Cell Cycle- and Chromatin Binding State-dependent
Phosphorylation of Human MCM Heterohexameric Complexes
A ROLE FOR cdc2 KINASE*
Masatoshi
Fujita
§,
Chieko
Yamada
,
Tatsuya
Tsurumi
,
Fumio
Hanaoka¶,
Kaori
Matsuzawa
**, and
Masaki
Inagaki
From the
Laboratories of Viral Oncology and
Biochemistry, Research Institute, Aichi Cancer Center,
Chikusa-ku, Nagoya, 464, Japan, the ¶ Institute for Molecular and
Cellular Biology, Osaka University, Suita, Osaka 565, and the
** Institute of Immunological Science, Hokkaido University,
Sapporo 060, Japan
 |
ABSTRACT |
The mammalian MCM protein family, presently with
six members, exists in the nuclei in two forms, chromatin-bound and
unbound. The former dissociates from chromatin with progression through the S phase. Recently, we have established a procedure to isolate chromatin-bound and unbound complexes containing all six human MCM
(hMCM) proteins by immunoprecipitation. In the present study, we
applied this procedure to HeLa cells synchronized in each of the
G1, S, and G2/M phases and could detect
hMCM heterohexameric complexes in all three. In addition, depending on
the cell cycle and the state of chromatin association, hMCM2 and 4 in
the complexes were found to variously change their phosphorylation
states. Concentrating attention on G2/M phase
hyperphosphorylation, we found hMCM2 and 4 in the complexes to be good
substrates for cdc2/cyclin B in vitro. Furthermore, when
cdc2 kinase was inactivated in temperature-sensitive mutant murine
FT210 cells, the G2/M hyperphosphorylation of the murine
MCM2 and MCM4 and release of the MCMs from chromatin in the
G2 phase were severely impaired. Taken together, the data suggest that the six mammalian MCM proteins function and undergo cell
cycle-dependent regulation as heterohexameric complexes and that phosphorylation of the complexes by cdc2 kinase may be one of
mechanisms negatively regulating the MCM complex-chromatin association.
 |
INTRODUCTION |
The MCM protein family, presently with six members, was originally
identified from its involvement in the initiation of DNA replication at
autonomously replicating sequences in budding yeast (1-7) and later
found to be conserved through eukaryotes (8-16). Although definite
functions of the MCM proteins remain largely unknown, they have been
implicated in the regulatory machinery allowing DNA to replicate only
once during the S phase (reviewed in Refs. 17 and 18).
In mammalian cells, MCM proteins are present in the nuclei in two
different forms, one extractable by nonionic detergents and the other
tightly associated with chromatin, which is resistant to such
extraction. The level of mammalian MCM does not greatly vary during the
cell cycle, but the chromatin-bound form gradually becomes dissociated
with progression through the S phase (19-23). It is now assumed that
the bound form is associated with prereplicative chromatin and released
at the time of replication; the soluble form existing abundantly in
G2 nuclei is considered inactive and no longer capable of
binding to chromatin. At least in budding yeast, the chromatin regions
to which MCM7 binds during the G1 phase contain the
replication origins (24). However, details of the mode of MCM-chromatin
binding remain unclear. In budding yeast and the Xenopus egg
extract system, it has been shown that MCM-chromatin binding is
regulated through multiple mechanisms, while MCM binds to chromatin
depending on CDC6 and the origin recognition complex (24-27), where
the binding is negatively regulated by both S phase and mitotic CDKs
(24, 28-30). However, there is so far no direct evidence as to whether
these regulators for MCM also function in the mammalian somatic cell
cycle.
Whereas the MCM proteins share substantial homology, it has been
suggested that each of the six is indispensable for DNA replication in
budding yeast (2-7), Xenopus egg extracts (31, 32), and mammalian somatic cells (19, 21, 33). On the other hand, physical
interactions among MCM proteins have been found in budding yeast (34),
Drosophila and Xenopus egg extract (13-15, 29, 31, 32, 35, 36), and murine and human cells (20, 37-41). Recently, we
have established a procedure to isolate chromatin-bound and unbound
complexes containing all six human MCM
(hMCM)1 proteins by
immunoprecipitation with anti-hMCM antibodies (41). However, it
remained to be elucidated whether there was any change in the complex
formation profile during the cell cycle. In the present study, we
therefore applied the immunoprecipitation procedure to synchronized
HeLa cells. The results indicate that the six hMCM members may exist as
heterohexameric complexes throughout the cell cycle. In addition, it
was also found that, depending on the cell cycle and the states of
chromatin association, hMCM4 and 2 proteins in the complexes variously
change their phosphorylation states. Furthermore, we could show that
cdc2 kinase plays a role in the G2/M phase
hyperphosphorylation of mammalian MCM2 and 4 and in regulation of the
MCM-chromatin association.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Synchronization--
HeLa cells were grown in
Dulbecco's modified Eagle's medium with 5% fetal calf serum. The
cells were synchronized at G2/M phase by treatment with 50 ng/ml nocodazole for 16-18 h. Cells arrested in early S phase were
obtained by 2.5 mM hydroxyurea or 15 µM
aphidicolin treatment for 18-20 h. G1 HeLa cells were obtained as follows. Cells in the log phase were first arrested in
early S phase by aphidicolin treatment and then released. After 10-12
h, mitotic cells were collected by mitotic shake-off, replated, and
cultured for a further 4-6 h.
FM3A and FT210 cells (42) were maintained at 32 °C in RPMI 1640 buffered with 25 mM Hepes and supplemented with 10% fetal calf serum. For synchronization experiments, the cells (5 × 105/ml) were first arrested in early S phase by 15 µM aphidicolin treatment for 16 h at 32 °C and
then released to progress through S phase in the presence of 50 ng/ml
nocodazole, either at 32 or 39 °C.
To monitor the cell cycle, cells were stained with propidium iodide
using a CycleTest kit (Becton Dickinson) according to the instructions
of the manufacturer. Cells (1 × 104) were analyzed
with a FACScan (Becton Dickinson).
Preparation of Cell Extracts--
If not stated otherwise, the
buffer used for preparation of cell lysates and the immunoprecipitation
procedure was modified CSK buffer containing 0.1% Triton X-100 and 0.1 mM ATP (0.1%TX-100mCSK; Ref. 41). Where necessary, 20 mM
-glycerophosphate, 200 µM Na3VO4, 10 mM NaF, and 0.2 µM calyculin A were added to the buffer as phosphatase
inhibitors, and 1 µM staurosporine was added as a kinase
inhibitor.
The Triton X-100-extractable and nuclear pellet fractions were prepared
as follows. HeLa cells cultured in 100-mm plates or FM3A or FT210 cells
in 20 ml of culture were washed with ice-cold phosphate-buffered
saline, lysed for 20 min on ice with 1 ml of ice-cold 0.1%TX-100mCSK,
and subjected to low speed centrifugation. The supernatants were
further clarified by centrifugation at 15,000 rpm for 5 min to obtain
Triton X-100-extractable fractions. Triton X-100-extracted nuclei were
washed once with the buffer and either directly added to SDS-sample
buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 5%
-mercaptoethanol, 10% glycerol, 0.01% bromphenol blue) or digested
with 1000 units/ml DNase I (10 units/µl, RNase-free, Boehringer
Mannheim) in 1 ml of 0.1%TX-100mCSK containing 1 mM ATP at
25 °C for 30 min to obtain DNase I-released fractions (41).
Immunoprecipitation--
Immunoprecipitation with anti-hMCM7
(hCDC47) or anti-murine MCM3 (mMCM3) antibodies was performed as
described previously (41). The immunoprecipitates were boiled in
SDS-sample buffer and subjected to 10% (acrylamide 10, bis-acrylamide 0.1) SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) followed by silver staining or immunoblotting.
Immunoblotting--
Immunoblotting was performed as described
previously (22, 41). The protein blots were incubated with appropriate
first antibodies for 1 h at room temperature; purified anti-hMCM7
antibodies at 1 µg/ml (22); rabbit anti-mMCM4 antiserum (20) at 1:500 dilution; rabbit anti-Xenopus MCM2 (XMCM2) antiserum (43) at 1:500 dilution; and anti-cdc2-phosphorylated vimentin mouse monoclonal antibody (4A4; Ref. 44) at 1:3000 dilution of the culture supernatant. The blots were then probed with peroxidase-labeled goat anti-rabbit IgG
or anti-mouse IgG antibodies (Zymed) and visualized using the ECL
system (Amersham Pharmacia Biotech).
-Phosphatase or cdc2 Kinase Treatment of the
Immunoprecipitates--
Immunoprecipitates were resuspended in 25 µl
of phosphatase buffer (50 mM Tris-HCl, pH 7.8, 5 mM dithiothreitol, 2 mM MnCl2, 0.5 mM phenylmethylsulfonyl fluoride) or kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 1 mM dithiothreitol, 100 µM ATP, 1 µM calyculin A, 0.5 mM phenylmethylsulfonyl fluoride) and incubated with 80 units of
-phosphatase (New England Biolabs) for 30 min at 30 °C
or 50 units of cdc2/cyclin B (New England Biolabs) for 60 min at
30 °C. The reactions were stopped by the addition of 2× SDS-sample
buffer and processed for SDS-PAGE.
Phosphorylation Analysis of G2/M Phase FT210 Cells by
Orthophosphate Labeling--
FT210 cells (2.5 × 106)
arrested in early S phase as described above were released in 2 ml of
phosphate-free RPMI 1640 medium supplemented with 10% dialyzed fetal
calf serum and 50 ng/ml nocodazole either at 32 or 39 °C. After
8 h, 1 mCi [32P]orthophosphate was added, and the
cells were further incubated for 2 h either at 32 °C or
39 °C. The Triton X-100-extractable fractions were prepared from the
cells as described above with 0.1%TX-100mCSK containing phosphatase
inhibitors and subjected to immunoprecipitation with anti-hMCM7 or
anti-mMCM3 antibodies. In these experiments, the washing buffer was
changed to 200 mM NaCl TET buffer (50 mM
Tris-HCl, pH 7.5, 1 mM EDTA, 0.1% Triton X-100, 200 mM NaCl) containing phosphatase inhibitors.
32P radiography was performed with a Bio-Imaging
analyzer BAS2000 (Fuji, Japan).
 |
RESULTS |
Six hMCM Members Participate in Heterohexameric Complexes
throughout the Cell Cycle, whether Bound to Chromatin or
Not--
Triton X-100-extractable fractions prepared from HeLa cells
synchronized in G1, S, and G2/M phase, and
DNase I-released fractions from G1 and S phase cells were
subjected to immunoprecipitation with anti-hMCM7 antibodies, and the
precipitates were subjected to SDS-PAGE followed by silver staining
(Fig. 1) and to Western blotting with
antibodies against each of the hMCMs (data not shown except for hMCM2
and 4 in Fig. 2; see also Ref 41). All
six hMCM proteins were detectable in the precipitates of all the
fractions tested. On the other hand, the hMCM2 and 4 proteins variously changed their electrophoretic mobility depending on the cell cycle and
the state of chromatin association. Because such changes in the
mobility could be due to phosphorylation, we treated the
immunoprecipitates with
-phosphatase, by which hMCM2 and 4 showing
aberrant mobility disappeared and the levels of p125 hMCM2 and p97
hMCM4 concomitantly increased. Mobility of the other hMCM proteins was
not changed, and treatment of the precipitates with phosphatase buffer
alone did not alter the mobility of any hMCM proteins (data not shown). As we reported previously (22, 41), hMCM7 resolved into a doublet on
SDS-PAGE, by an unknown mechanism. The results after
-phosphatase
treatment demonstrated that in all the fractions tested, anti-hMCM7
antibodies co-precipitated the other hMCMs with hMCM7 with roughly
equivalent stoichiometry (Fig. 1). Therefore, we suggest that the six
hMCM members may exist as heterohexameric complexes throughout cell
cycle, whether bound to chromatin or not.

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Fig. 1.
Detection of hMCM heterohexameric complexes
in HeLa cells synchronized in G1, S, and G2/M
phase. A, G1 phase HeLa cells were obtained
4-6 h after replating mitotic cells. Triton X-100-soluble fractions
and chromatin-bound fractions solubilized with DNase I digestion in the
presence of ATP were prepared and immunoprecipitated with a control
rabbit immunoglobulin or anti-hMCM7 antibodies. The precipitates were
separated by SDS-PAGE directly or after treatment with -phosphatase
(PPase), followed by silver staining. B, S phase
HeLa cells were obtained by treatment with hydroxyurea or aphidicolin
for 18-20 h. Triton X-100-soluble fractions and chromatin-bound
fractions were processed as in panel A.
C, G2/M phase HeLa cells were obtained by
treatment with nocodazole for 16-18 h. Triton X-100-soluble fractions
were processed as in panel A.
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Fig. 2.
Cell cycle- and chromatin binding
state-dependent phosphorylation of hMCM4 and 2 proteins in
hMCM hexamers. The immunoprecipitates obtained with anti-hMCM7
antibodies as described in Fig. 1 were immunoblotted with anti-mMCM4
(A) or anti-XMCM2 (B) antibodies. Results from
short exposure of blots with anti-mMCM4 are also shown in panel
A to demonstrate hMCM4 doublet bands detected in soluble fractions
from the G1 and S phase cells. PPase;
-phosphatase treatment.
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hMCM2 and 4 Proteins in the Complexes Undergo Complicated
Phosphorylation Depending on the Cell Cycle and the State of Chromatin
Association--
As described above, the hMCM4 protein in the
complexes was found to variously change its electrophoretic mobility
depending on the cell cycle and the state of chromatin association
(Figs. 1 and 2A). In the G1 cells, most of the
chromatin-bound hMCM4 migrated to the same position as that of
dephosphorylated hMCM4, whereas most of the soluble hMCM4 displayed
slower mobility. In the S phase cells, although the migration pattern
of the soluble hMCM4 was unchanged compared with that in G1
phase cells, the migration pattern of the chromatin-bound hMCM4 was
obviously changed; the forms showing slower mobility were increased. In
the G2/M phase cells, most of the hMCM4 displayed the
slowest mobility (apparent molecular mass was ~115 kDa). All of these
extra bands disappeared with
-phosphatase treatment. Although an
unchanged mobility pattern does not always mean an unchanged
phosphorylation state, these data suggest the following for the
phosphorylation of hMCM4 in hMCM complexes: (1) in the G1
phase, most of the chromatin-bound hMCM4 is in the underphosphorylated
form (if not unphosphorylated) while most soluble hMCM4 is in the more
phosphorylated form; (2) during progression from G1 to S
phase, although the phosphorylation state of the soluble hMCM4 is
apparently unchanged, the chromatin-bound form becomes more extensively
phosphorylated; and (3) most of the hMCM4 in G2/M phase is
in the hyperphosphorylated form. The buffer used for these
immunoprecipitation studies contained ATP to facilitate
chromatin-complex binding and complex stability, and the
chromatin-bound complexes were solubilized by incubation with DNase I
at 25 °C (41). Therefore, it is possible that the observed
phosphorylation pattern of hMCM4 is modified in vitro. We
therefore repeated the experiments in the presence of phosphatase inhibitors and a kinase inhibitor and in the absence of ATP, and we
solubilized the bound complexes by salt extraction on ice. Although the
hMCM heterohexameric complexes are partially disrupted under such
conditions (41), the same results for hMCM4 phosphorylation were
obtained (data not shown).
As previously reported (21), change in mobility because of
phosphorylation during the cell cycle was also observed for hMCM2 in
the complexes. In this case, phosphorylated forms displayed faster
mobility on SDS-PAGE (Figs. 1 and 2B). The changing pattern of the hMCM2 phosphorylation appeared to be essentially similar to that
of hMCM4.
Identification of cdc2/cyclin B as a Regulator of Mammalian MCM2
and 4 Hyperphosphorylation in G2/M Phase--
As an
initial step to analyze complex phosphorylation, we focused on the
G2/M phase hyperphosphorylation of the MCM2 and 4 proteins,
leading to their remarkable mobility shift. One possible candidate for
the responsible kinase(s) is cdc2/cyclin B, which is maximally active
in late G2/M phase. As shown in Fig.
3, it was found that recombinant
cdc2/cyclin B phosphorylated in vitro the hMCM2 and hMCM4 in
the hMCM complexes immunoprecipitated from S phase HeLa cells and
promoted a shift in their mobility. The positions of the shifted bands
were almost the same as those of hMCM2 and 4 in the G2/M
phase (see also Figs. 1C and 2). Mobility of the other hMCMs
was not changed by the treatment. These data show that hMCM2 and hMCM4
proteins in the hMCM complexes are excellent substrates for cdc2/cyclin
B in vitro.

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Fig. 3.
In vitro phosphorylation of hMCM4 and 2 proteins in the hMCM complexes by recombinant cdc2/cyclin B. Chromatin-bound or unbound hMCM hexameric complexes were
immunoprecipitated with anti-hMCM7 antibodies from S phase HeLa cells.
The precipitates were incubated with or without recombinant cdc2/cyclin
B and were subjected to SDS-PAGE followed by silver staining or
immunoblotting with anti-mMCM4 or anti-XMCM2 antibodies.
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We examined the in vivo role of cdc2/cyclin B in
G2/M hyperphosphorylation of mammalian MCM2 and 4 using
murine cdc2 kinase temperature-sensitive mutant FT210 cells and their
parental FM3A cells (42, 45-47). Cells were arrested at early S phase
by aphidicolin, and then released at a permissive (32 °C) or a
nonpermissive temperature (39 °C) in the presence of nocodazole to
prevent cells entering the next G1 phase. Analysis of the
DNA content and the mitotic index showed that there were no obvious
differences in cell cycle progression among FM3A cells, and FT210 cells
cultured at a permissive temperature; approximately 70-80% of the
cells were arrested in early S phase. 4 h after release, they were
in mid S phase; at 8 h, they were in late S/G2 phase;
and at 12 h, they were arrested in M phase (Fig.
4). However, when FT210 cells were
cultured at a nonpermissive temperature, although they similarly
reached late S/G2 phase 8 h after release, even at
12 h there were few mitotic cells (Fig. 4), in agreement with the
previously reported G2-arrest phenotype (45-47).

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Fig. 4.
S phase progression of murine cdc2
temperature-sensitive mutant FT210 and parental FM3A cells at
permissive or nonpermissive temperatures. Cells were synchronized
in early S phase by treatment with aphidicolin at 32 °C for 16 h. After washing, cells were divided and cultured at 32 or 39 °C in
the presence of nocodazole. Cells were collected at indicated times
after release and subjected to flow cytometry to determine cell cycle
progression. The percentages of cells with mitotic chromosomes were
also determined by visual inspection.
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Total cell lysates, Triton X-100-extractable fractions, and nuclear
pellets were prepared from these cells. First, the total cell lysates
were immunoblotted with monoclonal antibody 4A4 specific for
cdc2-phosphorylated vimentin (44) (Fig.
5A). The data showed cdc2
activation to occur around the G2 phase (8 h after release) and become maximum in the M phase (12 h after release) in FM3A cells,
and FT210 cells cultured at a permissive temperature. However, in FT210
cells cultured at a nonpermissive temperature, virtually no activation
of cdc2 was detected, as expected. Then the Triton X-100-extractable
and the nuclear fractions were immunoblotted with anti-mMCM4 antibodies
(Fig. 5B). In addition to the main band of mMCM4, the
antibodies also recognized two bands showing different mobility in FM3A
cells, and FT210 cells cultured at a permissive temperature; one was
observed in the chromatin-bound form in the S phase cells and displayed
slower mobility, and the other was observed in the soluble form in the
G2/M phase cells and displayed the slowest mobility. Both
of these two bands disappeared with phosphatase treatment, and the
former was undetectable in G1 phase cells (data not shown).
These data show that mMCM4 also has at least two counterparts of the
isoforms because of phosphorylation observed for hMCM4, the S phase-
and chromatin-bound form-specific phosphorylated form and the
hyperphosphorylated form in the G2/M phase. Interestingly,
the latter became almost completely undetectable when FT210 cells were
cultured at a nonpermissive temperature (Fig. 5B).
Similarly, immunoblot analysis with anti-XMCM2 antibodies demonstrated
the presence of hyperphosphorylated mMCM2 showing fastest mobility in
the G2/M phase cells and its disappearance in
cdc2-inactivated FT210 cells (Fig. 5C).

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Fig. 5.
Inhibition of G2/M phase
hyperphosphorylation of mMCM2 and 4 and of dissociation of mMCM
proteins from chromatin in FT210 cells at a nonpermissive
temperature. A, vimentin phosphorylation by cdc2 during
the cell cycle progression of FT210 and FM3A cells at permissive or
nonpermissive temperatures. Cells prepared as described in Fig. 4 were
lysed in SDS-sample buffer, and the lysates were subjected to
immunoblotting with cdc2-phosphorylated vimentin-specific monoclonal
antibody 4A4. B and C, cells prepared as in Fig.
4 were extracted with Triton X-100 to separate chromatin-bound and
-unbound fractions. mMCM4 and mMCM7 in both fractions (B)
and mMCM2 in the soluble fractions (C) were detected by
immunoblotting with anti-mMCM4, anti-hMCM7, and anti-XMCM2 antibodies.
Single asterisks in panels B and C
indicate the positions of G2/M type phospho-mMCM4 and
phospho-mMCM2, respectively. Double asterisks in panel
B indicate the position of S-phase- and bound form-specific
phospho-mMCM4.
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We further examined the phosphorylation of the mMCM proteins in
G2/M phase FT210 cells by metabolic labeling with
orthophosphate. Cells arrested in early S phase were released in
phosphate-free medium supplemented with nocodazole either at a
permissive or a nonpermissive temperature. After 8 h,
[32P]orthophosphate was added and the cells were labeled
for a further 2 h. Even in the absence of phosphate, the cells
reached G2 phase 8 h after the release as usual (data
not shown). The Triton X-100-extractable fractions prepared from the
labeled cells were subjected to immunoprecipitation with anti-hMCM7 or
anti-mMCM3 antibodies, and the immunoprecipitates were analyzed by
SDS-PAGE followed by silver staining, radiography or immunoblotting. In
these experiments, the washing buffer was changed to one containing 200 mM NaCl. Under such conditions, the hMCM heterohexameric
complexes are partially disrupted (41), making it easier to detect each
mMCM protein on SDS-PAGE; namely, anti-hMCM7 antibodies precipitated
mMCM7, 6, 4, and 2, whereas anti-mMCM3 did mMCM3 and 5 (Fig.
6, A and B). In the
precipitates from G2/M FT210 cells cultured at a permissive
temperature, mMCM2 showing the fastest mobility and predominantly
labeled by 32P was detected (Fig.
6A). However, it was remarkably decreased at a nonpermissive
temperature (Fig. 6A). However, the slow migrating mMCM2 was
still labeled by 32P at a nonpermissive temperature (Fig.
6A). Western blot analysis clearly showed the presence of
hyperphosphorylated mMCM4 showing the slowest mobility at a permissive
temperature and its disappearance at a nonpermissive temperature,
although its detection by silver staining or radiography was difficult
because of its overlapping with mMCM6 (Fig. 6A). Similar to
the situation for mMCM2, mMCM4 was also labeled by 32P even
at a nonpermissive temperature (Fig. 6A). mMCM3 and 6 were also labeled by 32P at a permissive temperature, and
seemingly, the rate of the phosphorylation was not changed at a
nonpermissive temperature (Fig. 6, A and B).
However, further analyses are required to establish conclusively
whether cdc2/cyclin B phosphorylates mMCM3 or 6 in vivo or
not. For mMCM5 and 7, virtually no incorporation of 32P was
found (Fig. 6, A and B). These in vivo
data with FT210 cells, together with the above described in
vitro data, indicate that cdc2/cyclin B plays an indispensable
role in G2/M hyperphosphorylation of mammalian MCM4 and
MCM2 proteins. On the other hand, the data from the labeling
experiments suggest that mMCM2, 3, 4, and 6 are also phosphorylated at
G2/M phase by kinase(s) other than cdc2.

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Fig. 6.
Phosphorylation analysis of mMCM proteins in
G2/M phase FT210 cells by metabolic labeling. Cells
arrested in early S phase were released in phosphate-free medium
supplemented with nocodazole either at a permissive or a nonpermissive
temperature. After 8 h, [32P]orthophosphate was
added, and the cells were labeled for a further 2 h. The Triton
X-100-extractable fractions prepared from the labeled cells were
subjected to immunoprecipitation with a control rabbit immunoglobulin,
anti-hMCM7 (A) and anti-mMCM3 antibodies (B).
After washing with the buffer containing 200 mM NaCl, the
immunoprecipitates were analyzed by SDS-PAGE followed by silver
staining, radiography, or immunoblotting with anti-mMCM4
antibodies.
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A Role for cdc2/cyclin B in Regulation of the Mammalian MCM
Protein-Chromatin Association--
Using this system, we also examined
possible roles of cdc2 kinase in regulation of mammalian MCM-chromatin
associations. As expected from previous observations (19-23), Western
blot analysis showed that the levels of the chromatin-bound mMCM4 and 7 proteins in the nuclei were decreased during the S phase, becoming
minimal in the G2 phase (8 h after release) in FM3A and
FT210 cells cultured at a permissive temperature (Fig. 5B).
The decrease in the level of the chromatin-bound mMCMs in
G2 was also observed in FM3A cells cultured at 39 °C.
However, reassociation of the mMCMs to the nuclei was observed in the M
phase (12 h after release) in these cells (Fig. 5B). The
reason for this reassociation is currently unknown. This unexpected
behavior of mMCMs at 39 °C made it difficult to estimate the effect
of cdc2 inactivation on mMCM-chromatin association. Nevertheless, the
levels of the chromatin-bound mMCMs in G2 phase FT210 cells
(8 h after release) cultured at a nonpermissive temperature decreased
to a lesser extent than in FM3A cells (Fig. 5B), suggesting
that cdc2 kinase plays a role in negatively regulating the
mMCM-chromatin association.
 |
DISCUSSION |
hMCM Complex Formation and Its Complicated Phosphorylation during
the Cell Cycle--
Our recent report concerning human cells (41) and
two regarding Xenopus egg extracts (31, 32) have provided
evidence of the existence of complexes containing all six MCMs.
Especially, we have also shown by chromatin immunoprecipitation that
chromatin-bound hMCM heterohexameric complexes are found (41). These
findings are in agreement with the demonstration that hMCM resides in
500-600 kDa complexes (38-40). However, the problem of whether there
is any change in the complex composition during the cell cycle
remained. In this regard, native gel electrophoresis analyses of
500-600 kDa MCM complexes in Xenopus and
Drosophila eggs showed that they are stably present
throughout the cell cycle, although their exact composition was not
examined (29, 36). Our present results suggest that the six hMCM
members may exist as hexameric complexes throughout the cell cycle,
whether bound to chromatin or not. It seems likely that they function
and undergo cell cycle-regulation as heterohexameric complexes. The
finding that all six XMCM proteins bind to chromatin and are displaced
synchronously in a Xenopus egg system (31, 32) is also
consistent with this notion. Very recently, it has been reported that
hMCM4/6/7 complexes have helicase and ATPase activity, which is
inhibited by hMCM2 (48). It remains possible, therefore, that more
complicated regulation of the complex composition occurs in specific
situations.
Our results also show that the hMCM2 and 4 proteins in the complexes
variously change their phosphorylation state depending on the cell
cycle and the condition of chromatin association. Therefore, changes in
phosphorylation of the hMCM complexes may not induce remarkable
rearrangement of the composition, although complex stability seems to
differ between bound and unbound forms (41). Hyperphosphorylation of
hMCM2 and 4 in the mitotic phase has already been reported (21, 39) and
confirmed here. In addition, the present study suggested that, in the
G1 phase, unbound hMCM2 and 4 are more phosphorylated than
chromatin-bound forms and that, during progression from G1
to S phase, bound forms become phosphorylated. Although investigation
of phosphorylation of the other mammalian MCMs during the cell cycle
other than in the G2/M phase was not performed in this
study, cell cycle-dependent regulation of phosphorylation
of mMCM3 has been reported previously; unbound mMCM3 is more
phosphorylated than the bound form, and the phosphorylation rate
increases during G1-S progression (19). In the present study, mMCM3 was shown to be phosphorylated also in G2/M
phase. Mammalian MCM5 (19) and
MCM72 in asynchronous cells
are not remarkably labeled in vivo by orthophosphate. This
was also the case in G2/M cells.
A Role for cdc2/cyclin B in G2/M Hyperphosphorylation
of Mammalian MCM2 and 4 and in Regulation of Mammalian MCM
Complex-Chromatin Association--
Our finding that the
G2/M phase hyperphosphorylation of mammalian MCM4 is
directed by cdc2/cyclin B is consistent with previous results. hMCM4
and mMCM4 proteins have a cluster of potential target sites for CDK in
the N terminus (37, 39), which is conserved through eukaryotes (29,
49). Furthermore, in a Xenopus egg system, XMCM4 was
suggested to be hyperphosphorylated by cdc2 kinase in the M phase (29,
49). In addition, the present data also suggest that mammalian MCM2 in
MCM complexes is another target of cdc2/cyclin B. It has so far not
been clarified whether XMCM2 could be a target of phosphorylation by
cdc2 kinase in Xenopus egg extracts, although it has been
shown that XMCM3 and 5 are not phosphorylated by cdc2 in
vitro (29).
The findings obtained with FT210 cells also indicate a role for cdc2 in
negative regulation of mammalian MCM-chromatin interaction although we
have no direct data as to whether phosphorylation of the MCM complexes
by cdc2/cyclin B per se is required for the regulation. The
findings are consistent with results obtained with a Xenopus
egg extract system and budding yeast (24, 29). In vitro
cdc2/cyclin B treatment of chromatin isolated from the egg extract
system induces dissociation of XMCM proteins from chromatin (29). Very
recently, it was shown in budding yeast that suppression of mitotic CDK
activity by a CDK inhibitor leads to reloading of MCM7 to chromatin
fragments containing the origin regions (24). Our data, therefore,
demonstrate that cdc2 is an important kinase for the regulation of
MCM-chromatin association also in the mammalian somatic cell cycle. The
function of cdc2/cyclin B to prohibit rereplication (50, 51) may be
achieved partly via its inhibition of MCM-chromatin interaction. This
may be also the case in mammalian cells.
Whereas cdc2 kinase is strongly implicated in G2/M
hyperphosphorylation of mammalian MCM2 and 4 and in regulation of
mammalian MCM complex-chromatin association, our data also suggest that mMCM2, 3, 4, and 6 are phosphorylated at G2/M phase by
kinase(s) other than cdc2. However, the biological roles and the
responsible kinase(s) are currently unknown.
S Phase Phosphorylation of Mammalian MCM2 and
4--
Phosphorylation of the chromatin-bound hMCM2 and 4 during the S
phase also seems intriguing. The observations are reminiscent of the
Xenopus egg system finding that nuclear XMCM4 is converted to a partially phosphorylated form in early S phase (49). However, in
contrast to the Xenopus system where most nuclear XMCM4 is phosphorylated (49), in human cells, only a part of the bound hMCM2 and
4 appeared phosphorylated during the S phase. This might be
attributable to differences in the temporal pattern of firing of
replication origins between the two; i.e. whereas origins
initiate synchronously in the Xenopus embryonic cells, they
fire in a temporally staggered fashion in the mammalian somatic cells.
S phase phosphorylation might occur only in hMCM complexes associated
with chromatin regions that are in the process of being fired. As the S
phase phosphorylation of XMCM4 seems independent on cdc2 kinase (49),
that of MCM4 in mammalian somatic cells may be irrelevant to cdc2
activity. Also, the S phase phosphorylation of hMCM2 and 4 proteins
per se does not seem to result in dissociation of the
complexes from chromatin, as is the case in the Xenopus egg
system (49). Whatever the exact regulation of the phosphorylation of
the mammalian MCM complexes during the cell cycle, the complicated
regulation and its effects on MCM function will require extensive study
for complete understanding.
 |
ACKNOWLEDGEMENTS |
We thank Dr. A. Matsukage for helpful
discussion and critical reading of the manuscript; Dr. H. Kimura for
anti-mMCM3 and 4 antibodies; Dr. S. Miyake for anti-XMCM2 antibodies;
and T. Yoshida and Y. Matsumura for technical assistance.
 |
FOOTNOTES |
*
This work was supported in part by a grant from the Ministry
of Education, Science and Culture of Japan.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: Laboratory of Viral
Oncology, Research Institute, Aichi Cancer Center, Chikusa-ku, Nagoya
464, Japan. Tel.: 81-52-762-6111; Fax: 81-52-763-5233; E-mail:
mfujita{at}aichigw.aichi-cc.pref.aichi.jp.
1
The abbreviations used are: hMCM, human MCM
protein; PAGE, polyacrylamide gel electrophoresis; TX-100mCSK, modified
CSK buffer containing Triton X-100.
2
M. Fujita and M. Ishibashi, unpublished
observations.
 |
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