MAP Kinases Mediate UVB-induced Phosphorylation of Histone H3
at Serine 28*
Shuping
Zhong
,
Yiguo
Zhang
,
Cheryl
Jansen
,
Hidemasa
Goto§,
Masaki
Inagaki§, and
Zigang
Dong
¶
From the
Hormel Institute, University of Minnesota,
Austin, Minnesota 55912 and the § Laboratory of
Biochemistry, Aichi Cancer Center Research Institute, 1-1 Kanokoden,
Chikusa-ku, aichi464-8681, Japan
Received for publication, December 4, 2000, and in revised form, January 23, 2001
 |
ABSTRACT |
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 28 in 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 in
Jnk1
/
cells but not in
Jnk2
/
cells. These results suggest that
UVB-induced H3 phosphorylation at serine 28 may be mediated by
mitogen-activated protein kinases.
 |
INTRODUCTION |
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 (1). The five types of histones (H1, H2A, H2B, H3,
and H4) fall into two main groups: core histones and linker histones.
Core histones are wrapped by DNA as octamers, consisting of two H2A-H2B
dimers and a tetramer of H3-H4 (2-4). A number of studies previously
reported that histones can be modified by acetylation and
phosphorylation (5-7), and the subsequent function(s) of these
modifications has begun to be understood (8-16). The histone
modifications may alter chromatin structure by influencing histone-DNA
and histone-histone contacts (17-19). The level of acetylated histone
is regulated by histone acetyltransferases and histone deacetylases
(10, 20, 21). The p300/CREB-binding protein histone
acetyltransferase was described initially as a transcriptional
coactivator that functions by interacting with a wide variety of
enhancer-binding proteins (22), and the histone deacetylase/Rpd3 family
of histone deacetylases is correlated with transcriptional regulatory
proteins (23, 24).
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 (15,
25, 26). 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 (5, 10, 15, 27-29). H3 phosphorylation at serine
10 occurs concurrently with the transcriptional activation of the early
genes c-fos and c-jun (25, 26), and induction of
ras 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 (6, 25, 26, 32). The pathway
responsible for mediating H3 phosphorylation at serine 10 depends on
the type of stimulation (25, 26, 32), and phosphorylation of serine 10 in histone H3 is also linked functionally in vitro and
in 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 (34). 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.
 |
MATERIALS AND METHODS |
Reagents and Antibodies--
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 (34). Active ERK1, ERK2, p38 kinase, JNK1, and
JNK2 were from Upstate Biotechnology, Inc.
Phosphorylation Assay of Histone H3 in
Vitro--
Phosphorylation of histone H3 by activated ERK1, ERK2, p38
kinase, JNK1, or JNK2 was carried out as described previously (32, 35).
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
-glycerophosphate, 2 mM dithiothreitol,
0.1 mM Na3VO4, 10 mM
MgCl2) 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 (32, 34).
UVB Irradiation--
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.
Acid-soluble Protein Extraction--
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 (32), 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 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 (36), and the
acid-soluble proteins were stored at
20 °C.
Assay of Phosphorylated H3--
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.).
 |
RESULTS |
Phosphorylation of Histone H3 in Vitro--
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 (34). 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 (32, 35). Phosphorylated H3 at serine 28 was
detected by a specific monoclonal antibody as before (32, 34). The
results show that pure histone H3 at serine 28 was phosphorylated
strongly by ERK1 (Fig. 1A),
ERK2 (Fig. 1B), or p38 kinase (Fig. 1C) and to a
comparatively lesser degree by JNK1 (Fig. 1D) or JNK2 (Fig.
1E) in vitro. Similar results were found also by
using chromatin as substrate for these MAP kinases (data not
shown).

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Fig. 1.
Phosphorylation of H3 at serine 28 by active
MAP kinases, ERK1, ERK2, p38 kinase, JNK1, or JNK2 occurs in
vitro. ERK1, ERK2, p38 kinase, JNK1, or JNK2 was
incubated with pure histone H3 protein at 30 °C for 45 min in the
presence of 200 µM ATP to facilitate phosphorylation.
Phosphorylated H3 at serine 28 was detected with a specific antibody
(34). Total H3 protein was detected in a parallel blot with
anti-histone H3 from Upstate Biotechnology, Inc. A, H3 at
serine 28 was phosphorylated by active ERK1. B, H3 at serine
28 was phosphorylated by active ERK2. C, H3 at serine 28 was
phosphorylated by active p38 kinase. D, H3 at serine 28 was
phosphorylated by active JNK1. E, H3 at serine 28 was
phosphorylated by active JNK2.
|
|
Phosphorylation of Histone H3 at Serine 28 after UVB
Irradiation--
Our previous study showed that UVB could induce
phosphorylation of histone H3 at serine 10 in JB6 Cl 41 cells (32). 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 (32, 34). 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.
2A). H3 phosphorylation at serine 28 was greater at 30 or 60 min than at 15 or 120 min after UVB irradiation (Fig. 2B). We found previously that phosphorylation of H3 at serine 10 was higher
at 15 or 30 min than at 60 min (32), and the level of phosphorylated H3
at serine 28 was higher at 60 min compared with phosphorylation at
serine 10 at 60 min (32) 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.

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Fig. 2.
UVB induces phosphorylation of H3 at
serine 28 in vivo. A, dose-response
study: JB6 Cl 41 cytomegalovirus-neo cells were starved by
incubating them 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% FBS MEM, after which time they were exposed to 1, 2, 4, or 6 kJ/m2 of UVB and then incubated for an additional 30 min.
Phosphorylation of H3 at serine 28 was determined by Western blot
analysis of acid-soluble nuclear proteins resolved by SDS-PAGE using a
specific antibody as in Fig. 1. Total H3 protein was detected in a
parallel blot with anti-histone H3 from Upstate Biotechnology, Inc.
B, time-course study: cells were treated as in A
but were exposed to UVB (4 kJ/m2) and incubated an
additional 15, 30, 60, or 120 min. Phosphorylation of H3 at serine 28 and total H3 protein were determined as indicated above. The
arrows denote the position of phospho-H3 at serine 28 and
total H3 protein.
|
|
Inhibition of UVB-induced Phosphorylation of Histone H3 at Serine
28 by PD 98059 and SB 202190--
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 (32).
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 (37-39). Previous studies demonstrated that PD 98059 specifically inhibits the activation and phosphorylation of ERKs (6,
37-40), and 50 µM PD 98059 totally blocks activation of
ERKs (22) but not JNKs or p38 kinases (40, 41). Our results showed that
25 µM PD 98059 markedly inhibited UVB-induced
phosphorylation of H3 at serine 28 (Fig.
3A). This result implies that
ERKs may be involved in the UVB-induced phosphorylation of H3 at serine
28. SB 202190 is a specific inhibitor of p38 kinase (26, 40, 41), and
pretreatment of cells with 0.5-4 µM SB 202190 almost
totally blocked UVB-induced phosphorylation of H3 at serine 28 (Fig.
3B). A high concentration of SB 202190 (40 µM)
can inhibit activation of ERKs, but 10 µM SB 202190 has
almost no effect on the phosphorylation of ERKs (39). Therefore, we used low concentrations of SB 202190 (0.5-4 µM), which
selectively blocks activation of p38 kinase, to inhibit p38-mediated H3
phosphorylation at serine 28. The above data indicate that UVB-induced
phosphorylation of H3 at serine 28 may be mediated by ERKs and p38
kinase in vivo.

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Fig. 3.
PD 98059 and SB 202190 inhibit the
UVB-induced phosphorylation of H3 at serine 28 in JB6 Cl 41 cells.
Cells were treated with various concentrations of PD 98059 or SB 202190 for 1 h and then exposed to UVB (4 kJ/m2).
Phosphorylated and total histone H3 proteins were detected as indicated
in Fig. 1. A, PD 98059, a specific inhibitor for
MEK1 kinase, blocked UVB-induced phosphorylation of H3 at serine 28. B, SB 202190, a specific inhibitor for p38 kinase, blocked
UVB-induced phosphorylation of H3 at serine 28. The arrows
denote the position of phospho-H3 at serine 28 and total H3
protein.
|
|
Inhibition of UVB-induced Phosphorylation of Histone H3 at Serine
28 by Expression of DNM ERK2, DNM p38 Kinase, and DNM
JNK1--
Previous studies showed that overexpression of DNM ERK2, DNM
p38 kinase, or DNM JNK1 markedly inhibited activation of endogenous ERKs (35, 40, 42, 43), p38 kinase (44, 45), or JNKs (46), 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.
4A), 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. 5A) was stronger than that by DNM p38 (Fig.
5B) or DNM JNK1 cells (Fig. 5C).

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Fig. 4.
UVB-induced phosphorylation of H3 at serine
28 is blocked by DNM ERK2, DNM p38 kinase, or DNM JNK1.
Phosphorylated and total histone H3 proteins were detected as indicated
in Fig. 1. A, phosphorylation of H3 at serine 28 was induced
strongly by UVB in JB6 Cl 41 cytomegalovirus-neo cells, but UVB-induced
H3 phosphorylation at serine 28 was completely blocked in JB6 Cl 41 DNM
ERK2 cells. UVB-induced H3 phosphorylation at serine 28 was blocked
markedly in JB6 Cl 41 DNM p38 cells (B) and in JB6 Cl 41 DNM
JNK1 cells (C) compared with JB6 Cl 41 cytomegalovirus-neo
cells. The arrows denote the position of phospho-H3 at
serine 28 and total H3 protein.
|
|

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Fig. 5.
Dose 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.
|
|
Inhibition of Phosphorylation of Histone H3 at Serine 28 in Jnk1
but Not Jnk2 Knockout Cells--
We also used Jnk1
(Jnk1
/
) and Jnk2
(Jnk2
/
) knockout cells and Jnk
wild-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 in
Jnk1
/
cells (Fig.
6, A and C) but not
in Jnk2
/
cells (Fig. 6, B and
D) 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 in
Jnk1
/
and Jnk2
/
cells (32).

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Fig. 6.
UVB-induced phosphorylation of H3 at serine
28 is blocked in Jnk1 / cells but not
in Jnk2 / cells.
Jnks+/+, Jnk1 / , and
Jnk2 / cells were starved by incubating in
0.1% FBS Dulbecco's modified Eagle's medium for 48 h at
37 °C in a 5% CO2 atmosphere. Cells were incubated for
2 h in fresh 0.1% FBS Dulbecco's modified Eagle's medium, after
which time they were exposed to UVB (4 kJ/m2) and incubated
an additional 15, 30, or 60 min for the time-course studies
(A and B) or exposed to UVB at 1, 2, or 4 kJ/m2 for dose-response studies (C and
D). Phosphorylation of H3 at serine 28 and total H3 protein
were determined by Western blot analysis of acid-soluble nuclear
proteins resolved by SDS-PAGE as described for Fig. 1. Phosphorylation
of H3 at serine 28 was induced strongly by UVB in
Jnks+/+ (A-D).
Jnk1 / cells seem to inhibit UVB-induced H3
phosphorylation at serine 28 markedly (A and C),
but Jnk2 / cells have little effect on
UVB-induced H3 phosphorylation at serine 28 (B and
D) compared with Jnks+/+ cells. The
arrows denote the position of phospho-H3 at serine 28 and
total H3 protein.
|
|
 |
DISCUSSION |
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 in
Jnk2
/
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
(19-22), and acetylation of H3/H4 is related closely to
transcriptional regulation (10, 23, 24). However, mechanisms regarding
phosphorylation of histone H3 at serine 10 and serine 28 have attracted
a great deal of interest in recent years. Phosphorylation of histone H3
at serine 10 is correlated tightly with mitotic chromosome condensation
and segregation in mammals (28, 47, 48), tetrahymena (15, 30), and
Xenopus (27). Chromosome segregation is required for
phosphorylation of histone H3 at serine 10 mediated by IpI1/aurora
kinase and Glc7/PP1 in Saccharomyces cerevisiae and
Caenorhabditis elegans (11). Histone H3 at serine 10 is
phosphorylated by NIMA kinase in Aspergillus nidulans
during mitosis (12). Histone H3 phosphorylation at serine 10 is related
closely to transcriptional activation of mitogen-stimulated
immediate-early response genes such as c-fos and
c-jun in mammalian cells (6, 25, 26). This
mitogen-stimulated phosphorylation of histone H3 at serine 10 was shown
to be mediated by RSK2 or MSK1 (25, 26), whereas UVB-induced histone H3
phosphorylation at serine 10 was found to be mediated by ERKs and p38
kinases (32). A recent study showed that phosphorylation of histone H3
at serine 28 also occurred in mitotic chromosome condensation in
mammalian cells (34). However, the pathway that mediates phosphorylation of histone H3 at serine 28 is unknown. In this study we
investigated the role of MAP kinases in mediating UVB-induced phosphorylation of histone H3 at serine 28. ERK2 was more effective than ERK1, p38 kinases, or JNKs in phosphorylating histone H3 at serine
28 in vitro (Fig. 1, A-E). PD 98059, a specific inhibitor of MEK1 (37-39), and SB 202190, a specific
inhibitor of p38 kinase (26, 40, 41), inhibited UVB-induced
phosphorylation of histone H3 at serine 28 in JB6 Cl 41 cells (Fig. 3,
A and B, respectively); expression of DNM ERK2
completely blocked the phosphorylation of histone H3 at serine 28 (Fig.
4A). Inhibition of phosphorylation of histone H3 at serine
28 by expression of DNM ERKs (Fig. 4A) is more marked than
inhibition by either DNM p38 kinase (Fig. 4B) or DNM JNK1
(Fig. 4C). This implies that ERKs may play a more important
role in UVB-induced phosphorylation of histone H3 serine 28 than p38
kinase or JNKs. JNK1 and JNK2 also phosphorylated histone H3 at serine
28 in vitro, but compared with JNK2, JNK1 phosphorylation of
histone H3 at serine 28 was weaker (Fig. 1, D and
E). Moreover, expression of DNM JNK1 inhibited UVB-induced phosphorylation of histone H3 at serine 28 (Figs. 4C and
5C), and phosphorylation of histone H3 was blocked in
Jnk1
/
cells (Fig. 6, A and
C) but not in Jnk2
/
cells (Fig.
6, B and D) compared with
Jnk+/+ cells (Fig. 6,
A-D). These results indicate that JNK1 indeed is
involved in UVB-induced phosphorylation of histone H3 at serine 28 in 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.
2B), whereas UVB-induced phosphorylation of histone H3 at
serine 10 is highest at 30 min after UVB irradiation (32). 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. 10), our data indicate that the kinases responsible for phosphorylation of each of these serine residues of histone H3 are
different. These phosphorylation responses to different signals are
likely to have distinct effects on H3 function during chromatin remodeling and gene expression.
 |
ACKNOWLEDGEMENT |
We thank Ann Bode for scientific discussion
and editorial advice.
 |
FOOTNOTES |
*
This work was supported by the Hormel Foundation and Grants
CA77646 and CA74916 from the NCI, National Institutes of Health.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.
¶
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, January 24, 2001, DOI 10.1074/jbc.M010931200
 |
ABBREVIATIONS |
The abbreviations used are:
MAP, mitogen-activated protein;
MEM, minimal Eagle's essential
medium;
FBS, fetal bovine serum;
ERK, extracellular
signal-regulated protein kinase;
JNK, c-Jun NH2-terminal
kinase;
PAGE, polyacrylamide gel electrophoresis;
DNM, dominant
negative mutant.
 |
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