Báo cáo khoa học: Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells pot

Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: a phos-phorylation that is greatly enhanced in the G2 an

cycle in mammalian cells

Yuki Komamura-Kohno1, Kumiko Karasawa-Shimizu1, Takako Saitoh1, Michio Sato1,

Fumio Hanaoka2, Shoji Tanaka1and Yukio Ishimi1,3

1 Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan

2 Graduate School of Frontier Biosciences, Osaka University, Japan

3 Faculty of Science, Ibaraki University, Mito, Japan

MCM2–7 proteins are essential for eukaryotic DNA

replication and are the most likely candidates for the

replicative DNA helicase responsible for unwinding

DNA at the replication forks [1–3] Consistent with

their primary amino acid sequences, a subcomplex of

MCM4⁄ 6 ⁄ 7 functions as DNA helicase in vitro [4] It

has been suggested that MCM2, -3 and -5 play a

regu-latory role in the function of MCM4⁄ 6 ⁄ 7 DNA

heli-case, because addition of MCM2 or MCM3⁄ 5 to

MCM4⁄ 6 ⁄ 7 complex resulted in inhibition of the MCM4⁄ 6 ⁄ 7 DNA helicase [5,6] Thus MCM2–7 com-plex, a major MCM complex on chromatin during the

G1 phase, has to be activated to show DNA helicase activity It is possible that several proteins, including CDC7 kinase and CDC45 are involved in this activa-tion Evidence suggests that MCM2–7 proteins may have additional functions during the cell cycle [3] Cyclin-dependent kinases (CDK), which play a critical

Keywords

CDK; cell cycle; DNA replication; MCM

proteins; phosphorylation

Correspondence

Y Ishimi, Faculty of Science, Ibaraki

University, 2-1-1 Bunkyo, Mito 310-8512,

Ibaraki, Japan

Fax: +81 29 228 8439

Tel: +81 29 228 8439

E-mail: ishimi@mx.ibaraki.ac.jp

(Received 5 September 2005, revised 6

January 2006, accepted 18 January 2006)

doi:10.1111/j.1742-4658.2006.05146.x

MCM4, a subunit of a putative replicative helicase, is phosphorylated dur-ing the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication However, detailed characterization of the phosphorylation of MCM4 remains to be per-formed We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phos-phorylation that is greatly enhanced in the G2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3) We present data indicating that phos-phorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2 Based on these results and those from in vitro phosphorylation of MCM4 with CDK2⁄ cyclin A,

we discuss the kinases responsible for MCM4 phosphorylation Phosphor-ylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not gener-ally colocalized with replicating DNA Unexpectedly, MCM4 phosphoryl-ated at site 32 was enriched in the nucleolus through the cell cycle These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell cycle

Abbreviations

CDK, cyclin-dependent kinases.

role in the G1 to S and G2 to M transitions in the

cell cycle are also required to prevent re-replication of

DNA in a single cell cycle Inactivation of CDK1 leads

to re-replication of DNA in eukaryotic cells including

human cells [7] Targets of the kinase in the regulation

of DNA replication include ORC2, Cdc6 and Mcm

proteins in Saccharomyces cerevisiae [8], and

disregula-tion of these proteins leads to limited over-replicadisregula-tion

of the genome In higher eukaryotic cells, MCM4

is phosphorylated extensively in the M phase, and

CDK1⁄ cyclin B appears to be responsible for the

phosphorylation [9–11] It has been proposed that

phosphorylation of MCM4 in the M phase may be

required to prevent binding of the MCM complex to

chromatin in Xenopus [6] Partly consistent with this

notion, it has been shown that CDK2⁄ cyclin A

phos-phorylates MCM4 to prevent binding of MCM

com-plex to chromatin [12] In contrast, a recent finding

suggests that an intermediate level of phosphorylation

of MCM4 is required for chromatin binding during

interphase [11] We showed that the MCM4⁄ 6 ⁄ 7

com-plex purified from HeLa cells in the G2 and M phase

shows a lower level of DNA helicase activity compared

with complex purified from logarithmically growing

cells [13] Thus, phosphorylation of MCM4, together

with the presence of geminin, which inhibits the ability

of CDT1 to load MCM proteins onto chromatin, may

help prevent re-replication of DNA in the G2 and M

phase During interphase, chromatin-bound MCM4 is

partially phosphorylated and its level is higher than

that of MCM4 that is not bound to chromatin [10,11]

The characterization and functional significance of

MCM4 phosphorylation during interphase remains to

be clarified

We report that in vitro phosphorylation of human

MCM4⁄ 6 ⁄ 7 complex with CDK2 ⁄ cyclin A leads to

inactivation of the DNA helicase activity of the

complex [14] We identified six Ser or Thr residues (3,

7, 19, 32, 53, 109) in the N-terminal region of mouse MCM4 as the sites required for phosphorylation with CDK2⁄ cyclin A and CDK1 ⁄ cyclin B in vitro [13] Con-version of these six Ser or Thr residues to Ala resulted

in the MCM4⁄ 6 ⁄ 7 complex showing resistance to inhi-bition with CDK2⁄ cyclin A, indicating that phos-phorylation at these six sites is responsible for the inactivation of MCM4⁄ 6 ⁄ 7 DNA helicase We charac-terized the phosphorylation of MCM4 during the cell cycle in human and mouse cells using antiphospho sera against these sites We show that phosphorylation at sites Thr7, Thr19, Ser32, Ser87 and Thr109 requires CDK1 in the M phase of mouse FM3A cells, and phosphorylation at sites 3 and 32 requires CDK2 dur-ing interphase in human HeLa cells Changes in the phosphorylation level during the cell cycle and the nuclear localization of phosphorylated MCM4 suggest that MCM4 phosphorylated at these sites has several distinct and site-specific roles in MCM functions

Results Characterization of antiphosphoMCM4 sera

We identified six SP or TP sites (Ser3, Thr7, Thr19, Ser32, Ser53 and Thr109) in the N-terminal region of mouse MCM4 as being required for the phosphoryla-tion of MCM4 with CDK2⁄ cyclin A in vitro [13] All six sites are conserved between mouse and human MCM4, although the numbers of sites 53 and 109 in mouse MCM4 was changed to 54 and 110 in human MCM4 (Fig 1) We prepared antiphosphoMCM4 sera against these sites of human MCM4 in addition to those against Ser88 Figure 2 shows the specificity of the antibodies as measured by ELISA The data indi-cate that all six antiphosphoMCM4 sera (P-3, -7, -19,

Fig 1 Amino acid alignment of human and mouse MCM4 in the N-terminal region Amino acid sequences in the N-terminal region of human and mouse MCM4 in which SP and TP sites are clustered are aligned These CDK sites are indicated by bold and italicized letters Among these sites, those that are required for phosphorylation with CDK2 ⁄ cyclin A (13) in addition to site 88 are indicated by their amino acid numbers.

-32, -54 and -110) bound almost specifically to their

corresponding phosphopeptides The binding

depend-ency of the antibodies on phosphorylation is examined

in Fig 3A Human MCM4⁄ 6 ⁄ 7 complexes were

incu-bated in the presence or absence of purified CDK2⁄

cyclin A in vitro and MCM4 proteins were then

analyzed by western blotting using six

phosphoantibod-ies In addition to wild-type MCM4⁄ 6 ⁄ 7 complex, a

mutant complex in which six Ser or Thr residues (3, 7,

19, 32, 54, 100) in MCM4 were converted to Ala was

also incubated with CDK2⁄ cyclin A All the antibodies

reacted to MCM4 in the wild-type complex but not in

the mutant complex The signals from the wild-type

complex were detected with P-3, -32 and -54 antibodies

even in the absence of CDK2⁄ cyclin A, indicating that

MCM4 is phosphorylated at these sites during

pre-paration from insect cells Incubation of wild-type

MCM4⁄ 6 ⁄ 7 complex with CDK2 ⁄ cyclin A enhanced

the signals detected with the antibodies (P-32 and -54)

or induced the signals with the antibodies (P-7, -19 and

-110) However, the kinase barely stimulated the signal

with P-3 antibodies under these conditions The signal

detected with P-3 antibodies in the absence of Cdk2⁄

cyclin A disappeared after incubation of the complex

with k phosphatase (Fig 3B) These results indicate that

all the signals detected with the six phosphoantibodies

are dependent on the phosphorylation of MCM4

Binding of these antibodies to human MCM4 in

log-arithmically growing cells and cells synchronized at the

G2 and M phase was examined (Fig 4) Because the

MCM proteins, including MCM4, are almost

exclu-sively detached from chromatin in the G2 and M

phase, they were recovered in a Triton-soluble (S)

frac-tion, which was detected using anti-MCM4 sera At

this stage, MCM4 was extensively phosphorylated, as

revealed by the retarded mobility of the protein in

SDS gel, compared with the mobility of protein

pre-pared from logarithmically growing HeLa cells All seven antiphosphoMCM4 sera recognized the retarded MCM4 prepared from cells in the M phase, indicating that these sites are indeed phosphorylated in the M phase in HeLa cells We classified the mode of phos-phorylation into two groups: (a) phosphos-phorylation is greatly enhanced in phases G2 and M (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphoryla-tion is firmly detected at interphase (Ser3) Phosphoryl-ated MCM4 in cells at interphase was weakly detected

by the P-32, -54 and -88 antibodies

Phosphorylation of MCM4 during the HeLa cell cycle

Changes in the levels of MCM4 phosphorylation at sites 3 and 32 during the HeLa cell cycle were analyzed (Fig 5) Logarithmically growing HeLa cells, pulse-labeled with BrdU, were stained with antiphospho-MCM4 sera [P-3 (A) and P-32 (B)] and anti-BrdU sera

We quantified the fluorescence intensity in the nucleus and cytoplasm separately In the M phase, we meas-ured fluorescence in regions surrounding total con-densed chromosomes and showed it to be the same as

in the nucleus Phosphorylation at site 3 increased in the nucleus during phases G1 and S, and was detected

in the cytoplasm during the M phase Phosphorylation

at site 32 increased gradually in the nucleus during phases S and G2, and was detected maximally in the cytoplasm in the M phase The timing of phosphoryla-tion and dephosphorylaphosphoryla-tion of MCM4 during phases

G2 and M was compared among the sites (Ser3, Thr7, Ser32, Ser54 and Thr110) (Fig 6) Figure 6A shows a typical example of the staining pattern seen using con-forcal microscopy The data suggest that all the phos-phorylated MCM4 is not bound with mitotic chromosomes The level of staining during phases G2

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

P-3Ab P-7Ab P-19Ab P-32Ab P-54Ab P-110Ab

A450

P-Ser3 P-Thr7 P-Thr19 P-Ser32 P-Ser54 P-Thr110

phosphopeptides

Fig 2 Specificity of binding of phospho-MCM4 antibodies The binding specificity of six antiphosphoMCM4 sera (P-3, -7, -19, -32, -54 and -110) to phosphopeptides was examined by ELISA The six phosphoanti-bodies were incubated with six correspond-ing phosphopeptides (P-Ser3, -Thr7, -Thr19, -Ser32, -Ser54 and -Thr110) and binding was detected by absorbance at 450 nm.

and M was quantified and compared among the five

sites (3, 7, 32, 54, 110) (Fig 6B) Phosphorylation at

sites 3 and 110 was maximal in the G2 phase, in

con-trast, phosphorylation at the other sites (7, 32, 54) was

maximal in the M phase Phosphorylation of MCM4 at

sites 7 and 32 decreased during anaphase Changes in

phosphorylation at sites 7 and 32 during the M phase

appear to parallel changes in CDK1⁄ cyclin B activity

These results indicate that the CDK sites in MCM4 are

differently phosphorylated and dephosphorylated

dur-ing the HeLa cell cycle in a site-specific manner

Cyclin-dependent protein kinase is involved in

the phosphorylation of MCM4

To determine which kinase is involved in the

phos-phorylation of MCM4 in phases G2 and M, we used

mouse FT210 cells in which CDK1 activity is tempera-ture sensitive [15] After synchronizing the cells at the

G1⁄ S boundary, they were allowed to progress to phases S and G2 At permissive temperatures, cells accumulated in the M phase in the presence of noco-dazole (mitotic index: 30%) At nonpermissive temper-atures, cells are arrested in the G2phase, this is caused

by inactivation of CDK1 which is crucial for entry into the M phase We compared the phosphorylation level of MCM4 between these two cells (Fig 7A) Only Triton-soluble fractions were examined for MCM4 phosphorylation We confirmed that all the antiphos-phoMCM4 sera (P-3, -7, -19, -32, -54, -88 and -110) recognized mouse MCM4 that had been prepared from baculovirus-infected insect cells and then

phosphorylat-ed with CDK2⁄ cyclin A in vitro (data not shown) Extensively phosphorylated MCM4, which showed

P-32 P-54 P-110

A

B

1 2 3 4 5 6

1 2 3

1 2 3

-wild 6A

CDK2

Fig 3 In vitro phosphorylation of MCM4

with CDK2 ⁄ cyclin A (A) Wild-type human

MCM4 ⁄ 6 ⁄ 7 complex (wild) (lanes 1–3) and

a mutant complex (6A) (lanes 4–6) in which

six Ser or Thr residues (3, 7, 19, 32, 54 and

110) of MCM4 had been converted to Ala

were incubated in the presence or absence

of increasing amounts of CDK2 ⁄ cyclin A.

Proteins were analyzed by W estern blot

using anti-phospho and anti-MCM4 sera as

indicated (B) Wild-type MCM4 ⁄ 6 ⁄ 7

com-plex was incubated in the presence or

absence of increasing amounts of k

phos-phatase under recommended conditions

(New England Biolabs) The proteins were

analyzed by western blot using anti-P-3 and

anti-MCM4 sera Arrows on the right-hand

side of the gel indicate the 95 kDa position.

retarded mobility, was detected in extracts from cells

cultured at a permissive temperature but not in cells

cultured at a nonpermissive temperature, which was

detected by anti-MCM4 sera The P-7 and P-19

phos-phoantibodies recognized MCM4 with retarded

mobil-ity as well as MCM4 at a nonretarded position in the

extracts prepared from cells cultured at a permissive

temperature In contrast, only MCM4 at the

nonre-tarded position was detected using these two

antibod-ies in extracts from cells cultured at a nonpermissive

temperature The phosphoantibodies (P-32, -88 and

-110) recognized almost exclusively MCM4 with

retar-ded mobility in the cells cultured at a permissive

tem-perature Weak bands recognized with P-88 and P-110

were detected near the nonretarded position but no

bands were recognized with P-32 antibodies in cells

cultured at a nonpermissive temperature Overall, the

intensity of the bands detected with these antibodies

(P-7, -19, -32, -88, and -110) decreased in cells cultured

at a nonpermissive temperature, because the intensity

ratio (39⁄ 33) was calculated as 0.2–0.58 In contrast,

bands detected with the P-3 and P-54 antibodies were

almost unchanged between cells cultured at a

permis-sive temperature and those cultured at a nonpermispermis-sive

temperature, because the intensity ratio (39⁄ 33) was

calculated as 0.91 and 1.1 These results suggest that

CDK1 is involved in the phosphorylation of mouse

MCM4 at five sites (Thr7, Thr19, Ser32, Ser87 and

Thr109) but not phosphorylation at the other two

(Ser3 and Ser53) in the M phase Involvement of

CDK1 for MCM4 phosphorylation at sites 32, 87 and

109 in the M phase is almost absolute However, involvement at sites 7 and 19 may be partial, because signals detected at the nonretarded position were not decreased at the nonpermissive temperature

To address the question of whether CDK2 is res-ponsible for the phosphorylation of MCM4 at CDK sites during interphase, a dominant-negative mutant of CDK2 [16] was expressed in HeLa cells, and the effect

of its expression on the phosphorylation of MCM4 was examined (Fig 7B) The level of MCM4 phosphory-lation at sites 3 and 32 was compared between cells that express the mutant CDK2 and those that do not Phos-phorylation of MCM4 at these two sites was signifi-cantly decreased in cells expressing mutant CDK2, as shown in Fig 7B For quantification, we separated the immunofluorescence intensity from each cell into two (strong and weak) In cells that do not express CDK2, strong signals detected with P-3 antibodies were observed in 30% (100⁄ 328) of cells, and in those that

do express the CDK2, strong signals were observed in 1.6% (2⁄ 129) of cells For P-32 antibodies, strong sig-nals were detected in 42% (100⁄ 238) of cells that do not express CDK2, and 15% (14⁄ 92) of cells that do express CDK2 Thus, CDK2 is almost exclusively involved in phosphorylation at site 3 and is partly involved in phosphorylation at site 32 during inter-phase in HeLa cells We also examined the effect of the expression of mutant CDK2 on phosphorylation of MCM4 at site 54 (data not shown) The results

MCM4

P-3

P-32

P-54

P-7

P-19

P-110

P-88

Fig 4 Detection of phosphorylated MCM4 using antiphospho sera by western blot ana-lyses (A) Logarithmically growing HeLa cells were incubated with nocodazole Cells detached from the bottle by shaking were collected and named mitotic (M) cells Residual cells were collected and named G 2

cells These cells and logarithmically grow-ing cells were separated into Triton-soluble (S) and Triton-insoluble (P) fractions After electrophoresis, proteins in these fractions were analyzed by using anti-MCM4 or an-tiphosphoMCM4 (P-3, -7, -19, -32, -54, -88 and -110) sera as indicated Arrows on the right-hand side of gel indicate the 95 kDa position.

indicated that phosphorylation at site 54 was almost

entirely resistant to the expression of mutant CDK2,

suggesting that CDK is not involved in the

phosphory-lation

Phosphorylated MCM4 on chromatin

HeLa cells were separated into Triton-soluble and

Tri-ton-insoluble fractions and the insoluble fraction was

further separated into soluble and

DNaseI-insoluble fractions Proteins in these fractions were

analyzed using antiphosphoMCM4 sera (Fig 8)

West-ern blotting analysis using anti-MCM4 sera showed

the distribution of total MCM4 proteins in these

frac-tions under these condifrac-tions MCM4 phosphorylated

at sites 3 and 54 was preferentially detected in the

Triton-soluble fraction In contrast, MCM4

phos-phorylated at sites 7 and 32 was mainly detected in the chromatin-bound fractions Although the phosphoanti-body against site 7 recognizes mainly MCM4 in the M phase (Fig 4), it can detect MCM4 during interphase

to a lesser extent These results suggest that MCM4 phosphorylated at different sites shows different affin-ity for chromatin To study the relationship between chromatin-bound phosphorylated MCM4 and DNA synthesis, logarithmically growing HeLa cells, pulse-labeled with BrdU, were treated with Triton and then stained with antiphosphoMCM4 sera (P-3 and P-32) (Fig 9A,B) BrdU-negative cells were differentiated into G1 and G2 cells using nuclear mass, and BrdU-positive cells were differentiated into the three phases

of early S (eS), middle S (mS) and late S (lS) from the pattern of nuclear staining with BrdU MCM4 phos-phorylated at sites 3 and 32 was not largely colocalized

Fig 5 Changes in MCM4 phosphorylation during the HeLa cell cycle Logarithmically growing HeLa cells that had been pulse-labeled with BrdU were incubated with antiphosphoMCM4 sera [P-3 (A) and P-32 (B)] and anti-BrdU sera, and were observed using a CCD camera In each cell, the fluorescence intensities of secondary antibodies were measured Using image cytometry, intensities in the cytoplasm and nucleus were individually quantified In mitotic cells, the intensity in a region containing whole chromosomes was quantified and shown to

be that in nucleus From the intensity of DAPI fluorescence and the reactivity to anti-BrdU sera, the cell-cycle stage was determined in each cell.

B

G1

Intensity of

fluorescence

Fig 6 Immunostaining of mitotic cells with phosphoantibodies (A) Logarithmically growing HeLa cells were stained with antiphosphoMCM4 sera (P-3,-7, -32, -54 and -110) and propidium iodide (10 l M ), and observed using a confocal laser scanning microscope Cells in the M phases (pro-phase, prometa(pro-phase, meta(pro-phase, anaphase and telophase) were collected in addition to those in the G1and G2phases A typical example of these cells is shown Binding of antiphosphoMCM4 sera and PI is shown in green and red, respectively (B) The fluores-cence intensities of antiphosphoMCM4 sera

in cells in phases G1, G2and M were meas-ured, and their averages (and standard devi-ation) are shown as relative values.

to BrdU-incorporated DNA in the three periods of the

S phase Similar results were obtained with

anti-MCM4 sera (data not shown) They are in agreement

with previous findings [17–19] The fluorescence

inten-sity generated by these antibodies was quantified

(Fig 9C) Phosphorylation of MCM4 at sites 3 and 32

on chromatin greatly increased in the S phase com-pared with the G1 phase MCM4 phosphorylation on chromatin at these sites began to decrease during the late S phase and was greatly reduced in the G2 phase

MCM4

92K

P-88

33°C

CDK1 inactivation(G2 arrest) CDK1 activation(M entry)

Aphidicolin Aphidicolin removal

Nocodazole addition

(G1/S)

Recovery of cells

(M)

33°C 39°C

phospho-MCM4

92K

P-3

P-54

0.91 1.1 0.40 0.58 0.31 0.20 0.42 1.0

P-32

A

B

Fig 7 CDKs are mainly responsible for the

phosphorylation of MCM4 (A) The

experi-mental design is presented at the top.

Mouse FT210 cells were synchronized at

the G1⁄ S boundary by incubating the cells

with aphidicolin for 16 h After removal of

the drug, cells were cultured for 12 h in the

presence of nocodazole at permissive

(33
C) or nonpermissive (39
C)

tempera-tures Cells were lyzed and Triton-soluble

fraction was examined for the presence of

phosphorylated MCM4 using western blot

analyses, which is shown at the bottom.

Antibodies used and temperature for culture

are indicated at the top In the bottom, the

ratio (39 ⁄ 33
C) of the intensity of the

signals is shown (B) A dominant-negative

mutant of human CDK2 was expressed as

fusion proteins with HA in HeLa cells The

effect of the expressed CDK on the

phosphorylation of MCM4 was examined by

costaining with anti-HA and

antipho-sphoMCM4 sera (P-3 or P-32) Each of the

single stainings and their merged image are

presented Arrows indicate HeLa cells

expressing HA–CDK2 proteins.

These changes were essentially similar to those with

anti-MCM4 sera The finding that the amounts of

phosphorylated MCM4 (Ser3 and Ser32) in the

chro-matin-bound form decrease during phases S and G2 is

in contrast to results shown in Fig 5, in which the

phosphorylated MCM4 in a cell increases during these

periods, indicating that phosphorylated MCM4 is

detached from chromatin as the cell cycle progresses

Unexpectedly, it has been shown that

chromatin-bound MCM4 phosphorylated at site 32 was

concen-trated in the nucleus during the cell cycle (Fig 9B)

Similar results were also observed to a lesser extent

with anti-MCM4 sera, but not with anti-MCM3 sera

(data not shown) This finding on P-32 antibodies may

be consistent with the notion that the fluorescence

intensity detected by the antibodies in the G2 phase

was slightly higher than detected by other antibodies

(Fig 9C) Nuclear localization of MCM4

phosphoryl-ated at site 32 was examined by costaining Triton-trea-ted HeLa cells with the P-32 antibodies and antibodies

to C23 nucleolar protein (Fig 10A) The data suggest that these two proteins are colocalized, indicating that MCM4 phosphorylated at site 32 is enriched in a nucleolar region Localization of MCM4 phosphory-lated at site 32 in HeLa cells was also immunochemi-cally examined using electron microscopy (Fig 10B) Signals with P-32 antibodies were detected in the entire nucleus but were clustered in several regions including the nucleolus In the nucleolus, signals were detected near densely stained structures However, as enrich-ment of the signals in the nucleolus is not obvious in this system it may indicate that the immnunoreactions are not saturated under these conditions From these results, it is suggested that MCM4 phosphorylated at different CDK sites shows a unique affinity for chro-matin

MCM4

P-32 P-7

S1 S3 P’

S1 S3 P’

S1 S3 P’

S1 S3 P’

S1 S3 P’

S1 S3 P’

105

85 105

85

kDa

kDa

histone

A

C

B

Fig 8 Detection of phosphorylated MCM4 in chromatin-bound fractions Logarithmically growing HeLa cells were separated into Triton-sol-uble (S) and Triton-insolTriton-sol-uble fractions The insolTriton-sol-uble fraction was further separated into DNaseI-solTriton-sol-uble (S3) and DNaseI-insolTriton-sol-uble (P¢) fractions Proteins in these fractions were stained with Coomassie Brilliant Blue (A) They were examined by western blot analysis using anti-MCM4 (B) or antiphosphoMCM4 sera (P-3, -7, -32 and -54) (C).

We showed that seven SP and TP sites in the N-terminal

region of MCM4 are uniquely phosphorylated during

the cell cycle CDK1 is required for phosphorylation at

five sites (Thr7, Thr19, Ser32, Ser87 and Thr109) during

the M phase in mouse FM3A cells, and CDK2 is

required for phosphorylation at least at two sites (Ser3

and Ser32) during interphase in HeLa cells, suggesting

that CDK is involved in phosphorylation at these sites

Changes in the phosphorylation level during the cell

cycle and the different affinities for chromatin suggest

that phosphorylation of MCM4 plays several distinct

roles in MCM function in mammalian cells The finding

that phosphorylated MCM4 is not largely colocalized

to replicated DNA may be consistent with the notion that the phosphorylation of MCM4 at CDK sites has a negative role in MCM function

All MCM2–7 members have an essential role in the initiation and elongation of DNA replication [20], possibly as a replicative DNA helicase [21,22] It is possible that MCM4⁄ 6 ⁄ 7 DNA helicase [4,23,24] is generated from the MCM2–7 complex as the function

of the MCM complex We have reported that the MCM4⁄ 6 ⁄ 7 DNA helicase activity is inhibited by phosphorylation of MCM4 with CDK2⁄ cyclin A at the six SP or TP sites [13] Our data indicate that phosphorylation at these sites is not equivalent in terms of cell cycle changes, localization in the nuclei or the role of CDK Phosphorylation at sites 7 and 32

Fig 9 Immunostaining of chromatin-bound MCM4 (A) Logarithmically growing HeLa cells that had been pulse-labeled with BrdU were trea-ted with Triton They were stained with anti-BrdU sera and antiphosphoMCM4 sera (P-3 and 32) Cells at G 1 , early S (eS) middle S (mS), late

S (lS) and G2phases were collected BrdU-negative cells were differentiated into G1and G2cells from total area of TOTO-stained nucleus Cells at the S phase are differentiated using their staining pattern with anti-BrdU sera Fluorescent signals detected by antiphosphoMCM4 sera are shown in red and BrdU staining is shown in green, respectively, and these signals are presented individually and combined (B) The intensity of fluorescence detected with antibodies (P-3, -32 and MCM4) was quantified in each cell at G1, eS, mS, lS and G2 Averages of the intensity in each cell are presented (with standard deviations) and they are shown as relative to values from the G1phase.