JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M909934199 on May 9, 2000

J. Biol. Chem., Vol. 275, Issue 28, 20980-20984, July 14, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/28/20980    most recent
M909934199v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Zhong, S.-P.
Right arrow Articles by Dong, Z.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zhong, S.-P.
Right arrow Articles by Dong, Z.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

ERKs and p38 Kinases Mediate Ultraviolet B-induced Phosphorylation of Histone H3 at Serine 10*

Shu-Ping Zhong, Wei-Ya Ma, and Zigang DongDagger

From the Hormel Institute, University of Minnesota, Austin, Minnesota 55912

Received for publication, December 9, 1999, and in revised form, May 5, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (1-3). 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 MgCl2, 10 mM KCl, 1.5 mM phenylmethylsulfonyl fluoride, 0.5 mM dithiothreitol), and then H2SO4 was added to a final concentration of 0.2 M (0.4 N) and left for 60 min on 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 -20 °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 MgCl2, 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 MgCl2, 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 -70 °C until used.

Protein Phosphorylation Assay in Vitro-- Phosphorylation of histone H3, Elk1, ATF2, or c-Jun by activated ERK2, p38 kinase, or JNK2 was carried out as described previously (16). In brief, pure histone H3, chromatin of JB6 Cl 41 cells or other kinase substrates (Elk1, ATF2, or c-Jun fusion protein) were incubated with ERK2, p38 kinase, or JNK2, respectively, and 200 µM ATP in 50 µl of kinase buffer (25 mM Tris, pH 7.5, 5 mM beta -glycerolphosphate, 2 mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2) for 45 min at 30 °C. The samples were resolved by 15% SDS-polyacrylamide gel electrophoresis, and phosphorylated H3 was detected by Western blot with H3-phospho-specific antibodies (6, 14, 16, 17). Western blot analysis of ERK2, JNK2, or p38 kinase and their phosphorylated substrates was carried out as described previously (18).

Km 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 µl of assay dilution buffer (20 mM MOPS, pH 7.2, 25 mM beta -glycerolphosphate, 5 mM EGTA, 1 mM Na3VO4, 1 mM dithiothreitol); 10 µl of various concentrations of pure histone H3; 10 µl of ERK2 or p38 kinase; and 10 µl of [gamma -32P]ATP mixture (1 µCi/µl). Tubes were incubated at 30 °C for 30 min, and then 30 µl of the mixture was spotted on individual pieces of p81 paper (2 cm2). 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. Km values for H3 phosphorylation by ERK2 or p38 kinase were then determined.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 anti-histone H3 antibody. Results showed that UVB (4 kJ/m2) 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/m2 (Fig. 1B). The results indicate that UVB-induced phosphorylation of H3 at serine 10 is time- and dose-dependent.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1.   UVB-induced phosphorylation of H3 at serine 10 in vivo. A, time course study. JB6 C1 41 CMV-neo cells were starved by incubating in 0.1% FBS MEM for 48 h at 37 °C in a 5% CO2 atmosphere. Cells were incubated for 2 h in fresh 0.1% FCS MEM, after which time they were exposed to UVB (4 kJ/m2) 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/m2 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.

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 µM SB 202190 (Fig. 2A), a specific inhibitor of p38 kinase (12, 13), and by 25-100 µM 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 UVB-induced 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. 


View larger version (18K):
[in this window]
[in a new window]
  		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/m2). 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.

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-/-, Jnk2-/-)-- 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 UVB-induced 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). Compared with wild type cells (Fig. 4A), the cells expressing DNM ERK2 (Fig. 4B) or DNM p38 kinase (Fig. 4C) markedly blocked H3 phosphorylation at serine 10 at 15 and 30 min (Fig. 4, B and C). However, a weak signal indicating H3 phosphorylation appears in DNM p38 cells at 60 min (Fig. 4C). In contrast, expression of DNM JNK1 did not reveal significant inhibition of H3 phosphorylation at serine 10 at 30 and 60 min; however, UVB-induced H3 phosphorylation was delayed in DNM JNK1 cells compared with JB6 Cl 41 cells (Fig. 4D). Further, we used Jnk1 and Jnk2 knockout cells (Jnk1-/-, Jnk2-/-) and Jnks wild-type cells (Jnks+/+) to study the role of JNKs in the UVB-induced H3 phosphorylation at serine 10. The results showed that UVB-induced H3 phosphorylation at serine 10 was not affected in Jnk1-/- or Jnk2-/- cells as compared with Jnks+/+ cells (Fig. 5). The dose-response studies with UVB exposure (1, 2, 4, and 6 kJ/m2) further indicated that ERKs and p38 kinase are mediators of UVB-induced H3 phosphorylation at serine 10 (Fig. 6).


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   Expression of DNM ERK2, p38, and JNK1 blocks UVB-induced ERKs, p38, and JNK activities in JB6 C1 41 cells. Cells were cultured in 5% FBS MEM until 85% confluent and then starved in 0.1% FBS MEM for 48 h. Cells were incubated for 2 h in fresh 0.1% FBS MEM, after which time they were exposed to UVB (4 KJ/m2) and then incubated an additional 15, 30, 60, or 120 min. Media were removed, and cells were washed two times with cold phosphate-buffered saline. Phosphorylated ERKs, p38, and JNK kinases were detected with rabbit anti-phospho-p42/44 MAP kinase, phospho-p38 kinase, and phospho-stress-activated protein kinase/JNK, respectively. Phospho-ERKs (A), phospho-p38 (B), and phospho-JNKs (C) were strongly expressed in JB6 C1 41-neo vector cells after UVB irradiation, especially at 15 or 30 min as shown in Fig. 1 previously. Phospho-ERKs, phospho-p38, and phospho-JNKs were inhibited in JB6 C1 41 cells expressing DNM ERK2 (D), DNM p38 (E), and DNM JNK1 (F) following UVB irradiation. The arrows denote the positions of phospho-ERKs, p38, and JNKs.


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 4.   UVB-induced phosphorylation of H3 at serine 10 is blocked by DNM ERK2 or DNM p38 kinases but not by DNM JNK1. A, phosphorylation of H3 at serine 10 was strongly induced by UVB in JB6 C1 41 CMV-neo cells. B, UVB-induced H3 phosphorylation at serine 10 was completely blocked in JB6 C1 41 DNM ERK2 cells. C, UVB-induced H3 phosphorylation at serine 10 was markedly blocked in JB6 C1 41 DNM p38 cells (14). D, UVB-induced H3 phosphorylation at serine 10 was not blocked in JB6 C1 41 DNM JNK1 cells. The arrows denote the phospho-H3 at serine 10.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 5.   UBV-induced phosphorylation of H3 at serine 10 is not blocked by Jnk1-/- or Jnk2-/-. Jnks+/+, Jnk1-/-, and Jnk2-/- cells were starved by incubating in 0.1% FCS MEM for 48 h at 37 °C in a 5% CO2 atmosphere. Cells were incubated for 2 h in fresh 0.1% FBS MEM, after which time they were exposed to UVB (4 kJ/m2) and incubated an additional 15, 30, or 60 min. Phosphorylation of H3 at serine 10 was determined by Western blot analysis of acid-soluble nuclear proteins resolved by SDS-polyacrylamide gel electrophoresis. Phosphorylation of H3 at serine 10 was strongly induced by UVB (4 kJ/m2) in Jnks+/+ (A), Jnk1-/- (B), and Jnk2-/- (C). UVB-induced H3 phosphorylation at serine 10 was not inhibited in Jnk1-/- or Jnk2-/- cells, compared with Jnks+/+ cells. The arrows denote the phospho-H3 at serine 10.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 6.   Dose-response curve of UVB-induced phosphorylation of H3 at serine 10 in JB6 C1 41, DNM ERK2, DNM p38, and DNM JNK JB6 C1 41 cells. Cells of JB6 Cl 41 (A), JB6 Cl 41 DNM ERK2 (B), JB6 Cl 41 DNM p38 (C), and JB6 Cl 41 DNM JNK1 (D) were exposed to UVB 1 kJ/m2, 2 kJ/m2, 4 kJ/m2 and 6 kJ/m2 and incubated an additional 30 min after UVB irradiation. Phosphorylation of H3 at serine 10 was detected. The arrows denote the 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 µM 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 UVB-induced H3 phosphorylation at serine 10. 


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 7.   Phosphorylation of H3 at serine 10 in vitro by active ERK2 and p38 kinase but not by active JNK2. Phosphorylation of H3 at serine 10 (A-D) or the phosphorylation of Elk1 at serine 389, ATF2 at thronine 71, and c-Jun at serine 63 (A-C) was carried out at 30 °C for 45 min in the presence of specific substrate, kinase buffer, 200 µM ATP, and one of the MAP kinase family. The phosphorylated proteins were detected by Western blot with specific antibodies. A, pure histone H3 and Elk1 were phosphorylated by active ERK2. B, pure histone H3 and ATF2 were phosphorylated by p38 kinase. C, c-Jun but not pure histone H3 was phosphorylated by active JNK2. D, histone H3 of chromatin from JB6 Cl 41 cells was phosphorylated by active ERK2 and p38 kinase but not by JNK2.

Km for Histone H3 Phosphorylation-- To determine the H3 phosphorylation by MAP kinases, we performed the kinase assay by using [gamma -32P]ATP and pure histone H3 protein. Phosphate incorporation was 3.89 × 105 µmol of phosphate/µmol of ERK2 and 2.68 × 105 µmol of phosphate/µmol of p38 kinase. The Km values of H3 phosphorylation by ERK2 and p38 kinase were calculated, and results showed that Km values for H3 phosphorylation by ERK2 and p38 kinase were 2.04 × 10-5 and 4.89 × 10-6 mM, respectively. Different Km 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 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 UVB-induced phosphorylation of H3 at serine 10.

Phosphorylation of H3 is highly associated with the G2 to M transition (23-26). 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-O-tetradecanoylphorbol-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 µM PD 98059 completely inhibited UVB-induced phosphorylation of H3 at serine 10 (Fig. 2B). 50 µM 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, 41-44). Pretreatment of cells with 1-4 µM 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 µM) 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 (~80%) 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-/- or Jnk2-/- cells as compared with Jnks+/+ 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 UVB-induced 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.

    ACKNOWLEDGEMENTS

We thank Dr. Harald H. O. Schmid and Dr. Ann Bode for scientific discussion and editorial advice.

    FOOTNOTES

* 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. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: The Hormel Inst., University of Minnesota, 801 16th Ave. NE, Austin, MN 55912. Tel.: 507-437-9640; Fax: 507-437-9606; E-mail: zgdong@smig.net.

Published, JBC Papers in Press, May 9, 2000, DOI 10.1074/jbc.M909934199

    ABBREVIATIONS

The abbreviations used are: MAP, mitogen-activated protein; UVB, ultraviolet B; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated protein kinase; MEM, Eagle's minimal essential medium; FBS, fetal bovine serum; DNM, dominant negative mutant; MOPS, 3-(N-morpholino)propanesulfonic acid; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Van Holde, K. E. (1988) in Chromatin (Rich, A., ed) , pp. 111-148, Springer-Verlag, New York
2. Mahadevan, L. C., Willis, A. C., and Barratt, M. J. (1991) Cell 65, 775-783
3. Honda, B. M., Dixon, G. H., and Candido, E. P. (1975) J. Biol. Chem. 250, 8681-8685
4. Lee, D. Y., Hayes, J. J., Pruss, D., and Wolffe, A. P. (1993) Cell 72, 73-84
5. Luo, R. X., Postigo, A. A., and Dean, D. C. (1998) Cell 92, 463-473
6. Wei, Y., Mizzen, C. A, Cook, R. G., Gorovsky, M. A., and Allis, C. D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7480-7484
7. Tlkoo, K., and Ali, Z. (1997) Biochem. J. 322, 281-287
8. Marshall, C. J. (1994) Curr. Opin. Genet. Dev. 4, 82-89
9. Cano, E., Hazzalin, C. A., Kardalinou, E., Buckle, R. S., and Mahadevan, L. C. (1995) J. Cell Sci. 108, 3599-3609
10. Hibi, M., Lin, A., Smeal, T., Minden, A., and Karin, M. (1993) Genes Dev. 7, 2135-2148
11. Kyriakis, J. M., Banerjee, P., Nikolakaki, E., Dai, T., Rubie, E. A., Ahmad, M. F., Avruch, J., and Woodgett, J. R. (1994) Nature 369, 156-160
12. Huang, C., Ma, W.-Y., and Dong, Z. (1999) Oncogene 18, 2828-2835
13. Thomson, S., Clayton, A. L., Hazzalin, C. A., Rose, S., Barratt, M. J., and Mahadevan, L. C. (1999) EMBO J. 18, 4779-4793
14. Sassone-Corsi, P., Mizzen, C. A., Cheung, P., Crosio, C., Monaco, L., Jacquot, S., Hanauer, A., and Allis, C. D. (1999) Science 285, 886-891
15. Pumo, D. E., Wierzbicki, R., and Chiu, J. F. (1980) Biochemistry 19, 2362-2367
16. Wei, Y., Yu, L., Bowen, J., Gorovsky, M. A., and Allis, C. D. (1999) Cell 97, 99-109
17. Altheim, B. A., and Schultz, M. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 1345-1350
18. Huang, C., Ma, W.-Y., Maxiner, A., Sun, Y., and Dong, Z. (1999) J. Biol. Chem. 274, 12229-12235
19. Derijard, B., Hibi, M., Wu, I. H., Barrett, T., Su, B., Deng, T., Karin, M., and Davis, R. J. (1994) Cell 76, 1025-1037
20. Minden, A., Lin, A., McMahon, M., Lange-Carter, C., Derijard, B., Davis, R. J., Johnson, G. L., and Karin, M. (1994) Science 266, 1719-1723
21. Huang, C., Ma, W.-Y., Hanenberger, D., Cleary, M. P., Bowden, G. T., and Dong, Z. (1997) J. Biol. Chem. 272, 26325-26331
22. Dong, Z., Huang, C., Ma, W.-Y., Malewicz, B., Baumann, W. J., and Kiss, Z. (1998) Oncogene 17, 1845-1853
23. Ajiro, K., and Nishimoto, T. (1985) J. Biol. Chem. 260, 15379-15381
24. Ajiro, K., Nishimoto, T., and Takahashi, T. (1983) J. Biol. Chem. 258, 4534-4538
25. Balhorn, R., Jackson, V., Granner, D., and Chalkey, R. (1975) Biochemistry 14, 2504-2511
26. Gurley, L. R., Walters, R. A., and Tobey, R. A. (1975) J. Biol. Chem. 250, 3936-3944
27. Guo, X. W., Th'ng, J. P., Swank, R. A., Anderson, H. J., Tudan, C., Bradbury, E. M., and Roberge, M. (1995) EMBO J. 14, 976-985
28. Ajiro, K., Yoda, K., Utsumi, K., and Nishikawa, Y. (1996) J. Biol. Chem. 271, 13197-13201
29. Ajiro, K., Yasuda, H., and Tsuji, H. (1996) Eur. J. Biochem. 241, 923-930
30. Hendzel, M. J., Wei, Y., Mancini, M. A., Van Hooser, A., Ranalli, T., Brinkley, B. R., Bazett-Jones, D. P., and Allis, C. D. (1997) Chromosoma 106, 348-360
31. Gurley, L. R., D'Anna, J. A., Barham, S. S., Deaven, L. L., and Tobey, R. A. (1978) Eur. J. Biochem. 84, 1-15
32. Paulson, J. R., and Taylor, S. S. (1982) J. Biol. Chem. 257, 6064-6072
33. Davie, J. R., and Spencer, V. A. (1999) J. Cell. Biochem. Suppl. 32/33, 141-148
34. Chadee, D. N., Hendzel, M. J., Tylipski, C. P., Allis, C. D., Bazett-Jones, D. P., Wright, J. A., and Davie, J. R. (1999) J. Biol. Chem. 274, 24914-24920
35. Alessi, D. R., Cuenda, A., Cohen, P., Dudley, D. T., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 27489-27494
36. Smalley, K. S., Feniuk, W., Sellars, L. A., and Humphrey, P. P. (1999) Biochem. Biophys. Res. Commun. 263, 239-243
37. Lazar, D. F., Wiese, R. J., Brady, M. J., Mastick, C. C., Waters, S. B., Yamauchi, K., Pessin, J. E., Cuatrecasas, P., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 20801-20807
38. Oliver, R. H., Khan, S. M., Leung, B. S., and Yeh, J. (1999) Biochem. Biophys. Res. Commun. 263, 143-148
39. Pang, L., Sawada, T., Decker, S. J., and Saltiel, A. R. (1995) J. Biol. Chem. 270, 13585-13588
40. Garnovskaya, M. N., Mukhin, Y., and Raymond, J. R. (1998) Biochem. J. 330, 489-495
41. Oh-hashi, K., Maruyama, W., Yi, H., Takahashi, T., Naoi, M., and Isobe, K. (1999) Biochem. Biophys. Res. Commun. 263, 504-509
42. Tan, Y., Rouse, J., Zhang, A., Cariati, S., Cohen, P., and Comb, M. J. (1996) EMBO J. 15, 4629-4642
43. Lee, J. C., Laydon, J. T., McDonnell, P. C., Gallagher, T. F., Kumar, S., Green, D., McNulty, D., Blumenthal, M. J., Heys, J. R., Landvatter, S. W., Strickler, J. E., McLaughlin, M. M., Siemens, I. R., Fisher, S. M., Livi, G. P., White, J. R., Adams, J. L., and Young, P. R. (1994) Nature 372, 739-746
44. Robinson, M. J., Harkins, P. C., Zhang, J., Baer, R., Haycock, J. W., Cobb, M. H., and Goldsmith, E. J. (1996) Biochemistry 35, 5641-5646
45. Watts, R. G., Huang, C., Young, M. R., Li, J. J., Dong, Z., Pennie, W. D., and Colburn, N. H. (1998) Oncogene 17, 3493-3498
46. Bianchini, L., L'Allemain, G., and Pouyssegur, J. (1997) J. Biol. Chem. 272, 271-279
47. Huang, C., Ma, W. Y., Li, J., Goranson, A., and Dong, Z. (1999) J. Biol. Chem. 274, 14595-14601
48. Raingeaud, J., Gupta, S., Rogers, J. S., Dickens, M., Han, J., Ulevitch, R. J., and Davis, R. J. (1995) J. Biol. Chem. 270, 7420-7426
49. Rincon, M., Enslen, H., Raingeaud, J., Recht, M., Zapton, T., Su, M. S., Penix, L. A., Davis, R. J., and Flavell, R. A. (1998) EMBO J. 17, 2817-2829

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
Q. Zhao, B. M. Barakat, S. Qin, A. Ray, M. A. El-Mahdy, G. Wani, E.-S. Arafa, S. N. Mir, Q.-E. Wang, and A. A. Wani
The p38 Mitogen-activated Protein Kinase Augments Nucleotide Excision Repair by Mediating DDB2 Degradation and Chromatin Relaxation
J. Biol. Chem., November 21, 2008; 283(47): 32553 - 32561.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. S. Lange, D. L. Mitchell, and K. M. Vasquez
High mobility group protein B1 enhances DNA repair and chromatin modification after DNA damage
PNAS, July 29, 2008; 105(30): 10320 - 10325.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
Q. Ke, Q. Li, T. P. Ellen, H. Sun, and M. Costa
Nickel compounds induce phosphorylation of histone H3 at serine 10 by activating JNK-MAPK pathway
Carcinogenesis, June 1, 2008; 29(6): 1276 - 1281.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. V. Kondadasula, J. M. Roda, R. Parihar, J. Yu, A. Lehman, M. A. Caligiuri, S. Tridandapani, R. W. Burry, and W. E. Carson III
Colocalization of the IL-12 receptor and Fc{gamma}RIIIa to natural killer cell lipid rafts leads to activation of ERK and enhanced production of interferon-{gamma}
Blood, April 15, 2008; 111(8): 4173 - 4183.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
E. Roze, S. Betuing, C. Deyts, E. Marcon, K. Brami-Cherrier, C. Pages, S. Humbert, K. Merienne, and J. Caboche
Mitogen- and stress-activated protein kinase-1 deficiency is involved in expanded-huntingtin-induced transcriptional dysregulation and striatal death
FASEB J, April 1, 2008; 22(4): 1083 - 1093.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
T. Fujita, S. Ryser, I. Piuz, and W. Schlegel
Up-Regulation of P-TEFb by the MEK1-Extracellular Signal-Regulated Kinase Signaling Pathway Contributes to Stimulated Transcription Elongation of Immediate Early Genes in Neuroendocrine Cells
Mol. Cell. Biol., March 1, 2008; 28(5): 1630 - 1643.
[Abstract] [Full Text] [PDF]

Home page
 FASEB J.Home page
H. S. Choi, B. S. Kang, J.-H. Shim, Y.-Y. Cho, B. Y. Choi, A. M. Bode, and Z. Dong
Cot, a novel kinase of histone H3, induces cellular transformation through up-regulation of c-fos transcriptional activity
FASEB J, January 1, 2008; 22(1): 113 - 126.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
B. A. Burkhart, S. B. Kennett, and T. K. Archer
Osmotic Stress-dependent Repression Is Mediated by Histone H3 Phosphorylation and Chromatin Structure
J. Biol. Chem., February 16, 2007; 282(7): 4400 - 4407.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
O. Tenhunen, J. Rysa, M. Ilves, Y. Soini, H. Ruskoaho, and H. Leskinen
Identification of Cell Cycle Regulatory and Inflammatory Genes As Predominant Targets of p38 Mitogen-Activated Protein Kinase in the Heart
Circ. Res., September 1, 2006; 99(5): 485 - 493.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
P. Kannan-Thulasiraman, E. Katsoulidis, M. S. Tallman, J. S. C. Arthur, and L. C. Platanias
Activation of the Mitogen- and Stress-activated Kinase 1 by Arsenic Trioxide
J. Biol. Chem., August 11, 2006; 281(32): 22446 - 22452.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
W. Huang, S. Batra, S. Korrapati, V. Mishra, and K. D. Mehta
Selective Repression of Low-Density Lipoprotein Receptor Expression by SP600125: Coupling of Histone H3-Ser10 Phosphorylation and Sp1 Occupancy
Mol. Cell. Biol., February 15, 2006; 26(4): 1307 - 1317.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
M. H. Aziz, D. L. Wheeler, B. Bhamb, and A. K. Verma
Protein Kinase C {delta} Overexpressing Transgenic Mice Are Resistant to Chemically but not to UV Radiation-Induced Development of Squamous Cell Carcinomas: A Possible Link to Specific Cytokines and Cyclooxygenase-2
Cancer Res., January 15, 2006; 66(2): 713 - 722.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
R. Parihar, R. Trotta, J. M. Roda, A. K. Ferketich, S. Tridandapani, M. A. Caligiuri, and W. E. Carson III
Src Homology 2-Containing Inositol 5'-Phosphatase 1 Negatively Regulates IFN-{gamma} Production by Natural Killer Cells Stimulated with Antibody-Coated Tumor Cells and Interleukin-12
Cancer Res., October 1, 2005; 65(19): 9099 - 9107.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
B. Schmeck, W. Beermann, V. van Laak, J. Zahlten, B. Opitz, M. Witzenrath, A. C. Hocke, T. Chakraborty, M. Kracht, S. Rosseau, et al.
Intracellular Bacteria Differentially Regulated Endothelial Cytokine Release by MAPK-Dependent Histone Modification
J. Immunol., September 1, 2005; 175(5): 2843 - 2850.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
A. Nagai, T. Sato, N. Akimoto, A. Ito, and M. Sumida
Isolation and Identification of Histone H3 Protein Enriched in Microvesicles Secreted from Cultured Sebocytes
Endocrinology, June 1, 2005; 146(6): 2593 - 2601.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
Y.-Y. Cho, Z. He, Y. Zhang, H. S. Choi, F. Zhu, B. Y. Choi, B. S. Kang, W.-Y. Ma, A. M. Bode, and Z. Dong
The p53 Protein Is a Novel Substrate of Ribosomal S6 Kinase 2 and a Critical Intermediary for Ribosomal S6 Kinase 2 and Histone H3 Interaction
Cancer Res., May 1, 2005; 65(9): 3596 - 3603.
[Abstract] [Full Text] [PDF]