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

Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells Yuki Komamura-Kohno1, Kumiko Karasawa-Shimizu1, Takako Saitoh1, Michio Sato1, Fumio Hanaoka2, Shoji Tanaka1 and Yukio Ishimi1,3 Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan Graduate School of Frontier Biosciences, Osaka University, Japan Faculty of Science, Ibaraki University, Mito, Japan 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 September 2005, revised 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 during 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 performed 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) phosphorylation 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 phosphorylation 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 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 Phosphorylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites and 32 suggested that they are not generally colocalized with replicating DNA Unexpectedly, MCM4 phosphorylated 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 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 ⁄ ⁄ functions as DNA helicase in vitro [4] It has been suggested that MCM2, -3 and -5 play a regulatory role in the function of MCM4 ⁄ ⁄ DNA helicase, because addition of MCM2 or MCM3 ⁄ to MCM4 ⁄ ⁄ complex resulted in inhibition of the MCM4 ⁄ ⁄ DNA helicase [5,6] Thus MCM2–7 complex, 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 activation Evidence suggests that MCM2–7 proteins may have additional functions during the cell cycle [3] Cyclin-dependent kinases (CDK), which play a critical Abbreviations CDK, cyclin-dependent kinases 1224 FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Komamura-Kohno et al 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 disregulation of these proteins leads to limited over-replication 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 phosphorylates MCM4 to prevent binding of MCM complex 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 ⁄ ⁄ complex 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 ⁄ ⁄ complex with CDK2 ⁄ cyclin A leads to inactivation of the DNA helicase activity of the MCM4 phosphorylation in mammalian cells 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] Conversion of these six Ser or Thr residues to Ala resulted in the MCM4 ⁄ ⁄ complex showing resistance to inhibition with CDK2 ⁄ cyclin A, indicating that phosphorylation at these six sites is responsible for the inactivation of MCM4 ⁄ ⁄ DNA helicase We characterized 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 and 32 requires CDK2 during 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 phosphorylation 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 shows the specificity of the antibodies as measured by ELISA The data indicate that all six antiphosphoMCM4 sera (P-3, -7, -19, Fig 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 FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS 1225 MCM4 phosphorylation in mammalian cells Y Komamura-Kohno et al 1.6 A450 1.4 1.2 P-3Ab P-7Ab 0.8 P-19Ab 0.6 P-32Ab 0.4 P-54Ab P-110Ab 0.2 -0.2 P-Ser3 P-Thr7 P-Thr19 P-Ser32 P-Ser54 P-Thr110 -0.4 phosphopeptides -32, -54 and -110) bound almost specifically to their corresponding phosphopeptides The binding dependency of the antibodies on phosphorylation is examined in Fig 3A Human MCM4 ⁄ ⁄ complexes were incubated in the presence or absence of purified CDK2 ⁄ cyclin A in vitro and MCM4 proteins were then analyzed by western blotting using six phosphoantibodies In addition to wild-type MCM4 ⁄ ⁄ 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 preparation from insect cells Incubation of wild-type MCM4 ⁄ ⁄ 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 logarithmically growing cells and cells synchronized at the G2 and M phase was examined (Fig 4) Because the MCM proteins, including MCM4, are almost exclusively detached from chromatin in the G2 and M phase, they were recovered in a Triton-soluble (S) fraction, 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 pre1226 Fig Specificity of binding of phosphoMCM4 antibodies The binding specificity of six antiphosphoMCM4 sera (P-3, -7, -19, -32, -54 and -110) to phosphopeptides was examined by ELISA The six phosphoantibodies were incubated with six corresponding phosphopeptides (P-Ser3, -Thr7, -Thr19, -Ser32, -Ser54 and -Thr110) and binding was detected by absorbance at 450 nm 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 phosphorylation into two groups: (a) phosphorylation is greatly enhanced in phases G2 and M (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation is firmly detected at interphase (Ser3) Phosphorylated 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 and 32 during the HeLa cell cycle were analyzed (Fig 5) Logarithmically growing HeLa cells, pulselabeled with BrdU, were stained with antiphosphoMCM4 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 measured fluorescence in regions surrounding total condensed chromosomes and showed it to be the same as in the nucleus Phosphorylation at site 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 phosphorylation and dephosphorylation 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 conforcal microscopy The data suggest that all the phosphorylated MCM4 is not bound with mitotic chromosomes The level of staining during phases G2 FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Komamura-Kohno et al MCM4 phosphorylation in mammalian cells A wild - 6A - CDK2 23 P-3 MCM4 34 P7 Fig In vitro phosphorylation of MCM4 with CDK2 ⁄ cyclin A (A) Wild-type human MCM4 ⁄ ⁄ 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 Western blot using anti-phospho and anti-MCM4 sera as indicated (B) Wild-type MCM4 ⁄ ⁄ complex was incubated in the presence or absence of increasing amounts of k phosphatase 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 P-32 B and M was quantified and compared among the five sites (3, 7, 32, 54, 110) (Fig 6B) Phosphorylation at sites and 110 was maximal in the G2 phase, in contrast, phosphorylation at the other sites (7, 32, 54) was maximal in the M phase Phosphorylation of MCM4 at sites and 32 decreased during anaphase Changes in phosphorylation at sites 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 during 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 phosphorylation of MCM4 in phases G2 and M, we used - 23 P-54 23 P-19 234 P-110 phosphatase 3 P-3 MCM4 mouse FT210 cells in which CDK1 activity is temperature 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 nocodazole (mitotic index: 30%) At nonpermissive temperatures, cells are arrested in the G2 phase, 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 antiphosphoMCM4 sera (P-3, -7, -19, -32, -54, -88 and -110) recognized mouse MCM4 that had been prepared from baculovirus-infected insect cells and then phosphorylated with CDK2 ⁄ cyclin A in vitro (data not shown) Extensively phosphorylated MCM4, which showed FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS 1227 MCM4 phosphorylation in mammalian cells log G2 S P S P S P M Y Komamura-Kohno et al log G2 S P S M P S MCM4 P P-7 P-3 P-19 P-32 P-110 P-54 P-88 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 phosphoantibodies recognized MCM4 with retarded mobility 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 nonretarded position was detected using these two antibodies in extracts from cells cultured at a nonpermissive temperature The phosphoantibodies (P-32, -88 and -110) recognized almost exclusively MCM4 with retarded mobility in the cells cultured at a permissive temperature 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 permissive temperature and those cultured at a nonpermissive 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 1228 Fig Detection of phosphorylated MCM4 using antiphospho sera by western blot analyses (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 G2 cells These cells and logarithmically growing 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 antiphosphoMCM4 (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 CDK1 for MCM4 phosphorylation at sites 32, 87 and 109 in the M phase is almost absolute However, involvement at sites 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 responsible 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 phosphorylation at sites and 32 was compared between cells that express the mutant CDK2 and those that not Phosphorylation of MCM4 at these two sites was significantly 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 not express CDK2, strong signals detected with P-3 antibodies were observed in 30% (100 ⁄ 328) of cells, and in those that express the CDK2, strong signals were observed in 1.6% (2 ⁄ 129) of cells For P-32 antibodies, strong signals were detected in 42% (100 ⁄ 238) of cells that not express CDK2, and 15% (14 ⁄ 92) of cells that express CDK2 Thus, CDK2 is almost exclusively involved in phosphorylation at site and is partly involved in phosphorylation at site 32 during interphase 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 FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Komamura-Kohno et al A MCM4 phosphorylation in mammalian cells B Fig 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 indicated that phosphorylation at site 54 was almost entirely resistant to the expression of mutant CDK2, suggesting that CDK is not involved in the phosphorylation Phosphorylated MCM4 on chromatin HeLa cells were separated into Triton-soluble and Triton-insoluble fractions and the insoluble fraction was further separated into DNaseI-soluble and DNaseIinsoluble fractions Proteins in these fractions were analyzed using antiphosphoMCM4 sera (Fig 8) Western blotting analysis using anti-MCM4 sera showed the distribution of total MCM4 proteins in these fractions under these conditions MCM4 phosphorylated at sites and 54 was preferentially detected in the Triton-soluble fraction In contrast, MCM4 phos- phorylated at sites and 32 was mainly detected in the chromatin-bound fractions Although the phosphoantibody against site 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 affinity for chromatin To study the relationship between chromatin-bound phosphorylated MCM4 and DNA synthesis, logarithmically growing HeLa cells, pulselabeled 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 BrdUpositive 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 phosphorylated at sites and 32 was not largely colocalized FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS 1229 MCM4 phosphorylation in mammalian cells A G1 Y Komamura-Kohno et al Prophase Metaphase Telophase Anaphase G2 Prometaphase B Intensity of fluorescence Fig 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 lM), and observed using a confocal laser scanning microscope Cells in the M phases (prophase, prometaphase, metaphase, anaphase and telophase) were collected in addition to those in the G1 and G2 phases A typical example of these cells is shown Binding of antiphosphoMCM4 sera and PI is shown in green and red, respectively (B) The fluorescence intensities of antiphosphoMCM4 sera in cells in phases G1, G2 and M were measured, and their averages (and standard deviation) are shown as relative values 1230 FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Komamura-Kohno et al MCM4 phosphorylation in mammalian cells A CDK1 inactivation(G2 arrest) 39°C CDK1 activation(M entry) 33°C 33°C 16hr Aphidicolin 28hr Aphidicolin removal Nocodazole addition (M) (G1/S) P-7 P-19 33 39 33 39 Recovery of cells P-32 P-88 33 39 33 39 P-110 33 39 MCM4 33 39 0.42 1.0 phosphoMCM4 92K 0.40 0.58 0.31 0.20 P-3 P-54 33 39 33 39 92K Fig CDKs are mainly responsible for the phosphorylation of MCM4 (A) The experimental 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) temperatures 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 antiphosphoMCM4 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 0.91 B 1.1 DN-CDK2(HA) P-3 merged DN-CDK2(HA) P-32 merged to BrdU-incorporated DNA in the three periods of the S phase Similar results were obtained with antiMCM4 sera (data not shown) They are in agreement with previous findings [17–19] The fluorescence intensity generated by these antibodies was quantified (Fig 9C) Phosphorylation of MCM4 at sites and 32 on chromatin greatly increased in the S phase compared 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 FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS 1231 MCM4 phosphorylation in mammalian cells Y Komamura-Kohno et al S1 S3 P’ A B S1 S3 P’ 105 85 kDa histone MCM4 C S1 S3 P’ S1 S3 P’ S1 S3 P’ S1 S3 P’ 105 85 kDa P-3 P-7 P-32 P-54 Fig Detection of phosphorylated MCM4 in chromatin-bound fractions Logarithmically growing HeLa cells were separated into Triton-soluble (S) and Triton-insoluble fractions The insoluble fraction was further separated into DNaseI-soluble (S3) and DNaseI-insoluble (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) These changes were essentially similar to those with anti-MCM4 sera The finding that the amounts of phosphorylated MCM4 (Ser3 and Ser32) in the chromatin-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 chromatinbound MCM4 phosphorylated at site 32 was concentrated 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 phosphoryl1232 ated at site 32 was examined by costaining Triton-treated 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 phosphorylated at site 32 in HeLa cells was also immunochemically 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 enrichment 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 chromatin FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Komamura-Kohno et al MCM4 phosphorylation in mammalian cells Fig Immunostaining of chromatin-bound MCM4 (A) Logarithmically growing HeLa cells that had been pulse-labeled with BrdU were treated with Triton They were stained with anti-BrdU sera and antiphosphoMCM4 sera (P-3 and 32) Cells at G1, early S (eS) middle S (mS), late S (lS) and G2 phases were collected BrdU-negative cells were differentiated into G1 and G2 cells 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 G1 phase Discussion 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 ⁄ ⁄ 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 ⁄ ⁄ 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 and 32 FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS 1233 MCM4 phosphorylation in mammalian cells Y Komamura-Kohno et al C Intensity of fluorescence Fig (Continued) begins to increase in the G2 phase and to decrease during anaphase, the kinetics of which seems to parallel the change in CDK1 ⁄ cyclin B activity These results are consistent with the notion that CDK1 ⁄ cyclin B is required for the phosphorylation of mouse MCM4 at these sites Because changes in the level of phosphorylation at other sites during phases G2 and M differ from those at sites and 32, other factors may be involved in the phosphorylation of MCM4 During interphase, phosphorylation at sites and 32 was 1234 sensitive to the expression of a dominant-negative mutant of CDK2 in HeLa cells However, in vitro studies (Fig 3) showed that CDK2 ⁄ cyclin A barely phosphorylates MCM4 at site 3, suggesting that CDK2 ⁄ cyclin A is indirectly involved in phosphorylation Phosphorylation at site 54 was relatively resistant to the expression of mutant CDK2 during interphase, and occurs in phases G2 and M even in the absence of CDK1 activity, suggesting that kinase(s) other than CDK may be involved in phosphorylation at site 54 during the cell cycle Consistently, it appears that CDK2 ⁄ cyclin A does not efficiently phosphorylate site 54 in vitro One unique feature of the nuclear localization is that MCM4 phosphorylated at site 32 was enriched in the nucleolus Using electron microscopy, the signals detected with P-32 antibodies were near densely stained structures that probably correspond to dense fibrillar components in which transcription by RNA polymerase I occurs [25] It is possible that MCM4 phosphorylated at site 32 has a unique role in the function of the nucleolus, including ribosomal RNA transcription It has been reported that MCM proteins rebind to chromatin at late telophase and DNA replication licensing is completed at the G1 phase [26,27] Unexpectedly, the level of chromatinbound MCM4 protein was greatly reduced in the G1 phase compared with the S phase (Fig 9C) Because the extraction conditions used are relatively stringent, it is possible that a large part of the MCM proteins on licensed chromatin in the G1 phase was detached from chromatin Phosphorylation of MCM4 and other factors may be involved in stronger binding of MCM4 protein to chromatin in the S phase compared with the G1 phase This assumption seems to be consistent with the finding that MCM4 phosphorylated at sites and 32 is enriched in the chromatin fraction (Fig 8) In HeLa cells, MCM4 phosphorylation at site 110 was detected not only in the M phase, but also in the G2 phase (Figs and 6B) These results appear to be inconsistent with those shown in Fig 7A, in which signals detected with P-110 antibodies decreased in G2-arrested FT210 cells cultured at a nonpermissive temperature This discrepancy remains to be resolved but may be explained by the difference between normal G2 and arrested G2 For example, the balance of phosphorylation and dephosphorylation at the site might differ between the two G2 conditions Another apparent inconsistency concerns the level of phosphorylation at site in the M phase The level of phosphorylation at the site is relatively high in the M phase in Fig 5A, but relatively low in Fig 6B Immunofluorescence with P-3 antibodies was detected using a CCD camera in Fig 5A but was detected using a confocal laser scanning micro- FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Komamura-Kohno et al A MCM4 phosphorylation in mammalian cells P-32 C23 merged B Fig 10 Nuclear localization of phosphorylated MCM4 (A) HeLa cells treated with Triton were costained with anti-P-32 and anti-C23 nucleolar protein sera The images of each staining are shown both singly and combined (B) HeLa cells were immunostained with P-32 antibodies and the signals, enhanced using silver, were detected by electron microscopy Images showing an entire nucleus (left) and the nucleolus (right) of the same section are presented scope in Fig Because quantification of total fluorescence in a cell using a CCD camera is more accurate, the data in Fig 5A are more reliable for understanding any change in phosphorylation at site during the cell cycle With regards to the role of MCM4 phosphorylation on MCM function, it has been suggested that a high level of phosphorylation in phases G2 and M might inhibit binding of the MCM complex to chromatin in G2 and M [9] To address this possibility, we expressed mutant MCM4 in which six amino acids (Ser or Thr) were converted to Ala or Glu to examine their binding to chromatin (data not shown) These two mutant MCM4s were recovered in the chromatin-bound fraction to a similar extent to wild-type MCM4, suggesting that phosphorylation of MCM4 may not directly affect its chromatin binding It is also possible that glutamic acid substitution may not mimic phosphorylation, however, further analysis is required to address this Our findings of cell-cycle changes in phosphorylation and a unique affinity for chromatin suggest that phosphorylation of MCM4 has different roles in MCM function They present a starting point from which to explore the molecular mechanisms underlying these phenomena and their functional significances Ten SP and TP sites were present in the N-terminal region of Drosophila MCM4 and three (3, 54, 110) of the six human CDK sites were conserved in Drosophila Essentially, no sequence conservation was detected between human and yeast, but nine and four SP or TP sites are clustered in this region of MCM4 in Schizosaccharomyces pombe and S cerevisiae, respectively These findings would be consistent with the notion that the interplay between CDK and MCM4 plays an important function in eukaryotic cells Experimental procedures Cell culture and antibodies HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% bovine serum FT210 cells were cultured in RPMI-1640 with 10% fetal bovine serum Logarithmically growing HeLa cells were incubated with FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS 1235 MCM4 phosphorylation in mammalian cells Y Komamura-Kohno et al 50 ngỈmL)1 nocodazole for 20 h Cells at mitosis were recovered after shaking a culture bottle Those that remained attached to the bottle were recovered after treatment with trypsin and were named G2 cells Anti-MCM4 and anti(phospho-human MCM4) (P-7, -19, -32, -54 and -110) rabbit sera were prepared as reported previously [28] and those against sites and 88 (P-3 and P-88) were also prepared reported previously [29] Anti-C23 mouse sera (MS-3) and anti-HA mouse sera (Y-11) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) ELISA Each of six peptides containing phospho-residue at the sites (Ser3, Thr7, Thr19, Ser32, Ser54, Thr110) was suspended at lgỈmL)1 in 0.05 m sodium cabonate buffer, pH 9.6 They were added to 96-well plate (100 lLỈwell)1) and the plate was incubated at °C overnight After washing the wells with NaCl ⁄ Pi containing 0.2% Tween-20, six antiphosphoMCM4 sera (200–500 lgỈmL)1) diluted 3000-fold with NaCl ⁄ Pi containing 0.05% Tween-20 were added to the wells (100 lLỈwell)1) and the plate was incubated at 37 °C for 1.5 h After washing the wells, peroxidase-conjugated anti-(rabbit IgG) sera (Bio-Rad Laboratories, Hercules, CA) diluted 10 000-fold were added to the wells and the plate was incubated at 37 °C for 1.5 h After washing, tetramethylbenzidine liquid substrate (Sigma, St Louis, MO) was added to each well (100 lLỈwell)1) The reaction was carried out for 10–20 at room temperature and stopped by adding 100 lL of m sulfuric acid Absorbance was measured at 450 nm Preparation of cell fractions and western blotting HeLa cells were lysed at · 106 cells per 100 lL in modified CSK buffer (10 mm Pipes, pH 6.8, 100 mm NaCl, mm MgCl2 and mm EGTA) containing 0.1% Triton X-100, mm ATP, proteinase inhibitors (Pharmingen) and phosphatase inhibitors (10 mm sodium b-glycerophosphate, mm sodium pyrophosphate, mm sodium orthovanadate and 50 mm sodium fluoride) (solution A) and placed on ice for 15 The cell suspension was centrifuged (2000 g for in a microcentrifuge), and its supernatant was saved (S1) Recovered precipitate was suspended in solution A, and the supernatant after centrifugation was saved (S2) Fractions S1 and S2 were combined and used as an S fraction in some cases The precipitate was suspended in a volume of solution A to yield · 106 cells per 100 lL (P) and the suspension was briefly sonicated in the presence of a loading buffer for SDS ⁄ PAGE When DNA in the precipitate was digested with DNaseI, cells were lyzed as described, except that the phosphatase inhibitors were replaced with phosphatase inhibitor cocktails I and II (Sigma) The precipitated fraction was suspended and then incubated with DNaseI (Takara, Japan) at 200 lgỈmL)1 at 30 °C for 1236 15 min, and then soluble (S3) and insoluble (P¢) fractions were recovered after centrifugation The proteins in these fractions were electrophoresed through 10% acrylamide gels containing SDS and then transferred to membranes (Immobilon, Millipore Corp., Bedford, MA) Approximately 30 lg of total proteins in the S fraction was loaded on to the gels After membranes had been incubated with a blocking solution (Blockace, Dai-nippon Pharmaceuticals, Japan) for h at room temperature, they were incubated at °C overnight with primary antibodies in the blocking solution (for antiMCM4 and anti-HA sera) or 5% bovine serum albumin in Tris-buffered saline (TBS; 50 mm Tris ⁄ HCl, pH 7.5 0.15 m NaCl) plus 0.1% Triton X-100 (for antiphosphoMCM4 sera) After washing with TBS plus 0.1% Triton X-100, membranes were incubated with peroxidase-conjugated secondary anti-rabbit sera (Bio-Rad) in the blocking solution The immunoreacted proteins were detected by Cool Saver AE-6935 (Atto) using a chemiluminescent detection system (SuperSignal West Pico or Femto Maximum Sensitivity Substrate, Pierce, Rockford, IL) In vitro phosphorylation Human MCM4 ⁄ ⁄ complex, in which six histidines were attached to MCM4 at the N-terminus, was purified from baculovirus-infected High cells using Ni-NTA chromatography and then by glycerol gradient centrifugation [30] A mutant MCM4 ⁄ ⁄ complex in which six Ser or Thr residues (3, 7, 19, 32, 54, 110) in the N-terminal region of MCM4 had been converted to Ala using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was also prepared These complexes were incubated with purified CDK2 ⁄ cyclin A complex as reported [14] Proteins were analyzed as described above Immunostaining HeLa cells were cultured on eight-well chambers (Falcon Becton Dickinson Labware, Franklin Lakes, NJ) or coverslips They were pulse-labeled with BrdU (50 lm) for 15 (Fig 9) After washing with NaCl ⁄ Pi, cells were fixed by incubation with 4% paraformaldehyde in NaCl ⁄ Pi for at room temperature To extract soluble proteins, cells were immersed in buffer containing Triton X-100 used for cell fractionation and then incubated at 37 °C for 15 in the same buffer before fixation (Figs and 10) Cells were washed with NaCl ⁄ Pi and then permeabilized and blocked by incubation with 0.1% Triton X-100, 0.02% SDS and 2% nonfat dried milk in NaCl ⁄ Pi for h at 37 °C Incubation of the cells with antiphosphoMCM4, anti-C23 or anti-HA sera (2.5 lgỈmL)1) was performed by incubation overnight at °C in the above blocking solution Cells were washed with the same solution and then incubated with Cy3-conjugated anti-rabbit or -mouse sera (Jackson Immuno-Research, West Grove, PA) and FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Komamura-Kohno et al FITC-conjugated anti-(mouse IgG) or anti-(rabbit IgG) sera (Cappel, Durham, NC) for 1.5 h at 37 °C in the blocking solution Washed cells were stained with lgỈmL)1 DAPI for 15 at room temperature After washing with NaCl ⁄ Pi, cells were mounted in 90% glycerol and 10% NaCl ⁄ Pi solution containing 1,4-diazabicyclo[2,2,2]-octane (DABCO, Sigma) (2.3%) and observed using fluorescence microscopy (AX-80, Olympus, Tokyo, Japan) In the experiment shown in Fig 9, cells that had been incubated with Cy3-conjugated anti-rabbit sera were refixed, treated with m HCl for 30 at room temperature and incubated with rat anti-BrdU sera (clone BU1 ⁄ 75; Harlan Sera Laboratory, Bicester, UK) followed by the incubation with FITC-conjugated anti-(rat IgG) sera (Cappel) Cells labeled with BrdU (50 lm) for 15 were fixed and permeabilized as reported for the experiments shown in Fig To quantify nuclear DNA content, cells were stained with DAPI solution (5.7 mm DAPI, ⁄ 10 concentrated McIlvaine buffer, pH 7.0, 0.15 m NaCl, 0.004 m KCl) for 30 min, rinsed in McIlvaine buffer and mounted with McIlvaine buffer ⁄ glycerol mixture (1 : v ⁄ v) To detect incorporated BrdU, cells were pretreated with the following reactions of a DNA nicking with 0.5 n HCl for at room temperature and a mild digestion with ExoIII nuclease (0.5 mL)1, Toyobo, Osaka, Japan) for 90 at 37 °C Linearity of DAPI-stained DNA content of a nucleus was preserved throughout such treatments; i.e the DNA content of G2, M cells are almost double that of G1 cells and the DNA content of S cells ranges between that of G1 cells and G2 or M cells (data not shown) BrdU was detected by the incubation with anti-(BrdU rat IgG) sera (Sera Laboratory) and then with anti-(rat IgG) sera conjugated with FITC (Cappel) Phosphorylated MCM4 proteins were detected by incubating with antiphosphoMCM4 sera and then with anti-(rabbit IgG) conjugated with Cy3 (Jackson Immuno-Research) Microfluorometry An improved method for multiparametric microfluorometry [31,32] was used to measure dual parameters on an identical cell for the experiments in Fig Cells were selected under phase-contrast illumination, and each was brought to the center of the field of the microfluorometer First, the intensity of DAPI fluorescence of a nucleus was measured using a UG 306-380 excitation filter and a LP 410 barrier filter (Zeiss, Jena, Germany) The position of the cell in reference to the x- and y-axes of the scanning stage was recorded using a microcomputer (PC-9801UX, NEC, Tokyo, Japan) Second, the fluorescence intensity of the immunocytochemically stained phosphoMCM4 protein was measured using a BP 450-490 excitation filter and a LP 520 barrier filter (Zeiss) Data were processed using two microcomputers (PC9801 UX, NEC; Macintosh Quadra950, Apple Computers) with software, some of which was designed for this study MCM4 phosphorylation in mammalian cells Image cytometry To independently quantify nuclear and cytoplasmic phosphoMCM4 protein content, an imaging method was developed for the experiments shown in Fig In addition, the cell-cycle phases of individual cells were identified based on the dual parameters of DNA content and BrdU incorporation Images of triple-stained cells on a slide were successively captured using a cooled CCD camera (SenSys, NIPPONROPER, Tokyo, Japan) equipped to an AX-80 fluorescence microscope (Olympus), controlled by a software (IpLab, Scananalytic, Rockville, MD) installed on a microcomputer (Power Mac G3, Apple Computers) DAPI image was measured using a BP360-370 excitation filter and a BA420-460 barrier filter (Carl Zeiss Japan, Tokyo, Japan) The FITC image was measured using a UG BP470490 excitation filter and a BA515-550 barrier filter Cy3 image was measured using a BP520-550 excitation filter and a BA580 barrier filter By using personally developed scripts of iplab software, images were processed to correct shading effects and gray value fluctuation Individual cell shapes were obtained from the images stained with antiphosphoMCM4 sera following the interactive segmentation procedure The nuclear image was extracted from DAPIstained image A particle analysis program was carried on the nuclear and cell image, respectively To make a correspondence between data from a cell and that of a nucleus, the nucleus measurement number was renumbered according to that of the cell Confocal laser scanning microscope Confocal laser scanning microscope observations were performed using a MRC 1024 (Bio-Rad) mounted on an Axioplan microscope (Zeiss) Two stains (FITC and Cy3) were excited simultaneously at wavelengths of 488 and 568 nm emitted from a Kr ⁄ Ar ion laser followed by detection at 522 and 605 nm, respectively Three stains (FITC ⁄ BrdU, Cy3 ⁄ MCM and TOTO-3 ⁄ DNA) were excited simultaneously at wavelengths of 488, 552 and 642 nm emitted from a Kr ⁄ Ar ion laser followed by detection at 522, 570 and 660 nm Electron microscope observation HeLa cells treated with Triton were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 m sodium phosphate buffer (pH 7.2) for h in an ice bath and the suspension was mixed with an equal volume of 1.5% lowmelting-point agarose (Sigma type VII) Hardened agarose was incubated with NaCl ⁄ Pi[-] (Mitsubishi Kagaku Iatron Inc., Tokyo, Japan) containing 15% sucrose and then 25% sucrose overnight The agarose was dropped in a Tissue-Tek OCT compound (Sakura Finetechnical Co Ltd, Japan) and frozen using dry ice Frozen sections were obtained by taking FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS 1237 MCM4 phosphorylation in mammalian cells Y Komamura-Kohno et al 10 lm slices of the compounded agarose using a Leica CM3050 Cryomicrotome Sections were incubated with 20% Blockace in NaCl ⁄ Pi They were incubated with P-32 antibodies ( lgỈmL)1) diluted with 5% Blockace (Dai-nippon Pharmaceuticals, Osaka, Japan) in NaCl ⁄ Pi for 48 h at °C and then incubated for 48 h at °C with anti-(rabbit IgG) sera linked to nm gold (Ultra-small; Aurion, Netherland) that had been diluted 40-fold with 5% Blockace in NaCl ⁄ Pi After washing with NaCl ⁄ Pi for h, the sections were incubated with 2% glutaraldehyde in phosphatebuffered saline for 30 and then washed with phosphatebuffered saline The sections were incubated with 50 mm Hepes–NaOH, pH 5.8 for 45 and then with distilled water for 30 They were incubated with an HQ silver kit (Nanoprobe, Gibson Research, CA) for 8–10 at room temperature in the dark and then washed with distilled water Sections were re-fixed with 0.5% OsO4 in distilled water for 20 After dehydration, the sections were embedded in Epoxy resin (Epon 812, TAAB, Aldermaston, England) Thin sections were obtained by cutting on a LeicaUltracut UCT ultramicrotome and collected on grids Sections were contrasted by exposure with 4% uranyl acetate and then observed using a JEOL JEM-1230 transmission microscope Acknowledgements We thank Dr Taku Chibazakura for his useful suggestions This study was supported in port by a grantin-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan References Tye BK (1999) MCM proteins in DNA replication Annu Rev Biochem 68, 649–686 Bell SP & Dutta A (2002) DNA replication in eukaryotic cells Annu Rev 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of successive multiparametric cytochemistry and microfluorometry on identical cells with special reference to cell cycle phases in a chick embryo Exp Cell Res 186, 6–14 32 Tanaka S, Ueda T, Nakajima K & Higashinakagawa T (1996) Replication patterns of repetitive DNA sequences on the W chromosome are altered during development of the chick embryo Exp Cell Res 223, 233–241 Supplementary material The following supplementary material is available online: Fig S1 Staining with P-3 antibodies of HeLa cells pulse-labeled with BrdU Logarithmically growing HeLa cells pulse-labeled with BrdU were stained with P-3 antibodies Staining with P-3 antibodies, anti-BrdU sera and DAPI, and a cell image to determine area are shown These are original data for quantification in Fig 5A This material is available from http://www.blackwellsynergy.com FEBS Journal 273 (2006) 1224–1239 ª 2006 The Authors Journal compilation ª 2006 FEBS 1239 ... 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 phosphorylation of MCM4 in phases... CDK2 during 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... 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 phosphorylation