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(Journal of Leukocyte Biology. 2000;68:293-300.)
© 2000 by Society for Leukocyte Biology

Chronic PMA treatment of Jurkat T lymphocytes results in decreased protein tyrosine phosphorylation and inhibition of CD3- but not Ti-dependent antibody-triggered Ca2+ signaling

Charaf E. Ahnadi*,{dagger}, Patrick Giguère{ddagger}, Serge Gravel*, Danièle Gagné§, Anne-Christine Goulet§, Tamàs Fülöp, Jr§, Marcel D. Payet{dagger} and Gilles Dupuis*

* Departments of Biochemistry and
{dagger} Physiology and Biophysics,
{ddagger} Clinical Research Center and
§ Centre de Recherche en Gérontologie et Gériatrie, Faculty of Medicine, University of Sherbrooke, Quebec, Canada

Correspondence: Dr. Gilles Dupuis, Department of Biochemistry, Faculty of Medicine, University of Sherbrooke, 3001 12th Avenue North, Sherbrooke, Quebec, Canada, J1H 5N4. E-mail: gdupui01{at}courrier.usherb.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have treated Jurkat T lymphocytes with a concentration (160 nM) of phorbol myristyl acetate (PMA) that down-regulates conventional and novel protein kinase C (PKC) isozymes and we have investigated the effects on Ca2+ signaling and protein tyrosine phosphorylation using mAb (C305) directed against the ß-subunit of the Ti heterodimer or the {varepsilon}/{delta}-component of the CD3 complex (mAb Leu 4 or OKT 3). The levels of expression of PKC {alpha}, ßI, ßII, and {delta} were reduced by 90% or more in PMA-treated cells, whereas the expression of PKC{theta} decreased by ~30%. In contrast, the chronic treatment with PMA increased the expression of PKC{varepsilon} and PKC{zeta}. There was a lack of Ca2+ response and myo-inositol trisphosphate (IP3) production in PMA-treated cells when they were exposed to mAb Leu 4 but the cells responded to mAb C305. The treatment with PMA did not affect the surface expression of Ti or CD3. The overall levels of tyrosine-phosphorylated proteins were markedly reduced in PMA-treated cells. We investigated whether these observations were related to defects in signal transduction related to protein tyrosine kinase (PTK) of the src and syk families. The electrophoretic mobilities of p59fyn or ZAP-70 were not changed in PMA-treated cells but p56Ick migrated as a large band of Mr 60–62 kDa. The decreased mobility of p56Ick was related to a state of hyperphosphorylation. The activity of modified p56Ick was not up-regulated in activated Jurkat cells. Our data suggest that clonotypic Ti can trigger Ca2+ mobilization independently of conventional PKC isoforms. Our observations further suggest that conventional PKC isoforms are involved early in the cascade of events associated with Jurkat T lymphocyte activation.

Key Words: protein kinase C • down-regulation • phorbol myristyl acetate • signal transduction • protein tyrosine kinase


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T cell response to antigenic stimulation is initiated by major histocompatibility complex (MHC)-restricted antigen presentation by antigen-presenting cells (APC) and recognition by the clonotypic Ti heterodimer of the TcR [1 ]. The TcR is composed of an {alpha}/ß (or {gamma}/{delta} in some cells) clonotypic heterodimer (Ti) associated with the invariant {gamma}, {delta}, and {varepsilon} components of CD3 and with the homodimeric {zeta} protein or its alternatively spliced {eta} murine isoform [2 ]. Sequence data have revealed that Ti does not possess motifs characteristic of PTK and has not been reported to be associated with cytoplasmic proteins [2 ]. However, it has been shown that the {varepsilon} [3 , 4 ] component of CD3 and the {zeta} protein [4 5 6 ] can transduce signaling when engineered as chimeric proteins, lending support to the notion that the Ti-associated CD3 complex and the {zeta} homodimer provide the necessary link between the occupation Ti and signal transduction [7 ].

Occupation of the TcR induces, within seconds, the activation of protein tyrosine kinase (PTK) of the src and syk families [8 , 9 ]. Two members of the src family of PTK, the CD4-associated p56Ick and the CD3-associated p59fyn, are largely responsible for the initial steps of T cell signaling [2 , 8 , 10 11 12 ]. A deletion of exon 7 in the sequence of p56Ick in the J.CaM1 Jurkat cell mutant [13 ] or knocking out the gene of p59fyn in mice [14 ] leads to impaired Ca2+ mobilization and tyrosine-phosphorylated proteins. Targets of p56Ick and p59fyn include tyrosine residues located in specific sequences, the ITAM motifs that are present in each component of CD3 and in the {zeta}/{eta} protein [15 ]. Phosphorylation of these motifs serves to anchor SH2 domain-containing proteins that participate in the assembly of the cellular machinery of signal transduction [16 ]. Occupation of the TcR also triggers the tyrosine phosphorylation and activation of phospholipase C (PLC)-{gamma}1 that generates myo-inositol 1, 4, 5-trisphosphate (InsP3) and diacylglycerol (DAG) [17 ]. InsP3 binding to its receptor Ca2+ channel [18 ] initiates the release of Ca2+ [19 ] that is essential for the activation of cytoplasmic and nuclear enzymes, some transcription factors [20 21 22 23 24 25 ], and some PKC isozymes [26 , 27 ].

Evidence has accumulated to support the view that PKC is essential to the process of T cell activation [reviewed in 27]. A combination of PKC activator and Ca2+ ionophores can bypass signal transduction through the TcR/CD3 complex [28 ]. In addition, down-regulation of PKC or the use of PKC inhibitors impairs T cell activation [29 30 31 ]. PKC can exert a positive and a negative effect on T cell signaling. The positive regulatory action of PKC involves the activation of c-Raf-1 [32 , 33 ] and transcription factors such as AP-1 [34 ] and NF-{kappa}B [35 ], whereas the negative regulatory effect of PKC is characterized by a decrease in the activity of PLC [36 ] and an increase in the rate of internalization of the TcR/CD3 complex [37 and references therein]. The site of action of PKC in the cascade of cell signaling is not fully elucidated, although it has been reported to act upstream of Shc [38 ] or downstream of p21ras [34 , 39 ] and p56Ick [40 ].

In this report, we showed that treating Jurkat T cells with PMA under conditions that are known to down-regulate some PKC isoforms of the conventional and novel families [41 ] did not affect the production of IP3 and the Ca2+ response to Ti-directed (ß-subunit) mAb C305. In contrast, mAb directed against CD3 (Leu 4 and OKT 3) failed to induce the production of IP3 or a Ca2+ response. In addition, our results showed that the chronic treatment of the cells with PMA was associated with a marked decrease in the patterns of tyrosine-phosphorylated proteins, the hyperphosphorylation of p56Ick and a lack of up-regulation of its activity in activated Jurkat cells. Our data suggest that clonotypic Ti can trigger Ca2+ mobilization independently of conventional PKC isoforms. In contrast, these PKC isoforms appear to be essential for the TcR/CD3-dependent up-regulation of PTK activity and the Ca2+ signal initiated by ligation of the CD3 complex.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
Fura 2/AM was obtained from Molecular Probes (Eugene, OR). RPMI 1640 culture medium, antibiotics, reference proteins used as markers in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, anti-phosphotyrosine mAb (clone PT-66), and peroxidase-coupled anti-mouse IgG pAb raised in the sheep were purchased from Sigma Chemical (St. Louis, MO). Anti-p56Ick and anti-PLC-{gamma}1 (Western blotting) mAb were from PharMingen (San Diego, CA). The anti-p56Ick (immunoassays) and anti-p59fyn mAb were from Transduction Laboratories (Lexington, KY). Anti-ZAP-70 pAb was the generous gift of Dr André Veillette (McGill Cancer Center, McGill University, Montreal). mAb C305 [42 ] and OKT3 (American Type Culture Collection, Rockville, MD) were obtained from pooled culture supernatants of the respective hybridoma and partially purified by ammonium sulfate precipitation, as published [43 ]. mAb Leu 4 was purchased from Becton Dickinson (Montreal). The following anti-PKC pAb were from Life Technologies (Gaithersburg, MD; PKC{alpha}), Sigma (PKCßI and ßII) and Transduction Laboratories (PKC{delta}, {varepsilon}, {theta}, and {zeta}). The peroxovanadium derivative bpV(pic) was a gift from Dr. Barry Posner (Department of Medicine, McGill University, Montreal). The chemiluminescence detection kit used in Western blotting was purchased from Boehringer Mannheim (Montreal) or Amersham (Montreal). Other chemicals were from Sigma or local suppliers.

Cells
Jurkat E6.1 cells and the mAb C305-producing murine hybridoma (C305.2) were the generous gifts of Dr. Arthur Weiss (Howard Hughes Medical Institute, Department of Medicine, University of California, San Francisco, CA). All the cells used in this study were maintained in complete culture medium that consisted of RPMI 1640 medium, heat-inactivated (56°C, 30 min) fetal bovine serum (10%, v/v), penicillin (100 units/mL), streptomycin (100 µg/mL), and garamycin (40 ng/mL). The cells were passaged as described [43 ].

Chronic treatment of Jurkat cells with PMA
These experiments were done as before [43 ] following the protocol of Tsutsumi et al. [41 ]. Jurkat cells were cultured in the presence of PMA (160 nM) or 4{alpha}-PMA (160 nM) dissolved in freshly distilled DMSO (stock solution, 160 µM) or DMSO (55 µL, controls), for a period of 24 h, washed, and used in the experiments.

Quantification of [Ca2+]i and IP3
Jurkat cells (5 x 106 lymphocytes/mL) in Gey’s balanced salt solution were loaded with a DMSO solution of Fura 2/AM (1 mM) at a final concentration of 3 µM [43 ]. Changes in fluorescence were recorded with a spectrofluorimeter (SPF 500C; SLM Aminco, Urbana, IL) in the slow time-based mode. [Ca2+]i was calculated from the equation, [Ca2+]i = KD [(F - Fmin)/(Fmax - F)], after calibration with Triton X-100 and EGTA [43 ], respectively, and using a KD of 224 nM at 37°C [44 ]. Inositol trisphosphate derivatives were measured as described [45 ], with some modifications. Jurkat E6.1 cells (3 x 106 lymphocytes) were cultured for 24 h in 1 mL of myo-inositol-free RPMI 1640 medium, 10% FBS, and myo-[3H]inositol (5 µCi/mL). The cells were washed, incubated for an additional 30 min in RPMI 1640 medium, and washed. They were transferred to Hanks’ balanced salt solution, incubated for 15 min in the presence of LiCl (10 mM), and assays were performed. The incubations were stopped by addition of HClO4 (5%, v/v), and 200 µL of a solution of BSA (20 mg/mL) were added. The pH was adjusted to 7.0 and the various myo-inositol phosphate derivatives were separated on Dowex 1-X8 columns (200–400 mesh, formate form).

Western blotting and quantification
Jurkat lymphocytes (4 to 5 x 106 cells) cultured under various conditions were solubilized by treatment with NP-40 (1% v/v) in 200 µL of a Tris (50 mM)-sodium chloride (125 mM) buffer (pH 7.5) containing (mM), EDTA 5, EGTA 10, phenylmethylsulfonyl fluoride (PMSF) 1, benzamidine 10, aprotinin 0.1 mg/mL; and protein phosphatase inhibitors (mM), sodium orthovanadate 0.5, sodium pyrophosphate 10, and sodium fluoride 10. The mixture was briefly mixed and left on ice for 15 min, with occasional shaking. The extract was centrifuged (15 min, 16,000 g) and protein concentration determined with the Bio Rad Protein Assay kit (Bio Rad, Richmond, CA). The proteins (50 µg/well) were separated by SDS-PAGE according to Laemmli [46 ] on 10% polyacrylamide gels using a Mini-PROTEAN II apparatus (Bio Rad). The proteins were transferred to a nitrocellulose membrane (Hybond-C Extra; Amersham Pharmacia Biotech, Montreal, Quebec) using a Mini Trans-Blot apparatus (Bio Rad). Efficiency of transfer was monitored with the Ponceau red stain [47 ]. After washing with a TBS buffer (Tris, 20 mM; NaCl, 137 mM; pH 7.6) to remove the stain, the membrane was placed in a heat-sealable plastic bag and treated with a TBS-T buffer [TBS buffer containing 0.1% (v/v) Tween 80] and 5% (w/v) powdered skimmed milk to block nonspecific binding sites. The mixture was incubated by rotary mixing with a Roto-Torque apparatus (Cole Palmer, Niles, IL) for 1 h at room temperature or overnight at 4°C. The membrane was then washed twice (5 min each) in a bath containing TBS-T buffer and then incubated in the same buffer with the appropriate mAb. mAb were used at the following concentrations: 40 ng/mL (anti-p56Ick), 1 µg/mL (anti-p59fyn), 50 ng/mL (anti-ZAP-70) and a dilution of 1:2000 (anti-phosphotyrosine), for 1 h at room temperature. The membrane was washed twice in TBS-T buffer and incubated with the peroxidase-conjugated secondary Ab (dilution 1:10,000). After two washes in TBS-T buffer, the antigens were revealed by chemiluminescence. Protein bands were scanned (Scanman, Logitech, Fremont, CA) and quantitated by volume integration using the ImageQuant software (Molecular Dynamics, Sunnyvale, CA). An exposed area of film outside each lane was used as background and subtracted from experimental values.

Western blotting of PKC isoforms was performed with Jurkat cell lysates obtained by solubilization with NP-40 (1% v/v), as above, except that the Tris buffer contained a commercial mixture of protease inhibitors (Boehringer Mannheim), PMSF, the mixture of EDTA and EGTA but did not contain protein phosphatase inhibitors. Protein sizing on SDS-PAGE gels, transfer, and detection (chemiluminescence) were as above. The primary antibodies were used at 1:1000–1:2000 dilutions and the peroxidase-conjugated secondary antibodies were used at a 1:2000 dilution.

Immunoprecipitations
Jurkat cells were lysed by treatment with NP-40 (1% v/v) as above, the volume of cell extract (300 µg proteins) was completed to 250 µL with PBS, and the required dilution of primary antibody was added. The mixture was incubated overnight at 4°C under rotary mixing, then mixed with a suspension (25 µL) of PBS-washed Protein A-Sepharose beads (Sigma) and incubated under rotary mixing for 3 h at 4°C. The beads were centrifuged, washed three times (10 min each, 4°C) with a Tris buffer (pH 7.5) containing (mM), Tris 50, NaCl 150, and NP-40 (0.05% v/v), and beef gelatin (0.25%, w/v), and then centrifuged. The immunoprecipitates were used for Western blotting or PTK assays.

Determination of p56Ick activity
The pellet obtained as above was washed twice (10 min, 4°C) with kinase assay buffer (MOPS, 20 mM, MgCl2, 10 mM, pH 7.0) and enzyme activity determined as described by Flint et al. [48 ] using acid-denatured enolase [49 ]. The reaction mixture was separated on 12% polyacrylamide SDS-PAGE, the gel was dried under vacuum, and exposed to an X-Omat RP XRP-1 (Kodak) film. The relevant protein bands were cut out and counted.

Statistical analysis of data
Data were analyzed with the Sigma Stat computer program (Jandel Scientific, San Rafael, CA) using Student’s t-test for paired data. Significance was set at the 95% confidence level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Relative levels of PKC isozymes in PMA-treated Jurkat lymphocytes
Chronic overnight treatment with PMA has been shown to induce the down-regulation of PKC{alpha}, ß, {delta}, and {varepsilon} in Jurkat lymphocytes [41 ] but not {zeta} [41 , 50 ]. In the present study, the levels of expression of PKC isozymes in PMA-treated cells relative to control (untreated Jurkat cells) were determined by densitometric analysis of Western blots. Data showed that the treatment of Jurkat cells with PMA resulted in significantly decreased levels of PKC isozymes of the conventional and novel families (Fig. 1 ). For instance, the relative level of expression of PKCßI was approximately 20% of control, whereas this was 10% in the case of PKC{delta}. There was a near absence of expression of PKC{alpha} (1.6%) and PKCßII (0.7%). The PMA treatment had less of an effect on the relative level of PKC{theta}, which was reduced by ~30%. In contrast, the relative levels of PKC{varepsilon} (133%) and {zeta} (150%) were increased in cells treated with PMA.



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  		Figure 1. Western blots of PKC isozymes in Jurkat cells. The cells were left untreated (C) or were treated (T) for 24 h in the presence of PMA (160 nM). They were lysed, the proteins resolved by SDS-PAGE (10% polyacrylamide), electrotransfered to a membrane, and PKC isozymes were revealed by chemiluminescence using isozyme-specific pAb and appropriate peroxidase-conjugated secondary pAb. The X-ray films were scanned and quantitated by densitometric analysis. Ratios refer to values recorded in PMA-treated cells with respect to untreated cells. Sizes were determined by calibration with colored marker proteins.

 
Comparative Ca2+ responses in untreated, 4{alpha}-PMA- and PMA-treated Jurkat cells exposed to mAb C305 and Leu 4
Untreated Jurkat cells responded to mAb C305 with the typical biphasic response characterized by an initial transient rise in [Ca2+]i followed by a plateau (Fig. 2A ). Jurkat cells treated with PMA (Fig. 2B) or inactive 4{alpha}-PMA (not shown) also displayed the typical biphasic Ca2+ profile when challenged with mAb C305. When the external medium was replaced with a low (EGTA-buffered) Ca2+-containing medium (<100 nM), the response to mAb C305 was transient in each case (not shown). mAb Leu 4 that is directed against the {delta} and {varepsilon} components of CD3 [51 , 52 ] triggered a Ca2+ response in untreated Jurkat lymphocytes (Fig. 2C) . The amplitude of the response was smaller than in the case of cells challenged with mAb C305. There was a similar response in cells treated with the inactive 4{alpha}-PMA analog (not shown). When the experiments were done with cells bathing in a low (EGTA-buffered) Ca2+-containing medium (<100 nM), the Ca2+ profile was transient (not shown). Unexpectedly, Jurkat cells treated with PMA failed to respond to mAb Leu 4. However, they responded to a subsequent challenge with mAb C305 (Fig. 2D) . An absence of Ca2+ response was also observed when mAb OKT3 was used (not shown), suggesting that the lack of response was related to the PMA treatment rather than the mAb used. Furthermore, cross-linking anti-CD3 mAb with a secondary Ab did not generate a Ca2+ response (not shown). The increases in [Ca2+]i are summarized in Table 1 .



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  		Figure 2. Time-course of the Ca2+ responses of Jurkat E6.1 lymphocytes to anti-Ti (C305) or anti-CD3 (Leu 4) mAb. Fura 2-loaded Jurkat T cells (5 x 106 lymphocytes/mL) were incubated in a 1 mM Ca2+-containing medium. Control (A, C) corresponds to experiments performed with untreated Jurkat cells. PMA-treatment (B, D) refers to Jurkat cells treated with PMA (24 h, 160 nM). Changes in fluorescence were recorded in a slow time-base mode at 37°C. The point of addition of each mAb is indicated by the arrows.

 

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  		Table 1. Summary of the Increases in [Ca2+]i in Untreated, 4{alpha}-PMA- and PMA-Treated Jurkat T Lymphocytes Exposed to mAb C305 and Leu 4

 
Production of IP3
Untreated Jurkat cells responded to mAb C305 with an increased production of IP3 that was 2.44 ± 0.03-, 1.98 ± 0.26-, and 2.70 ± 0.12-fold that of unstimulated cells, in the case of untreated, 4{alpha}-PMA- and PMA-treated cells (Fig. 3 ), respectively. Untreated Jurkat cells or cells treated with the inactive 4{alpha}-PMA analog responded to mAb Leu 4 with a 2.08 ± 0.05 and 1.74 ± 0.05-fold (with respect to unstimulated cells) increase in IP3 but the response was absent (1.05 ± 0.01) in cells chronically treated with PMA (Fig. 3) .



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  		Figure 3. IP3 production by Jurkat E6.1 lymphocytes. Untreated and PMA-treated cells were loaded with myo-[3H]inositol as described in Materials and Methods and then exposed to mAb C305 (open bars) or to mAb Leu 4 (filled bars) for 15 min. The content of IP3 was determined by ion-exchange chromatography and is represented by dividing the value (cpm) of labeled IP3 measured under experimental conditions by the value determined in unstimulated (basal) samples (basal values were arbitrarily set to 1.0). Ratio (average ± SEM, vertical bars) are shown in the case of untreated Jurkat cells (control) and cells treated for 24 h with the inactive 4{alpha}-PMA analog (160 nM) or with PMA (160 nM). The figure is representative of two separate experiments done in triplicate.

 
Surface expression of Ti and CD3 in untreated and PMA-treated Jurkat cells
A trivial explanation of the preceding results would be that the phorbol ester treatment affected the expression of the CD3 complex or that the {delta} and {varepsilon} antigens were not available to mAb Leu 4 (or OKT 3). However, indirect immunofluorescence cytofluorimetric analysis (FACScan instrument; Becton Dickinson, Montreal) revealed that the level of expression of Ti (mAb C305) or CD3 (mAb Leu 4) were unchanged in the PMA-treated cells in comparison to untreated lymphocytes (Fig. 4 ).



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  		Figure 4. Cytofluorimetric analysis of Ti and CD3 expression in untreated and PMA-treated Jurkat cells. Jurkat cells were left untreated (A, B) or were cultured for 24 h in the presence of PMA (160 nM; C, D). The lymphocytes were exposed to the anti-TcR ß-subunit mAb C305 (A, C) or to the anti-CD3 mAb Leu 4 (B, D) for 30 min at 4°C, washed, and incubated with a FITC-labeled rabbit anti-mouse Ig polyclonal Ab. A minimum of 10,000 cells were analyzed. Data are representative of two separate experiments.

 
Patterns of tyrosine-phosphorylated proteins (Western blots) of untreated, 4{alpha}-PMA- and PMA-treated Jurkat cells exposed to mAb C305 and Leu 4
Results of immunodetection using a mAb directed against the phosphotyrosine hapten showed that the overall intensities were similar in the case of untreated cells and cells treated with the inactive 4{alpha}-PMA analog (Fig. 5 , lanes 1–3 and 7–9). In contrast, the treatment with PMA resulted in an overall decrease of 30–40% (densitometric scanning) of the levels of tyrosine-phosphorylated proteins (Fig. 5 , lanes 4–6). Densitometric analysis revealed that mAb C305 and Leu 4 increased the levels of tyrosine phosphorylation in protein bands of Mr 30–35, 40–43, 56, 60–62, 70, 77–80, 95–100, and 105–115 kDa in untreated and in 4{alpha}-PMA-treated lymphocytes (Fig. 5 , lanes 2, 3, 8, and 9). These results were similar to those reported in Jurkat cells exposed to anti-Ti [13 ] or anti-CD3 [13 , 53 ] mAb. The effect of PMA treatment in unstimulated cells (basal conditions) was assessed by dividing the values of densitometric data of PMA-treated cells by the values of untreated cells and the results are shown in Table 2 . In this case, the ratio of protein bands of Mr 53, 60–62 and 70 kDa was not changed but it was decreased in the other proteins. Ratios in the case of cells exposed to mAb C305 or Leu 4 were corrected to account for the lower levels of tyrosine phosphorylation in PMA-treated cells. This was done by dividing the densitometric values for each protein band in PMA-treated lymphocytes by the ratio value for the corresponding protein band determined under basal conditions. Stimulation with mAb C305 was found to trigger tyrosine phosphorylation to the same extent as in untreated cells in most of the protein bands, except for those of Mr 56, 70, and 105–115 kDa (Table 2) . Similar observations were made in the case of Jurkat cells exposed to mAb Leu 4, although the level of tyrosine phosphorylation was decreased in protein bands of Mr 40–43, 90, and 95–100 kDa.



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  		Figure 5. Effect of PMA treatment on the pattern of tyrosine-phosphorylated proteins in Jurkat cells. The cells were exposed to the anti-Ti (ß-subunit) mAb C305 or to the anti-CD3 mAb Leu 4. Jurkat T cells were left untreated (lanes 1–3) or were cultured for 24 h in the presence of PMA (160 nM; lanes 4–6) or inactive 4{alpha}-PMA (160 nM; lanes 7–9). The cells (5 x 106 lymphocytes) were left unstimulated (lanes 1, 4, and 7) or were exposed to mAb C305 (lanes 2, 5, and 8) or Leu 4 (lanes 3, 6, and 9) for 2 min at 37°C. The cells were lysed, the proteins resolved by SDS-PAGE (10% polyacrylamide), electrotransfered to a membrane, and the phosphotyrosine-containing proteins revealed by Western blotting with an anti-phosphotyrosine mAb and a chemiluminescence assay kit. The Mr of reference proteins (kDa) are shown on the left. Data are representative of five separate experiments.

 

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  		Table 2. Densitometric Ratios of Tyrosine-Phosphorylated Proteins in PMA-Treated Jurkat Lymphocytes Relative to Untreated Cells

 
Ca2+ mobilization in activated Jurkat T cells requires a PTK-dependent phosphorylation of PLC-{gamma}1 that results in its activation [18 ]. Based on the results of Figures 2 and 5 , we investigated whether the treatment with PMA was associated with a decrease in the level of tyrosine phosphorylation of PLC-{gamma}1. Jurkat cells were cultured for 24 h in the absence (control)/presence of PMA (160 nM), washed, and were then left untreated or exposed to mAb C305 or Leu 4. Results of Western blots (anti-phosphotyrosine) of immunoprecipitates (anti-PLC-{gamma}1) revealed no significant differences in the ratio of untreated cells to PMA-treated lymphocytes (not shown).

Electrophoretic behavior and immunoassays of p56Ick
Western blotting of untreated and PMA-treated cells using an anti-p56Ick mAb revealed that the mAb recognized two isoforms of the protein that migrated as a major entity of Mr 56 kDa and a faint band of 60 kDa in untreated cells (Fig. 6A , lane 1) and in cells treated with 4{alpha}-PMA (Fig. 6A , lane 7), independently of stimulus (Fig. 6A , lanes 2, 3, 8, and 9). However, the p60–62 isoform was the major protein band found in unstimulated (Fig. 6A , lane 4) and in stimulated (Fig. 6A , lanes 5 and 6) PMA-treated Jurkat cells. The ratio (densitometry, average ± SEM) of p60–62 to p56 was 0.07 ± 0.01 (n = 3) in the case of untreated and 4{alpha}-PMA-treated cells and 3.09 ± 0.39 (n = 3) in PMA-treated Jurkat lymphocytes.



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  		Figure 6. Western blots of p56Ick in Jurkat cells. (A) Untreated (lanes 1–3), PMA- (24 h, 160 nM; lanes 4–6), or 4{alpha}-PMA- (24 h, 160 nM; lanes 7–9) treated cells (5 x 106 lymphocytes) were left unstimulated (lanes 1, 4, and 7) or were exposed (2 min) to the anti-Ti C305 (lanes 2, 5, and 8) or to the anti-CD3 Leu 4 (lanes 3, 6, and 9) mAb. Cell lysates were sized by electrophoresis (10% polyacrylamide) and electrotransfered to a membrane. Western blots were performed with an anti-p56Ick mAb and proteins were revealed by chemiluminescence using a peroxidase-conjugate anti-mouse Ig pAb. The relative molecular sizes (kDa) of the protein bands are indicated on the left. (B) Effect of the phosphotyrosine phosphatase inhibitor peroxovanadium bpV(pic) on p56Ick. Untreated (lanes 1–3), PMA- (24 h, 160 nM; lanes 4–6) and bpV(pic)- (15 min, 10 µM; lanes 7–9) treated Jurkat (5 x 106) cells were left unstimulated (lanes 1, 4, and 7) or were exposed (2 min) to the anti-Ti C305 (lanes 2, 5, and 8) or the anti-CD3 Leu 4 (lanes 3, 6, and 9) mAb. Cell lysate processing and detection were as above. The relative molecular sizes (kDa) of the protein bands are indicated on the left. The figure is representative of five similar experiments.

 
The electrophoretic behavior of p56Ick was reminiscent of reported observations [54 ] that p56Ick can exist in a phosphorylated form of reduced electrophoretic mobility ({approx}60 kDa). We investigated whether the shift in electrophoretic mobility of p56Ick observed here was due to hyperphosphorylation. Jurkat cells were treated with bpV(pic), a peroxovanadium derivative that is a specific inhibitor of phosphotyrosine phosphatases [55 ]. Western blotting revealed that bpV(pic) dramatically increased the proportion of the p60–62 band (Fig. 6B , lanes 7–9) with respect to untreated cells (Fig. 6B , lanes 1–3), in a manner similar to the effect of the PMA treatment (Fig. 6A , lanes 4–6). The ratio of p60–62 to p56 in pbV(pic)-treated cells was 5.71 ± 0.18 (n = 3). The electrophoretic mobilities of p59fyn and ZAP-70 were not modified in PMA-treated Jurkat cells (not shown).

We investigated whether the decreased level of protein tyrosine phosphorylation observed in PMA-treated Jurkat cells (Fig. 5) could be related in part to a decreased activity of hyperphosphorylated p56Ick that would result in defective signal transduction in stimulated Jurkat cells. The activity of p56Ick was determined in immunoprecipitates from untreated and PMA-treated Jurkat lymphocytes that were left unstimulated or exposed to mAb C305 or Leu 4. Quantitation of incorporated label (Fig. 7 ) showed that mAb C305 increased the level of incorporated label approximately 1.6-fold in p56Ick and enolase (with respect to unstimulated cells, ratio set to 1.0), whereas activating the cells through the CD3 complex resulted in an approximate 2.4-fold increase in both substrates. In marked contrast, p56Ick activity was not up-regulated in the case of Jurkat cells that had been treated with PMA and exposed to mAb C305 or Leu 4.



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  		Figure 7. Quantification of the activity of p56Ick. The activity of p56Ick was determined in immunoprecipitates using [{gamma}-32P]ATP and enolase as substrates. Phosphorylated proteins were sized by SDS-PAGE (12% polyacrylamide), the gel dried under vacuum, and then exposed to a Kodak RP XRP-1 film. Bands corresponding to enolase (Mr 30 kDa) and p56Ick were cut out and counted. Results are expressed as the ratio of incorporated label in Jurkat cells exposed to cross-linked anti-Ti ß-subunit mAb C305 (open bars) or to cross-linked anti-CD3 mAb Leu 4 (hatched bars) with respect to unstimulated (secondary pAb only) cells (value set to 1.0, filled bars). *Significant differences (Student’s t test for paired data; P < 0.01) with respect to unstimulated cells. The figure is representative of two separate experiments done in duplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ligation of the TcR/CD3 complex initiates the activation of protein kinases and phosphatases, the turnover of phospholipids, the mobilization of Ca2+, the activation of PKC, and the expression of specific genes [reviewed in 56]. PKC is involved in the activation of factors of transcription [34 , 35 ] and the MAP kinase pathway [32 , 33 , 40 ] but its role in the cascade of events that leads to T cell activation is still not fully understood. PMA-sensitive PKC isozymes can be down-regulated when Jurkat cells are exposed to a pharmacological concentration of PMA [41 ]. Experimental conditions used in the present study resulted in the down-regulation of PKC isoforms of the conventional family ({alpha}, ßI, ßII), whereas isoforms of the novel family were significantly ({delta}) or partially ({theta}) decreased or up-regulated ({varepsilon}). As expected [50 ], the atypical PKC{zeta} isoform was not down-regulated but its expression was up-regulated (Fig. 1) . Up-regulation of PKC isozymes as a result of cell treatment with PMA has been reported. For instance, PKC{delta} is up-regulated in the T84 human epithelial cell line exposed to a pharmacological concentration of PMA [57 ], an observation that has been attributed to a phenomenon of cross-talk between the different isoforms of PKC [58 ].

Chronic treatment of Jurkat cells with PMA did not affect the Ca2+ response to ligation of Ti (Figs. 2) , which showed the same characteristic as untreated cells (Fig. 2 , Table 1 ). It is interesting that cells that responded initially to mAb C305 failed to respond to a subsequent challenge with anti-CD3 mAb and reciprocally (not shown), suggesting that the Ti- and CD3-dependent Ca2+ signal targeted the same InsP3-sensitive Ca2+ store. It is interesting that PMA-treated cells failed to generate a Ca2+ response to mAb Leu 4 (Fig. 2) and did not produce IP3 (Fig. 3) . The lack of Ca2+ response was independent of the mAb used (Leu 4 or OKT 3) or their physical state (monomeric or cross-linked) and was not due to a reduced level of expression of Ti or CD3 (Fig. 4) . Data shown in Figures 2 and 3 suggested an absence of up-regulation of PLC-{gamma}1 activity that can be assessed by increased tyrosine phosphorylation [18 ]. Here, Western blotting experiments failed to show significant differences (three separate experiments) in the levels of tyrosine-phosphorylated PLC-{gamma}1 in PMA-treated and untreated cells (not shown). These observations are in agreement with the report of Schröder et al. [59 ] who have presented indirect evidence for the involvement of a PLC isoform different than PLC-{gamma}1, which would act downstream of PMA-sensitive PKC in activated T cells.

We investigated whether the treatment with PMA influenced the patterns of tyrosine-phosphorylated proteins in Jurkat cells exposed to anti-Ti or anti-CD3 mAb. In this connection, it has been reported that down-regulation of PKC in Jurkat cells does not prevent tyrosine phosphorylation of the components of CD3 [60 ]. Our data showed an overall decrease in the levels of tyrosine phosphorylation of most of the proteins in PMA-treated lymphocytes (Fig. 5 , Table 2 ) independently of mAb (anti-Ti or anti-CD3) stimulation. The reduced levels of tyrosine phosphorylation were not related to cytotoxicity, differences in cell growth, or unequal protein loads on the gels. These results were consistent with the interpretation that conventional (PMA-sensitive) PKC were involved at an early step in the cascade of events leading to the TcR/CD3-dependent activation of Jurkat lymphocytes.

Strauss and Weiss [13 ] have generated a mutant of Jurkat T cells (J.CaM1) that is devoid of p56Ick activity due to a defect in alternative splicing of the p56Ick gene. This mutant possesses some characteristics that are relevant to observations reported here. For instance, the J.CaM1 mutant displayed a biphasic Ca2+ response to an anti-CD3 mAb but failed to respond to mAb C305 [13 ], in contrast with our findings in the case of PMA-treated Jurkat cells (Fig. 2) . In addition, the mutant was severely defective in the induction of tyrosine-phosphorylated proteins in response to stimulation through Ti or the CD3 complex [13 ] in agreement with our data (Fig. 5) . Close examination of the tyrosine-phosphorylated protein patterns of Figure 5 showed that a protein band of Mr 60–62 kDa was present to a greater extent in PMA-treated cells than in controls. This band was not revealed by an anti-p59fyn mAb but by an anti-p56Ick mAb that also recognized a faint band of Mr 56 kDa (Fig. 6A) . This result contrasted with the observation that the same anti-p56Ick mAb recognized a major phosphotyrosine-positive band of Mr 56 kDa and a faint band of Mr {approx}60 kDa in untreated and in 4{alpha}-PMA-treated cells (Fig. 6A) . The reduced electrophoretic mobility of p56Ick suggested a state of hyperphosphorylation [54 ] that was confirmed by treating the cells with the phosphotyrosine phosphatase inhibitor pbV(pic) [55 ]. In this case, p56Ick shifted to a major phosphotyrosine-positive band of Mr 60–62 kDa, accompanied by a faint band of Mr 56 kDa (Fig. 6B) . We tested the hypothesis that the hyperphosphorylated state of p60–62Ick interfered with its up-regulation, thus inhibiting the p56Ick-dependent arm of the signal transduction pathways in a manner similar to that observed in the case of the J.CaM1 mutant [13 ]. Whereas stimulating untreated Jurkat lymphocytes with mAb C305 or Leu 4 up-regulated p56Ick activity, these TcR/CD3-directed mAb failed to up-regulate the activity of the enzyme in PMA-treated cells (Fig. 7) .

The role of PKC in T cell activation is slowly emerging. For example, the PKC{theta} isoform that is predominantly expressed in hematopoietic and skeletal cells plays a critical role in the regulation of transcriptional activation of early-activation genes [reviewed in 61] and activation-induced apoptosis in T cells [62 ]. Furthermore, PKC{theta} is the only PKC isoform to localize at the contact site between the TcR/CD3 complex of antigen-responding T cells and the MHC of antigen-presenting cells [63 , 64 ]. Other PKC isoforms are involved early in signal transduction as well. For instance, Miranti et al. [38 ] have used transfection experiments in Cos7 cells to obtain evidence that classical and novel PKC isoforms act upstream of the Shc adaptor protein and Schröder et al. [59 ] have recently reported that PKC is involved in a modulation of the MAP kinase pathway associated with the up-regulation of p56Ick in activated T cells, in agreement with data obtained with the J.CaM1 p56Ick-deficient mutant [40 ]. Data presented here are in agreement with these reports of an early involvement of PKC in T cell signaling but do not exclude the possibility of further involvement of PKC downstream of p21ras or p56Ick, as reported by several laboratories [34 , 39 , 40 ]. Our results suggest that members of the classical and novel families of PKC isozymes play a critical role in the tight coupling of Ti to the CD3 signal-transducing complex in Jurkat cells and the up-regulation of p56Ick activity. However, the possibility remains that these PKC isoforms could also be involved in the regulation of protein tyrosine phosphatase activity associated with the regulation of signaling in Jurkat T cells.


   ACKNOWLEDGEMENTS
 
This work was supported in part by a grant (to G. D. and M. D. P.) from the Medical Research Council of Canada. We thank Dr. A. Weiss for providing the Jurkat E6.1 variant and the mAb C305-producing hybridoma, Dr. André Veillette for a generous gift of the an anti-ZAP-70 pAb and the protocols for immunoprecipitations, and Dr. Barry Posner for a sample of pbV(pic). We are also indebted to Dr. Nicole Gallo-Payet for her collaboration in the quantification of IP3 and Ms Valérie Breton for technical assistance.

Received August 28, 1998; revised March 2, 2000; accepted March 4, 2000.


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