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Originally published In Press as doi:10.1074/jbc.M002713200 on June 8, 2000

J. Biol. Chem., Vol. 275, Issue 37, 29042-29052, September 15, 2000
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Human Cdc7-related Kinase Complex

IN VITRO PHOSPHORYLATION OF MCM BY CONCERTED ACTIONS OF Cdks AND Cdc7 AND THAT OF A CRITICAL THREONINE RESIDUE OF Cdc7 BY Cdks*

Hisao MasaiDagger §, Etsuko MatsuiDagger , Zhiying You||, Yukio Ishimi||, Katsuyuki Tamai**, and Ken-ichi AraiDagger

From the Dagger  Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639,  CREST, Japan Science and Technology Corporation (JST), the || Mitsubishi Kasei Institute of Life Sciences, Machida 194-0031, and ** Medical and Biological Laboratories Co., Ltd., Nagano 1063-103, Japan

Received for publication, March 30, 2000, and in revised form, May 11, 2000

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

huCdc7 encodes a catalytic subunit for Saccharomyces cerevisae Cdc7-related kinase complex of human. ASK, whose expression is cell cycle-regulated, binds and activates huCdc7 kinase in a cell cycle-dependent manner (Kumagai, H., Sato, N., Yamada, M., Mahony, D., Seghezzi, W., Lees, E., Arai, K., and Masai, H. (1999) Mol. Cell. Biol. 19, 5083-5095). We have expressed huCdc7 complexed with ASK regulatory subunit using the insect cell expression system. To facilitate purification of the kinase complex, glutathione S-transferase (GST) was fused to huCdc7 and GST-huCdc7-ASK complex was purified. GST-huCdc7 protein is inert as a kinase on its own, and phosphorylation absolutely depends on the presence of the ASK subunit. It autophosphorylates both subunits in vitro and phosphorylates a number of replication proteins to different extents. Among them, MCM2 protein, either in a free form or in a MCM2-4-6-7 complex, serves as an excellent substrate for huCdc7-ASK kinase complex in vitro. MCM4 and MCM6 are also phosphorylated by huCdc7 albeit to less extent. MCM2 and -4 in the MCM2-4-6-7 complex are phosphorylated by Cdks as well, and prior phosphorylation of the MCM2-4-6-7 complex by Cdks facilitates phosphorylation of MCM2 by huCdc7, suggesting collaboration between Cdks and Cdc7 in phosphorylation of MCM for initiation of S phase. huCdc7 and ASK proteins can also be phosphorylated by Cdks in vitro. Among four possible Cdk phosphorylation sites of huCdc7, replacement of Thr-376, corresponding to the activating threonine of Cdk, with alanine (T376A mutant) dramatically reduces kinase activity, indicative of kinase activation by phosphorylation of this residue. In vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylates the Thr-376 residue of huCdc7, suggesting possible regulation of huCdc7 by Cdks.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Saccharomyces cerevisiae Cdc7 encodes a serine/threonine kinase essential for G1/S transition (1, 2). DBF4, originally isolated as a temperature-sensitive mutant defective in initiation of DNA replication (3), was later identified as a multicopy suppressor of cdc7(ts) (4). Furthermore, Dbf4 was shown to associate with Cdc7 and to activate its kinase activity (5). Kinases related to Cdc7 are widely conserved in eukaryotes, suggesting conserved important functions of this class of kinase in initiation of S phase (6-10). Recently, functional homologues of Dbf4 were identified in fission yeast and human (11-17). Genetic studies in fission yeast clearly demonstrate essential role of Hsk1-Dfp1/Him1 kinase complex (fission yeast homologue of Cdc7-Dbf4) in S phase initiation (11, 14).

In human, cellular Cdc7 kinase activity appears to be largely determined by the level of ASK protein, the human homologue of Dbf4, which fluctuates during the cell cycle. ASK protein as well as huCdc7 kinase activity are kept high during S phase and decreases by the next G1 phase. Microinjection of anti-ASK antibody into human primary fibroblast inhibited progression into S phase, indicating essential roles of ASK in mammalian DNA replication (13). Cdk2-Cyclin E is also essential for G1-S transition in mammalian cells, although its crucial target for S phase initiation has not been elucidated. It is not known if Cdc7 and Cdk2-Cyclin E function in a parallel pathway or in the same pathway for S phase initiation. Results in budding yeast have suggested that Cdc7 may be regulated by phosphorylation of the conserved threonine present near the APE motif (Thr-281), although the kinase responsible for this phosphorylation has not been known (18, 19, 41).

Similar to prokaryotic DNA replication, initiation of DNA replication in eukaryotes requires assembly of a series of replication proteins at the origins (20). These include ORC (origin recognition complex), Cdc6, MCM (minichromosomal maintenance), Cdc45, DNA polymerases, and single-stranded DNA-binding protein (RFA). One-hybrid assays in yeast indicated that Dbf4 protein is tethered at the origins (21), strongly suggesting that Cdc7 kinase complex is also present on chromatin in association with replication complexes at the origin. Biochemical studies have indicated that MCM complex may be an important target of Cdc7 kinase (7, 12, 14). Furthermore, genetic studies have shown that MCM2 may be a physiologically important substrate of Cdc7 (22).1

To characterize mammalian Cdc7 kinase complex in more detail, we have overexpressed and purified active human Cdc7-ASK kinase complex. Using a purified Cdc7 kinase preparation, we have examined phosphorylation of MCM complex and other various replication proteins by huCdc7-ASK kinase complex in vitro. We have shown that MCM2, either by itself or in a complex, serves as an efficient substrate of Cdc7 and that other replication proteins, including SV40 Tag, are also phosphorylated by huCdc7-ASK complex to different degrees. We have shown that Cdk-mediated phosphorylation of a MCM complex facilitates phosphorylation of MCM2 by huCdc7. We have also shown that Cdks, including Cdk2-Cyclin E, can phosphorylate huCdc7 and ASK proteins in vitro and identified a possible phosphorylation site mutation (T376A) of huCdc7 that significantly affects the kinase activity of huCdc7. We have shown that the critical Thr-376 residue is phosphorylated by Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B in vitro, providing the possible functional links between Cdks and Cdc7.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Construction of Insect Cell Expression Vectors for huCdc7 and ASK-- NotI-linearized pKU3-HA-short huCdc7 (7) was partially digested with XbaI and the 2.3-kilobase pair fragment containing the HA-tagged 562-amino acid (residues 13-574) huCdc7 coding frame was subcloned at NotI-XbaI sites of pVL1392, resulting in pVL1392-huCdc7(WT). The kinase-negative mutants, KE and KR (Lys-90 replaced by Glu and Arg, respectively), were similarly subcloned from pKU3-HAhuCdc7 into pVL1392. For expression of GST2-fused huCdc7 proteins in insect cells, the GST fragment, amplified by a set of primers (5' ATA AGA ATG CGG CCG CAT ATG TCC CCT ATA CTA GGT TAT 3'and 5' ATA AGA ATG CGG CCG CCA CGA CCT TCG ATC AGA TCC 3') on pGEX-5X-3, was digested with NotI and was inserted at the NotI site of pVL1392-huCdc7 (wild-type or mutants), resulting in in-frame fusion of GST and huCdc7 coding frames. For expression of ASK, pME18S-mycASK (13) was digested with StuI (present in the vector at 40 base pairs upstream of the myc tag) and NotI and subcloned at SmaI-NotI sites of pVL1393, resulting in myc-tagged ASK protein (containing 63 extra amino acids derived from the 5'-noncoding region).

Purification of GST-huCdc7-ASK Complex-- Three days after infection of Sf9 cells with recombinant virus solutions of GST-huCdc7 and myc-ASK (multiplicity of infection 2 of each), cells were scraped off from the plates and harvested. 109 cells were resuspended in 100 ml of IP buffer (40 mM Hepes·KOH (pH 7.6), 40 mM potassium glutamate, 10% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Nonidet P-40, and protease inhibitors). Cells were lysed in a glass hand homogenizer, and supernatant was obtained by centrifugation at 8000 rpm at 4 °C. After addition of glutathione-Sepharose 4B resin, the extract was incubated for 1 h with gentle rotation. The resin was thoroughly washed with the IP buffer, and washed resin could be directly used for in vitro kinase assays. To obtain purified soluble preparations of huCdc7 kinase complex, bound proteins were eluted from the resin with IP buffer containing 10 mM glutathione.

Purification of MCM2, MCM Complexes, SV40 Tag and p27-- Baculoviruses for preparation of mouse MCM2-4-6-7 complex were constructed as reported (23). For preparation of the MCM4-6-7 complex, Sf9 cells were coinfected with baculoviruses containing MCM7, cloned onto pVL1392, and those containing His-tagged MCM4 and MCM6. Baculoviruses containing MCM3, cloned onto pVL1392, and those containing His-tagged MCM5, cloned onto pAcHLT-A (PharMingen), were used for coinfection to prepare MCM3-5 complex. Mouse MCM2 gene was cloned into pAcHLT-A. Free His-tagged MCM2 and MCM complexes (MCM2-4-6-7, MCM4-6-7 and MCM3-5) were purified as described above except that cells expressing His-tagged MCM2 were resuspended in IP buffer containing 0.5 M NaCl. Nickel resin was added to the extracts, was extensively washed with IP buffer, and proteins were eluted from the resin by IP buffer containing 10 mM imidazole. Proteins were further purified by Mono Q column on SMART system (Amersham Pharmacia Biotech). The peak fractions containing the MCM2-4-6-7 complex, which eluted at 0.3 M NaCl, were dialyzed against IP buffer and were used for in vitro kinase assays. SV40 T antigen was purified from insect cells by using anti-Tag monoclonal antibody column, as described previously (42). Histidine-tagged mouse p27 protein, expressed in insect cells (gift from Dr. Junya Kato), was purified by nickel column, as described above for the MCM2-4-6-7 complex.

In Vitro Kinase Assays for huCdc7-ASK Kinase Complex-- The standard reaction mixtures (25 µl) for huCdc7 in vitro kinase assays contained 40 mM Hepes·KOH (pH 7.6), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM beta -glycerophosphate, 1 mM NaF, 2 mM dithiothreitol, 10 mM magnesium acetate, 80 µg/ml bovine serum albumin, 0.1 mM ATP, 1 µCi of [gamma -32P]ATP, 0.1-0.5 µg of MCM2 (or MCM2-4-6-7 complex) unless otherwise stated, and 50 ng of huCdc7-ASK kinase complex or immunoprecipitates. The reaction mixtures were normally incubated at 30 °C for 30 min. After addition of SDS-PAGE sample buffer, the reaction mixtures were incubated at 75 °C for 3 min and applied on 8% SDS-PAGE.

Expression and Purification of Cdk-Cyclin Complexes in Insect Cells-- Insect cell vectors for expression of mouse Cdk2, mouse Cdk4, human Cdc2, mouse Cyclin D1, human Cyclin E, and human Cyclin A were generously provided by Dr. H. Matsushime (24) and were used for coexpression with huCdc7 and ASK. Expression vectors for purification of mouse Cdk-Cyclin complexes, Cdk4, Cdk2, Cdc2, GST-Cyclin D1, GST-Cyclin E, GST-Cyclin A, and GST-Cyclin B were generous gifts from Dr. H. Yasuda, and Cdk-Cyclins were purified with glutathione-Sepharose 4B column, as described for purification of GST-huCdc7 kinase complexes.

Generation of Dephosphorylated MCM Proteins-- Affinity-purified preparations of free MCM2 or MCM2-4-6-7 complex were treated with lambda -phosphatase (New England BioLabs, Inc; 4000 units/1 ml of affinity-purified preparation), and dephosphorylation of MCM subunits was confirmed on SDS-PAGE. Then, it was further purified on Mono Q column to separate MCM from the phosphatase.

Two-step Kinase Reaction-- In the first stage, dephosphorylated or untreated MCM2-4-6-7 was incubated in the standard kinase reaction mixtures (lacking radioactive ATP) with Cdks for 20 min at 30 °C, and p27 (0.5 microgram) was added and was further incubated at 30 °C for 5 min. The second stage reaction was initiated by adding huCdc7-ASK and [gamma -32p]ATP (1 µCi), and aliquots were mixed with SDS-PAGE sample buffer at the times indicated. In control, p27 protein was present in the first stage reaction mixture.

Mutagenesis of huCdc7-- To generate mutant huCdc7 harboring an alanine substitution at four individual threonine or serine residue in TP or SP sequence, polymerase chain reaction-directed mutagenesis was conducted. Four sets of primers (5' ATG GCT TTT gCT CCC CAG CGT G-3'and 5'-CAC GCT GGG GAG cAA AAG CCA T 3', 5' ATT TCA CAT GAG gcC CCT GCA GTG AAA 3' and 5' TTT CAC TGC AGG Ggc CTC ATG TGA AAT 3', 5' TAG GGC AGG TgC ACC AGG ATT CA 3' and 5' TGA ATC CTG GTG cAC CTG CCC TA 3', 5' GGA TTC TAG CgC TCC CAA GTT AA 3' and 5' TTA ACT TGG GAG cGC TAG AAT CC 3') were used to introduce alanine substitution at Ser-16, Ser-302, Thr-376 and Thr-472, respectively. The first and the second of the pair was combined, respectively, with 5' CGG ATT TCC TTG AAG AGA GTG 3' and 5' CCT ATA AAT ATT CCG GAT TAT TC 3' (vector primers), for polymerase chain reaction amplification of C-terminal and N-terminal halves of the coding region. The polymerase chain reaction fragments were recovered from polyacrylamide gel and were mixed with the vector primers for amplification of the entire coding region with an expected mutation. The fragment was digested with NotI + SmaI, and inserted at NotI-SmaI site of pVL1392.

Cyanogen Bromide (CNBr) Digestion of Phosphorylated huCdc7 Protein-- After in vitro phosphorylation in the presence of 10-fold excess of labeled nucleotide, the products were run on 8% SDS-PAGE and were transferred to a nitrocellulose filter. The transferred protein band was excised, and the filter was incubated in 100 mg/ml CNBr in formic acid for 90 min at room temperature. The solution containing digested polypeptides was recovered and evaporated. After addition of 500 µl of water, it was further lyophilized and resuspended in SDS-PAGE sample buffer. The polypeptides were analyzed on a 24% low bis-Tricine gel.

Development of Phosphorylated Thr-376-specific Antibody-- A synthetic oligopeptide, CVAPRAGT(PO3H2)PGFRA (residues 370-381 of huCdc7), was synthesized and used as antigen to develop rabbit antisera. Antibody was affinity-purified using the nonphosphorylated peptide and the antigen peptide.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Active HA-tagged huCdc7-myc-tagged ASK Kinase Complex in Insect Cells-- The huCdc7 coding frame, either the wild-type or kinase negative, lacking the first 12 amino acids and tagged with the 7-amino acid hemaglutinin epitope tag (7), was cloned under the polyhedrin promoter on the insect cell expression vector pVL1393. Similarly, the ASK coding frame containing extra 63 amino acids derived from the 5'-noncoding region and fused to the myc epitope tag was cloned on pVL1393 (13). Both plasmids were transfected into insect cell Sf9, and recombinant viruses were recovered, which were coinfected into Sf9 cells. The huCdc7 and ASK formed a complex in insect cells, as indicated by coimmunoprecipitation of huCdc7 protein with anti-myc antibody (Fig. 1, lanes 1-3, lower panel). The immunoprecipitate containing wild-type huCdc7 and ASK exhibited two smeared phosphorylated bands on SDS-PAGE, which were identified as ASK, as previously shown with mammalian cell extracts overexpressing the both subunits (Ref. 13; Fig. 1, lane 1). This phosphorylation was not detected with KE or KR mutant huCdc7, in which the conserved lysine residue at position 90 was replaced with glutamic acid or arginine, respectively, although they associated with ASK (Fig. 1, lanes 2 and 3). Autophosphorylation of huCdc7 was not detected, when huCdc7 alone was expressed (Fig. 1, lane 4). Gel filtration analyses of the extract expressing both huCdc7 and ASK indicated that both proteins migrated at around 150 kDa, consistent with formation of a heterodimer (data not shown).


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  		Fig. 1.   In vitro phosphorylation assays with insect cell-expressed huCdc7-myc-ASK complex. The wild-type, KE, or KR mutant of huCdc7 was coexpressed with myc-tagged ASK (lanes 1-3), or only the wild-type huCdc7 was expressed (lane 4) in insect cells, and extracts were prepared. Proteins were immunoprecipitated by anti-myc antibody (lanes 1-3) or with anti-huCdc7 antibody (lane 4) and used for in vitro kinase assays as described under "Experimental Procedures." Products were analyzed on 10% SDS-PAGE and were autoradiographed (upper panel) or blotted with anti-huCdc7 antibody (lower panel). Lane 1, wild-type huCdc7 and myc-ASK; lane 2, KE huCdc7 and myc-ASK; lane 3, KR huCdc7 and myc-ASK; and lane 4, wild-type huCdc7 alone.

Purification and Characterization of GST-fused huCdc7 Protein Complexed with myc-ASK-- To facilitate purification of an active huCdc7 kinase complex, we have fused GST tag at the N terminus of huCdc7 and expressed the GST-huCdc7 fusion protein in Sf9. In separate experiments, we have fused the GST tag to the N terminus of Hsk1 (6), the fission yeast homologue of Cdc7, and showed the resulting GST-Hsk1 can complement the growth defect of the hsk1 null mutant,3 strongly suggesting that the presence of the GST tag at the N terminus of Cdc7-related kinases may not affect their functions. Sf9 cells, coinfected with recombinant viruses encoding GST-huCdc7 (wild-type or KE mutant) and myc-ASK, were harvested 3 days later, and extracts were prepared. GST-huCdc7-myc-ASK complex was affinity-purified onto glutathione-Sepharose 4B resin, and these preparations attached to the resin exhibited strong autophosphorylation activity in vitro (data not shown). The kinase complex could be eluted from the resin with buffer containing 10 mM glutathione. Since ASK is extensively phosphorylated in insect cells when coexpressed with huCdc7 and appears as smeared bands on SDS-PAGE (Fig. 2A, lane 2), the preparations containing the complex was pretreated with lambda -phosphatase prior to loading onto gel (Fig. 2A, lane 1). A major band corresponding to GST-huCdc7 and a cluster of several bands corresponding to myc-ASK were detected after silver staining in addition to several other contaminating bands (Fig. 2A, lanes 1 and 3). Only the huCdc7 band was detected in the preparations from the extract singly infected by wild-type GST-huCdc7 or KE GST-huCdc7 (Fig. 2A, lanes 4 and 5). These preparations were used for in vitro kinase assays using GST-MCM2N containing the N-terminal 206 amino acids of human MCM2 protein generated in Escherichia coli. Autophosphorylation of GST-huCdc7 and myc-ASK as well as phosphorylation of GST-huMCM2N was detected with the wild-type huCdc7 complex, but not with the kinase-negative mutant huCdc7 nor with free GST-huCdc7 proteins (Fig. 2B, lanes 1-4), consistent with requirement of ASK protein for expression of huCdc7 kinase activity. The purified protein in solution was more efficient in phosphorylating an exogenous substrate than the resin-bound form or the immunoprecipitates (data not shown; see Fig. 7B for comparison), probably due to absence of steric hindrance caused by the resin or associated antibody, which may inhibit the access of the substrate to the kinase.


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  		Fig. 2.   In vitro kinase assays with purified GST-fused huCdc7-myc-ASK complexes. Either wild-type or KE mutant GST-fused huCdc7 was coexpressed with myc-ASK in insect cells, and the complex was purified by glutathione-Sepharose 4B column. A, purified proteins were analyzed on 8% SDS-PAGE and were stained with silver. Lanes 1 and 2, wild-type huCdc7 and myc-ASK; lane 3, KE huCdc7 and myc-ASK; lane 4, wild-type huCdc7 alone; and lane 5, KE huCdc7 alone. In lane 1, purified protein fraction was treated with lambda -phosphatase prior to electrophoresis to eliminate phosphorylation, which causes mobility shift on the gel. The smear appearing between the 80- and 110-kDa markers in lane 2 represents autophosphorylated forms of myc-ASK protein. B, kinase assays were conducted with the purified proteins in the presence of GST-huMCM2N protein as a substrate and products were analyzed on 8% SDS-PAGE, followed by autoradiogram. Lane 1, wild-type huCdc7 and myc-ASK; lane 2, KE huCdc7 and myc-ASK; lane 3, wild-type huCdc7 alone; and lane 4, KE huCdc7 alone.

The amount of the incorporation could be quantified by spotting an aliquot on P81 paper, which was extensively washed in 75 mM phosphoric acid. At 30 °C, the reaction continued linearly for 10 min and leveled off after 60 min (Fig. 3A). With 5 pmol of the full-length MCM2 protein, expressed in and purified from insect cells, as a substrate, the incorporation increased linearly up to 0.7 pmol of huCdc7 kinase complex and reached 20 pmol of incorporation of phosphate (Fig. 3B), indicating that the kinase functions in a catalytic manner. The presence of salt was generally inhibitory for kinase activity. The level of incorporation was inhibited by 50% at 100 mM KCl (data not shown). The kinase reaction required the presence of a divalent cation, and the reaction reached plateu at over 4 mM Mg(OAc) and stayed at the similar level at up to 20 mM. Manganese could support only less than 20% of the incorporation of that attained with magnesium (Fig. 3C). The reaction was not significantly affected by pH between the range of 6.0 and 8.5 and slightly decreased at pH 9.0 (data not shown). The reaction was most efficient at 37 °C and was inhibited by 30% at 48 °C. Incubation of the purified protein at 55 °C for 5 min resulted in loss of 50% activity. Titration of ATP concentration indicated the apparent Km for ATP in phosphorylation of MCM2 (in the MCM2-4-6-7 complex purified similarly from insect cells, see below) by huCdc7 is 1-2 µM, indicating high affinity of huCdc7 kinase complex for ATP. GTP, CTP, and UTP at 1 mM did not support any detectable level of phosphorylation (Fig. 3D).


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  		Fig. 3.   Characterization of phosphorylation reaction by purified Cdc7 kinase complex. A, 5 × reaction mixture (125 µl) of the standard in vitro kinase assay for huCdc7 was incubated at 30 °C. B, standard in vitro phosphorylation reactions were conducted in the presence of various amount of purified huCdc7 kinase complex as indicated. C, in vitro phosphorylation assays were conducted in the presence of various concentrations of magnesium acetate (open circles) and manganese chloride (closed circles). In A and B, open and closed squares indicate the reaction with wild-type GST-huCdc7 + myc-ASK and KE GST-huCdc7 + myc-ASK, respectively. In A-C, full-length MCM2 protein (5 pmol/25-µl reaction mixture) was used as a substrate, and 10 µl was withdrawn at each time point or from each reaction mixture, and incorporation of 32P into proteins was measured by spotting onto P81 filter paper followed by extensive washing in 75 mM phosphoric acid. The values represent those for 25-µl reaction. D, in vitro phosphorylation assays were conducted in the presence of various concentrations of ATP or 1 mM GTP, CTP, or UTP with purified MCM2-4-6-7 complex as a substrate. The MCM2-4-6-7 complex was affinity-purified and used without prior phosphatase treatment and purification with Mono Q. The reactions did not contain labeled nucleotides and the products were analyzed on 8% SDS-PAGE, followed by silver staining of the gel. The phosphorylation of MCM2 and MCM4 was monitored by the intensities of the mobility-shifted bands on SDS-PAGE, which are phosphorylated forms. The ratio of the mobility-shifted forms (P-MCM2 or P-MCM4) to the unphosphorylated form (MCM2 or MCM4) was calculated and plotted for titration of ATP.

Phosphorylation of MCM2 Complex and T-antigen by huCdc7 Kinase Complex in Vitro-- Previous biochemical and genetic evidence indicated that MCM complex may be the physiological substrate of Cdc7-related kinases (7, 12, 22). Among the MCM components, MCM2 protein appears to be a conserved target of Cdc7 in various eukaryotes examined so far. We have purified various subassemblies of mouse MCM complex expressed in insect cells and used them as substrates for in vitro kinase assays.

Uncomplexed full-length MCM2 served as an excellent substrate for huCdc7-ASK (Fig. 4A, lane 3), and quantification of 32P incorporated into MCM2 indicated 2-2.5 molecules of phosphates were transferred to one molecule of MCM2 (Fig. 4B). MCM2-4-6-7 also served as an excellent substrate, and MCM2 was efficiently phosphorylated (Fig. 4A, lane 5). MCM4 and -6 also were phosphorylated by huCdc7, albeit with much less efficiency (Fig. 4A, lane 5). MCM4 and MCM6 in the MCM4-6-7 complexes were also phosphorylated to a similar extent (Fig. 4A, lane 7). Phosphorylation of the MCM3-5 complex by huCdc7 was not evident due to the presence of insect cell-derived unknown kinases in the preparation (Fig. 4A, lanes 9 and 10).


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  		Fig. 4.   Phosphorylation of various substrates by purified huCdc7 kinase complex in vitro. A, standard reaction mixtures for in vitro phosphorylation by huCdc7-ASK kinase complex were incubated in the presence of various substrates shown for 30 min at 30 °C, and products were analyzed on 8% SDS-PAGE. The gel was stained with silver (upper panel) and dried for autoradiogram (lower panel). Lanes 1 and 2, no substrate; lanes 3 and 4, uncomplexed MCM2; lanes 5 and 6, MCM2-4-6-7 complex; lanes 7 and 8, MCM4-6-7 complex; and lanes 9 and 10, MCM3-5 complex; and lanes 11 and 12, SV40 T-antigen. B, quantification of 32P incorporation into MCM2. In vitro phosphorylation assays were conducted with various amounts of the full-length MCM2 protein as substrate. After the products were run on 8% SDS-PAGE and transferred to 3MM paper, the phosphorylated MCM2 bands were excised, and radioactivity was measured by scintillation counter. The lower panel is the autoradiogram of the SDS-PAGE on which products were run. The upper panel shows MCM2 protein in each reaction mixture visualized by silver staining on a separate SDS-PAGE. The specific activity of [gamma -32P]ATP in the reaction mixtures were 860 cpm/pmol of ATP. Below the panels, the amounts of MCM2 protein and incorporation of 32P into MCM2 in each reaction are indicated. The values for incorporation have been corrected by subtraction of background incorporation on the lane 1 (without MCM2 protein), which represents a portion of the autophosphorylation of myc-tagged ASK protein.

It should be noted that phosphorylation of free MCM2 causes upward mobility shift on SDS-PAGE (Fig. 4A, lane 3), while MCM2 in the MCM2-4-6-7 complex shifts downward upon phosphorylation (Fig. 4A, lane 5). This indicates the presence of specific phosphorylation site(s) on MCM2 that become available for phosphorylation by huCdc7-ASK only in the complex.

Among other replication proteins examined, p180, p68 and p54 subunits of DNA polymerase alpha  (in collaboration with Drs. T. Mizuno and F. Hanaoka), ORC4 protein (Ref. 25; in collaboration with Dr. A. Dutta), Geminin (Ref. 26; in collaboration with Dr. T. McGarry) were phosphorylated by huCdc7 in vitro (data not shown). Viral initiators, SV40 Tag and bovine papilloma virus E1 proteins (in collaboration with Dr. J. Borowiec) were also phosphorylated by huCdc7-ASK (Fig. 4A, lane 11 and data not shown). SV40 Tag was phosphorylated to the level comparable with that of MCM2. The functional significance of this phosphorylation is being investigated.

Cdks and huCdc7 May Cooperate for Phosphorylation of MCM2-- The recombinant MCM proteins purified from Sf9 have undergone phosphorylation at the time of harvest presumably by a varieties of kinases present in the insect cells, which is estimated from the presence of mobility shifts on SDS-PAGE. This phosphorylation may affect its efficacy as a substrate for Cdc7 kinase. Sugino et al.4 have shown that phosphorylation of MCM2 with budding yeast Cdc7-Dbf4 in vitro depends on the presence of "prephosphorylation" of MCM2 by some other kinases. Therefore, we have generated "dephosphorylated" substrates by treating them with a phosphatase and further purifying them on a column and compared Cdc7-mediated phosphorylation of these dephosphorylated substrates with that of untreated ones. Treatment of the MCM complex with lambda -phosphatase resulted in disappearance of mobility-shifted bands on MCM2 and MCM4 (Fig. 5A, compare the silver staining pattern of the rightmost lanes of left and middle panels).


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  		Fig. 5.   Effect of prior dephosphorylation of MCM complex on efficacy of phosphorylation by huCdc7 kinase. Affinity-purified MCM2 and MCM2-4-6-7 were further purified by Mono Q column. One portion of MCM2-4-6-7 was treated with phosphatase prior to purification with Mono Q. A, in vitro phosphorylation assays were conducted with increasing amounts of each substrate in the reaction mixtures, and the products were analyzed on 8% SDS-PAGE, which was silver-stained (upper panels), dried, and autoradiographed (lower panels). The numbers indicate the positions of each subunit of MCM. B, the levels of MCM2 phosphorylation on the autoradiogram in A were quantified by Fuji image analyzer. The amount of MCM2 protein in each reaction mix was also measured by scanning the intensities of silver stained bands. Both measurements were expressed as relative values and they were plotted. Open squares, untreated MCM2-4-6-7; closed squares, phosphatase-treated MCM2-4-6-7; open circles, untreated full-length MCM2.

Interestingly, the efficiency of phosphorylation of dephosphorylated MCM complex, as detected by the appearance of the fully mobility-shifted form, was lower than that of untreated substrate (Fig. 5, A and B). MCM2 in the dephosphorylated MCM complex can be converted to this mobility-shifted form with 100% efficiency, if sufficient amount of huCdc7 is added, indicating that prephosphorylation of MCM2 only contributes to the efficiency of phosphorylation but not to the phosphorylation pattern by huCdc7. This may indicate that the presence of prephosphorylated residues on MCM2 may be required for efficient phosphorylation by huCdc7.

Some MCM proteins are known to be phosphorylated by Cdks. We have examined whether known Cdks can phosphorylate the MCM complex in vitro. Among Cdks examined, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B, but not Cdk4-Cyclin D1, phosphorylated MCM2 and MCM4 to a similar extent (Fig. 6A, lanes 5-7). Biochemical and genetic evidence which showed Cdc2-mediated phosphorylation of MCM4 was reported previously (27, 28). Ishimi et al. (43) recently reported that Cdk2-Cyclin A phosphorylates MCM4 and that this phosphorylation inhibits DNA helicase activity of MCM4-6-7 complex. Phosphorylation of MCM2 by Cdks did not cause any additional mobility shift on SDS-PAGE (Fig. 6A, compare lane 1 and lanes 5-7), whereas huCdc7 caused downward mobility shift (Fig. 6A, lane 2), indicating that huCdc7 phosphorylates sites on MCM2 distinct from those by Cdks. MCM4 is also mobility-shifted by phosphorylation with huCdc7-ASK but not with Cdks (Fig. 6, compare lane 2 and lanes 5-7), indicative of phosphorylation of distinct sites on MCM4 by huCdc7-ASK.


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  		Fig. 6.   Phosphorylation of MCM by various Cdks in vitro and its effect on phosphorylation by huCdc7 kinase. A, affinity-purified MCM2-4-6-7 was phosphorylated in vitro by huCdc7-ASK or by various Cdks (lanes 1-7). Lanes 8-12 did not contain any MCM. Kinases used are: none (lane 1), wild-type huCdc7-myc-ASK (lanes 2 and 12), KE huCdc7-myc-ASK (lane 3), Cdk4-Cyclin D1 (lanes 4 and 8), Cdk2-Cyclin E (lanes 5 and 9), Cdk2-Cyclin A (lanes 6 and 10), Cdc2-Cyclin B (lanes 7 and 11). Upper and lower panels show, respectively, the silver staining and autoradiogram of the 8% SDS-PAGE on which products were analyzed. B, effect of Cdk on rate of MCM2 phosphorylation by huCdc7. Lanes 1-14, dephosphorylated MCM2-4-6-7 complex as substrate; lanes 15 and 16, untreated MCM2-4-6-7 complex as substrate. Lanes 1 and 2, no Cdk; lanes 3-5; Cdk4-Cyclin D1; lanes 6-8; Cdk2-Cyclin E; lanes 9-11, Cdk2-Cyclin A; lanes 12-14, Cdc2-Cyclin B. Lanes 1, 4, 7, 10, 13, and 15, wild-type GST-huCdc7 + myc-ASK; lanes 2, 5, 8, 11, 14, and 16, KE GST-huCdc7 + myc-ASK. The lower panel is the short exposure of the same gel, in which only the protein bands of MCM2 are visible. C, p27 protein was incubated in the standard kinase assays for Cdks and huCdc7, and incorporation was measured by spotting an aliquot on P81 paper. Histone H1 (200 pmol) and MCM2-4-6-7 complex (3 pmol) were used as substrates for Cdks and huCdc7, respectively. One hundred percent incorporation in the absence of p27 was 81 pmol, 56 pmol, and 11 pmol for Cdk2-Cyclin E, Cdk1-Cyclin A, and huCdc7-ASK, respectively. D, two-step kinase reactions were conducted as described under "Experimental Procedures." p27 protein was included in the second stage (lanes 1-15) or in the first stage (lanes 16-26). Lanes 1-21, dephosphorylated MCM2-4-6-7 as substrate; lanes 22-26, phosphorylated MCM2-4-6-7 as substrate. Cdk2-Cyclin E (lanes 6-10, 18, and 19), Cdk2-Cyclin A (lanes 11-15 and 21) or none (lanes 1-5, 16, 17, 22-26) was added to the first stage reaction. Aliquots were withdrawn at t = 0 min (at the time of addition of huCdc7-ASK and radioactive ATP; lanes 1, 6, 11, 16, 18, 20, and 22), 5 min (lanes 2, 7, 12, and 23), 10 min (lanes 3, 8, 13, and 24), 20 min (lanes 4, 9, 14, and 25), 30 min (lanes 5, 10, 15, 17, 19, 21, and 26) for analysis on 8% SDS-PAGE. The levels of phosphate incorporation into histone H1 (400 pmol) by Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B used in A, B, and D above were 280, 190, and 210 pmol, respectively. The levels of phosphate incorporation into Rb protein (residue 769-921 of mouse Rb protein fused to GST, 150 pmol; purchased from Santa Cruz) by Cdk4-Cyclin D1 and Cdk2-Cyclin E were 61 and 93 pmol, respectively.

Therefore, it would be interesting to examine whether phosphorylation of MCM by Cdks affects efficiency of phosphorylation by hyCdc7. We have conducted kinase assays on dephosphorylated MCM2-4-6-7 complex in the presence of both huCdc7-ASK and a Cdk. The reaction mixtures contained suboptimal concentration of huCdc7 kinase, and less than half of MCM2 in dephosphorylated MCM2-4-6-7 complex was converted to the mobility-shifted form, whereas nearly all the MCM2 protein in the untreated MCM2-4-6-7 complex was converted to the shifted form (Fig. 6B, lanes 1 and 15). Under this condition, the presence of Cdk2-Cyclin A significantly stimulated huCdc7-mediated phosphorylation, as judged by the amount of the downward mobility-shifted form generated (Fig. 6B, compare lanes 1 and 10). Stimulation was observed also with Cdk2-Cyclin E and Cdc2-Cyclin B (Fig. 6B, lanes 7 and 13). Autophosphorylation of huCdc7 and ASK does not appear to be significantly affected by the presence of Cdk, suggesting that effect of Cdk is not due to modulation of Cdc7 activity per se. These results indicate that phosphorylation of MCM2 by huCdc7 may be stimulated by cophosphorylation by Cdks.

To show more directly that the effect of Cdks is mediated by their actions on MCM, not on huCdc7-ASK, we have conducted the reactions in two steps by taking advantage of the fact that p27 protein inhibits Cdk2-Cyclin E and Cdk2-Cyclin A, but has no effect on huCdc7 kinase activity (Fig. 6C). We first incubated the phosphatase-treated MCM2-4-6-7 with Cdk2-Cyclin E or Cdk2-Cyclin A and then added p27 protein to inactivate Cdks. huCdc7-ASK, together with radioactive [gamma -32P]ATP, was then added, and phosphorylation of MCM2 was analyzed on SDS-PAGE. Both Cdk2-Cyclin E and Cdk2-Cyclin A significantly increased the rate of MCM2 phosphorylation and the amount of the mobility-shifted form (Fig. 6D, lanes 6-15). When p27 was present in the first stage, no effect of the Cdks was observed (Fig. 6D, lanes 17, 19 and 21). With the untreated MCM2-4-6-7 complex, which has presumably been phosphorylated by insect cell-derived Cdks, efficient phosphorylation of MCM2 was observed, resulting in the appearance of mobility-shifted form after as early as 5-min incubation (Fig. 6D, lanes 22-26). The results clearly indicate that Cdks affect the efficacy of MCM2 phosphorylation by huCdc7 through phosphorylation of the MCM complex.

Threonine 376, Corresponding to Activation Threonine of Cdk, Is Required for Full Kinase Activity of huCdc7-- Cdc7-ASK mainly functions in G1/S transition and S phase presumably downstream of G1 Cdks, Cdk4-Cyclin D1 and Cdk2-Cyclin E. However, functional interactions, if any, between Cdc7 and Cdks are not known. There are four SP or TP sequences (Ser-16, Ser-302, Thr-376 and Thr-472), possible Cdk target sites, on huCdc7 (Fig. 7A). We have replaced each of them with alanine and expressed the resulting mutant huCdc7 in insect cells. We then coexpressed the mutant huCdc7-ASK complexes, which were coimmunoprecipitated with huCdc7 antibody for kinase assays. The mutations did not affect the complex formation between Cdc7 and ASK, as far as immunoprecipitation could measure (Fig. 7B, middle and lower panels). All the mutants, except for T376A, displayed kinase activity comparable with the wild-type (Fig. 7B, upper panel). T376A mutant huCdc7 exhibited significantly reduced phosphorylation activity. The extent of mobility shift of ASK and huCdc7 was significantly reduced in T376A mutant (data not shown), indicating that only subset of autophosphorylation sites on huCdc7 and ASK are phosphorylated with the T376A mutant.


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  		Fig. 7.   Kinase activities of huCdc7 mutants lacking possible Cdk phosphorylation sites. A, schematic representation of the positions of possible Cdk phosphorylation sites and that of the conserved lysine residue mutated in KE and KR mutants. Roman letters indicate the conserved kinase motifs proposed by Hanks et al. (40). B, wild-type or mutant huCdc7 was coexpressed with myc-tagged ASK in insect cells, and extracts were prepared. Proteins were immunoprecipitated by anti-huCdc7 antibody and were used for in vitro phosphorylation assays in the presence of GST-huMCM2N protein as a substrate. The huCdc7 used in the assays were: wild-type (lane 1), KE (lane 2); S16A (lane 3), S302A (lane 4), T376A (lane 5), and T472A (lane 6). Upper panel, autoradiogram of products of kinase assays run on 8% SDS-PAGE; middle panel, huCdc7 protein in the immunoprecipitates detected by Western blotting with anti-huCdc7 antibody; lower panel, myc-ASK protein in the immunoprecipitates detected by Western blotting with anti-myc antibody. Note that with wild-type, S16A, S302A, and T472A, myc-ASK is autophosphorylated and appears as a smear as a result of mobility shift. C, phosphorylation of MCM2 by the purified T376A GST-huCdc7-myc-ASK kinase complex. Upper panel, silver staining of the gel; lower panel, autoradiogram of the same gel. Kinases used were: no kinase (lanes 1 and 5), wild-type huCdc7 + myc-ASK (lanes 2 and 6), KE huCdc7 + myc-ASK (lanes 3 and 7), and T376A huCdc7 + myc-ASK (lanes 4 and 8). Substrates used were: free full-length MCM2 (lanes 1-4), and MCM2-4-6-7 complex (lanes 5-8). M, molecular weight marker. The affinity-purified substrates were further purified with Mono Q after lambda -phosphatase treatment.

We have purified GST-fused huCdc7 T376A mutant complexed with myc-tagged ASK and examined its kinase activity. The T376A supported only 10% level of phosphorylation of MCM2 protein compared with the wild-type (Fig. 7C, lower panel; compare lanes 2 and 4 and 6 and 8). Whereas the upward mobility shift in free form of MCM2 and downward mobility shift of MCM2 in the MCM2-4-6-7 complex are obvious after phosphorylation with the wild-type huCdc7-ASK (Fig. 7C, upper panel, lanes 2 and 6), they were barely detected with T376A mutant (Fig. 7C, upper panel, lanes 4 and 8). These results clearly show that phosphorylation activity is impaired in the T376A mutant. Thr-376 corresponds to Thr-160 of Cdk, whose phosphorylation by CAK is required for activation of Cdk (44). Therefore, huCdc7 may also be activated by phosphorylation of Thr-376 by some unknown kinase.

Phosphorylation of Thr-376 of huCdc7 by Cdks-- To explore possible interplay between Cdks and Cdc7, we examined whether Cdks can phosphorylate huCdc7-ASK kinase complex. When purified Cdks were incubated with purified GST-huCdc7 in vitro, Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B phosphorylated huCdc7 (Fig. 8A, upper panel, lanes 6-8). No significant phosphorylation of huCdc7 was detected with Cdk4-Cyclin D1 (Fig. 8A, lane 5). The T376A mutant was also phosphorylated by the three Cdks (Fig. 8A, upper panel, lanes 10-12). However, the level of phosphorylation by Cdks on the T376A mutant was slightly lower than that on wild-type. Both huCdc7 and ASK subunits in the huCdc7-ASK complex appear to be phosphorylated by the three Cdks (Fig. 8A, upper panel, lanes 18-20). Phosphorylation of the Thr-376 residue of huCdc7 by Cdk2-Cyclin E was also indicated by comparing the peptide mapping pattern of wild-type and T376A huCdc7 phosphorylated by Cdk2-Cyclin E in vitro. The polypeptide band indicated by an arrow in Fig. 8B is seen only in the sample from wild-type huCdc7 (Fig. 8B, lane 2).


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  		Fig. 8.   Phosphorylation of huCdc7 and ASK by Cdks. A, in vitro phosphorylation of huCdc7-ASK with Cdks. Purified huCdc7 proteins were phosphorylated by various Cdks in vitro in the standard kinase reaction. Reaction was run on 8% SDS-PAGE in duplicate, and one was dried and autoradiographed (upper panel), while the other was blotted by anti-phospho-Thr-376 peptide antibody (middle panel). The latter was deprobed and reprobed by the huCdc7 monoclonal sntibody4A8 (lower panel). Lanes 1-4, no substrate; lanes 5-8, wild-type GST-huCdc7; lanes 9-12, T376A GST-huCdc7; lane 13-16, KR GST-huCdc7; lanes 17-20, KR GST-huCdc7 complexed with myc-ASK. Lanes 1, 5, 9, 13, and 17, no Cdk added; lanes 2, 6, 10, 14, and 18, Cdk2-Cyclin E; lanes 3, 7, 11, 15, and 19, Cdk2-Cyclin A; lanes 4, 8, 12, 16, and 20, Cdc2-Cyclin B. The levels of Cdk activities used in this experiment are similar to those used in Fig. 6. B, the in vitro phosphorylated T376A (lane 1) and wild-type (lane 2) GST-huCdc7 proteins were digested with CNBr and analyzed on a 24% Tricine gel. huCdc7 preparations used in the above experiments (A and B) were pretreated with lambda -phosphatase prior to affinity purification.

To further confirm phosphorylation of the Thr-376 residue by Cdks, we have developed an antibody specific for the phosphorylated Thr-376 residue. The wild-type GST-huCdc7 protein phosphorylated by Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B reacted with the phosphopeptide antibody (Fig. 8A, middle panel, lanes 6-8), whereas T376A GST-huCdc7 protein did not react with the antibody under the same condition (Fig. 8A, middle panel, lanes 10-12). The KR mutant of GST-huCdc7, either free form or in a complex with myc-ASK, also reacted with the phosphopeptide antibody after phosphorylation by Cdks (Fig. 8A, middle panel, lanes 14-16 and 18-20). Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B could phosphorylate the Thr-376 residue to similar levels, although extra mobility shift was observed only with Cdk2-Cyclin E (Fig. 8A, middle panel, lanes 14 and 18). These results indicate that the critical Thr-376 residue of huCdc7 could be a target of phosphorylation by Cdks.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cdc7 kinase plays a crucial role in activation of preRC at each origin (29, 30). Genetic and biochemical evidence indicates that the MCM complex is an important target of Cdc7 kinase. Among the six components of MCM, MCM2 appears to be the conserved target of Cdc7-related kinases (7, 12, 22). Cdc7 and its related kinases require a regulatory subunit for expression of its kinase activity (16). Dbf4, Him1/Dfp1, and ASK form complexes with Cdc7, Hsk1, and huCdc7, respectively, and activate or stimulate the kinase activity of the latter subunits. In this report, we have purified the huCdc7-ASK kinase complex and characterized its biochemical properties and phosphorylation reactions. We attempt to explore the functional interactions among MCM, Cdc7, and Cdks, using in vitro kinase assays with purified proteins.

ASK Subunit Is Absolutely Required for Kinase Activity of huCdc7-- We previously reported that coexpression of ASK is required for kinase activity of huCdc7 in transient transfection assays in mammalian cells (13). We have confirmed this using purified protein preparations. The purified GST-huCdc7 protein was completely inert as a kinase. The complex of GST-huCdc7 and ASK was active and autophosphorylated both subunits in vitro. S. cerevisiae Cdc7 kinase also totally depends on association with its regulatory subunit Dbf4 protein for expression of its kinase activity.5 In contrast, fission yeast Hsk1 kinase possesses intrinsic kinase activity on its own, which is highly stimulated by the presence of Him1/Dfp1 protein (12, 14). Whereas purified Him1/Dfp1 protein is capable of activating purified Hsk1 protein in vitro (12),3 our attempt to reconstitute the active huCdc7-ASK kinase complex from purified proteins failed. An assembly factor may be required for formation of the active huCdc7-ASK kinase complex, as was reported for assembly of Cdk7-CyclinH complex (31). Alternatively, prior modification of either or both subunits may be required for stable association of the two proteins.

Substrates of huCdc7-ASK Kinase Complex in Vitro-- We previously reported that MCM subunits could be phosphorylated by huCdc7 in vitro (7). Subsequently, MCM2 was reported to be phosphorylated by Cdc7-related kinases in yeasts both in vivo and in vitro (12, 22).1 We therefore tested whether the purified huCdc7 kinase complex could phosphorylate MCM proteins in vitro. We purified various subassemblies of MCM protein complexes and used them in in vitro phosphorylation assays. Free MCM2, as well as MCM2 in the MCM2-4-6-7 complex, served as an excellent substrate for huCdc7. Quantification of in vitro phosphorylated MCM2 protein indicated the presence of multiple phosphorylation sites on MCM2 by huCdc7. The phosphorylation sites on MCM2 in the complex may not be identical with those on free MCM2, as indicated by differential mobility shift on SDS-PAGE as well as by two-dimensional peptide mapping.6

Prior phosphorylation of MCM2 in insect cells may affect the efficacy of Cdc7-mediated phosphorylation. In fact, prior dephosphorylation of the MCM complex reduced the efficiency of huCdc7-mediated phosphorylation by about 50%, although the identical mobility shift was observed on both untreated and dephosphorylated MCM2 proteins. Cophosphorylation of the MCM2-4-6-7 complex with Cdk2-Cyclin A/Cdk2-Cyclin E/Cdc2-Cyclin B, and huCdc7-ASK resulted in enhanced phosphorylation of MCM2 by huCdc7 kinase. Most notably, the appearance of the mobility-shifted form of MCM2 was significantly stimulated by prior phosphorylation of the MCM complex by Cdks (Fig. 6D), suggesting the presence of a specific phosphorylation site(s) on MCM2, the recognition of which by huCdc7 is highly facilitated by Cdk-mediated phosphorylation of the complex. This phosphorylation appears to be specific to MCM2 in the complex, since stimulation of Cdc7-mediated phosphorylation of MCM2 by Cdks occurs only with MCM2-4-6-7 complex, not with free MCM2 protein (data not shown). Cdk2-Cyclin E and Cdk2-Cyclin A can phosphorylate both MCM2 and MCM4 in the MCM2-4-6-7 complex. These phosphorylation events may induce conformational change of the MCM2-4-6-7 complex, facilitating the phosphorylation of MCM2 by huCdc7 kinase. Our results suggest the importance of concerted actions of Cdks and Cdc7 on MCM in regulation of S phase.

Although ample genetic and biochemical evidence demonstrates that MCM is essential for initiation of DNA replication, the precise role of MCM complex in origin activation is still not clear. Recent demonstration that DNA helicase activity is associated with the MCM4-6-7 complex (23, 32) may suggest that MCM plays essential roles in duplex melting at the origin and/or duplex unwinding at the replication forks. The possibility that phosphorylation of MCM by huCdc7 modulates DNA helicase activity of MCM complex is currently being explored. It is intriguing that SV40 T antigen, which is known to act as DNA helicase at the forks (33), can be efficiently phosphorylated by huCdc7-ASK in vitro (Fig. 4A). Bovine papillomavirus-encoded E1 protein, a viral helicase (34), can also be phosphorylated by huCdc7 in vitro (data not shown). DNA helicase activity of SV40 T antigen is known to be activated through phosphorylation of specific residues by multiple kinases (35), and the possibility that huCdc7 kinase is capable of activating T antigen is currently being examined.

A number of other replication proteins were phosphorylated in vitro by purified huCdc7-ASK protein, including p180, p68, and p54 subunits of DNA polymerase alpha  (36) and ORC4 (25, 37). The physiological significance of these phosphorylation events in vivo needs to be examined with genetic characterization in yeasts.

huCdc7 May Be a Target of Cdks-- Although huCdc7 kinase activity appears to be largely determined by the level of ASK subunit, which increases during S phase, posttranslational regulation of its kinase activity may also operate. The timing of kinase activation places this kinase downstream of G1 and G1/S Cdk. There are four SP/TP sites on huCdc7, which are potential phosphorylation sites by Cdks. Among the four serine/threonine-to-alanine mutants of huCdc7, only the T376A mutant displayed significantly reduced kinase activity in vitro. This Thr residue corresponds to Thr-160 of Cdk, an activation threonine, whose phosphorylation by CAK is essential for the kinase activity (38). Alanine substitution of the corresponding threonine of budding yeast Cdc7 resulted in a mutant with attenuated activity both in vivo and in vitro (18, 19).5

We have shown that huCdc7 protein can be phosphorylated in vitro by Cdk2-Cyclin A, Cdk2-Cyclin E, and Cdc2-Cyclin B, whereas Cdk4-Cyclin D1 did not phosphorylate it to any significant level. We have developed a specific antibody that recognizes the phosphorylated Thr-376 residue and have presented evidence that Cdk2-Cyclin E, Cdk2-Cyclin A, and Cdc2-Cyclin B are able to phosphorylate Thr-376 in vitro. It is especially an intriguing possibility that Cdk2-Cyclin E targets huCdc7 for activation by phosphorylating the Thr-376 residue. However, as seen in Fig. 6B, the presence of Cdks did not stimulate the kinase activity of huCdc7-ASK complex in vitro as judged by the level of autophosphorylation. This could be because the Thr-376 residue of huCdc7 within the complex may have already been phosphorylated by insect cell-derived kinase(s). In fact, significant level of phosphorylation of Thr-376 in GST-huCdc7(WT)-myc-ASK preparation is detected by the phospho-T376-specific antibody (data not shown). Therefore, fully dephosphorylated huCdc7-ASK complex needs to be prepared to examine the effect of Thr-376 phosphorylation on its kinase activity. Furthermore, analyses of in vivo phosphorylation of huCdc7 kinase during cell cycle would be needed to clarify the physiological role of this phosphorylation.

Although other three TA/SA mutations did not affect the intrinsic kinase activity of huCdc7, it is possible that phosphorylation of these residues by Cdks may regulate interaction of huCdc7-ASK complex with chromatin or other replication proteins. Multiple mobility-shifted bands of huCdc7 after phosphorylation by Cdks indicate the presence of more than one phosphorylation sites on huCdc7. In yeasts, Cdc7-Dbf4 was reported to be associated with chromatin only during S phase (39). The possibility that cell cycle-dependent association and dissociation of Cdc7 kinase complex with chromatin may be regulated by phosphorylation by Cdks is now being examined.

    ACKNOWLEDGEMENT

We thank Hiromi Iiyama and Chika Taniyama for excellent technical assistance on construction of various plasmid DNAs as well as for various aspects of this study. We are grateful to Hideyo Yasuda, Hitoshi Matsushime, and Junya Kato for providing us with recombinant insect cell baculoviruses expressing Cdk-cyclins and those expressing histidine-tagged p27. We also thank Satoshi Asano and Taku Tanaka for help and instructions in the use of SMART system. We also thank Akio Sugino for communicating unpublished results; Tadayuki Takada for critical reading of the manuscript; and Noriko Sato, Cho Ming Kwan, and other members of our laboratory for useful discussion.

    FOOTNOTES

* 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. Tel.: 81-3-5449-5661; Fax: 81-3-5449-5424; E-mail: hisao@ims.u-tokyo.ac.jp.

Published, JBC Papers in Press, June 8, 2000, DOI 10.1074/jbc.M002713200

1 T. Takeda, K. Ogino, K. Tatebayashi, H. Ikeda, K. Arai, and H. Masai, submitted for publication.

3 H. Masai, K. Ogino, and K. Arai, unpublished data.

4 M. Kihara, W. Nakai, S. Asano, A. Suzuki, K. Kitada, Y. Kawasaki, L. H. Johnston, and A. Sugino, submitted for publication.

5 H. Masai, unpublished results.

6 M. K. Cho, E. Matsui, N. Sato, K. Arai, and H. Masai, unpublished data.

    ABBREVIATIONS

The abbreviations used are: GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HA, hemagglutinin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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