Selective Inhibition of Protein Kinase C Isozymes by Fas Ligation
1999; Elsevier BV; Volume: 274; Issue: 22 Linguagem: Inglês
10.1074/jbc.274.22.15320
ISSN1083-351X
AutoresChangyan Chen, Douglas V. Faller,
Tópico(s)Retinal Development and Disorders
ResumoActivation of protein kinase C (PKC) can protect cells from apoptosis induced by various agents, including Fas ligation. To elucidate a possible interaction between Fas-mediated apoptotic signals and activation-related protective signals, we investigated the impact of Fas ligation on PKC activity. We demonstrate that engagement of Fas on human lymphoid Jurkat cells triggered apoptosis, and Fas ligation resulted in partial blockade of cellular PKC activity. The phorbol 12-myristate 13-acetate-mediated translocation of PKCθ from the cytoplasm to the membrane was inhibited by treatment with anti-Fas antibody, whereas the translocation of PKCα or ε was not affected.In vitro kinase assay of PKCα or ε phosphotransferase activity demonstrated that Fas ligation inhibited the ability of PKCα to phosphorylate histone H1 as substrate but did not inhibit ε isozyme activity. This inhibition of PKCα activity mediated by Fas ligation was reversed by okadaic acid, a phosphatase inhibitor, suggesting the involvement of a member of the protein phosphatase 2A subfamily in this component of Fas signaling. Identical patterns of PKC isozyme inhibition were obtained using mouse thymoma cells overexpressing the fas gene (LF(+)). These results suggest that the selective inhibition of a potentially protective, PKC-mediated pathway by Fas activation may, to some extent, contribute to Fas-induced apoptotic signaling. Activation of protein kinase C (PKC) can protect cells from apoptosis induced by various agents, including Fas ligation. To elucidate a possible interaction between Fas-mediated apoptotic signals and activation-related protective signals, we investigated the impact of Fas ligation on PKC activity. We demonstrate that engagement of Fas on human lymphoid Jurkat cells triggered apoptosis, and Fas ligation resulted in partial blockade of cellular PKC activity. The phorbol 12-myristate 13-acetate-mediated translocation of PKCθ from the cytoplasm to the membrane was inhibited by treatment with anti-Fas antibody, whereas the translocation of PKCα or ε was not affected.In vitro kinase assay of PKCα or ε phosphotransferase activity demonstrated that Fas ligation inhibited the ability of PKCα to phosphorylate histone H1 as substrate but did not inhibit ε isozyme activity. This inhibition of PKCα activity mediated by Fas ligation was reversed by okadaic acid, a phosphatase inhibitor, suggesting the involvement of a member of the protein phosphatase 2A subfamily in this component of Fas signaling. Identical patterns of PKC isozyme inhibition were obtained using mouse thymoma cells overexpressing the fas gene (LF(+)). These results suggest that the selective inhibition of a potentially protective, PKC-mediated pathway by Fas activation may, to some extent, contribute to Fas-induced apoptotic signaling. The Fas/APO-1 antigen, a member of the tumor necrosis factor receptor family, is a transmembrane molecule and is expressed by a variety of cells, including transformed cell lines and activated T lymphocytes (1Itoh N. Onehara S.Y. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. Cell. 1991; 66: 233-240Abstract Full Text PDF PubMed Scopus (2678) Google Scholar, 2Oehm A. Behrmann J. Falk W. Pawlita M. Maier G. Klas C. Li-Weber M. Richards S. Dhein J. Trauth B.C. Ponstingl H. Krammer P.H. J. Biol. Chem. 1992; 267: 10709-10714Abstract Full Text PDF PubMed Google Scholar, 3Smith C.A. Farrah T. Goodwin R.G. Cell. 1994; 76: 959-965Abstract Full Text PDF PubMed Scopus (1839) Google Scholar). The function of Fas/APO-1 appears to be the induction of apoptosis, and a growing number of Fas-associated molecules and signal pathways have been discovered (1Itoh N. Onehara S.Y. Ishii A. Yonehara M. Mizushima S. Sameshima M. Hase A. Seto Y. Nagata S. 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Although CAPP has been demonstrated to mediate some of the cellular actions of ceramide, the link between ceramide-induced phosphatase activation and subsequent intracellular events is still not clear. Protein kinase C (PKC) has been implicated as one of the critical components of multiple signaling pathways, including T cell activation processes (28Genot E.M. Parker P.J. Cantrell D.A. J. Biol. Chem. 1995; 270: 9833-9839Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 29Baier-Bitterlich G. Uberall F. Bauer B. Fresser F. Wachter H. Grunicke H. Utermann G. Altman A. Baier G. Mol. Cell. Biol. 1996; 16: 1842-1850Crossref PubMed Google Scholar, 30Monks C.R.F. Kupfer H. Tamir I. Barlow A. Kupfer A. Nature. 1997; 385: 83-86Crossref PubMed Scopus (494) Google Scholar). At least 11 isotypes of PKC have been discovered, and these isotypes can be classified according to their structure and cofactor requirements for activation (31Berry N. Nishizuka Y. Eur. J. 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Sci. 1994; 345: 257-263Crossref PubMed Scopus (27) Google Scholar, 12Singer G.G. Abbas A.K. Immunity. 1994; 1: 365-371Abstract Full Text PDF PubMed Scopus (704) Google Scholar). Ligation of Fas significantly suppresses TcR/CD3 complex-mediated early signal transduction events, including inhibition of TcR/CD3-triggered tyrosine phosphorylation of cellular proteins (50Kovacs B. Tsokos G.C. J. Immunol. 1995; 155: 5543-5549PubMed Google Scholar). Thus, Fas engagement may attenuate T cell activation events, whereas T cell activation-related events, such as activation of PKC, may reciprocally protect cells against apoptosis. This opportunity for cross-talk between signaling pathways led us to examine the potential interrelationship between these two seemingly opposing processes: apoptosis and activation. Here, we demonstrate that engagement of Fas triggers the apoptotic process in human Jurkat and mouse thymoma cells stably expressing the fas gene (LF(+)) but not in mouse thymoma cells stably transfected with antisensefas gene (LF(−)). Fas ligation selectively inhibits the activation of different isotypes of PKC in both Jurkat and LF(+) cells. The translocation of PKCθ in response to phorbol 12-myristate 13-acetate (PMA) stimulation is inhibited by prior Fas ligation. PKCα-mediated phosphorylation of histone H1 is blocked by prior Fas activation. This inhibition of PKC activity by Fas activation could be prevented by pretreatment with okadaic acid, indicating an involvement of a protein phosphatase in Fas signaling. The activity of PKCε was not affected by Fas stimulation. Therefore, these data suggest that the integration of multiple pro- and anti-apoptotic signals resulting from Fas activation may be required to execute the apoptotic program successfully. The human lymphoblastoid cell line Jurkat (American Tissue Culture Collection, Rockville, MD) was maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated newborn calf serum (Hazelton Research Products, Inc., Lenexa, KA), 2 mml-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. LF1210 mouse thymoma cells were transfected with the fas gene (]LF(+)) or an antisensefas gene (LF(−)) (generous gifts from Dr. S.-T. Ju, Boston University) and cultured in RPMI 1640 medium containing 10% heat-inactivated newborn calf serum plus 0.7 mg/ml Geneticin. Jurkat, LF(+), or LF(−) cells (0.5 × 106 cells/ml) were cultured in six-well plates with 5 ml of medium containing 10% newborn calf serum plus 1.5 μg/ml anti-human Fas Ab (Pan Vera Corp., Madison, WI) for Jurkat and PH1 cells or 1.5 μg/ml IgM Ab as control. The mouse thymoma cells (LF) were cultured under the same conditions with the addition of 5 μg/ml mouse anti-mouse Fas Ab (Pharmingen, San Diego). Cells were collected at the time points indicated and enumerated using trypan blue dye exclusion to assess viability. Error bars represent the S.D. over five independent experiments. The cells (1 × 106/ml) were cultured with 1.5 μg/ml anti-human Fas Ab for 12 h and resuspended in 1 ml of 1% sodium citrate, 0.1% Triton X-100, 50 μg of propidium iodide, and 10 μl of RNase (1 mg/ml). The stained samples were kept in the dark at 4 °C overnight before DNA fragmentation analysis by FACScan (Becton Dickinson, Mountain View, CA). Human and mouse cells (1 × 106 cells/ml) were cultured in five replicate wells of a six-well plate with 10 ml of medium containing 1.5 μg/ml anti-human Fas Ab or 5 μg/ml anti-mouse Fas Ab for 60 min and subsequently treated with 100 nm PMA for 15 min. For single stimulation experiments, the cells were exposed to either anti-Fas Ab or PMA alone for 60 or 15 min, respectively. For PKC enzyme activity inhibitor experiments, cells were exposed to PMA for 15 min after the addition of 0.1 μm staurosporine for 15 min. After lysing the cells in 25 mm Tris-HCl (pH 7.5), 1% Triton X-100, 20 mm MgCl2, 150 mm NaCl (9Takahashi T. Tanaka M. Brannan C.I. Jenkins N.A. Copeland N.G. Suda T. Nagata S. Cell. 1994; 76: 969-976Abstract Full Text PDF PubMed Scopus (1474) Google Scholar), the lysates were normalized for protein concentration, and 150-μg aliquots of protein were analyzed for PKC activity using a PKC assay kit containing a specific substrate peptide for PKC and an inhibitor mixture that blocks the activity of PKA and calmodulin kinase (Upstate Biotechnology Inc., Lake Placid, NY). Subsequently, the32P-incorporating substrate from each treatment was separated from the residual [32P]ATP using p81 phosphocellulose paper, and the radioactivity incorporated into the substrate was measured by scintillation counting. Cells (50 × 106), following the different treatments described above, were washed twice with 1 × phosphate-buffered saline and resuspended in 1 ml of buffer B (20 mm Tris-HCl (pH 7.5), 2 mm EDTA, 5 mm EGTA, 10 mmβ-mercaptoethanol, 10 μg/ml leupeptin, 10 μg/ml aprotinin) (39Meller N. Liu Y.-C. Collins T.L. Bonnefoy-Berard N. Baier G. Isakov N. Altman A. Mol. Cell. Biol. 1996; 16: 5782-5791Crossref PubMed Google Scholar). The cell suspensions were transferred to a 1-ml syringe and sheared by being passed 40 times through 25-gauge needle. The lysates were centrifuged at 280 × g for 10 min to precipitate nuclei, and the supernatants were collected. One-third of the whole cell extract was saved, and the remainder was centrifuged at 16,000 × g for 30 min. The supernatant (cytosol) was collected, and the pellet was washed in buffer B containing 1% Nonidet P-40 for 1 h on ice and centrifuged again at 16,000 ×g. The supernatant representing the membrane fraction was saved. Each fraction (whole cell lysate, cytosol fraction, and membrane fraction) was normalized and separated on 8% of acrylamide gel. Subsequently the gel was immunoblotted with the anti-mouse (Santa Cruz Biotechnology, Santa Cruz, CA) or human (Transduction Laboratories, Lexington, KY) PKC Abs, which recognize the isoforms of PKCθ, α, and ε. The blot was developed with an anti-mouse Ig alkaline phosphatase reagent (Oncogene Science, Uniondale, NY). The cells (20 × 106) were incubated with PMA at 100 nm for 15 min prior to 60 min of exposure to anti-Fas Abs. For phosphatase inhibitor experiments, okadaic acid was added to a final concentration of 50 nm 15 min before the next treatments. The cell lysates were then prepared with the lysis buffer (150 mmNaCl, 50 mm Tris-HCl (pH 8.0), 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 5 mm EGTA, 10 mm NaF, 10 μg/ml leupeptin, 1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride) (27Lee J.Y. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1996; 271: 13169-13174Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The lysates were normalized for protein concentration, and each sample (containing approximately 500 μg of protein) was immunoprecipitated with the corresponding anti-PKC Ab and collected by absorption to protein A-Sepharose. The immunocomplexes bound to protein A-Sepharose were washed with lysis buffer twice and kinase buffer twice (50 mm Tris-HCl (pH 7.4), 10 mm NaF, 1 mm Na3VO4, 0.5 mm EDTA, 0.5 mm EGTA, 2 mm MgCl2, 10 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride). Subsequently, the immunocomplexes bound to the beads were resuspended in reaction buffer (20 mm Tris-HCl (pH 7.4), 10 mm MgCl2, 10 μm cold ATP, 0.4 mg/ml histone H1, 2.5 μCi of [γ-32P]ATP (6,000 Ci/mmol)) and incubated at 30 °C for 10 min. The reactions were terminated by the addition of protein loading buffer. Proteins were separated on 10% SDS-polyacrylamide gel, and the gel was subjected to autoradiography. Jurkat cells express a significant amount of Fas antigen on their surfaces (51Chen C.-Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The sensitivity of these cells to treatment with anti-Fas Ab was examined in medium containing 10% serum (Fig.1 a). The number of viable Jurkat cells did not begin to decline until 5 h after exposure to the Ab, and the death proportion of dead cells increased steadily from that time on. By 24 h, approximately 65% of the Jurkat cells lost viability. Exposure to an unrelated, isotype-matched (IgM) antibody did not induce cell death in Jurkat cells; rather, the numbers of cells started to increase 6 h after the addition of the unrelated Ab, reflecting normal cell growth. The cell death observed was caused by apoptosis, as confirmed by cytofluorometric analysis of nuclear DNA fragmentation, as described previously (Fig. 1 b). The percentage of cells with fragmented DNA 15 h after the addition of anti-Fas Ab was more than 20%, and by 24 h more than 30% of the cells contained fragmented DNA. This time course of apoptosis was also confirmed by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay (data not shown). We demonstrated previously that down-regulation or inhibition of PKC could induce Jurkat cells expressing oncogenic ras to undergo apoptosis and that Fas-mediated signals may be involved in this apoptotic process (51Chen C.-Y. Liou J. Forman L.W. Faller D.V. J. Biol. Chem. 1998; 273: 16700-16709Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 52Chen C.-Y. Faller D.V. Oncogene. 1995; 11: 1487-1498PubMed Google Scholar). Several other groups have documented that PKC can regulate or modulate both proliferative and anti-apoptotic pathways in diverse cell types (28Genot E.M. Parker P.J. Cantrell D.A. J. Biol. 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