The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation
2008; Elsevier BV; Volume: 283; Issue: 47 Linguagem: Inglês
10.1074/jbc.m803963200
ISSN1083-351X
AutoresQun Zhao, Bassant M. Barakat, Song Qin, Alo Ray, Mohamed A. El‐Mahdy, Gulzar Wani, El‐Shaimaa A. Arafa, Safita N. Mir, Qi-En Wang, Altaf A. Wani,
Tópico(s)Genomics and Chromatin Dynamics
ResumoThe p38 MAPK is a family of serine/threonine protein kinases that play important roles in cellular responses to external stress signals, e.g. UV irradiation. To assess the role of p38 MAPK pathway in nucleotide excision repair (NER), the most versatile DNA repair pathway, we determined the efficiency of NER in cells treated with p38 MAPK inhibitor SB203580 and found that p38 MAPK is required for the prompt repair of UV-induced DNA damage CPD. We further investigated the possible mechanism through which p38 MAPK regulates NER and found that p38 MAPK mediates UV-induced histone H3 acetylation and chromatin relaxation. Moreover, p38 MAPK also regulates UV-induced DDB2 ubiquitylation and degradation via phosphorylation of the target protein. Finally, our results showed that p38 MAPK is required for the recruitment of NER factors XPC and TFIIH to UV-induced DNA damage sites. We conclude that p38 MAPK regulates chromatin remodeling as well as DDB2 degradation for facilitating NER factor assembly. The p38 MAPK is a family of serine/threonine protein kinases that play important roles in cellular responses to external stress signals, e.g. UV irradiation. To assess the role of p38 MAPK pathway in nucleotide excision repair (NER), the most versatile DNA repair pathway, we determined the efficiency of NER in cells treated with p38 MAPK inhibitor SB203580 and found that p38 MAPK is required for the prompt repair of UV-induced DNA damage CPD. We further investigated the possible mechanism through which p38 MAPK regulates NER and found that p38 MAPK mediates UV-induced histone H3 acetylation and chromatin relaxation. Moreover, p38 MAPK also regulates UV-induced DDB2 ubiquitylation and degradation via phosphorylation of the target protein. Finally, our results showed that p38 MAPK is required for the recruitment of NER factors XPC and TFIIH to UV-induced DNA damage sites. We conclude that p38 MAPK regulates chromatin remodeling as well as DDB2 degradation for facilitating NER factor assembly. Living organisms are incessantly exposed to a variety of DNA-damaging agents with potentially devastating consequences. To overcome the deleterious effects of such exposures, cells respond with a variety of defensive strategies to eliminate damage and maintain the integrity of their genome. Among them, nucleotide excision repair (NER) 3The abbreviations used are: NER, nucleotide excision repair; MAPK, mitogen-activated protein kinase; CPD, cyclobutane pyrimidine dimer; 6-4PP, pyrimidine-pyrimidone (6-4) photoproduct; DDB, damaged DNA-binding protein; GG-NER, global genomic NER; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal kinase; DMSO, dimethyl sulfoxide; MNase, micrococcal nuclease; ISB, immuno-slot blot. is the most important repair system that removes UV-induced photolesions, including cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone photoproducts (6-4PP), as well as other bulky DNA adducts caused by different chemical carcinogens (1De Laat W.L. Jaspers N.G. Hoeijmakers J.H. Genes Dev. 1999; 13: 768-785Crossref PubMed Scopus (922) Google Scholar). There are two subpathways of NER: global genome NER (GG-NER), which removes lesions from the entire genome, and transcription-coupled NER, which eliminates DNA damage from the transcribed strand of the actively transcribed genes (1De Laat W.L. Jaspers N.G. Hoeijmakers J.H. Genes Dev. 1999; 13: 768-785Crossref PubMed Scopus (922) Google Scholar, 2Hanawalt P.C. Oncogene. 2002; 21: 8949-8956Crossref PubMed Scopus (357) Google Scholar). The two pathways differ mainly at the initial damage recognition steps. For transcription-coupled NER, the lesion is detected by stalled RNA polymerase during transcription (3Donahue B.A. Yin S. Taylor J.-S. Reines D. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8502-8506Crossref PubMed Scopus (310) Google Scholar, 4Hanawalt P. Mellon I. Curr. 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Repair of both CPD and 6-4PP is affected by their location and accessibility because their repair is faster in the nucleosome-free and linker regions than in the nucleosome core (30Smerdon M.J. Thoma F. Cell. 1990; 61: 675-684Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 31Wellinger R.E. Thoma F. EMBO J. 1997; 16: 5046-5056Crossref PubMed Scopus (127) Google Scholar). Earlier studies have also revealed a relationship of NER to UV-induced chromatin accessibility occurring through histone acetylation (32Ramanathan B. Smerdon M.J. Carcinogenesis. 1986; 7: 1087-1094Crossref PubMed Scopus (75) Google Scholar, 33Ramanathan B. Smerdon M.J. J. Biol. Chem. 1989; 264: 11026-11034Abstract Full Text PDF PubMed Google Scholar, 34Smerdon M.J. Lan S.Y. Calza R.E. Reeves R. J. Biol. Chem. 1982; 257: 13441-13447Abstract Full Text PDF PubMed Google Scholar). It is reported that histone acetylation increases the chromatin accessibility and facilitates NER in mammalian cells. Furthermore, DNA repair synthesis is enhanced in hyperacetylated nucleosome (33Ramanathan B. Smerdon M.J. J. Biol. Chem. 1989; 264: 11026-11034Abstract Full Text PDF PubMed Google Scholar, 34Smerdon M.J. Lan S.Y. Calza R.E. Reeves R. J. Biol. Chem. 1982; 257: 13441-13447Abstract Full Text PDF PubMed Google Scholar). Interestingly, the tumor suppressor protein p53, which enhances GG-NER, is also involved in UV-induced histone H3 and H4 acetylation and chromatin relaxation (35Rubbi C.P. Milner J. EMBO J. 2003; 22: 975-986Crossref PubMed Scopus (203) Google Scholar). Another family of tumor suppressors, ING1 and ING2, are reported to enhance NER through histone acetylation and increased chromatin accessibility in a p53-dependent manner (36Cheung Jr., K.J. Mitchell D. Lin P. Li G. Cancer Res. 2001; 61: 4974-4977PubMed Google Scholar, 37Wang J. Chin M.Y. Li G. Cancer Res. 2006; 66: 1906-1911Crossref PubMed Scopus (64) Google Scholar, 38Kuo W.H. Wang Y. Wong R.P. Campos E.I. Li G. Exp. Cell Res. 2007; 313: 1628-1638Crossref PubMed Scopus (36) Google Scholar). All of these studies suggest that chromatin structure modification is an important parameter in DNA lesion accessibility and consequently efficient NER. The p38 MAPK, a serine/threonine kinase, is known to play important roles in cellular responses to various external stress signals. It has been shown that p38 MAPK is an important mediator of phosphorylation of histone H3 at serine 10 in response to UV (39Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). This phosphorylation is involved in the alteration of chromatin condensation during transcription activation and cell division (40Cheung P. Allis C.D. Sassone-Corsi P. Cell. 2000; 103: 263-271Abstract Full Text Full Text PDF PubMed Scopus (824) Google Scholar). Furthermore, p38 MAPK has been found to mediate H3 phosphorylation at serine 10 induced by cisplatin, which binds to DNA to form a covalent platinum-DNA adduct that is removed by NER (41Wang D. Lippard S.J. J. Biol. Chem. 2004; 279: 20622-20625Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Thus far, no report has attributed the UV-induced acetylation and global chromatin relaxation to the action of p38 MAPK, and it is still not known whether p38 MAPK plays any role in facilitating NER in response to UV. In the present report, we have studied the function of p38 MAPK in NER in cells exposed to UV irradiation. Our studies indicate that p38 MAPK is involved in UV-induced histone H3 acetylation at lysine 14 and chromatin relaxation. Our results also show that p38 MAPK, by regulating DDB2 degradation and consequently the recruitment of XPC and TFIIH, plays a pivotal role in the removal of UV lesion CPD through NER. Cell Lines and Treatments—Normal human fibroblasts (OSU-2) were established in our laboratory as described previously (42Venkatachalam S. Denissenko M. Wani A.A. Oncogene. 1997; 14: 801-809Crossref PubMed Scopus (48) Google Scholar). HeLa cells stably transfected with NH2-terminal FLAG-hemagglutinin-tagged DDB2 (HeLa-DDB2) were a gift from Dr. Yoshihiro Nakatani (Dana-Farber Cancer Institute, Boston, MA). These cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO2. To study the function of p38 MAPK, the cells were pretreated with p38 MAPK inhibitor SB203580 (Calbiochem, San Diego, CA) or DMSO for 30 min before UV irradiation. To measure ubiquitylation of DDB2, HeLa-DDB2 cells were cultured in medium supplemented with MG132 (Calbiochem, San Diego, CA) 30 min before UV treatment. For UV irradiation, the cells were washed twice with phosphate-buffered saline, irradiated at varying UV doses, and incubated in appropriate medium for the desired time period. The irradiation was done with a germicidal lamp at a dose rate of 0.8 J/m2/s as measured by a Kettering model 65 radiometer (Cole Palmer Instrument, Co., Vernon Hill, IL). In Vivo Ubiquitylation and Phosphorylation Detection—DDB2 ubiquitylation and phosphorylation were detected as described before (43Li J. Wang Q.E. Zhu Q. El-Mahdy M.A. Wani G. Praetorius-Ibba M. Wani A.A. Cancer Res. 2006; 66: 8590-8597Crossref PubMed Scopus (85) Google Scholar). Briefly, HeLa-DDB2 cells were treated with MG132 (10 μm) 30 min before UV irradiation at 40 J/m2 and kept in the same medium for indicated time periods. Whole cell lysates were prepared by boiling in SDS lysis buffer (2% SDS, 10% glycerol, 10 mm dithiothreitol, 62 mm Tris-HCl, pH 6.8, and protease inhibitor mixture) for 10 min. After centrifugation, the supernatant was diluted with radioimmune precipitation assay buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitor mixture) and subjected to immunoprecipitation with anti-FLAG M2 affinity gel (Sigma). The immunoprecipitates were then subjected to Western blotting and detected with anti-ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-serine, anti-phospho-tyrosine (Cell Signaling Technology, Danvers, MA), and anti-DDB2 antibodies. Western Blot Analysis of Proteins—The proteins were quantified and separated by SDS-PAGE, and the immunoblot analysis was done by using chemiluminescent detection. Phosphorylated MK2, phospho-Akt (Ser473), and phospho-H3 (Ser10) were detected by using rabbit polyclonal antibodies purchased from Cell Signaling Technology (Danvers, MA). Phospho-ERK (Santa Cruz Biotechnology) and phospho-JNK (Promega) antibodies were used to detect the activated ERK and JNK, respectively. Acetyl-H3 (K14), acetyl-H3 (K9, 14), acetyl-H3 (K18), and acetyl-H4 were detected using rabbit polyclonal antibodies purchased from Upstate Biotechnology (Billerica, MA). Rabbit anti-DDB2 antibody was generated in our lab as described before (44Wang Q.E. Zhu Q. Wani G. El-Mahdy M.A. Li J. Wani A.A. Nucleic Acids Res. 2005; 33: 4023-4034Crossref PubMed Scopus (134) Google Scholar). To serve as a loading control, total lamin B or actin levels in the cell lysates were detected using anti-lamin B or anti-actin antibody (Santa Cruz Biotechnology). Micrococcal Nuclease (MNase) Digestion Assay—The cells were trypsinized and resuspended in hypotonic cell lysis buffer (10 mm Tris-HCl, pH 8.0, 10 mm MgCl2, 1 mm dithiothreitol, 25% glycerol, 0.2% Nonidet P-40, 0.5 μm spermidine, 0.15 μm spermine, and protease inhibitor mixture). The cells were incubated on ice for 10 min followed by pipetting 10 times to release nuclei and low speed centrifugation. The nuclei were resuspended in 200 μl of MNase buffer (10 mm Tris-HCl, pH 8.0, 50 mm NaCl, 300 mm sucrose, 3 mm MgCl2, and 1 mm CaCl2) and digested with a concentration range of MNase (Sigma) for 5 min at room temperature. The reaction was stopped by adding 5× stop solution (0.1 m EDTA, 0.01 m EGTA, pH 8.0). DNA was extracted with phenol/chloroform, and 2 μg of DNA was separated on a 2% agarose gel. Quantification of CPD and 6-4PP by Immuno-Slot Blot (ISB) Analysis—A noncompetitive ISB assay, described earlier (45Wani A.A. D'Ambrosio S.M. Alvi N.K. Photochem. Photobiol. 1987; 46: 477-482Crossref PubMed Scopus (67) Google Scholar), was used to quantify the initial formation of UV-induced CPD or 6-4PP and those remaining in cells after desired time of repair. Briefly, after different treatments, the cells were lysed immediately for DNA isolation. The same amount of DNA was loaded on nitrocellulose membranes, and the amounts of CPD or 6-4PP were detected with monoclonal anti-CPD TDM-2 or anti-6-4PP 64 M-2 antibody (MBL International Corporation, Woburn, MA). Fractionation of Cellular Proteins—OSU-2 cells were pretreated with either SB203580 or DMSO for 30 min before UV irradiation at 20 J/m2. After further incubation for 30 min, the cells were harvested and subjected to cellular protein fractionation procedure as described earlier (46Wang Q.E. Zhu Q. Wani G. Chen J. Wani A.A. Carcinogenesis. 2004; 25: 1033-1043Crossref PubMed Scopus (93) Google Scholar). Briefly, cytoplasm and nuclei were first separated by suspending cells in hypotonic buffer (10 mm HEPES, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, and protease inhibitor mixture) and treated with 0.1% Triton X-100. After centrifugation, the supernatant was saved as S, and the nuclear pellet was then treated with a higher concentration of Triton X-100 (1%) in LS buffer (10 mm Tris-HCl, pH 7.4, 0.2 mm MgCl2, and protease inhibitor mixture) to remove the nuclear envelope and recover the fraction of nucleoplasmic soluble proteins, designated as TW. The nuclear pellet was further extracted consecutively with increasing concentrations (0.3, 0.5, and 2.0 m) of NaCl in LS buffer to result in supernatant fractions, designated as 0.3, 0.5, and 2.0, respectively. Each protein fraction, corresponding to an equivalent cell number, was loaded on SDS-PAGE and analyzed by immunoblotting with anti-DDB2 antibody. Localized Micropore UV Irradiation and Immunofluorescent Staining—OSU-2 cells growing on glass coverslips were pretreated with either SB203580 or DMSO for 30 min, and the cells were then washed with phosphate-buffered saline and UV-irradiated through a 5-μm isopore polycarbonate filter (Millipore, Bedford, MA) as described previously (47Wang Q.E. Zhu Q. Wani M.A. Wani G. Chen J. Wani A.A. DNA Repair. 2003; 2: 483-499Crossref PubMed Scopus (83) Google Scholar). The cells were then double stained with rabbit anti-XPC and mouse anti-CPD (TDM-2, MBL International Corporation), or rabbit anti-XPB and mouse anti-CPD, or rabbit-anti-phospho-p38 and mouse anti-CPD antibodies. Fluorescein isothiocyanate and Texas Red conjugates of anti-rabbit and anti-mouse IgG were simultaneously used for the detection of primary antibody binding. Fluorescence images were obtained with a Nikon fluorescence microscope E80i (Nikon, Tokyo, Japan) fitted with appropriate filters for fluorescein isothiocyanate and Texas Red. The digital images were then captured with a cooled CCD camera and processed with the help of its SPOT software (Diagnostic Instruments, Sterling Heights, MI). p38 MAPK Is Required for Optimal Repair of UV-induced CPD—To study the role of p38 MAPK in NER, we inhibited p38 MAPK activity with its specific pharmacological inhibitor, SB203580. This pyridinyl imidazole inhibitor (structure shown in Fig. 1A) is now extensively used to understand the role of p38 MAPK. It has been shown to selectively inhibit p38 MAPK activity without significantly affecting the activity of other MAP kinases, e.g. JNK and ERK (48Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3945) Google Scholar). To determine the inhibition of p38 MAPK activity by SB203580, OSU-2 cells or HeLa-DDB2 cells were pretreated with SB203580 at different concentrations followed by UV irradiation at 20 J/m2. The cell lysates were subjected to Western blot analysis using phospho-MK2-specific antibody. MK2, often referred to as MAPK-activated protein kinase-2, is a physiological target of p38 MAPK (49Rouse J. Cohen P. Trigon S. Morange M. onso-Llamazares A. Zamanillo D. Hunt T. Nebreda A.R. Cell. 1994; 78: 1027-1037Abstract Full Text PDF PubMed Scopus (1503) Google Scholar). Therefore, the phosphorylation of MK2 indicates the activation of p38 MAPK. We found that the phospho-MK2 level was markedly elevated after UV irradiation in control DMSO-treated cells. In contrast, cells treated with 10 μm SB203580 exhibited a dramatic reduction of UV-induced MK2 activation in both OSU-2 (Fig. 1B) and HeLa-DDB2 (Fig. 1C) cell lines. Importantly, however, drug concentrations below 5 μm exhibited a markedly reduced inhibitory effect on p38 MAPK activity (Fig. 1, B and C). Thus, in this study we used 10 μm SB203580 to effectively suppress UV-induced p38 MAPK activity in both OSU and HeLa-DDB2 cells. At this concentration, we did not observe an evident inhibition of ERK and JNK activities in either OSU-2 (Fig. 1E) or HeLa-DDB2 cells (Fig. 1F). It should be noted that higher concentrations (>1 μm) of SB203580 are reported to also inhibit the interleukin-2-stimulated phosphorylation of Akt in T cells independent of p38 MAPK (50Lali F.V. Hunt A.E. Turner S.J. Foxwell B.M. J. Biol. Chem. 2000; 275: 7395-7402Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). We tested for this in our system and found that in OSU-2 cells UV irradiation at 20 J/m2 failed to exhibit any elevation of phosphorylated Akt (Ser473) within 24 h after UV irradiation (Fig. 1D). Moreover, within a short period of time (30 min) after UV treatment, an even higher dose of UV (100 or 200 J/m2) did not induce any activation of Akt (Fig. 1E). Similarly, in HeLa-DDB2 cells, we did not observe the activation of Akt after UV irradiation at 20 or 40 J/m2 (Fig. 1F). More importantly, 10 μm concentration of SB203580 did not have any inhibitory effect on the basal level of phospho-Akt in both OSU-2 and HeLa-DDB2 cells (Fig. 1, D–F). Therefore, Akt activity is not likely a candidate for the effects observed upon 10 μm SB203580 treatment of cell lines in this study. Next, we tested the effect of modulating p38 MAPK activity on the efficiency of NER. OSU-2 cells were treated with SB203580 or DMSO for 30 min and then UV-irradiated at a dose of 10 J/m2. The cells were allowed to repair for indicated time periods and analyzed for the activity of p38 MAPK and the removal of UV-induced CPD using ISB analysis with anti-CPD antibody. As shown in Fig. 2A, p38 MAPK activity was successfully inhibited by SB203580 at each time point. When p38 MAPK was inhibited by SB203580, efficiency of NER was severely compromised as reflected by a decreased removal rate of UV-induced CPD (Fig. 2B). In fact, 8 h after UV treatment, only 55% of CPD remained in control cells in comparison with 87% CPD remaining in cells with suppressed p38 MAPK activity. Similarly, 41% CPD was left in control cells after 24 h of repair in contrast to 70% CPD in cells with curtailed p38 MAPK activity. Notably, however, the repair of another UV-induced DNA lesion, 6-4PP, was not affected by inactivation of p38 MAPK (Fig. 2C). These results indicate that p38 MAPK is required for prompt repair of UV-induced CPD. p38 MAPK Affects UV-induced Chromatin Relaxation and Histone Acetylation—Upon UV irradiation of cells, p38 MAPK has been reported to phosphorylate histone H3 at serine 10 (39Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), indicating its potential role in modulating chromatin remodeling in response to UV treatment. Because repair of DNA damage occurs within highly condensed chromatin fibers, the relaxation of chromatin structure is necessary for the accessibility of damaged DNA to repair proteins (29Ura K. Araki M. Saeki H. Masutani C. Ito T. Iwai S. Mizukoshi T. Kaneda Y. Hanaoka F. EMBO J. 2001; 20: 2004-2014Crossref PubMed Scopus (155) Google Scholar, 51Wang Z.G. Wu X.H. Friedberg E.C. J. Biol. Chem. 1997; 272: 24064-24071Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Thus, it is reasonable to assume that increased efficiency of NER caused by p38 MAPK function results from enhanced chromatin relaxation. To ascertain this possibility, OSU-2 cells were grown in serum-free medium for 48 h before UV treatment to arrest cells in the G0/G1 phase. Flow cytometric analysis revealed that following serum starvation 89.4% of the cells were arrested in G0/G1 phase (Fig. 3A, upper panel) compared with 47.6% of cells grown in complete medium (Fig. 3A, lower panel). The serum-starved growth-arrested cells were pretreated with SB203580 to inhibit p38 MAPK activity or treated with DMSO as control and then UV-irradiated at 200 J/m2. After a further 30 min of incubation, the cell nuclei were gently isolated and subjected to digestion with appropriately titrated concentrations of MNase. Among the three different concentrations tested, lower and higher MNase concentrations were unable to clearly distinguish the effect of the inhibitor on nuclease digestibility (Fig. 3B). In contrast, intermediate MNase concentration (0.5 units/ml) provided easily discernable digestion patterns between unirradiated versus irradiated as well as in absence and presence of the inhibitor. Chromatin relaxation at 30 min after UV irradiation, observed in control cells, was reflected by dramatically increased DNA sensitivity to MNase. However, when p38 MAPK activity was suppressed, UV-mediated increased DNA sensitivity to MNase was dramatically reversed. (Fig. 3B). This result suggests that UV-induced chromatin relaxation is intimately modulated by the activation of p38 MAPK. Histone acetylation is believed to play an important role in chromatin remodeling during the NER process (32Ramanathan B. Smerdon M.J. Carcinogenesis. 1986; 7: 1087-1094Crossref PubMed Scopus (75) Google Scholar, 33Ramanathan B. Smerdon M.J. J. Biol. Chem. 1989; 264: 11026-11034Abstract Full Text PDF PubMed Google Scholar, 34Smerdon M.J. Lan S.Y. Calza R.E. Reeves R. J. Biol. Chem. 1982; 257: 13441-13447Abstract Full Text PDF PubMed Google Scholar). To assess whether the effect of p38 MAPK on chromatin relaxation is invoked through histone acetylation, growth-arrested OSU-2 cells were pretreated with SB203580 and then analyzed for the status of histone H3 acetylation immediately after UV irradiation. Fig. 3C shows the H3 acetylation at lysines 9, 14, and 18 by the use of specific anti-AcH3 antibodies. Histone H3 acetylation between p38 MAPK-active and p38 MAPK-inactive cells was essentially unaltered for lysines 9 and 18. On the other hand, acetylation at H3 lysine 14 showed a prompt increase within 10 min of UV treatment and subsequent return to basal levels within 30 min. However, upon p38 MAPK inhibition, histone H3 lysine 14 acetylation levels were negligible under both physiological conditions and after UV treatment. It is clear that specific histone acetylation occurring at specific lysine residues after UV irradiation is impaired in the absence of p38 MAPK, which could be instrumental in decreased chromatin relaxation and NER. We further tested the effect of SB203580 on UV-induced phosphorylation of H3 at serine 10 in growth-arrested OSU-2 cells. UV induced a dramatic increase of phospho-H3 (Ser10) in DMSO-treated cells, whereas in cells deprived of p38 MAPK activity, phospho-H3 (Ser10) level remained the same as nonirradiated cells (Fig. 3D). p38 MAPK Participates in UV-induced DDB2 Degradation—p38 MAPK is known to regulate degradation of many cellular proteins through their phosphorylation (52Poizat C. Puri P.L. Bai Y. Kedes L. Mol. Cell. Biol. 2005; 25: 2673-2687Crossref PubMed Scopus (98) Google Scholar, 53Li J.P. Yang J.L. J. Cell. Physiol. 2007; 212: 481-488Crossref PubMed Scopus (10) Google Scholar, 54Kida A. Kakihana K. Kotani S. Kurosu T. Miura O. 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