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Translocation of Protein Kinase Cε and Protein Kinase Cδ to Membrane Is Required for Ultraviolet B-induced Activation of Mitogen-activated Protein Kinases and Apoptosis

1999; Elsevier BV; Volume: 274; Issue: 22 Linguagem: Inglês

10.1074/jbc.274.22.15389

1083-351X

Nanyue Chen, Weiya Ma, Chuanshu Huang, Zigang Dong,

Protein Tyrosine Phosphatases

UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCε and PKCδ, but not PKCα, from cytosol to membrane. Dominant negative mutant (DNM) PKCε or PKCδ inhibited UVB-induced translocation of PKCε and PKCδ, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCε and PKCδ, whereas the DNM of PKCα was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCδ, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCα, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 μm) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 μm), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCδ and PKCε, but not PKCα, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs. UV-induced signal transduction may be involved in tumor promotion and induction of apoptosis. The role of protein kinase C (PKC) in UVB-induced signal transduction is not well understood. This study showed that UVB markedly induced translocation of membrane-associated PKCε and PKCδ, but not PKCα, from cytosol to membrane. Dominant negative mutant (DNM) PKCε or PKCδ inhibited UVB-induced translocation of PKCε and PKCδ, respectively. UVB-induced activation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs) was strongly inhibited by DNM PKCε and PKCδ, whereas the DNM of PKCα was less effective on the UVB-induced phosphorylation of Erks and JNKs. Among the PKC inhibitors used only rottlerin, a selective inhibitor of PKCδ, markedly inhibited the UVB-induced activation of Erks and JNKs, but not p38 kinases. Safingol, a selective inhibitor for PKCα, did not show any inhibitory effect on UVB-induced mitogen-activated protein kinase activation. GF109203X is a stronger inhibitor of classical PKC than novel PKC. Lower concentrations of GF109203X (<10 μm) had no effect on UVB-induced activation of Erks or JNKs. However, at higher concentrations (over 20 μm), GF109203X inhibited UVB-induced activation of JNKs, Erks, and even p38 kinases. Meanwhile, rottlerin and GF109203X markedly inhibited UVB-induced apoptosis of JB6 cells, whereas safingol had little inhibitory effect. DNM-Erk2 cells and PD98059, a selective inhibitor for mitogen-activated protein kinase/extracellular signal-regulated kinase 1 that directly activates Erks, inhibited UVB-induced apoptosis. DNM-JNK1 cells also blocked UVB-induced apoptosis, whereas SB202190, a specific inhibitor for p38 kinases, did not produce the inhibitory effect. These data demonstrate that PKCδ and PKCε, but not PKCα, mediate UVB-induced signal transduction and apoptosis in JB6 cells through activation of Erks and JNKs. UV radiation from the sun is the major environmental factor responsible for a high incidence of nonmelanoma skin cancer (1Fry R.J. Ley R.D. Carcinogenesis. 1989; 11: 321-337Google Scholar, 2Matsui M.S. Mintz E. DeLeo V.A. Mukhtar H. Skin Cancer: Mechanisms and Human Relevance. CRC Press, Boca Raton, FL1995: 21-30Google Scholar, 3Findlay G. Lancet. 1928; 2: 1070-1075Abstract Scopus (126) Google Scholar, 4Boutwell R.K. CRC Crit. Rev. Toxicol. 1974; 2: 419-443Crossref PubMed Scopus (686) Google Scholar). The electromagnetic spectrum of UV can be divided into three parts: UVA (320–400 nm), UVB (290–320 nm), and UVC (100–290 nm) (2Matsui M.S. Mintz E. DeLeo V.A. Mukhtar H. Skin Cancer: Mechanisms and Human Relevance. CRC Press, Boca Raton, FL1995: 21-30Google Scholar). In animal experiments, both UVB and UVC can act as complete carcinogens, whereas UVA can only act as a tumor promoter (2Matsui M.S. Mintz E. DeLeo V.A. Mukhtar H. Skin Cancer: Mechanisms and Human Relevance. CRC Press, Boca Raton, FL1995: 21-30Google Scholar, 3Findlay G. Lancet. 1928; 2: 1070-1075Abstract Scopus (126) Google Scholar, 4Boutwell R.K. CRC Crit. Rev. Toxicol. 1974; 2: 419-443Crossref PubMed Scopus (686) Google Scholar, 5Willis I. Menter J. Whyte H. J. Invest. Dermatol. 1981; 76: 404-408Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 6Strickland P.T. J. Invest. Dermatol. 1986; 86: 37-41Crossref PubMed Scopus (8) Google Scholar). Because UVC light does not penetrate the atmosphere (2Matsui M.S. Mintz E. DeLeo V.A. Mukhtar H. Skin Cancer: Mechanisms and Human Relevance. CRC Press, Boca Raton, FL1995: 21-30Google Scholar), UVB radiation is believed to be responsible for most of the carcinogenic effects of sun exposure (2Matsui M.S. Mintz E. DeLeo V.A. Mukhtar H. Skin Cancer: Mechanisms and Human Relevance. CRC Press, Boca Raton, FL1995: 21-30Google Scholar,7Fitzpatrick T.B. Sober A.J. Pearson B.J. Lew R. Castellani A. Research in Photobiology. 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Am. Acad. Dermatol. 1983; 9: 487-502Abstract Full Text PDF PubMed Scopus (154) Google Scholar). The mechanism behind the tumor-promoting ability of UV, however, is not well understood. A number of reports have established that UVC and UVB induce certain gene expression (13Adler V. Schaffer A. Kim J. Dolan L. Ronai Z. J. Biol. Chem. 1995; 270: 26071-26077Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 14Huang C. Ma W.-y. Ding M. Bowden G.T. Dong Z. J. Biol. Chem. 1997; 272: 27753-27757Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 15Huang C. Ma W.Y. Dong Z. Oncogene. 1997; 14: 1945-1954Crossref PubMed Scopus (36) Google Scholar, 16Dong Z. Huang C. Ma W.-y. Malewicz B. Baumann W.J. Kiss Z. Oncogene. 1998; 17: 1845-1853Crossref PubMed Scopus (20) Google Scholar). These "UV responses" activate several signal transduction pathways and transcription factors (14Huang C. Ma W.-y. Ding M. Bowden G.T. Dong Z. J. Biol. 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It was assumed that the epidermal growth factor receptor, but not protein kinase C (PKC), 1The abbreviations used are: PKC, protein kinase C; DNM, dominant negative mutant; EMEM, Eagle's minimal essential medium; Erk, extracellular signal-regulated protein kinase; FBS, fetal bovine serum; JNK, c-Jun NH2-terminal kinase; MAP, mitogen-activated protein1The abbreviations used are: PKC, protein kinase C; DNM, dominant negative mutant; EMEM, Eagle's minimal essential medium; Erk, extracellular signal-regulated protein kinase; FBS, fetal bovine serum; JNK, c-Jun NH2-terminal kinase; MAP, mitogen-activated protein plays a major role in the UV-induced response (23Sachsenmaier C. Radler-Pohl A. Zinck R. Nordheim A. Herrlich P. Rahmsdorf H.J. Cell. 1994; 78: 963-972Abstract Full Text PDF PubMed Scopus (405) Google Scholar). Very recently, we demonstrated that atypical PKC is involved in UV-induced AP-1 activation, whereas the epidermal growth factor receptor is not required for UV-induced signal transduction (15Huang C. Ma W.Y. Dong Z. Oncogene. 1997; 14: 1945-1954Crossref PubMed Scopus (36) Google Scholar, 24Huang C. Ma W.-y. Bowden G.T. Dong Z. J. Biol. Chem. 1996; 271: 31262-31268Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In the present study, we investigated whether other types of PKC isozymes of classical PKC or novel PKC are activated and/or involved in UVB-induced signal transduction and apoptosis. Eagle's minimum essential medium (EMEM) and fetal bovine serum (FBS) were from Whittaker Biosciences;l-glutamine was from Life Technologies, Inc.; gentamicin was from Quality Biological, Inc.; luciferase assay substrate was from Promega. Aprotinin and leupeptin were from Sigma; rottlerin, GF109203X, and safingol were from Calbiochem. The phosphospecific antibodies against phosphorylated sites of Erks, JNKs, and p38 kinase were from New England Biolabs; the antibodies against protein kinase C subtypes were from Santa Cruz. UVB irradiation was performed on serum-starved monolayer cultures utilizing a transluminator emitting UVB. Because the normal UVB lamp also generates a small amount of UVC light, the UVB irradiation was carried out in a UVB exposure chamber fitted with a Kodak Kodacel K6808 filter that eliminates all wavelengths below 290 nm (14Huang C. Ma W.-y. Ding M. Bowden G.T. Dong Z. J. Biol. Chem. 1997; 272: 27753-27757Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Mouse epidermal JB6 promotion sensitive Cl 41 and its dominant negative mutant cell lines for PKCα, PKCε, and PKCδ were grown at 37 °C in EMEM supplemented with 5% heat-inactivated FBS, 2 mml-glutamine, and 25 mg/ml gentamicin (25Huang C. Ma W.-y. Ryan C.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11957-11962Crossref PubMed Scopus (74) Google Scholar). Cl 41 PKCα-DNM and PKCε-DNM, the stable transfectants with dominant negative PKCα and PKCε, were established and reported previously (26Huang C. Ma W.-y. Dong Z. Cell. Signalling. 1998; 10: 185-190Crossref PubMed Scopus (21) Google Scholar). PKCδ mutants and vector plasmid PεMTH (26Huang C. Ma W.-y. Dong Z. Cell. Signalling. 1998; 10: 185-190Crossref PubMed Scopus (21) Google Scholar, 27Lehel C. Olah Z. Jakab G. Szallasi Z. Petrovics G. Harta G. Blumberg P.M. Anderson W.B. J. Biol. Chem. 1995; 270: 19651-19658Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 28Olah Z. Lehel C. Jakab G. Anderson W.B. Anal. Biochem. 1994; 221: 94-102Crossref PubMed Scopus (65) Google Scholar) were received from Dr. Peter Blumberg. JB6 promotion sensitive cells, Cl 41, were cultured in a 6-well plate until they reached 85–90% confluence. We used 0.3 μg of cytomegalovirus-neo vector and 2 μg of AP-1 luciferase plasmid with 6 μg of a dominant negative mutant of PKCδ plasmids or vector PεMTH plasmid DNA and 15 μl of LipofectAMINE reagent to transfect each well in the absence of serum. After 10–12 h, the medium was replaced with 5% FBS/MEM. Approximately 30–60 h after the beginning of the transfection, the cells were treated with 0.033% trypsin, and the cell suspensions were transferred to 75-ml culture flasks and cultured for 24–28 days with geneticin selection (300 μg/ml). Stable transfectants were screened by Western blotting with rabbit polyclonal IgG against PKCα, PKCε, and PKCδ. The stable transfectants and PKCδ-DNM were cultured in geneticin-free EMEM for at least two passages before each experiment (26Huang C. Ma W.-y. Dong Z. Cell. Signalling. 1998; 10: 185-190Crossref PubMed Scopus (21) Google Scholar). 2 × 105 cells were seeded in a 10-cm dish. After they reached 85–90% confluence, the cells were starved for 24–48 h in 0.1% FBS. After irradiation, the cells were washed one time with ice-cold phosphate-buffered saline (without Ca2+). Two hundred μl of homogenization buffer A (20 mm Tris-HCl, pH 8.0, 10 mm EGTA, 2 mm EDTA, 2 mm dithiothreitol, 1 mmphenylmethylsulfonyl fluoride, 25 μg/ml aprotinin, 10 μg/ml leupeptin) was added to each dish, and the cells were scraped into a 1.5-ml tube with a rubber policeman. The suspension was sonicated for 10 s at output 4 with a sonicator (Ultrasonics Inc., NY) and centrifuged at 100,000 × g for 1 h at 4 °C. The supernatant was collected as the cytosol fraction. The pellet was resuspended in 200 μl of homogenization buffer B (1% Triton X-100 in buffer A) and sonicated for 10 s. The suspension was centrifuged at 15,000 × g for 15 min at 4 °C. The supernatant was collected as a membrane fraction. Protein concentration of each sample was determined, and 100 μl of 3× Laemmli sample buffer (187.5 mm Tris-HCl, pH 6.8, 6% SDS, 30% glycerol, 150 mm dithiothreitol, 0.3% bromphenol blue) was added (29Olivier A.R. Parker P.J. J. Biol. Chem. 1994; 269: 2758-2763Abstract Full Text PDF PubMed Google Scholar). Samples containing equal amount of protein were loaded in each lane for 8% SDS-polyacrylamide gel electrophoresis. The gel was transferred and analyzed as described previously (30Dong Z. Huang C. Brown R.E. Ma W.Y. J. Biol. Chem. 1997; 272: 9962-9970Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Immunoblots for phosphorylated proteins of p38 kinases, Erks, and JNKs were carried out using phosphospecific mitogen-activated protein kinase antibodies against phosphorylated sites of p38, Erks and JNKs as described previously (30Dong Z. Huang C. Brown R.E. Ma W.Y. J. Biol. Chem. 1997; 272: 9962-9970Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Antibodies for phosphorylated MAP kinases were from New England Biolabs, and antibodies for PKC subtypes were from Santa Cruz. Antibody-bound proteins were detected by chemiluminescence (ECL of New England Biolabs or ECF of Amersham Pharmacia Biotech) and analyzed using the Storm 840 (Molecular Dynamics). Cells were grown in a 15 cm-dish and treated with various PKC inhibitors and UVB irradiation when cell density reached 50–70% confluence. Both detached and attached cells were harvested by scraping and centrifuging. Then the cells were lysed with lysis buffer (5 mm Tris-HCl, pH 8.0, 20 mmEDTA, 0.5% Triton X-100) on ice for 45 min. Fragmented DNA in the supernatant after centrifugation at 14,000 rpm (45 min at 4 °C) was extracted twice with phenol/chloroform/isopropanol (25:24:1, v/v) and once with chloroform and then precipitated with ethanol and 5m NaCl. The DNA pellet was washed once with 70% ethanol and resuspended in Tris-EDTA buffer (pH 8.0) with 100 μg/ml RNase at 37 °C for 2 h. The DNA fragments were separated by 1.8% agarose gel electrophoresis (31Sun Y. Bian J. Wang Y. Jacobs C. Oncogene. 1997; 14: 385-393Crossref PubMed Scopus (79) Google Scholar, 32Huang C.S. Ma W.-y. Li J.X. Hecht S.S. Dong Z. Cancer Res. 1998; 58: 4102-4106PubMed Google Scholar). JNKs, Erks, and p38 kinase activities were assayed as described previously (25Huang C. Ma W.-y. Ryan C.A. Dong Z. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11957-11962Crossref PubMed Scopus (74) Google Scholar). Translocation of PKC to a particulate fraction is the key step for the activation of this enzyme (33Newton A.C. Curr. Biol. 1995; 5: 973-976Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Determination of PKC content in membranes or the ratio of membranes to cytosol can reflect PKC activity. The following three PKCs can be dominantly detected in JB6 cells: PKCα, PKCε, and PKCδ (15Huang C. Ma W.Y. Dong Z. Oncogene. 1997; 14: 1945-1954Crossref PubMed Scopus (36) Google Scholar). To investigate whether these PKCs are involved in UVB-induced signal transduction, we analyzed the membrane/cytosol distribution of the three main subtypes of PKC in JB6 cells (15Huang C. Ma W.Y. Dong Z. Oncogene. 1997; 14: 1945-1954Crossref PubMed Scopus (36) Google Scholar). 12-O-tetradecanoylphorbol-13-acetate was used as a positive control for stimulating PKC translocation. Our results showed that UVB markedly induced the translocation of PKCε and PKCδ, but not PKCα, from cytosol to membrane. The UVB-induced PKCε and PKCδ translocation was dose-dependent (Fig.1) and reached a high level 5–15 min after UVB irradiation (Fig. 2).Figure 2The time course of PKC α, PKC ε, and PKC δ translocation by UVB. JB6 Cl 41 cells (2 × 105) were cultured in monolayer in 10-cm dishes until 90% confluent. Then the cells were starved for 48 h in 0.1% FBS/EMEM. The cells were harvested at 5, 15, and 30 min after UVB irradiation at 8 kJ/m2. The membrane and cytosol fractions were obtained as described under "Experimental Procedures" and were analyzed by 8% SDS-polyacrylamide gel electrophoresis and Western blotting. The same membrane was stripped and reprobed with the different isotype-specific antibodies. The immunoblots were visualized using the ECF detection reagents and scanned by Storm imager. The experiments were repeated three times and similar results were obtained. A, Western blotting. B, blots were scanned and quantified by Storm 840. Each value is the relative ratio of membrane to cytosol PKC.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The above results suggest that PKCε and PKCδ, but not PKCα, were involved in mediating UVB-induced signal transduction. To address this question, we used JB6 stable transfectants with the DNM of PKCε, PKCα, or PKCδ (26Huang C. Ma W.-y. Dong Z. Cell. Signalling. 1998; 10: 185-190Crossref PubMed Scopus (21) Google Scholar). These DNMs were constructed by site-directed mutagenesis of lysine residues in the ATP binding site (located in the catalytic domain) (26Huang C. Ma W.-y. Dong Z. Cell. Signalling. 1998; 10: 185-190Crossref PubMed Scopus (21) Google Scholar). The protein level of PKCδ in these transfectants was determined using rabbit polyclonal IgG against PKCδ. The results showed a high level of the introduced mutated protein of PKCδ in this transfectant (Fig. 3). Fig.4 showed that the DNM PKCε or PKCδ blocked UVB-induced translocation of PKCε and PKCδ, respectively. Further, we have analyzed the effect of these dominant negative PKC mutants on the UVB-induced activation of Erks, JNKs, and p38 kinases. We found that the total protein content of Erks, JNKs, and p38 kinases in three DNM PKC transfectants was lower than in Cl 41 cells, although the equal volume in each sample was loaded (Fig.5). Further, we demonstrated that the total protein in each sample was equal by dying the blotting membrane with Coomassie Blue (data not shown). However, UVB-induced phosphorylation of Erks and JNKs was strongly inhibited by DNM PKCε and PKCδ, whereas the DNM of PKCα was less effective on the UVB-induced phosphorylation of Erks and JNKs. The results suggest that the DNM of PKCε or PKCδ, but not the DNM of PKCα, inhibited UVB-induced activation of Erks and JNKs, but not p38 kinases.Figure 4Effect of DNM PKC δ and DNM PKC ε on UVB-induced PKC translocation. JB6 Cl 41 cells and Cl 41 stable transfectants with DNM PKCδ and DNM PKCε (2 × 105) were cultured in monolayer in 10-cm dishes until 90% confluent. Then the cells were starved for 48 h in 0.1% FBS/EMEM. The cells were harvested at 5, 15, and 30 min after UVB irradiation at 8 kJ/m2. The samples were fractionated as described under "Experimental Procedures." The samples were analyzed by 8% SDS-polyacrylamide gel electrophoresis and Western blotting. The same membrane was stripped and reprobed with the different isotype-specific antibodies. The immunoblots were visualized using the ECF detection reagents and scanned by Storm imager. Each value is the relative ratio of membrane to cytosol PKC. This is one of three similar experiments. A, relative ratio of membrane to cytosol PKCδ. B, relative ratio of membrane to cytosol PKCε.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Effect of DNM PKC α, PKC δ, and PKC ε on UVB-induced phosphorylation of Erks, JNKs, and p38 kinases. JB6 Cl 41 cells and Cl 41 stable transfectants with DNM PKCα, PKCδ, and PKCε (5 × 104/well) were cultured in monolayer in 6-well plates until 90% confluent. The cells were harvested at different times after UVB irradiation at 8 kJ/m2 as indicated. The samples were analyzed by Western blotting with antibodies for nonphosphorylated and phosphorylated Erk, JNK, and p38 proteins using a PhosphoPlus MAP kinase kit from New England Biolabs.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Further, we also compared protein phosphorylation and enzyme activity of MAP kinases after treatment with UVB. The results in Fig.6 showed that the phosphorylation of MAP kinases induced by UVB is well correlated with the activity of these kinases. It has been reported that some PKC inhibitors can selectively inhibit certain PKC isozymes without inhibiting other subtypes of PKC or other protein kinases (34Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (755) Google Scholar). The above results suggest that the role of PKC in mediating UVB stimulation is isozyme-specific. We therefore used several PKC inhibitors to investigate the role of these PKC subtypes on UVB-induced MAP kinase activity. GF109203X is an inhibitor mainly of classical PKC and novel PKC (35Toullec D. Pianetti P. Coste H. Bellevergue P. Grand-Perret T. Ajakane M. Baudet V. Boissin P. Boursier E. Loriolle F. Duhamel L. Charon D. Kirilovsky J. J. Biol. Chem. 1991; 266: 15771-15781Abstract Full Text PDF PubMed Google Scholar, 36Wilkinson S.E. Parker P.J. Nixon J.S. Biochem. J. 1993; 294: 335-337Crossref PubMed Scopus (490) Google Scholar); rottlerin is a selective inhibitor of PKCδ (34Gschwendt M. Muller H.J. Kielbassa K. Zang R. Kittstein W. Rincke G. Marks F. Biochem. Biophys. Res. Commun. 1994; 199: 93-98Crossref PubMed Scopus (755) Google Scholar), and safingol is only active for inhibition of PKCα (37Sachs C.W. Safa A.R. Harrison S.D. Fine R.L. J. Biol. Chem. 1995; 270: 26639-26648Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The results in Fig. 7 showed that rottlerin could markedly inhibit the UVB-induced phosphorylation of Erks and JNKs, but not p38 kinases. We also found that the protein levels of Erks and JNKs in rottlerin treatment groups were lower than in the control group, although the total p38 protein level was not affected. Safingol, the selective inhibitor for PKCα, did not inhibit UVB-induced activation of Erks, JNKs, or p38 kinases. At a low concentration of GF109203X ( 20 μm) inhibited activation of all three MAP kinases (Fig. 7). Because the affinity of GF109203X on classical PKC is stronger than on novel PKC and it inhibits PKCε and PKCδ at higher concentrations, the results obtained using this inhibitor provide additional evidence that PKCε and PKCδ, but not PKCα, are involved in UVB-induced signal transduction. Based on these results, together with the effect of DNM of PKC, we conclude that the activation of PKCδ and PKCε, but not PKCα, plays an important role in mediating UVB-induced phosphorylation of Erks, JNKs, and p38 kinases. UV radiation is a strong inducer of cell apoptosis. We have found that UVB-induced apoptosis in JB6 cells is dose-dependent (data not shown). To further assess the biological significance of PKC in mediating the UVB-induced signal transduction, we investigated the role of PKC in UVB-induced apoptosis in JB6 cells. The results in Fig.8 show that UVB-induced apoptosis of JB6 cells was markedly inhibited by rottlerin and GF109203X, whereas safingol had little inhibitory effect. These results suggest that the regulation by PKC of UVB-induced signal transduction may mediate UVB-induced apoptosis. Because rottlerin inhibits UVB-induced Erks and JNKs, but not p38 kinases, and inhibits UVB-induced apoptosis, we hypothesize that Erks and JNKs, but not p38 kinases, play a role in mediating UVB-induced apoptosis. We therefore used PD98059, a selective inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase that is a specific upstream activator of Erks as well as dominant negative mutant Erk2, to investigate the role of Erks in UVB-induced apoptosis. We also used dominant negative mutant JNK1 and a selective inhibitor of p38 kinase, SB202190, to study the role of JNK1 and p38 kinases in UVB-induced apoptosis, respectively (Fig.9). The results showed that inhibiting Erk activation by PD98059 and DNM-Erks inhibited UVB-induced apoptosis. Further, cells expressing the dominant negative mutant of JNK1 also blocked UVB-induced apoptosis, whereas SB202190 was not inhibitory. The concentration of SB202190 applied in the apoptosis assay has been shown to inhibit the UV-induced phosphorylation of p38 kinases in JB6 cells. 2N. Chen, W.-y. Ma, C. Huang, and Z. Dong, manuscript submitted. These results suggest that Erk2 and JNK1, but not p38 kinases, mediate UVB-induced apoptosis. In the present work, we found that UVB irradiation induced PKCδ and PKCε, but not PKCα, translocation from cytosol to membrane and that this translocation was partially blocked in the cells that express DNM PKCδ or DNM PKCε. In cells transfected with DNM PKCε and PKCδ, UVB-induced phosphorylation of JNKs and Erks was markedly attenuated. A selective inhibitor of PKCδ completely blocked UVB-induced phosphorylation of JNKs and Erks, but not p38 kinases. However, the PKCα-specific inhibitor had no effect on the UVB-induced phosphorylation of MAP kinases (Erks, JNKs, and p38 kinases). These findings indicate that the UVB-induced phosphorylation of Erks and JNKs requires PKCδ and PKCε activation. PKC belongs to a large kinase family consisting of at least 11 members, which are divided into three groups on the basis of their biochemical properties and sequence homologies (38Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1345) Google Scholar). The different PKC isotypes may have specific roles in signal transduction (39Schonwasser D.C. Marais R.M. Marshall C.J. Parker P.J. Mol. Cell. Biol. 1998; 18: 790-798Crossref PubMed Scopus (670) Google Scholar). It has been reported that PKC may be involved in UVA-induced signal transduction (40Petersen M. Hamilton T. Li H.L. Photochem. Photobiol. 1995; 62: 444-448Crossref PubMed Scopus (28) Google Scholar), but not UVC-induced signal transduction (23Sachsenmaier C. Radler-Pohl A. Zinck R. Nordheim A. Herrlich P. Rahmsdorf H.J. 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Commun. 1994; 199: 93-98Crossref PubMed Scopus (755) Google Scholar), UVB-induced phosphorylation of Erks and JNKs, but not p38 kinases, was markedly blocked. GF109203X, a potent inhibitor for PKCα and PKCε, produced an inhibitory effect on UVB-induced Erks or JNKs at a high concentration (20 μm). Safingol, a specific inhibitor of PKCα (37Sachs C.W. Safa A.R. Harrison S.D. Fine R.L. J. Biol. Chem. 1995; 270: 26639-26648Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), did not display any inhibitory effect on these three MAP kinases. Cells transfected with the dominant negative mutants of PKCε or PKCδ inhibited the stimulation of Erks, JNKs, and p38 kinases induced by UVB. These results indicate that activated PKCε and PKCδ are necessary in mediating UVB-induced signal transduction. Activation of PKC is associated with the translocation of enzymes from the cytosol to the cell particulate fraction (41Kraft A.S. Anderson W.B. Cooper H.L. Sando J.J. J. Biol. Chem. 1982; 257: 13193-13196Abstract Full Text PDF PubMed Google Scholar). UVB stimulation of membrane-associated PKC could result from several possible mechanisms. The translocation of PKC is triggered by diacylglycerol or 12-O-tetradecanoylphorbol-13-acetate interacting with the C1 domain of the PKC protein (33Newton A.C. Curr. Biol. 1995; 5: 973-976Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). It was reported that UVB induces phospholipase A2 activation and arachidonic acid release (42Blackshear P.J. Nairn A.C. Kuo J.F. FASEB J. 1988; 2: 2957-2969Crossref PubMed Scopus (159) Google Scholar), a reaction that also produces lysophospholipid. It has been found that lysophosphatidylcholine and arachidonic acid are also activators for PKC (42Blackshear P.J. Nairn A.C. Kuo J.F. FASEB J. 1988; 2: 2957-2969Crossref PubMed Scopus (159) Google Scholar). Punnonen and Yuspa (43Punnonen K. Yuspa S.H. J. Invest. Dermatol. 1992; 99: 221-226Crossref PubMed Scopus (44) Google Scholar) reported that UVB irradiation of cultured cells increases levels of diacylglycerol. On the other hand, UV energy generates oxygen radicals such as H2O2, which may activate PKC (44Cunningham M.L. Krinsky N.I. Giovanazzi S.M. Peak M.J. J. Free Radic. Biol. Med. 1985; 1: 381-385Crossref PubMed Scopus (101) Google Scholar, 45Hayes G.R. Lockwood D.H. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8115-8119Crossref PubMed Scopus (146) Google Scholar, 46Chan T.M. Chen E. Tatoyan A. Shargill N.S. Pleta M. Hochstein P. Biochem. Biophys. Res. Commun. 1986; 139: 439-445Crossref PubMed Scopus (46) Google Scholar). Indeed, it has been reported that reactive oxygen species directly activate purified PKC in vitro (47Gopalakrishna R. Anderson W.B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6758-6762Crossref PubMed Scopus (368) Google Scholar). It is interesting that DNM PKCs decrease the total protein level of MAP kinases. We also found that rottlerin inhibits the total protein levels of Erks and JNKs, but not p38 kinases. The mechanisms of the inhibitory effect on MAP kinase protein level are currently under investigation in our laboratory. Considerable attention has recently been focused on the role played by different kinase cascades in the control of apoptosis. In the present report, we found that both rottlerin and GF109203X inhibited UVB-induced apoptosis. However, rottlerin blocked UV-induced Erks and JNKs, but not p38, whereas GF109203X can block all three MAP kinases. MAP kinase signaling cascades are involved in the many cellular responses, including apoptosis, to extracellular stimuli. For example, Jimenez et al. (48Jimenez L.A. Zanella C. Fung H. Janssen Y.M. Vacek P. Charland C. Goldberg J. Mossman B.T. Am. J. Physiol. 1997; 273: L1029-L1035PubMed Google Scholar) reported that the mitogen-activated protein kinase/extracellular signal-regulated kinase 1 inhibitor PD98059 blocked asbestos-induced apoptosis in rat pleural mesothelial cells. Activation of JNKs plays a causal role in the induction of apoptosis in numerous cells when stimulated by some stresses, whereas the inhibition of the Erks pathway has been observed in a number of cell systems undergoing programmed cell death (49Berra E. Diaz-Meco M.T. Moscat J. J. Biol. Chem. 1998; 273: 10792-10797Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). In the present study, we found that the inhibition of Erks or JNKs, but not p38 kinases, blocks UVB-induced apoptosis. This suggests that activation of PKCδ and PKCε and phosphorylation of Erks and JNKs, but not p38 kinases, are important in mediating UVB-induced apoptosis. In summary, UVB induces activation of PKCδ, PKCε, and MAP kinases in JB6 cells. Inhibition of PKCδ and PKCε blocks UVB-induced MAP kinases and apoptosis. We conclude that UVB-induced apoptosis appears to be mediated by PKCδ, PKCε, Erks, and JNKs. We thank Dr. Harald H. O. Schmid and Patricia C. Schmid for critical reading.