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• W2019331336 abstract "Article17 June 2002free access Drf1, a novel regulatory subunit for human Cdc7 kinase A. Montagnoli A. Montagnoli Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author R. Bosotti R. Bosotti Department of Biology, DRO-Oncology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author F. Villa F. Villa Department of Biology, DRO-Oncology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author M. Rialland M. Rialland Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author D. Brotherton D. Brotherton Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author C. Mercurio C. Mercurio Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author J. Berthelsen J. Berthelsen Department of Biology, DRO-Oncology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author C Santocanale Corresponding Author C Santocanale Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author A. Montagnoli A. Montagnoli Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author R. Bosotti R. Bosotti Department of Biology, DRO-Oncology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author F. Villa F. Villa Department of Biology, DRO-Oncology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author M. Rialland M. Rialland Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author D. Brotherton D. Brotherton Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author C. Mercurio C. Mercurio Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author J. Berthelsen J. Berthelsen Department of Biology, DRO-Oncology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author C Santocanale Corresponding Author C Santocanale Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy Search for more papers by this author Author Information A. Montagnoli1, R. Bosotti2, F. Villa2, M. Rialland1, D. Brotherton1, C. Mercurio1, J. Berthelsen2 and C Santocanale 1 1Department of Pharmacology, Pharmacia Corp, 20014 Nerviano, Italy 2Department of Biology, DRO-Oncology, Pharmacia Corp, 20014 Nerviano, Italy *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:3171-3181https://doi.org/10.1093/emboj/cdf290 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Studies in model organisms have contributed to elucidate multiple levels at which regulation of eukaryotic DNA replication occurs. Cdc7, an evolutionarily conserved serine–threonine kinase, plays a pivotal role in linking cell cycle regulation to genome duplication, being essential for the firing of DNA replication origins. Binding of the cell cycle-regulated subunit Dbf4 to Cdc7 is necessary for in vitro kinase activity. This binding is also thought to be the key regulatory event that controls Cdc7 activity in cells. Here, we describe a novel human protein, Drf1, related to both human and yeast Dbf4. Drf1 is a nuclear cell cycle-regulated protein, it binds to Cdc7 and activates the kinase. Therefore, human Cdc7, like cyclin-dependent kinases, can be activated by alternative regulatory subunits. Since the Drf1 gene is either absent or not yet identified in the genome of model organisms such as yeast and Drosophila, these findings introduce a new level of complexity in the regulation of DNA replication of the human genome. Introduction In eukaryotic organisms, the duplication of the genome requires the action of at least two kinases: a cyclin-dependent kinase (CDK), Cdk2 in metazoans, and the Cdc7 kinase. Both of these kinases need to associate with a regulatory subunit in order to become fully activated. Two related proteins, cyclin E and cyclin A, bind and activate Cdk2 to regulate entry and progression through S phase, respectively. While the levels of Cdk2 do not change during the cell cycle, cyclin E and A have different timing of expression. Cell cycle fluctuation of these cyclins plays a major role in determining the timing of activation and substrate specificity of Cdk2 (reviewed in Sherr, 1993; Harper and Adams, 2001). The human Dbf4 gene was identified by a ‘two-hybrid’ screening using Cdc7 as bait. Physical interaction between Cdc7 and Dbf4 was confirmed further both in human cells and in insect cells infected with recombinant baculoviruses. Binding of Dbf4 to Cdc7 was shown to activate the kinase (Jiang et al., 1999; Kumagai et al., 1999). Cdc7 and Dbf4 proteins are well conserved during evolution (Johnston et al., 2000; Sclafani, 2000). In budding yeast, Dbf4 protein, like cyclins, oscillates during the cell cycle, it accumulates in S phase and is targeted for ubiquitin-mediated degradation by the anaphase-promoting complex (APC) at the end of mitosis (Oshiro et al., 1999; Weinreich and Stillman, 1999; Ferreira et al., 2000). In human cells, Dbf4 levels also oscillate during the cell cycle: in particular, Dbf4 accumulation, formation of a Cdc7–Dbf4 complex and Cdc7 kinase activity appear to fluctuate with approximately the same kinetics, suggesting that Cdc7 binding to Dbf4 is the main level of regulation of the kinase (Jiang et al., 1999; Kumagai et al., 1999). As in the case of CDKs, phosphorylation events may play an important regulatory role (Masai et al., 2000). Basic mechanisms that regulate origin activation appear to be highly conserved and have been characterized thoroughly in model organisms such as yeast and Xenopus. At the end of mitosis and in G1, a pre-replicative complex is formed around the DNA origin. This contains the origin recognition complex (ORC), Cdc6, Cdt1 and minichromosome maintenance (MCM) proteins (reviewed in Diffley and Labib, 2002). In budding yeast, Cdc7 kinase is required for the firing of DNA replication origins (Bousset and Diffley, 1998; Donaldson et al., 1998). It is thought that Dbf4 targets the kinase to pre-replicative complexes bound at origins and that, once in S phase, Cdc7 phosphorylates one or more subunits of the MCM complex, thus converting dormant pre-replicative complexes into two active replication forks (Dowell et al., 1994; Lei et al., 1997). Genetic evidence supports this notion, since the bob1-1 mutation in the MCM5 gene allows bypass of the requirement for both Cdc7 and Dbf4 for yeast DNA replication (Hardy et al., 1997). Importantly, once the activation step has occurred, neither Cdc7 nor Dbf4 appears to be necessary for ongoing replication (Bousset et al., 1998; Jares and Blow, 2000). The essential role of human Cdc7 and Dbf4 in the replication of the genome was established through microinjection of neutralizing antibodies (Jiang et al., 1999; Kumagai et al., 1999). Sequence analysis together with mutagenesis studies have defined a common architecture in the Dbf4-related protein family from different species. Three conserved amino acid boxes that, from the N- to the C-terminus of the protein, are called N, M and C motifs, respectively, can be identified (Masai and Arai, 2000). The M and C motifs define a bipartite module important for binding and activating the catalytic subunit (Masai et al., 2000; Ogino et al., 2001). The function of the N motif is not clear but, because of its similarity to a BRCT domain, it appears to mediate protein–protein interactions, possibly targeting Cdc7–Dbf4 complexes to origins of replication. The full sequencing of Saccharomyces cerevisiae and Drosophila melanogaster genomes has now revealed that only a single Dbf4-related gene exists in these organisms while, intriguingly, two genes showing significant sequence similarity, Dfp1/Him1 and Spo6, are present in Schizosaccharomyces pombe. Genetic and biochemical studies have now shown that while Dfp1/Him1 binds to the S.pombe Cdc7 homolog Hsk1 and is involved in DNA replication (Masai et al., 1995; Brown and Kelly, 1998), Spo6 associates with a different kinase, Spo4. This kinase is related to Hsk1 and is required for progression through meiosis II and sporulation (Nakamura et al., 2000, 2002). In order to understand fully the roles of the human Cdc7 kinase in the regulation of the cell cycle, we set out to look for new Cdc7 partners. Using a bioinformatic approach, we have identified a new gene coding for a protein, named Drf1, that has sequence similarity to Dbf4. This work provides experimental evidences that Drf1 is a novel, cell cycle-regulated protein that activates human Cdc7 kinase. Results Identification of the Drf1 gene To identify new partners for the Cdc7 kinase, we screened public and the LGtemplatesFEB2000 Incyte database using the whole human Dbf4 sequence as query. While at the time no matches were found in the public databases, two templates with low similarity to two different regions of Dbf4 were identified in the Incyte database (Incyte unique 150210.1 and Incyte unique 143458.1). Two clones belonging to the first template (3051528 and 477042) were fully sequenced. They were found to contain partially overlapping DNA fragments that defined a single full-length cDNA of 3006 bp. These sequence data have been submitted to the DDBJ/EMBL/GenBank database under accession No. AF448801. Analysis of the assembled sequence revealed the presence of an open reading frame (ORF) coding for a protein of 615 amino acids with a predicted mol. wt of 67 239 Da. Because of its similarity to Dbf4, we decided to give this new gene and the corresponding protein the name Drf1, for Dbf4-related factor 1. The similarity of Drf1 to Dbf4 is significant in the N-terminal regions of the proteins and within the three amino acid boxes that characterize the Dbf4 protein family. The similarity decreases in the C-terminal region (Figure 1A). Prosite scanning of the Drf1 sequence identifies a putative bipartite nuclear localization signal (PS50079) located just before the M box (amino acids 207–224). No other motifs within confidence levels were found, although a putative BRCT domain overlapping with the N box was detected at a low score (amino acids 43–96). Figure 1.Drf1 sequence analysis. (A) Sequence alignment of human Drf1 (top) and Dbf4 (bottom) proteins. Identical amino acids are on a black background; conserved substitutions are in gray. The N, M and C motifs conserved in the Dbf4 protein family are indicated by the boxes. The Drf1 putative bipartite nuclear localization signal is underlined. (B) Phylogenetic tree of Dbf4-related proteins: Hs: Homo sapiens, Mm: Mus musculus, Dm: Drosophila melanogaster, Sc: Saccharomyces cerevisiae, An: Aspergillus nidulans, Sp: Schizosaccharomyces pombe. Download figure Download PowerPoint Phylogenetic analysis shows a close evolutionary relationship between Drf1 and Dbf4 proteins, indicating that Drf1 belongs to the same protein family (Figure 1B). To date, however, we have not been able to identify other genes related to Drf1 in other species in the available databases. When the Drf1 cDNA sequence was used as bait to search the draft of the human genome, we found that Drf1 identifies the contig _010765 corresponding to a region of chromosome 17q21. We analyzed Drf1 expression in different tissues and cell lines by northern blotting. Drf1 generally is highly expressed in testis and in all tumor cell lines we analyzed, with the exception of the A549 (small lung cell carcinoma), while it is poorly expressed in most other tissues (data not shown). A similar expression pattern was observed previously for Dbf4 (Kumagai et al., 1999), suggesting that the two molecules might be co-regulated in cells. Reconstitution of recombinant Cdc7–Drf1 kinase The presence of amino acid motifs in the Drf1 protein similar to those that in Dbf4 are required for binding to Cdc7 (Masai and Arai, 2000) prompted us to test whether Drf1 could also directly bind to Cdc7 kinase. We therefore cloned Drf1, wild-type and mutant Cdc7(K90R) sequences into a baculovirus expression vector, thus obtaining a recombinant virus expressing these proteins as a fusion with GST. Lys90 is a critical residue for the ATP-binding domain, and mutation at this residue to arginine was shown to abolish the enzymatic activity of the Cdc7–Dbf4 complex (Jiang et al., 1999; Masai et al., 2000). Hi5 insect cells were infected with the Cdc7 virus alone or with the Drf1 virus in combination with either wild-type Cdc7 or mutant Cdc7(K90R) virus. Recombinant GST–Cdc7, GST–Cdc7/GST–Drf1 and GST–Cdc7(K90R)/GST–Drf1 proteins were purified in a two-step procedure involving glutathione–Sepharose affinity chromatography followed by size exclusion chromatography. This procedure allowed a purification of >90% and the separation of the Cdc7–Drf1 complex from free Cdc7. Purified proteins were separated on a polyacrylamide gel and visualized by silver staining. In these purified samples, we found that Drf1 and Cdc7 polypeptides were present in approximately the same amount and that mutation of Lys90 does not detectably affect the relative abundance of these proteins (Figure 2A). Figure 2.Purification and characterization of the Drf1–Cdc7 kinase complex. (A) Cdc7 (lane 1), Cdc7–Drf1 (lane 2) and Cdc7(K90R)/Drf1 (lane 3) recombinant proteins purified from insect cells as a fusion with GST were separated on a 10% polyacrylamide gel and stained with silver. (B) For each time point, 50 ng of recombinant Cdc7 (closed triangles), Cdc7–Drf1 complex (closed squares) or Cdc7(K90R)/Drf1 (open squares) were incubated in the presence of the Mcm2 N-terminal fragment (amino acids 1–285) and labeled ATP. Protein kinase activity of these proteins was measured using multiscreen plates as described in Materials and methods. (C) The Cdc7–Drf1 complex was incubated in the presence of 6 μM of the indicated proteins as substrate. After 30 (gray bars) or 60 (black bars) min, the reaction was stopped and proteins separated on a polyacrylamide gel. The amount of radioactivity in the different substrates was measured with a Storm 840 Phosphoimager. Mcm2 corresponds to the N-terminal 285 amino acid fragment of the Mcm2 protein, H1 and H3 to histone H1 and H3, respectively, MBP to myelin basic protein; and RB to the retinoblastoma protein. Download figure Download PowerPoint Next, we tested these samples for the presence of kinase activity using a fragment of the Mcm2 protein (amino acids 1–285) as substrate. Mcm2 (amino acids 1–285) was chosen for these experiments because it is known to be an excellent substrate for the Cdc7–Dbf4 complex in vitro (Sato et al., 1997). As shown in Figure 2, kinase activity was only detected in samples containing wild-type Cdc7 and Drf1 but not in samples containing either Cdc7 alone or a kinase-dead Cdc7–Drf1 complex. The specific activity of the Cdc7–Drf1 complex in this preparation was ∼6.5 nmol of phosphate transferred to the Mcm2 fragment/min/mg. ATP titration indicated that the apparent Km for ATP is ∼0.5 μM, similar to what was observed previously for the Cdc7–Dbf4 complex (Masai et al., 2000). When we used different proteins as substrate for Cdc7–Drf1 kinase at saturating concentration, we observed 4–5 times more activity with the Mcm2 fragment compared with histone H1 and myelin basic protein (MBP), and ∼8 times more when compared with casein; we failed to detect any activity using the retinoblastoma protein and histone H3 (Figure 2). This suggests that in vitro, Cdc7–Drf1, as previously observed for Cdc7–Dbf4, has substrate preference for Mcm2 (Masai et al., 2000; Sclafani, 2000). Drf1 interacts with Cdc7 kinase in mammalian cells To examine the ability of Drf1 to bind to Cdc7 in mammalian cells, the Drf1-coding sequence was cloned into the expression vector pCDNA-HA. With this construct, expression of Drf1 protein tagged with a single hemagglutinin (HA) epitope is driven by the cytomegalovirus (CMV) promoter. Flag-tagged wild-type or mutant Cdc7(K90R) expression plasmids were also prepared. These constructs were transfected in 293 cells in various combinations and cells were harvested 3 days post-transfection. Transfected HA-Drf1 protein normally migrates in polyacrylamide gels with an apparent mol. wt of ∼75 kDa. However, when it is co-transfected with Cdc7, the mobility of Drf1 is greatly reduced and it can be detected as a smear of bands between 75 and 105 kDa. The extent of this mobility shift appears to be variable, depending on the ratio of Drf1 and Cdc7 plasmids and on the time of cell collection after transfection (data not shown). Since Drf1 protein mobility shift is detected in the presence of a functional Cdc7 but not with kinase-dead Cdc7 (Figure 3A, lanes 1 and 2), we reasoned that the HA-Drf1 mobility shift may be due to Cdc7-dependent phosphorylation. This appears to be the case, since Drf1 mobility shift is reversed by treatment with intestinal calf phosphatase in a reaction that is blocked by addition of phosphatase inhibitors (Figure 3A, lanes 3–5). Figure 3.Characterization of Cdc7–Drf1 complexes in human cells. (A) Drf1 is phosphorylated when co-transfected with functional Cdc7. 293 cells were transiently transfected with HA-Drf1 in combination with either wild-type or kinase-deficient (K90R)Cdc7 constructs as indicated. Protein samples were either kept on ice (lanes 1 and 2) or incubated with or without calf intestinal phosphatase in the absence or presence of phosphatase inhibitors as indicated (lanes 3–5) before western blot analysis with anti-HA antibodies. (B and C) Formation of active Cdc7–Drf1 complexes in human cells. Protein extracts from 293 cells transfected with the indicated constructs were immunoprecipitated with either anti-HA (lanes 1–4) or anti-Flag (lanes 5–8) antibodies and subsequently incubated with radioactive ATP and a fragment of Mcm2 protein for 15 min before SDS–PAGE. In (B), Drf1 and Cdc7 were visualized with anti-HA or anti-Cdc7 antibodies, respectively. In (C), labeled proteins were visualized by autoradiography. Images correspond to different exposure times: a longer exposure was required to detect phosphorylated Drf1 and Dbf4 compared with phospho-MCM2. (D) Dbf4 does not immunoprecipitate with Drf1. Protein extracts from HeLa cells transfected with the indicated constructs were probed with anti-HA and anti-Cdc7 antibodies in western blot experiments in lanes 1 and 2. Drf1 and Dbf4, both tagged with the HA epitope, are indicated and migrate differentially in these gels. In lanes 3–5, extracts were immunoprecipitated with either anti-Drf1 mAb 5G4 or unrelated murine IgGs before western blot analysis. (E) Anti-Drf1 mAbs pull-down Cdc7 from extracts prepared from exponentially growing HeLa cells. In each lane, 4 mg of whole-cell extract were used in immunoprecipitation experiments with the anti-Drf1 mAbs 5G4 and 5H4 or control IgG. Western blot was performed with affinity-purified anti-Cdc7 polyclonal antibodies. Download figure Download PowerPoint Protein extracts prepared from transfected 293 cells as above were used in immunoprecipitation experiments with either anti-HA or anti-Flag antibodies. Before separating the proteins on acrylamide gel and western blot analysis, immunocomplexes were also incubated for 15 min in the presence of radioactive ATP and the Mcm2 fragment in kinase reaction buffer. The presence of HA-Drf1 and Cdc7 in the immunocomplexes was detected using anti-HA antibodies or anti-Cdc7 polyclonal antibodies, respectively. As shown in Figure 3B, anti-HA antibodies were able to immunoprecipitate Drf1 together with Flag-Cdc7. We also observed a strong immunoreactive band that migrates slightly faster than Flag-Cdc7 (lanes 1–3). This band corresponds to the endogenous Cdc7, since it cross-reacts with anti-Cdc7 antibodies and shows the same electrophoretic mobility as Cdc7 (data not shown). Anti-HA antibodies did not precipitate Cdc7 when HA-Drf1-expressing plasmid was not used in the transfection (lane 4). Reciprocally, anti-Flag antibodies efficiently precipitated transfected Flag-Cdc7 together with HA-Drf1 (lanes 6 and 7). As a negative control, immunoprecipitation was also performed from extract overexpressing only Cdc7 in the absence of Ha-Drf1 (lane 8). Both wild-type and mutant Cdc7 were able to co-precipitate with Drf1 with the same efficiency (Figure 3B, compare lanes 1 and 2, and 6 and 7), indicating that the interaction between these two proteins is not affected by the mutation in the catalytic site as previously observed with baculovirus-expressed proteins. We also observed that both modified and unmodified Drf1 were found in the anti-HA immunoprecipitate (lane 2) while only modified Drf1 is detected in the anti-Flag immunoprecipitate (lane 7). This result suggests that only when Drf1 is bound to Cdc7 can it be phosphorylated efficiently on those residues responsible for the change in the mobility. Finally, since we did not find endogenous Cdc7 in the anti-Flag immunoprecipitates (Figure 3B, lanes 5–8), dimerization of Cdc7 subunits does not occur in either the presence or absence of Drf1. Labeled phosphoproteins on the same filter were visualized subsequently by autoradiography. When both HA-Drf1 and wild-type Cdc7 were present in the immunocomplex, we observed efficient phosphorylation of Mcm2, irrespective of whether anti-HA antibody or anti-Flag antibody had been used for the immunoprecipitation (Figure 3C). As previously shown, phosphorylation of Mcm2 protein by Cdc7 kinase results in a reduced electrophoretic mobility of the substrate (compare lane 2 with lane 1, and lanes 5 and 7 with lane 6) (Masai et al., 2000). Since Cdc7 is not active in the absence of a regulatory subunit (Jiang et al., 1999; Kumagai et al., 1999) and overexpression of mutant Cdc7(K90R) strongly reduces the activity (compare lane 1 with lane 2, and lane 6 with lane 7), we reason that the kinase activity observed in the immunoprecipitates containing both Drf1 and wild-type Cdc7 is due to the formation of Cdc7–Drf1 active complexes as observed for Cdc7–Dbf4 (Figure 3C, lane 5; Jiang et al., 1999; Kumagai et al., 1999). Furthermore, a labeled phosphoprotein migrating at the same molecular weight as Drf1 could be detected only when Drf1 was transfected in combination with wild-type but not with the kinase-dead Cdc7. Altogether, these results indicate that Drf1 associates with Cdc7, that it activates the kinase and that Drf1 itself is a substrate of Cdc7 kinase. In order to understand whether Cdc7 can bind to both Drf1 and Dbf4 simultaneously, HeLa cells were transiently transfected with plasmids overexpressing Flag-Cdc7, HA-Drf1 and HA-Dbf4. HA-Drf1 and HA-Dbf4 proteins in these extracts were detected simultaneously using anti-HA antibodies and can be identified by their different molecular weights (Figure 3D, lanes 1 and 2). Immunoprecipitations were performed with either anti-Drf1 monoclonal antibody (mAb) 5G4 or control IgG. Using mAb 5G4, we were able to pull-down Drf1 together with Cdc7, independent of the presence of Dbf4 (Figure 3D, lanes 4 and 5). However, we failed to detect any Dbf4 protein in the immunoprecipitate when it was present in the extract (lane 5). This observation suggests that Drf1, Dbf4 and Cdc7 do not form ternary complexes efficiently and the binding of Drf1 and Dbf4 to Cdc7 is mutually exclusive under these conditions. When unrelated mouse IgGs were used instead, neither Drf1, Cdc7 nor Dbf4 were found in the beads (Figure 3D, lane 3). Further experiments with purified proteins will be required to understand fully the binding mode of Drf1 and Dbf4 to Cdc7. Finally, we asked if Drf1–Cdc7 interaction could be observed with endogenous proteins. To this end, extract prepared from exponentially growing HeLa cells was immunoprecipitated with either two different mAbs generated against Drf1 protein (mAb 5G5 and mAb 5H4) or unrelated mouse IgG. These immunocomplexes were then tested for the presence of Cdc7 protein using anti-Cdc7 polyclonal antibodies. Figure 3E shows that endogenous Cdc7 can be detected in both samples immunoprecipitated with the anti-Drf1 mAbs (lanes 1 and 2) but not in the control sample (lane 3), indicating that binding of endogenous Drf1 to endogenous Cdc7 occurs in HeLa cells. Cell cycle regulation of Drf1 expression It was observed previously that human Cdc7 kinase activity fluctuates during the cell cycle, peaking in S phase. It is believed that this cell cycle regulation occurs mainly at the level of binding with the Dbf4 regulatory subunit that is per se cell cycle regulated at both the mRNA and protein level (Jiang et al., 1999; Kumagai et al., 1999). We therefore investigated the possibility that Drf1 expression might also be cell cycle regulated. Levels of Drf1 mRNA during the cell cycle were studied by northern blot analysis in normal human dermal fibroblasts (NHDFs). Cells were arrested by serum starvation and stimulated to enter into the cell cycle by the addition of 10% serum. RNA samples were taken at different times after stimulation. Expression of both Dbf4 and Drf1 was low in G0-arrested cells and throughout G1; however, when cells reach the G1/S border, ∼14 h post-stimulation, both Dbf4 and Drf1 mRNAs begin to accumulate. Interestingly, we observed that while Drf1 mRNA keeps accumulating in S phase, Dbf4 levels begin to decrease at 20 h post-stimulation. Therefore, induction of Drf1 and Dbf4 upon re-entry into the cell cycle is a late G1 event that precedes initiation of S phase (Figure 4A and B). To expand the timing studies, we performed thymidine block and release experiment with the same fibroblasts. Samples for RNA, protein and fluorescence-activated cell sorting (FACS) analysis were taken. FACS analysis shows that thymidine-treated NHDFs are arrested in S phase, with a broad distribution of early, middle and late S phase cells. After release, cells recover DNA synthesis and synchronously go through the cell cycle. At 6 h post-release, most of the cells have a 4N DNA content; by 9 h, half of the population has completed mitosis; and by 12 h the majority are in G1. By 24 h post-release, cell synchrony appears to be lost. Under these conditions, Drf1 and Dbf4 mRNAs exhibit a moderate fluctuation during the cell cycle. Their levels peak at 9 h post-release, showing a 2- to 2.5-fold change between the lowest and highest levels (Figure 4C). Western blot analysis of protein extracts of the same experiment with the anti-Drf1 mAb 5H4 showed that Drf1 protein levels, in contrast to mRNA levels, first increase after the release from the thymidine block and then decrease when most of the cells appear to leave mitosis, with kinetics similar to the cyclin A decrease. In the same experiment, cyclin E protein fluctuates during the cell cycle as previously described (Ohtsubo et al., 1995; Ekholm and Reed, 2000). Figure 4.Cell cycle analysis of Drf1 and Dbf4 expression. (A and B) Drf1 and Dbf4 mRNAs are induced upon re-entry into the cell cycle. NHDFs were arrested in G0 by serum starvation and then stimulated by addition of 10% serum. At the indicated times, RNA was prepared and Drf1 and Dbf4 mRNA levels analyzed by northern blotting. (B) The DNA content at the indicated times measured by FACS. (C, D and E) Fluctuation of S-phase cyclins and Cdc7 regulatory subunits during the cell cycle in normal human fibroblasts. NHDFs were blocked in S phase with thymidine and released into fresh medium. (C) Drf1, Dbf4 and β-actin mRNA levels were analyzed by northern blotting. The bar chart represents the quantification of Drf1 and Dbf4 levels at different times after normalization with respect to β-actin. Arbitrary units are given on the y-axis. Data were obtained by analyzing radioactivity present in each lane for each hybridization using the Molecular imager FX phosphoimager and Quantity One software (Bio-Rad). (D) Western blot analysis of Drf1, cyclin E, cyclin A and cdk2 levels. (E) The DNA content at the indicated times measured by FACS. Download figure Download PowerPoint This experiment indicates that Drf1 protein is regulated during the cell cycle and suggests that, since Drf1 protein levels do not fully mirror mRNA levels, post-transcriptional mechanisms regulating Drf1 might exist. Drf1 is a protein with a short half-life Previous experiments show that Drf1 protein acts as a ‘cyclin’ for Cdc7 kinase: it binds to Cdc7, activates Cdc7 kinase and fluctuates during the cell cycle, although with different kinetics compared with its mRNA. Cyclin regulation is achieved both by cell cycle-dependent transcription and by controlled proteolysis (Ekholm and Reed, 2000; Tyers and Jorgensen, 2000). Dbf4, at least in budding yeast, has also been shown to be an unstable protein specifically degraded at the end of mitosis in an APC-dependent manner (Oshiro et al., 1999; Weinreich and Stillman, 1999; Ferreira et al., 2000). We therefore measured the half-life of Drf1 protein in human cells. HeLa cells were transiently transfected with HA-Drf1 plasmid, and 48 h later the protein synthesis inhibitor cycloheximide was added. Cells were collected at different times, and protein extracts were prepared and analyzed by western blot with anti-Drf1 antibodies. Figure 5 shows that Drf1 levels sharply decrease after addition of cycloheximide, while constant levels are seen in mock-treated samples. This result indicates that Drf1, at least when overexpressed, is a highly unstable protein. Densitometric analysis of the same blot suggests that Drf1 has a half life ≤1.5 h" @default.
• W2019331336 created "2016-06-24" @default.
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• W2019331336 date "2002-06-17" @default.
• W2019331336 modified "2023-09-30" @default.
• W2019331336 title "Drf1, a novel regulatory subunit for human Cdc7 kinase" @default.
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