. 4). Identical results concerning IL-2 gene expression were reported recently (35).

There are several possibilities explaining why Abs against PKC-β, -δ, or -ε inhibited IL-2 synthesis to different degrees. Although our control experiments suggested that the single Abs inhibited their specific corresponding PKC isotype after introducing them into the cells, we cannot rule out the possibility that differential degrees of inhibition by different anti-PKC Abs of IL-2 synthesis reflected different avidities to their Ags, i.e., to single PKC isoforms. Similarly, one should consider the possibility that some PKC was not reached by the Abs (because of compartmentalization) and thus remained active in Ab-treated cells. Furthermore, different PKCs have different half-lives; new synthesis and posttranslational phosphorylation of distinct isotypes might also be different, thus resulting in differences in the amounts and activities of special isoforms (3, 4).

PKC-dependent regulation of IL-2 gene expression is well documented (36), and the results of this study show that PKC-β especially was of great importance for the induction of IL-2 synthesis and secretion. IL-2 gene expression is, however, regulated by complex signal transduction pathways (2, 37, 38). Besides PKCs, the role of p21ras and subsequent activation of the cytoplasmic mitogen-activated protein kinase cascade is a well documented mechanism in the regulation of different transcription factors regulating IL-2 gene expression (39, 40, 41). On the other hand, it was suggested that signals from PKC and p21ras might converge to control the activity of the protein kinase, Raf-1. Calcium-dependent processes, especially dephosphorylation of transcription factor(s) by calcineurin, also seem to be critically involved in the regulation of cytokine gene expression (42, 43). More recent data indicated that Jun-N-terminal kinase(s), especially JNK1, might participate in the regulation and nuclear binding of specific transcription factors (44, 45). Thus, IL-2 synthesis might be a net effect of several regulatory mechanisms.

The fact that anti-PKC-β Abs resulted in a more than 70% inhibition of IL-2 synthesis strongly suggests, however, that PKC-β-regulated signaling pathways are of great importance for the synthesis and secretion of this cytokine. Recent observations in Jurkat T cells suggested that PKC-α and -ε might regulate parallel signaling pathways, and the point of convergence would be in the nucleus at the level of transcription factors in the course of T cell activation (19). Our results indicate that PKC-α was one of the major PKC isoforms regulating IL-2R expression, while PKC-ε participated in the regulation of IL-2 synthesis.

The functional role of PKC-ζ is controversially discussed in the literature. PKC-ζ has been shown to be involved in the activation of important transcription factors and was claimed to regulate cellular proliferation (33, 46, 47). On the other hand, overexpression of PKC-ζ had no influence on the activation of transcription factors regulating IL-2 gene expression, nor was the expression of early activation markers (CD69) influenced by the PKC isoform (19). In our hands in resting T lymphocytes PKC-ζ was not regulated by the TCR/CD3 complex; accordingly, induction of anti-PKC-ζ Abs was without any effect on IL-2 synthesis and receptor expression (data not shown). Thus, PKC-ζ seems to have different functions in different cells.

The present results confirm and extend recent observations showing that long-lasting activation of PKC(s) was a prerequisite of IL-2 synthesis, while for up-regulation of IL-2R, short term activation of PKC(s) was sufficient (48, 49). In human PBL, the different PKC isoenzymes were differentially regulated upon stimulation via the TCR; while PKC-α and -θ were translocated fast and transiently, with a peak at about 10 min, long-lasting activation of the β, δ, and ε isoforms was observed, with a delay following TCR stimulation. Accordingly, inhibition of short term activation of PKC-α and -θ resulted in nearly complete suppression of IL-2R.

The involvement of different PKC isoenzymes in the induction of IL-2 synthesis or IL-2R expression was also suggested by the effects of specific inhibitors. Thus, cholera toxin, which blocked degradation of phosphatidylinositol bisphosphate, the so-called “Pl response,” prevented activation of PKC-α and subsequently IL-2R expression without affecting IL-2 synthesis (50). On the other hand, cyclosporin A, which is known to suppress IL-2 synthesis but not the expression of its high affinity receptors, specifically inhibited activation of PKC-β, but left activation of PKC-α unchanged (8, 51). The results of this study strengthen the idea that PKC-β might be critically involved in the induction of IL-2 synthesis.

Taken together, the results clearly show that different PKC isoenzymes may have selective functions in the course of TCR-induced signal transduction and thus in the regulation of cellular functions in human lymphocytes.

The excellent technical assistance of Mrs. M. Schmidt, A. Garbe, S. Eikemeyer, J. v. d. Ohe, and M. Golombek is gratefully acknowledged. The excellent typing assistance of Mrs. K. Erdogan is gratefully acknowledged.

1

This work was supported by the Deutsche Forschungsgemeinschaft (Grants SFB 265 A9).

3

Abbreviations used in this paper: DAG, 1,2-diacylglycerol; PKC, protein kinase C; PE, phycoerythrin; DTAF, dichlorotriazinylaminofluorescein; nPKC, novel (calcium-independent) PKC.

1
Weiss, A., D. Littman.
1994
. Signal transduction by lymphocyte antigen receptors.
Cell
76
:
263
2
Weiss, A., M. Iwashima, B. Irving, N. van-Oers, T. Kadlecek, D. Straus, A. Chan.
1994
. Molecular and genetic insights into T cell antigen receptor signal transduction.
Adv. Exp. Med. Biol.
365
:
53
3
Dekker, L., P. Parker.
1994
. Protein kinase C: a question of specificity.
Trends Biochem. Sci.
19
:
73
4
Hug, H., T. Sarre.
1993
. Protein kinase C isoenzymes, divergence in signal transduction.
Biochem. J.
291
:
329
5
Dekker, L., R. Palmer, P. Parker.
1995
. The protein kinase C related gene family.
Curr. Opin. Struct. Biol.
5
:
396
6
Berry, N., Y. Nishizuka.
1990
. Protein kinase C and T cell activation.
Eur. J. Biochem.
189
:
205
7
Keenan, C., D. Kelleher, A. Long.
1995
. Regulation of non-classical protein kinase C isoenzymes in a human T cell line.
Eur. J. Immunol.
25
:
9833
8
Szamel, M., K. Resch.
1995
. T-cell antigen receptor induced signal transduction pathways: activation and function of protein kinases C in T lymphocytes.
Eur. J. Biochem.
228
:
1
9
Izquierdo, M., S. Leevers, D. Williams, C. Marshall, A. Weiss, D. Cantrell.
1994
. The role of protein kinase C in the regulation of extracellular signal regulated kinase by the T cell antigen receptor.
Eur. J. Immunol.
24
:
2462
10
Izquierdo, M., J. Downward, J. Graves, D. Cantrell.
1992
. Role of protein kinase C in T-cell antigen receptor regulation of p21ras: evidence that two p21ras regulatory pathways coexist in T cells.
Mol. Cell. Biol.
12
:
3305
11
Ways, D., C. Kukoly, J. deVente, J. Hooker, W. Bryant, K. Posekany, D. Fletcher, P. Cook, P. Parker.
1995
. MCF-7 breast cancer cells transfected with protein kinase C-α exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype.
J. Clin. Invest.
95
:
1906
12
Goldstein, D., A. Cacace, I. Weinstein.
1995
. Overexpression of protein kinase C-beta1 in the SW 480 colon cancer cell line causes growth suppression.
Carcinogenesis
16
:
1121
13
Hundle, B., T. McMahon, J. Dadgar, R. Messing.
1995
. Overexpression of epsilon-protein kinase C enhances nerve growth factor-induced phosphorylation of mitogen-activated protein kinases and neurite outgrowth.
J. Biol. Chem.
270
:
30134
14
La-Porta, C., R. Comolli.
1995
. Overexpression of protein kinase C δ is associated with a delay in preneoplastic lesion development in diethyl-nitrosamine-induced rat hepatocarcinogenesis.
Carcinogenesis
16
:
1233
15
Li, W., J. Yu, D. Shin, J. Pierce.
1995
. Characterisation of a protein kinase C-δ (PKC-δ) ATP binding mutant: an inactive enzyme that competitively inhibits wild type PKC δ enzymatic activity.
J. Biol. Chem.
270
:
8311
16
Moreton, K., R. Turner, M. Blake, A. Paton, N. Groome, M. Rumsby.
1995
. Protein expression of the alpha, gamma, delta and epsilon subspecies of protein kinase C changes as C6 glioma cells become contact inhibited and quiescent in the presence of serum.
FEBS Lett.
372
:
33
17
Limatola, C., D. Schaap, W. Moolenaar, W. van-Blitterswijk.
1994
. Phosphatidic acid activation of protein kinase C-zeta overexpressed in Cos cells: comparison with other protein kinase C isotypes and other acidic lipids.
Biochem. J.
304
:
1001
18
Walker, S., N. Murray, D. Burns, A. Fields.
1995
. Protein kinase C chimeras: catalytic domains of α and β II protein kinase C contain determinants for isotype specific function.
Proc. Natl. Acad. Sci. USA
92
:
9156
19
Genot, E., P. Parker, D. Cantrell.
1995
. Analysis of the role of protein kinase C-α, -ε, and -ζ in T cell activation.
J. Biol. Chem.
270
:
9833
20
Ways, D., K. Posekany, J. deVente, T. Garris, W. Chen, J. Hooker, W. Qin, P. Cook, D. Fletcher, P. Parker.
1994
. Overexpression of protein kinase C-ζ stimulates leukemic cell differentiation.
Cell Growth Differ.
5
:
1195
21
Chen, W., E. Schweins, X. Chen, O. Finn, M. Cheever.
1994
. Retroviral transduction of protein kinase C-γ into tumor-specific T cells allows antigen-independent long term growth in IL-2 with retention of functional selectivity in vitro and ability to mediate tumor therapy in vivo.
J. Immunol.
153
:
3630
22
Borner, C., M. Ueffing, S. Jaken, P. Parker, I. Weinstein.
1995
. Two closely related isoforms of protein kinase C produce reciprocal effects on the growth of rat fibroblasts: possible molecular mechanisms.
J. Biol. Chem.
270
:
78
23
Howe, L., A. Weiss.
1995
. Multiple kinases mediate T-cell-receptor signaling.
Trends Biochem. Sci.
20
:
59
24
van-Oers, N., N. Killeen, A. Weiss.
1994
. ZAP-70 is constitutively associated with tyrosine phosphorylated TCR ζ in murine thymocytes and lymph node T cells.
Immunity
1
:
675
25
Bartels, F., H. Bergel, H. Bigalke, J. Fevert, J. Halpern, J. Middlebrook.
1994
. Specific antibodies against the Zn2+-binding domain of clostridial neurotoxins restore exocytosis in chromaffin cells treated with tetanus or botulinum neurotoxin.
J. Biol. Chem.
269
:
8122
26
Szamel, M., H. Leufgen, R. Kurrle, K. Resch.
1995
. Differential signal transduction pathways regulating interleukin-2 synthesis and interleukin-2 receptor expression in stimulated human lymphocytes.
Biochim. Biophys. Acta
1235
:
33
27
Alexander, D., J. Graves, S. Lucas, D. Cantrell, M. Crumpton.
1990
. A method for measuring protein kinase C activity in permeabilized T lymphocytes by using peptide substrates.
Biochem. J.
268
:
303
28
Szamel, M., B. Rehermann, B. Krebs, R. Kurrle, K. Resch.
1989
. Activation signals in human lymphocytes: incorporation of polyunsaturated fatty acids into plasma membrane phospholipids regulates IL-2 synthesis via sustained activation of protein kinase C.
J. Immunol.
143
:
2806
29
Csermely, P., M. Szamel, K. Resch, J. Somogyi.
1988
. Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.
J. Biol. Chem.
263
:
6487
30
Bradford, M..
1976
. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72
:
248
31
Kracht, M., A. Heiner, K. Resch, M. Szamel.
1993
. Interleukin-1-induced signalling in T cells: evidence for the involvement of phosphatases PP1 and PP2A in regulating protein kinase C mediated protein phosphorylation and interleukin-2 synthesis.
J. Biol. Chem.
268
:
21066
32
Diaz-Meco, M., E. Berra, M. Municio, L. Sanz, J. Lozano, I. Dominguez, V. Diaz-Golpe, M. Lain-deLera, J. Alcami, C. Paya. C. Arenzana, J. Virelizier, J. Moscat.
1993
. A dominant negative protein kinase C ζ subspecies blocks NF-κB activation.
Mol. Cell. Biol.
13
:
4770
33
Berra, E., M. Diaz-Meco, T. Dominguez, M. Municio, L. Sanz, J. Lozano, R. Chapkin, J. Moscat.
1993
. Protein kinase C ζ isoform is critical for mitogenic signal transduction.
Cell
74
:
555
34
Monks, C. R., H. Kupfer, I. Tamir, A. Barlow, A. Kupfer.
1997
. Selective modulation of protein kinase C-θ during T-cell activation.
Nature
386
:
83
35
Saloga, J., R. Schwinzer, H. Renz, N. Terada, R. Or, E. Gelfand.
1993
. Characterization of the progression signal for human T-cell proliferation provided by monoclonal antibodies to the CD3-TCR complex.
Clin. Immunol. Immunopathol.
67
:
232
36
Rao, A..
1994
. NF-ATp, a transcription factor required for the coordinate induction of several cytokine genes.
Immunol. Today
15
:
274
37
Fraser, D., D. Straus, A. Weiss.
1993
. Signal transduction events leading to T-lymphokine gene expression.
Immunol. Today
14
:
357
38
Woodrow, M., N. Clipstone, D. Cantrell.
1993
. p21ras and calcineurin synergize to regulate the nuclear factor of activated T cells.
J. Exp. Med.
178
:
1517
39
Downward, J., J. Graves, D. Cantrell.
1992
. The regulation and function of p21ras in T cells.
Immunol. Today
13
:
89
40
Cantrell, D..
1994
. G proteins in lymphocyte signaling.
Curr. Opin. Immunol.
6
:
380
41
Izquierdo, M., S. Leevers, C. Marshall, D. Cantrell.
1993
. p21ras couples the T cell antigen receptor to extracellular signal-regulated kinase 2 in T lymphocytes.
J. Exp. Med.
178
:
1199
42
Clipstone, N., G. Crabtree.
1992
. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation.
Nature
357
:
695
43
Clipstone, N., G. Crabtree.
1993
. Calcineurin is a key signalling enzyme in T lymphocyte activation and target of the immunosuppressive drugs cyclosporin A and FK 506.
Ann. NY Acad. Sci.
696
:
20
44
Derijard, B., M. Hibi, I. Wu, T. Barrett, B. Su, T. Deng, M. Karin, R. Davis.
1994
. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain.
Cell
76
:
1025
45
Su, B., M. Hibi, T. Kallunki, M. Karin, Y. BenNeriah.
1994
. JNK is involved in signal integration during costimulation of T lymphocytes.
Cell
77
:
727
46
Lozano, J., E. Berra, M. Municio, M. Diaz-Meco, I. Dominguez, L. Sanz, J. Moscat.
1994
. Protein kinase C zeta isoform is critical for κ B-dependent promoter activation by sphingomyelinase.
J. Biol. Chem.
269
:
19200
47
Diaz-Meco, M., I. Dominguez, L. Sanz, P. Dent, J. Lozano, M. Municio, E. Berra, R. Hay, T. Sturgill, J. Moscat.
1994
. ζ PKC induces phosphorylation and inactivation of IκB-α in vitro.
EMBO J.
13
:
2842
48
Berry, N., K. Ase, A. Kishimoto, Y. Nishizuka.
1990
. Activation of resting human T cells requires prolonged stimulation of protein kinase C.
Proc. Natl. Acad. Sci. USA
87
:
2294
49
Nishizuka, Y..
1995
. Protein kinase C and lipid signaling for sustained cellular responses.
FASEB J.
9
:
484
50
Szamel, M., U. Ebel, P. Uciechowski, V. Kaever, K. Resch.
1997
. T cell antigen receptor dependent signalling in human lymphocytes: cholera toxin inhibits interleukin-2 receptor expression but not interleukin-2 synthesis by preventing activation of a protein kinase C isotype, PKC-α.
Biochim. Biophys. Acta
1356
:
237
51
Szamel, M., F. Bartels, K. Resch.
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-β.
Eur. J. Immunol.
23
:
3072