MAP Kinases Mediate UVB-induced Phosphorylation of Histone H3 at Serine 28
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m010931200
ISSN1083-351X
AutoresShuping Zhong, Yiguo Zhang, Cheryl Jansen, Hidemasa Goto, Masaki Inagaki, Zigang Dong,
Tópico(s)Cancer-related Molecular Pathways
ResumoHistone H3 phosphorylation is related closely to chromatin remodeling and chromosome condensation. H3 phosphorylation at serine 28 is coupled with mitotic chromosome condensation in diverse mammalian cell lines. However, the pathway that mediates phosphorylation of H3 at serine 28 is unknown. In the present study, ERK1, ERK2, or p38 kinase strongly phosphorylated H3 at serine 28in vitro. JNK1 or JNK2 was able also to phosphorylate H3 at serine 28 in vitro but to a lesser degree. UVB irradiation markedly induced phosphorylation of H3 at serine 28 in JB6 Cl 41 cells. PD 98059, a MEK1 inhibitor, and SB 202190, a p38 kinase inhibitor, efficiently repressed UVB-induced H3 phosphorylation at serine 28. Expression of dominant negative mutant (DNM) ERK2 in JB6 Cl 41 cells totally blocked UVB-induced phosphorylation of H3 at serine 28. Additionally, DNM p38 kinase or DNM JNK1 partially blocked UVB-induced H3 phosphorylation at serine 28. Furthermore, UVB-induced H3 phosphorylation at serine 28 was inhibited inJnk1 −/− cells but not inJnk2 −/− cells. These results suggest that UVB-induced H3 phosphorylation at serine 28 may be mediated by mitogen-activated protein kinases. Histone H3 phosphorylation is related closely to chromatin remodeling and chromosome condensation. H3 phosphorylation at serine 28 is coupled with mitotic chromosome condensation in diverse mammalian cell lines. However, the pathway that mediates phosphorylation of H3 at serine 28 is unknown. In the present study, ERK1, ERK2, or p38 kinase strongly phosphorylated H3 at serine 28in vitro. JNK1 or JNK2 was able also to phosphorylate H3 at serine 28 in vitro but to a lesser degree. UVB irradiation markedly induced phosphorylation of H3 at serine 28 in JB6 Cl 41 cells. PD 98059, a MEK1 inhibitor, and SB 202190, a p38 kinase inhibitor, efficiently repressed UVB-induced H3 phosphorylation at serine 28. Expression of dominant negative mutant (DNM) ERK2 in JB6 Cl 41 cells totally blocked UVB-induced phosphorylation of H3 at serine 28. Additionally, DNM p38 kinase or DNM JNK1 partially blocked UVB-induced H3 phosphorylation at serine 28. Furthermore, UVB-induced H3 phosphorylation at serine 28 was inhibited inJnk1 −/− cells but not inJnk2 −/− cells. These results suggest that UVB-induced H3 phosphorylation at serine 28 may be mediated by mitogen-activated protein kinases. Histones are relatively small proteins with a very high proportion of positively charged amino acids (lysine and arginine); the positive charge helps the histones bind tightly to DNA regardless of its nucleotide sequence (1Alberts B. Bray D. Lewis J. Raft M. Roberts K. Watson J.D. Robertson M. Adams R. Molecular Biology of the Cell. Garland Publishing, New York1994: 342Google Scholar). The five types of histones (H1, H2A, H2B, H3, and H4) fall into two main groups: core histones and linker histones. 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The phosphorylation of histone H3 is thought to be a highly conserved event among eukaryotes and probably is involved in transcriptional regulation and chromosome condensation during mitosis and meiosis (15Wei Y. Mizzen C.A. Cook R.G. Gorovsky M.A. Allis C.D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7480-7484Crossref PubMed Scopus (357) Google Scholar,25Sassone-Corsi P. Mizzen C.A. Cheung P. Crosio C. Monaco L. Jacquot S. Hanauer A. Allis C.D. Science. 1999; 285: 886-891Crossref PubMed Scopus (428) Google Scholar, 26Thomson S. Clayton A.L. Hazzalin C.A. Rose S. Barratt M.J. Mahadevan L.C. EMBO J. 1999; 18: 4779-4793Crossref PubMed Scopus (404) Google Scholar). Two phosphorylation sites are present in the N terminus of histone H3, serine 10, and serine 28. Previous studies showed that H3 phosphorylation at serine 10 was associated with mitosis in diverse types of eukaryotic cells and with chromosome condensation during mitosis and meiosis (5Van Hold K.E. Rich A. Chromatin. 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Science. 1999; 285: 886-891Crossref PubMed Scopus (428) Google Scholar, 26Thomson S. Clayton A.L. Hazzalin C.A. Rose S. Barratt M.J. Mahadevan L.C. EMBO J. 1999; 18: 4779-4793Crossref PubMed Scopus (404) Google Scholar), and induction ofras expression results in a rapid increase in H3 phosphorylation at serine 10 (30, 31). Various stimuli including epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, anesomycin, and okadaic acid, and stresses such as UV irradiation induce rapid H3 phosphorylation in mammalian cells (6Mahadevan L.C. Willis A.C. Barratt M.J. Cell. 1991; 65: 775-783Abstract Full Text PDF PubMed Scopus (375) Google Scholar, 25Sassone-Corsi P. Mizzen C.A. Cheung P. Crosio C. Monaco L. Jacquot S. Hanauer A. Allis C.D. Science. 1999; 285: 886-891Crossref PubMed Scopus (428) Google Scholar, 26Thomson S. Clayton A.L. Hazzalin C.A. Rose S. Barratt M.J. Mahadevan L.C. EMBO J. 1999; 18: 4779-4793Crossref PubMed Scopus (404) Google Scholar, 32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The pathway responsible for mediating H3 phosphorylation at serine 10 depends on the type of stimulation (25Sassone-Corsi P. Mizzen C.A. Cheung P. Crosio C. Monaco L. Jacquot S. Hanauer A. Allis C.D. Science. 1999; 285: 886-891Crossref PubMed Scopus (428) Google Scholar, 26Thomson S. Clayton A.L. Hazzalin C.A. Rose S. Barratt M.J. Mahadevan L.C. EMBO J. 1999; 18: 4779-4793Crossref PubMed Scopus (404) Google Scholar, 32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and phosphorylation of serine 10 in histone H3 is also linked functionally in vitro andin vivo to acetylation of histone at lysine 14 (33). H3 at serine 28 is phosphorylated during early mitosis and with mitotic chromosome condensation in various mammalian cell lines (34Goto H. Tomono Y. Ajiro K. Kosako H. Fujita M. Sakurai M. Okawa K. Iwamatsu A. Okigaki T. Takahashi T. Inagaki M. J. Biol. Chem. 1999; 274: 25543-25549Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). However, the kinase that is responsible for H3 phosphorylation at serine 28 remains unknown. Here we investigated the role of MAP1 kinases in phosphorylation of H3 at serine 28 in vitro and in vivo after UVB irradiation. Minimal Eagle's medium (MEM) and fetal bovine serum (FBS) were from BioWhittaker, Inc.l-Glutamine was from Life Technologies, Inc. Gentamicin was from Quality Biological. Bradford reagent was from Bio-Rad. PD 98059 and SB 202190 were from Calbiochem-Novabiochem. Phenylmethylsulfonyl fluoride was from Sigma. Pure histone H3 was from Roche Molecular Biochemicals. Antibody-conjugated alkaline phosphatase and antibodies for phosphorylated ERKs, p38 kinase, and JNKs were from New England Biolabs. Antibody for H3 was from Upstate Biotechnology, Inc. Antibody for phosphorylated H3 at serine 28 was produced and identified as described previously (34Goto H. Tomono Y. Ajiro K. Kosako H. Fujita M. Sakurai M. Okawa K. Iwamatsu A. Okigaki T. Takahashi T. Inagaki M. J. Biol. Chem. 1999; 274: 25543-25549Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Active ERK1, ERK2, p38 kinase, JNK1, and JNK2 were from Upstate Biotechnology, Inc. Phosphorylation of histone H3 by activated ERK1, ERK2, p38 kinase, JNK1, or JNK2 was carried out as described previously (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 35Huang C. Ma W.Y. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In brief, pure histone H3 or chromatin of JB6 Cl 41 cells was incubated with ERK1, ERK2, p38 kinase, JNK1 or JNK2, and 200 μm ATP in 50 μl kinase buffer (25 mm Tris, pH 7.5, 5 mm β-glycerophosphate, 2 mm dithiothreitol, 0.1 mm Na3VO4, 10 mmMgCl2) for 45 min at 30 °C. The samples were resolved by 15% SDS-PAGE, and phosphorylated H3 at serine 28 was detected by Western blotting with a specific antibody (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 34Goto H. Tomono Y. Ajiro K. Kosako H. Fujita M. Sakurai M. Okawa K. Iwamatsu A. Okigaki T. Takahashi T. Inagaki M. J. Biol. Chem. 1999; 274: 25543-25549Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). Equivalent numbers of cells were seeded in 10-cm dishes and cultured in 5% FBS MEM until they reached 85% confluence and then were starved in 0.1% FBS MEM for 48 h. Cells then were incubated for 2 h in fresh 0.1% FBS MEM, after which time they were exposed to UVB and then cultured for additional time periods. Because the normal UVB lamp also generates a small amount of UVC light, the UVB irradiation was carried out in a UVB exposure chamber with a Kodak Kodacel K6808® filter that eliminates all wavelengths below 290 nm. After UVB irradiation the media were removed. The cultured cells were harvested and washed two times with cold phosphate-buffered saline. Acid-solution protein extraction was carried out as described by the protocol of Upstate Biotechnology, Inc. In brief (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), acid-soluble proteins were extracted with lysis buffer (10 mm HEPES, pH 7.9, 1.5 mm MgCl2, 10 mm KCl, 1.5 mm phenylmethylsulfonyl fluoride, 0.5 mmdithiothreitol), and then H2SO4 was added to a final concentration of 0.2 m (0.4 n) and the protein solutions were left on ice for 60 min. Supernatant fractions were transferred to fresh microcentrifuge tubes after centrifugation at 14,000 rpm/10 min and precipitated on ice for 45 min with a final concentration of 20% trichloroacetic acid. These tubes were centrifuged at 14,000 rpm/10 min at 4 °C, and the pellets were washed once with acidic acetone and then once with acetone. The protein concentration was measured by the Bradford method (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217547) Google Scholar), and the acid-soluble proteins were stored at −20 °C. Acid-soluble proteins were resolved by 15% SDS-PAGE after boiling for 5 min in SDS sample buffer. Resolved acid-soluble proteins were transferred to polyvinylidene difluoride membranes. Polyvinylidene difluoride membranes were blocked with 5% nonfat dry milk in phosphate-buffered saline for 1 h at room temperature and incubated overnight at 4 °C with the polyclonal antibody against H3 or the monoclonal antibody against phosphorylated H3 at serine 28. The second antibody against rabbit or rat IgG-conjugated alkaline phosphatase, respectively, was incubated with the respective membrane for 4 h at 4 °C. Membrane-bound proteins were detected with chemiluminescence (Enzyme-catalyzed fluorescence of Amersham Pharmacia Biotech) and analyzed using the Storm 840 Scanner (Molecular Dynamics, Inc.). Similar to phosphorylation of histone H3 at serine 10, phosphorylation of histone H3 at serine 28 plays a key role during early mitosis and coincides with the initiation of mitotic chromosome condensation (34Goto H. Tomono Y. Ajiro K. Kosako H. Fujita M. Sakurai M. Okawa K. Iwamatsu A. Okigaki T. Takahashi T. Inagaki M. J. Biol. Chem. 1999; 274: 25543-25549Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). To determine the role of MAP kinases in mediating H3 phosphorylation at serine 28, we incubated pure histone H3 protein with each of the active MAP kinases (ERK1, ERK2, p38 kinase, JNK1, or JNK2) and 200 μm ATP (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 35Huang C. Ma W.Y. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Phosphorylated H3 at serine 28 was detected by a specific monoclonal antibody as before (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 34Goto H. Tomono Y. Ajiro K. Kosako H. Fujita M. Sakurai M. Okawa K. Iwamatsu A. Okigaki T. Takahashi T. Inagaki M. J. Biol. Chem. 1999; 274: 25543-25549Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). The results show that pure histone H3 at serine 28 was phosphorylated strongly by ERK1 (Fig. 1 A), ERK2 (Fig. 1 B), or p38 kinase (Fig. 1 C) and to a comparatively lesser degree by JNK1 (Fig. 1 D) or JNK2 (Fig.1 E) in vitro. Similar results were found also by using chromatin as substrate for these MAP kinases (data not shown). Our previous study showed that UVB could induce phosphorylation of histone H3 at serine 10 in JB6 Cl 41 cells (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). To investigate the signal transduction pathways responsible for H3 phosphorylation at serine 28 in vivo, we exposed mouse epidermal JB6 cells to UVB irradiation and then extracted acid-soluble proteins for detection of H3 phosphorylation at serine 28 by Western blot with a specific antibody (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 34Goto H. Tomono Y. Ajiro K. Kosako H. Fujita M. Sakurai M. Okawa K. Iwamatsu A. Okigaki T. Takahashi T. Inagaki M. J. Biol. Chem. 1999; 274: 25543-25549Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). The results show that UVB strongly induced H3 phosphorylation at serine 28 (Fig.2, A and B). The dose-response study showed that H3 phosphorylation at serine 28 increased with UVB exposure from 1 to 6 kJ/m2 (Fig.2 A). H3 phosphorylation at serine 28 was greater at 30 or 60 min than at 15 or 120 min after UVB irradiation (Fig. 2 B). We found previously that phosphorylation of H3 at serine 10 was higher at 15 or 30 min than at 60 min (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), and the level of phosphorylated H3 at serine 28 was higher at 60 min compared with phosphorylation at serine 10 at 60 min (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) after UVB irradiation. This difference in the phosphorylation time courses between H3 at serine 28 and serine 10 suggests that phosphorylation of H3 at serine 28 and serine 10 may be mediated by different pathways. These results indicate that UVB-induced H3 phosphorylation at serine 28 is dose- and time-dependent. MAP kinases including ERKs, p38 kinase, and JNKs are mediators of signal transduction from the cell surface to the nucleus. We showed previously that UVB strongly induced phosphorylation of ERKs, p38 kinase, and JNKs in JB6 Cl 41 cells (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). To determine the possible role of MAP kinases in mediating UVB-induced H3 phosphorylation at serine 28 in vivo, we first examined the influence of specific chemical inhibitors on UVB-induced H3 phosphorylation at serine 28 in JB6 Cl 41 cells. PD 98059 is a specific inhibitor of the activation of MEK1 in vivo and in vitro (37Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3259) Google Scholar, 38Smalley K.S. Feniuk W. Sellers L.A. Humphrey P.P. Biochem. Biophys. Res. 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Previous studies showed that overexpression of DNM ERK2, DNM p38 kinase, or DNM JNK1 markedly inhibited activation of endogenous ERKs (35Huang C. Ma W.Y. Maxiner A. Sun Y. Dong Z. J. Biol. Chem. 1999; 274: 12229-12235Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 40Huang C. Ma W.Y. Dong Z. Oncogene. 1999; 18: 2828-2835Crossref PubMed Scopus (64) Google Scholar, 42Watts R.G. Huang C.S. Young M.R. Li J.J. Dong Z.G. Pennie W.D. Colburn N.H. Oncogene. 1998; 17: 3493-3498Crossref PubMed Scopus (107) Google Scholar, 43Huang C. Ma W.Y. Li J. Goranson A. Dong Z. J. Biol. Chem. 1999; 274: 14595-14601Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), p38 kinase (44Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar, 45Rincon M. Enslen H. Raingeaud J. Recht M. Zapton T. Su M.S. Penix L.A. Davis R.J. Flavell R.A. EMBO J. 1998; 17: 2817-2829Crossref PubMed Scopus (359) Google Scholar), or JNKs (46Derijard B. Hibi M. Wu I.H. Barrett T. Su B. Deng T. Karin M. Davis R.J. Cell. 1994; 76: 1025-1037Abstract Full Text PDF PubMed Scopus (2957) Google Scholar), respectively. To identify the role of MAP kinases in UVB-induced H3 phosphorylation at serine 28 in vivo, we used cells expressing these mutant kinases. Compared with JB6 Cl 41 cells (Fig.4 A), cells expressing DNM ERK2 totally blocked UVB-induced H3 phosphorylation at serine 28 at 60 min after UVB irradiation, and DNM p38 or DNM JNK1 also markedly suppressed UVB-induced H3 phosphorylation at serine 28 by ∼70–80% (Fig. 4,B and C). In contrast, phosphorylation of H3 at serine 28 at 60 min increased ∼2-fold in UVB-treated JB6 Cl 41 cells (Fig. 4, A–C). The inhibition of UVB-induced phosphorylation of H3 at serine 28 in DNM-ERK2, DNM-p38, and DNM-JNK1 cells also was dependent on UVB dose (Fig.5, A–C). However, inhibition of phosphorylation of H3 at serine 28 by DNM ERK2 cells (Fig. 5 A) was stronger than that by DNM p38 (Fig.5 B) or DNM JNK1 cells (Fig. 5 C).Figure 5Dose response of UVB-induced phosphorylation of H3 at serine 28 in JB6 Cl 41, DNM ERK2, DNM p38, and DNM JNK JB6 Cl 41 cells. Cells of JB6 Cl 41 and JB6 Cl 41 DNM ERK2 (A), JB6 Cl 41 DNM p38 (B), and JB6 Cl 41 DNM JNK1 (C) were exposed to UVB at doses of 1, 2, 4, or 6 kJ/m2 and incubated an additional 30 min. Phosphorylation of H3 at serine 28 and total H3 protein were detected as indicated in Fig. 1. The arrows denote the position of phospho-H3 at serine 28 and total H3 protein.View Large Image Figure ViewerDownload (PPT) We also used Jnk1(Jnk1 −/−) and Jnk2(Jnk2 −/−) knockout cells and Jnkwild-type (Jnk +/+) cells to examine the role of JNKs in UVB-induced H3 phosphorylation at serine 28. The results showed that UVB-induced H3 phosphorylation at serine 28 was blocked inJnk1 −/− cells (Fig.6, A and C) but not in Jnk2 −/− cells (Fig. 6, B andD) compared with Jnk1 +/+ cells (Fig.6, A–D). These experiments further confirmed that ERK1, ERK2, p38 kinase, and JNK1 mediate UVB-induced phosphorylation of H3 at serine 28. In contrast, UVB-induced H3 phosphorylation at serine 10 was not affected inJnk1 −/− and Jnk2 −/−cells (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Our present study indicates that UVB irradiation activates MAP kinases (ERKs, p38 kinase, and JNKs) resulting in phosphorylation of H3 at serine 28. We found that active ERK1, ERK2, and p38 kinase strongly phosphorylated H3 at serine 28, whereas JNK1 and JNK2 phosphorylation of H3 at serine 28 was relatively weaker in vitro. Further, our data showed that PD 98059 and SB 202190 and the expression of DNM ERK2, p38 kinase, or JNK1 inhibited UVB-induced H3 phosphorylation at serine 28. UVB-induced phosphorylation of H3 at serine 28 was blocked also in Jnk1 −/− but not inJnk2 −/− cells. These data clearly indicate that UVB-induced phosphorylation of H3 at serine 28 is mediated mainly through ERKs, p38 kinase, and JNK1 pathways. The covalent modification of the amino-terminal tails of histone H3 has emerged as an important mechanism in regulation of transcriptional activation and chromatin condensation. The best understood histone modification is acetylation of lysine residues of H3/H4, which is mediated by histone acetyltransferases and histone deacetylases (19Mizzen C.A. Yang X.J. Kokubo T. Brownell J.E. Bannister A.J. Owen-Hughes T. Workman J. Wang L. Berger S.L. Kouzarides T. Nakatani Y. Allis C.D. Cell. 1996; 87: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (624) Google Scholar, 20Kornberg R.D. 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Moreover, expression of DNM JNK1 inhibited UVB-induced phosphorylation of histone H3 at serine 28 (Figs. 4 C and 5C), and phosphorylation of histone H3 was blocked inJnk1 −/− cells (Fig. 6, A andC) but not in Jnk2 −/− cells (Fig.6, B and D) compared withJnk +/+ cells (Fig. 6,A–D). These results indicate that JNK1 indeed is involved in UVB-induced phosphorylation of histone H3 at serine 28in vivo but not in phosphorylation of histone H3 at serine 10 (32). The difference between phosphorylation of H3 at serine 28 and serine 10 by JNKs suggests that H3 phosphorylation at distinct sites in the N terminus may be important in different physiological functions after UVB irradiation. Our results also show that the highest peak of UVB-induced phosphorylation of histone H3 at serine 28 is at 60 min (Fig.2 B), whereas UVB-induced phosphorylation of histone H3 at serine 10 is highest at 30 min after UVB irradiation (32Zhong S.P. Ma W.Y. Dong Z. J. Biol. Chem. 2000; 275: 20980-20984Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Because serine 10 of histone H3 is closer than serine 28 to the NH2-terminal tail of histone H3, serine 10 of histone H3 may be phosphorylated faster than serine 28 after UVB irradiation. This difference in phosphorylation time implies that outside serine residues of histone H3 are phosphorylated preferentially after UVB irradiation. This difference in phosphorylation time of H3 at serine 10 and serine 28 also suggested that MAP kinase may indirectly regulate phosphorylation of histone H3 at serine 28 through activation of as yet unidentified protein kinases. We are currently investigating the role of MSK1, a downstream kinase of MAP kinases, in the UVB-induced phosphorylation of H3 at serine 28 (26). Although serine 28 and serine 10 of histone H3 have identical surrounding sequences (that is, both are RKS; Ref. 10Strahl B.D. Allis C.D. 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