ERKs and p38 kinases mediate ultraviolet B-induced phosphorylation of histone H3 at serine 10.

Histone H3 is the core protein of the nucleosome. Phosphorylation of H3 involves immediate early gene expression, chromatin remodeling, and chromosome condensation during mitosis. Very recently, Rsk2 or MSK1 kinase-mediated phosphorylation of H3 at serine 10 was reported. In the present study, we show that both ERKs and p38 kinase may mediate ultraviolet B-induced phosphorylation of H3 at serine 10. PD 98059, a MEK1 inhibitor, and SB 202190, a p38 kinase inhibitor, efficiently inhibited ultraviolet B-induced phosphorylation of H3. Phosphorylation of H3 was also inhibited in cells expressing dominant negative mutant (DNM) ERK2 and DNM p38 kinase. In contrast, no inhibition of H3 phosphorylation in Jnk1 or Jnk2 knockout cells (Jnk1(-/-) or Jnk2(-/-)) and cells expressing DNM JNK1 was observed. More importantly, incubation of active ERK2 or p38 kinase with H3 protein resulted in phosphorylation of H3 at serine 10 in vitro. These results suggest that ERK and p38 kinase are at least two important mediators of phosphorylation of H3 at serine 10.

The nucleosome core particle consists of 146 base pairs of DNA wrapped in 1.75 turns around an octamer formed from two copies of each histone H2A, H2B, H3, and H4. Histone modification by acetylation, phosphorylation, and methylation plays an important role in gene transcription and cell division. Histone H3 can be acetylated at lysines 9, 14, 18, 23; phosphorylated at serines 10 and 28; and methylated at lysines 9 and 27 in the N-terminal domain (1)(2)(3). Acetylation of core histone (H3/H4) regulates interaction of the transcription factor TFIIIA with the Xenopus 5 S RNA gene (4). Major late promoter plasmid is associated with acetylated histone H3, and recruitment of a histone deacetylase-associated repressor to the promoter can decrease the level of H3 acetylation at the promoter in vivo (5). Phosphorylation of H3 at serine 10 is thought to be a highly conserved event among eukaryotes and is probably involved in both mitotic and meiotic chromosome condensation (6). Methylation of H3 and H4 is known to be associated with the arrest of liver cell growth (7).
Various stimuli including epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate and stresses such as UV irradiation induce a Ras-dependent mitogen-activated protein (MAP) 1 kinase cascade that results in the transcription of a subset of genes known as immediate early response genes (8 -11). These genes include members of the c-fos and c-jun families that comprise transcription factor activator protein 1.
Our previous study showed that UVB markedly induced activator protein 1 activity via the ERK pathway (12). In addition, UVB was shown to induce phosphorylation of H3 (2), and immediate early response gene induction was reported to correlate with phosphorylation of H3 at serine 10 (13). These data imply that phosphorylation of H3 may play an important role in transcriptional regulation, chromatin remodeling, and chromosome condensation during mitosis. However, the role of MAP kinases (ERKs, p38, and JNKs) in the process of H3 phosphorylation remains unclear. Very recently, Thomson et al. (13) indicated that MSK1 was a potential histone H3/ HMG-14 kinase, and Paolo et al. (14) reported that epidermal growth factor induces phosphorylation of H3 at serine 10 by the Rsk-2 pathway. In this study, we investigated the role of MAP kinases in phosphorylation of H3 at serine 10 in vitro and in vivo after UVB irradiation.

EXPERIMENTAL PROCEDURES
Materials-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 of phosphorylated ERKs, p38 kinase, and JNKs were from New England Biolabs; antibodies of H3 and phosphorylated H3 were from Upstate Biotechnology, Inc. (Lake Placid, NY); active ERK2, active JNK2, and active p38 kinase were from Upstate Biotechnology; Elk1, ATF2, and c-Jun fusion protein and MAP kinase (ERK2) were from New England Biolabs; antibodies of phosphorylated Elk1 serine 383, phosphorylated ATF2 thronine 71, and phosphorylated c-Jun serine 63 were from New England Biolabs; minimum essential medium (MEM) and fetal bovine serum (FBS) were from Biowhittaker Biosciences; L-glutamine was from Life Technologies, Inc.; gentamicin was from Quality Biological, Inc.
UVB Irradiation-Equivalent numbers of JB6 Cl 41 cells were seeded in six-well plates or 10-cm dishes and cultured in 5% FBS MEM until they were 85% confluent and then starved in 0.1% FBS MEM for 48 h. Cells were then 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. Since 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.
Acid-soluble Protein Extraction-After UVB irradiation, 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 (available on the World Wide Web). In brief, acid-soluble proteins were extracted with lysis buffer (10 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 1.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol), and then H 2 SO 4 was added to a final concentration of 0.2 M (0.4 N) and left for 60 min on * This work was supported by the Hormel Foundation and NCI, National Institutes of Health Grants CA77646 and CA81064. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertise- ice. Supernatant fractions were transferred to fresh microcentrifuge tubes after centrifugation at 14,000 rpm/10 min and precipitated on ice for 45 min with 50% trichloroacetic acid to 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 once with acetone, respectively. The acid-soluble proteins were stored at Ϫ20°C. Assay of Phosphorylated H3-Acid-soluble proteins were resolved by 15% SDS-polyacrylamide gel electrophoresis 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 with the first antibody against H3 or phospho-H3 overnight at 4°C. The second antibody against rabbit IgG-conjugated alkaline phosphatase was incubated with the membranes for 4 h at 4°C. Membrane-bound proteins were detected with chemiluminescence (ECF of Amersham Pharmacia Biotech) and analyzed using the Storm 840 Scanner (Molecular Dynamics, Inc., Sunnyvale, CA).
Preparation of Chromatin-Chromatin was prepared at 4°C according to the procedure of Pumo et al. (15). In brief, JB6 Cl 41 cells were seeded in 10-cm dishes and cultured in 5% FBS MEM until they were 85% confluent and then harvested. Cells were washed two times with cold phosphate-buffered saline, lysed by the addition of 4 volumes of buffer A (10 mM Tris-HCl, pH 7.9, 3 mM MgCl 2 , 0.5 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, 0.25 M sucrose), and homogenized. The suspension was centrifuged at 2500 rpm/15 min (Rotor 224, Centra-MP4R; International Equipment Company). Pellets of cells were suspended by the addition of 9 volumes of buffer B (10 mM Tris-HCl, pH 7.9, 3 mM MgCl 2 , 0.5 mM phenylmethylsulfonyl fluoride, 1.5 M sucrose). The 4.5-ml suspension was added to 1.5 ml of buffer B in 6-ml centrifuge tubes and centrifuged at 15,000 rpm/40 min (Ti 42, Beckman L5-75). The pellets were resuspended by the addition of 20 volumes of buffer C (10 mM Tris-HCl, pH 7.9, 1 mM EDTA), homogenized, and then centrifuged at 2500 rpm/15 min. Pellets were suspended by the addition of 50 volumes of buffer D (0.15 M NaCl, 15 mM sodium citrate). The suspension was centrifuged at 3500 rpm/20 min (Rotor 224, Centra-MP4R; international Equipment Company) to recover chromatin. The protein concentration of chromatin was determined by the Bradford method (Bio-Rad) after sonicating four times for 5 s each. The chromatin pellet was stored at Ϫ70°C until used.
K m Analysis of H3 Phosphorylation-Phosphorylation of pure histone H3 by ERK2 or p38 kinase was carried out as described by the protocol of Upstate Biotechnology. In brief, the following components were added to a series of tubes: 10 l of assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM ␤-glycerolphosphate, 5 mM EGTA, 1 mM Na 3 VO 4 , 1 mM dithiothreitol); 10 l of various concentrations of pure histone H3; 10 l of ERK2 or p38 kinase; and 10 l of [␥-32 P]ATP mixture (1 Ci/l). Tubes were incubated at 30°C for 30 min, and then 30 l of the mixture was spotted on individual pieces of p81 paper (2 cm 2 ). The assay squares were washed with 0.75% phosphoric acid and acetone and then transferred to scintillation vials with 5 ml of scintillation mixture and counted in a scintillation counter. K m values for H3 phosphorylation by ERK2 or p38 kinase were then determined.

UVB Induces
Phosphorylation of H3 at Serine 10 -Phosphorylation of H3 may play a very important role in transcriptional regulation by a variety of signals. To study whether UV irradiation induces phosphorylation of H3 at serine 10, we first exposed JB6 epidermal C1 41 cells to UVB and analyzed the phosphorylation of H3 by Western blot using a phosphospecific antibody against phosphorylated H3 at serine 10 (6, 14, 16, 17). In a parallel blot, equal amounts of acid-soluble protein were confirmed by Western blot to detect histone H3 with antihistone H3 antibody. Results showed that UVB (4 kJ/m 2 ) induced a rapid and transient phosphorylation of H3 at serine 10 in the JB6 C1 41 mouse epidermal cell line (Fig. 1A). Phosphorylation of H3 at serine 10 was greater at 15 or 30 min than at 60 min following UVB irradiation and decreased markedly by 120 min (Fig. 1A). A dose-response study showed that phosphorylation of H3 gradually increased with UVB exposures of 1-6 kJ/m 2 (Fig. 1B). The results indicate that UVB-induced phosphorylation of H3 at serine 10 is time-and dose-dependent.
SB 202190 and PD 98059 Inhibit UVB-induced Phosphorylation of H3 at Serine 10 -UVB irradiation causes activation of the MAP kinase superfamily, composed of ERKs, JNKs, and p38 kinase (11,20,21). To identify the possible role of MAP kinases in mediating UVB-induced phosphorylation of H3, we first examined the influence of specific chemical inhibitors on UVB-induced H3 phosphorylation at serine 10 in JB6 C1 41 cells. The results showed that UVB-induced phosphorylation of H3 was markedly blocked by pretreatment of cells with 1-4 M SB 202190 ( Fig. 2A), a specific inhibitor of p38 kinase (12,13), and by 25-100 M PD 98059 (Fig. 2B), a specific inhibitor of MEK1 (12,13,18). We previously showed that at these concentrations, SB 202190 and PD 98059 specifically inhibit UVBinduced p38 kinase and MEK1, respectively (12,18). These results suggest that ERKs and p38 kinase may be involved in UVB-induced H3 phosphorylation at serine 10.
UVB-induced Phosphorylation of H3 at Serine 10 Is Blocked by Expressing DNM ERK2 and DNM p38 Kinase but Not DNM JNK1 or in Jnk1 and Jnk2 Knockout Cells (Jnk1 Ϫ/Ϫ , Jnk2 Ϫ/Ϫ )-SB 202190 and PD 98059 block the UVB-induced phosphorylation of H3 at serine 10. A chemical inhibitor for JNKs is unknown at this time. Therefore, to further identify the role of MAP kinases in UVB-induced phosphorylation of H3, we used JB6 C1 41 cells expressing DNMs of ERK2, p38, and JNK1 (16,22). The results showed that UVB strongly induced phosphorylation of ERKs, p38 and JNK in JB6 C1 41 cells (Fig. 3, A-C), but the expression of DNM ERKs, p38 kinase, and JNK1 in JB6 C1 41 cells specifically blocked UVBinduced phosphorylation of ERKs, p38, and JNK kinases (Fig.  3, D-F). We then analyzed UVB-induced phosphorylation of H3 at serine 10 in wild type and mutant JB6 C1 41 cells (Fig. 4). Cells were incubated for 2 h in fresh 0.1% FCS MEM, after which time they were exposed to UVB (4 kJ/m 2 ) or not exposed to UVB (control) and incubated an additional 15, 30, 60, or 120 min. Phosphorylation of H3 at serine 10 was determined by Western blot analysis of acid-soluble nuclear proteins resolved by SDS-polyacrylamide gel electrophoresis. H3 was detected in a parallel blot with anti-histone H3 from Upstate Biotechnology. B, dose-response study. Cells were treated as in A but were exposed to 1, 2, 4, or 6 kJ/m 2 of UVB and then incubated an additional 30 min. Phosphorylation of H3 at serine 10 was determined as indicated. The arrow denotes the position of phospho-H3 at serine 10.
ERK2 and p38 Kinase Directly Phosphorylate H3 at Serine 10 in Vitro-The above results strongly suggest that ERKs and p38 kinase are mediators of UVB-induced phosphorylation of H3 at serine 10. Therefore, active ERKs and p38 kinases should phosphorylate H3 at serine 10 in vitro. To test this hypothesis, we incubated purified H3 protein with each of the MAP kinase family proteins (ERK2, p38, and JNK2) in the presence of 200 M ATP. The phosphorylation level of H3 at serine 10 was detected with specific antibodies by Western blot analysis (6,14,17,18). These results show that active ERK2 (Fig. 7A) and p38 kinase (Fig. 7B), but not JNK2 (Fig. 7C), can directly phosphorylate H3 at serine 10 in vitro, while active JNK2, ERK2, and p38 kinase were able to phosphorylate their substrate c-Jun, Elk1, and ATF2, respectively (Fig. 7, A-C). Further, by using chromatin isolated from Cl 41 cells as substrate for these kinases, we obtained similar results (Fig. 7D), showing that ERK2 and p38 kinase, but not JNK2, caused H3 protein phosphorylation at serine 10 in vitro. These results confirm that ERK2 and p38 kinase are mediators of UVBinduced H3 phosphorylation at serine 10.
K m for Histone H3 Phosphorylation-To determine the H3 phosphorylation by MAP kinases, we performed the kinase assay by using [␥-32 P]ATP and pure histone H3 protein. Phosphate incorporation was 3.89 ϫ 10 5 mol of phosphate/mol of ERK2 and 2.68 ϫ 10 5 mol of phosphate/mol of p38 kinase. The K m values of H3 phosphorylation by ERK2 and p38 kinase were calculated, and results showed that K m values for H3 phosphorylation by ERK2 and p38 kinase were 2.04 ϫ 10 Ϫ5 and 4.89 ϫ 10 Ϫ6 mM, respectively. Different K m values for ERK2 and p38 kinase indicated that p38 kinase is more effective than ERK2 for H3 phosphorylation.
To further investigate the interaction between H3 and MAP kinases in vivo, we used anti-phospho-H3 at serine 10, phospho-ERKs, or phospho-p38 kinase to co-immunoprecipitate the complex of H3 and ERKs or H3 and p38 kinase from JB6 Cl 41 cells following UVB irradiation. No H3-ERKs or H3-p38 kinase co-immunoprecipitation complex was found (data not shown). DISCUSSION In the present study, we provide evidence that UVB irradiation induced phosphorylation of H3 at serine 10 in vivo. Pretreatment of cells with SB 202190, a p38 kinase inhibitor (12,13), or PD 98059, a MEK1 kinase inhibitor (12, 13, 18), blocked UVB-induced phosphorylation of H3 at serine 10. Expression of FIG. 2. PD 98059 and SB 202190 inhibit the UVB-induced phosphorylation of H3 at serine 10 in JB6 C1 41 cells. Cells were treated with various concentrations of PD 98059 or SB 20490 for 1 h and then exposed to UVB (4 KJ/m 2 ). Cells were incubated an additional 30 min. H3 was detected in a parallel blot with anti-histone H3 from Upstate Biotechnology. A, SB 202190, a specific inhibitor for p38 kinase, blocked UVB-induced phosphorylation of H3 at serine 10 activated by p38 kinase. B, PD 98059, a specific inhibitor for MEK1 kinase, blocked UVB-induced phosphorylation of H3 at serine 10 activated by ERKs.
DNM ERK2 or DNM p38 inhibited UVB-induced phosphorylation of H3 at serine 10. MAP kinases, ERK2 and p38, directly phosphorylated H3 at serine 10 in vitro. These results clearly suggest that ERKs and p38 kinase are mediators of UVBinduced phosphorylation of H3 at serine 10.
Phosphorylation of H3 is highly associated with the G 2 to M transition (23)(24)(25)(26). Fostriecin and okadaic acid initiate premature chromatin condensation and induce H3 phosphorylation (27,28). In contrast, vanadate-induced dephosphorylation of H3 correlates with chromatin decondensation (29). H3 at serine 10 is specifically phosphorylated near its N terminus in mitotic and meiotic chromosome condensation (6,30). Histone H3 is not phosphorylated during interphase but becomes phosphorylated at serine 10 just prior to metaphase (27). Site-specific phosphorylation of H3 at serine 10 occurs during mitosis in mammalian cells (31,32) and various stimuli, including epidermal growth factor and 12-O-tetradecanoylphorbol-13-acetate, and stresses such as UV irradiation induce phosphorylation of H3 at serine 10 (13,14). Phosphorylation of H3 at serine 10 is also related to the expression of immediate early response genes (c-fos and c-jun gene families) (9,13). In addition, induction of ras expression results in a rapid increase in H3 phosphorylation (33,34). The above findings strongly imply that H3 phosphorylation plays an important role in gene expression and mitotic and meiotic chromatin condensation. However, H3 serine 10 is not phosphorylated by cyclin-dependent kinases (27). The H3 phosphorylation pathway remains poorly understood. Recently, epidermal growth factor was reported to induce phosphorylation of H3 at serine 10 by the RSK2 pathway (14), and MSK1 phosphorylates H3 at serine 10 after 12-Otetradecanoylphorbol-13-acetate and anisomycin treatment (13). In this study, we investigated the role of MAP kinases in mediation of UVB-induced phosphorylation of H3 at serine 10. UVB induced the activation of ERKs, JNKs, and p38 kinase. Fifteen minutes after UV irradiation, UV-induced activation of these kinases correlated well with the UVB-induced phosphorylation of H3 at serine 10 ( Figs. 1 and 3). However, the persistence of the H3 phosphorylation at 60 min suggests that H3 may be also phosphorylated by a kinase downstream of ERK or p38 kinase (Figs. 1 and 3). PD 98059 is a specific inhibitor of the activation of MEK1 in vivo and in vitro (35,36). Previous studies demonstrated that PD 98059 inhibited the activation and phosphorylation of ERKs specifically (12-13, 18, 35-40). Our results showed that 50 M PD 98059 completely inhibited UVB-induced phosphorylation of H3 at serine 10 (Fig. 2B). 50 M PD 98059 totally blocked activation of ERKs but not JNKs or p38 kinase. This result implies that ERKs are involved in the UVB-induced phosphorylation of H3 at serine 10. SB 202190 is a specific inhibitor of p38 kinase (12,13,(41)(42)(43)(44). Pretreatment of cells with 1-4 M SB 202190 totally inhibited UVB-induced phosphorylation of H3 at serine 10 ( Fig. 2A). This concentration range of SB 202190 has no effect on ERKs (18,41). These concentrations of SB 202190 (1-4 M) selectively block activation of p38 kinases (18). The above data indicated that UVB-induced phosphorylation of H3 at serine 10 may be mediated by ERKs and p38 kinase. Further studies using cells with dominant negative MAP kinases (DNM ERK2, DNM p38 kinase, or DNM JNK1) help clarify the role of MAP kinases in UVB-induced phosphorylation of H3 at serine 10. Previous studies showed that overexpression of DNM ERK2, DNM p38 kinase, or DNM JNK1 markedly inhibited activation of endogenous ERKs (12, 44 -47), p38 kinase (48,49) or JNKs (19), respectively. Our results showed that overexpression of dominant negative MAP kinases inhibited phosphorylation of endogenous ERKs, p38 kinase, or JNKs by about 60% (Fig. 3, D-F), compared with phosphorylation of ERKs, p38 kinase, or JNKs in wild type JB6 Cl 41 cells (Fig. 3, A-C). Time-dependent patterns of UVB-induced phosphorylation of MAP kinases were similar to UVB-induced phosphorylation of H3 at serine 10. Phosphorylation of ERK2 and p38 kinase at 60 min declined by about 25%, compared with the level at 15 min following UVB irradiation in JB6 Cl 41 cells (Fig. 3, A and B). However, phosphorylation of H3 at serine 10 at 60 min decreased by about 80% in JB6 Cl 41 cells (Fig. 1A). Compared with ERKS and p38 kinase, the persistence of the H3 phosphorylation at serine 10 at 60 min implies that a downstream effector kinase of MAP kinase may be involved in H3 phosphorylation. This suggests that UVB-induced phosphorylation of H3 at serine 10 may be dependent on the activation of MAP kinases. Expression of DNM ERK2 completely blocked phosphorylation of H3 at serine 10 ( Fig. 4B). This result confirmed that ERKs indeed mediate UVB-induced phosphorylation of H3 at serine 10. Expression of DNM p38 kinase also displayed a similar inhibition (ϳ80%) of H3 phosphorylation at 15 and 30 min (Fig. 4C), but little signal was observed at 60 min following UVB irradiation. Expression of DNM JNK1 did not appear to inhibit UVB-induced phosphorylation of H3 at serine 10 ( Fig. 4D), but the time course for phosphorylation of H3 at serine 10 was slightly delayed in DNM JNK1 cells following UVB irradiation. The reason for this delay is unclear and is under investigation in this laboratory. UVB-induced phosphorylation of H3 at serine 10 was not inhibited in the Jnk1 Ϫ/Ϫ or Jnk2 Ϫ/Ϫ cells as compared with Jnks ϩ/ϩ cells (Fig. 5). The in vitro experiments further confirmed, regardless of substrates for pure histone H3 or chromatin from JB6 Cl 41 cells, that ERK2 and p38 kinase, but not JNKs, phosphorylated H3 at serine 10. Notably, the assay of kinase activity showed that p38 kinase was more effective than ERK2 for H3 phosphorylation. Together, these results show that reduction of ERKs and p38 kinase leads to essentially complete cessation of UVBinduced H3 phosphorylation at serine 10, but JNKs do not appear to be involved.
In conclusion, we have determined that ERKs and p38 kinase mediate UVB-induced phosphorylation of H3 at serine 10. These data suggest that phosphorylation of H3, which is a basic component of the transcriptional machinery and a structural protein of the nucleosome, is mediated by different MAP kinases as a means to satisfy the needs of cells in response to various environmental stimuli.