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Copyright © 1998, The American Society for Cell Biology Negative Regulation of Cdc18 DNA Replication Protein by Cdc2 Mitsuhiro Yanagida, Monitoring Editor *Corresponding author. Received July 30, 1997; Accepted October 15, 1997. ![]() | |||||||||||||
Abstract Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle. | |||||||||||||
DNA replication must be stringently controlled to guarantee that the genome is duplicated exactly once during each cell cycle—failure to maintain this control would create havoc with the genome. Thus, once S phase is initiated, control mechanisms ensure that all chromosomal DNA is replicated and a new round of replication does not occur before chromosomes are segregated into the two daughter cells at mitosis. The mechanisms that limit DNA replication to once per cell cycle have been the focus of major research efforts in the last few years (Muzi-Falconi et al., 1996a The fission yeast Schizosaccharomyces pombe has served as an outstanding model organism for studying cell cycle controls. Recent studies of fission yeast have suggested that the Cdc18 protein plays an important role in regulating chromosomal DNA replication. The cdc18+ gene is essential for initiation of DNA replication (Nasmyth and Nurse, 1981 Many studies have implicated cyclin dependent kinases (CDKs) in the cell cycle controls regulating DNA replication (Muzi-Falconi et al., 1996a The Cdc2 substrates involved in control of initiation of DNA replication are unknown, although a clue was recently provided through the discovery of Orp2 as a protein that interacts with Cdc2 in vivo (Leatherwood et al., 1996 | |||||||||||||
Plasmids, Strains, and General Methods Schizosaccharomyces pombe strains expressing glutathione S-transferase (GST) or GST-Cdc18 were created by targeted integration of plasmids pAL24 and pAL27 at the ura4–294 locus. Plasmid pAL24 was constructed by inserting the 2.8-kb PstI–SacI fragment from pJL205 plasmid, containing the nmt1 promoter upstream of the GST open reading frame, into pJK210, an integrative vector (Keeney and Boeke, 1994 ![]() ![]() ![]() Expression from the nmt1 promoter was induced by thiamine depletion (Maundrell, 1993 Protein Methods Purification of GST fusion proteins expressed in fission yeast was performed as described previously (Leatherwood et al., 1996 ![]() ![]() In Vitro Mutagenesis Cdc18 threonine codons corresponding to residues 10, 46, 60, 104, 134, and 374 were changed to alanine and residue 104 to serine by Altered Sites II in vitro mutagenesis system (Promega, Madison, WI). Plasmid pALTER-cdc18 was constructed by inserting the 1.8-kb BamHI fragment from pAL27 into pATLER-1. This plasmid was used as template for the mutagenesis reaction. Primer sequences introducing the mutations were: AL39 (5′-GGTTGTCATG CACCTCGAAG-3′); AL40 (5′-ATTCCGACTG CACCC AGCAG-3′); A43 (5′-GCTCACATTT CCAAGCACCC ACAAAAAG-3′); AL33 (5′-ACTCCTAAAG CCCCCAAAAG-3′); AL35 (5′-TTGCAATCGG CACCTCACCG-3′); AL44 (5′-CAGAAAAAAA CAATCCTTTTG CTCCTATTAAA TCAATCTCTG-3′); and AL45 (5′-ACTCCTAAAT CCCCCAAAAGG-3′). DNA sequence analysis confirmed the mutations. The mutated genes were cloned into the integration plasmid pAL24, creating pAL27-T10A, -T46A, -T60A, -T104A, -T134A, T374A and, -T104S. These plasmids were integrated at the ura4–294 locus in OM1603 to create strains AL1988-AL1992 and AL2049. The strain AL2129 [cdc18-k9 leu1–32 ura4–294:nmt1-GST-cdc18T104A(ura4+)] was used for the mutant complementation studies.nmt1+ Promoter Turn-off Experiment The strains AL1854 and AL1991 were grown in minimal medium for 14 h before adding back 5 μl/ml thiamine to repress the expression of the nmt1+ promoter. Aliquots were taken at the indicated time points and processed for immunoblot analysis as described above. | |||||||||||||
High Expression of GST-Cdc18 Induces Continuous DNA Replication Biochemical investigations of Cdc18 were facilitated by construction of strains that used the nmt1 promoter to regulate expression of GST-Cdc18 fusion protein. Two experiments were carried out to analyze the functional properties of the GST-Cdc18 fusion protein in vivo. The first experiment tested the ability of GST-Cdc18 to rescue the cell cycle defect of a cdc18-K9 mutant. This analysis revealed that GST-Cdc18 expressed at very low levels from the repressed nmt1 promoter was sufficient to rescue the temperature-sensitive cell cycle defect of a cdc18-K9 mutant, indicating that GST-Cdc18 was functional in vivo. The second experiment evaluated whether GST-Cdc18 was able to induce cell cycle arrest and continuous DNA replication when highly overexpressed from the induced nmt1 promoter. Cells expressing unfused GST or GST-Cdc18 were incubated for 2 d on plates. Cells expressing GST appeared to be normal, whereas cells expressing GST-Cdc18 became extremely elongated, exhibiting a cell division cycle arrest phenotype (Figure 1A). Next, we measured the DNA content of these cells during the time course of induction. Cells expressing GST had 2C DNA content throughout the experiment, whereas DNA content increased steadily in cells expressing GST-Cdc18 (Figure 1B). These experiments show that GST-Cdc18 is functional and when produced at high levels it induces continuous replication without intervening mitosis.
GST-Cdc18 Interacts with Orp2, Cdc2 and B-type Cyclins A series of experiments were carried out to investigate the relationship between Cdc18 and Cdc2-cyclin B kinases. As a first step in this analysis, we asked whether GST-Cdc18 associates with Cdc2 and the replication factor Orp2. GST-Cdc18 and GST were purified from cell lysates using GSH-Sepharose and the bound proteins were separated by SDS-PAGE. Immunoblotting detection revealed that both Cdc2-Cdc13 and Cdc2-Cig2 kinases coprecipitated with GST-Cdc18, whereas none of these proteins associated with GST (Figure 2). Orp2 also coprecipitated specifically with GST-Cdc18. We previously reported an interaction between Cdc18 and Orp2 when both proteins were overexpressed (Leatherwood et al., 1996 ![]()
GST-Cdc18 Is Phosphorylated by a Rum1-sensitive Kinase We next explored the possibility that Cdc18 is a substrate of the kinase Cdc2. GST-Cdc18 was purified from S. pombe and incubated in the presence of [γ-32P]ATP. In this assay, GST-Cdc18 became heavily phosphorylated, indicating that GST-Cdc18 copurifies with a protein kinase that phosphorylates GST-Cdc18 efficiently (Figure 3, upper panel, lane 0). The potential involvement of Cdc2 was investigated by adding GST-Rum1 to the kinase reactions. Rum1 is a potent inhibitor of Cdc2-Cdc13 and a weaker inhibitor of Cdc2-Cig2 (Moreno and Nurse, 1994 ![]() ![]() ![]() ![]() ![]() ![]()
Cdc2 Phosphorylates Cdc18 The results described above supported the conclusion that Cdc2 is the kinase responsible for GST-Cdc18 phosphorylation. We further explored this question by determining whether GST-Cdc18 was phosphorylated by purified Cdc2 in vitro. Protein kinases that associated with GST-Cdc18 were inactivated by treatment with FSBA (ρ-fluorosulfonyl-benzoyl 5′-adenosine), an irreversible protein kinase inhibitor (Zoller and Taylor, 1979 ![]() ![]()
Cdc2 Phosphorylates Cdc18 on T104 Cdc2 prefers to phosphorylate serine or threonine in the motif S/T-P-X-K/R, in which X is any amino acid (Holmes and Solomon, 1996 ![]()
The T104A mutant phenotype may be attributable to increased activity or abundance of Cdc18. We addressed the latter possibility by immunoblot analysis of GST-Cdc18 and GST-Cdc18T104A following induction in thiamine-free medium. The abundance of GST-Cdc18 and GST-Cdc18T104A underwent a parallel and equal increase following induction in thiamine-free medium. Indeed, an nmt1 promoter turn-off experiment showed that GST-Cdc18 and GST-Cdc18T104A did not differ in stability (Figure 5D). This result strongly suggests that the T104A mutation enhances the intrinsic activity of GST-Cdc18 as opposed to altering GST-Cdc18 stability. Immunoblotting revealed that GST-Cdc18T104A coprecipitated with Orp2, Cdc2, Cdc13, and Cig2, indicating that the T104A mutation did not alter the association between Cdc18 and these interacting proteins (Figure 5E). Role of Cig2 in the Negative Regulation of Cdc18 Our findings are fully consistent with studies showing that Cdc2-Cdc13 kinase is required to prevent reactivation of replication origins during G2 phase (Hayles et al., 1994 ![]() ![]() ![]() ![]()
We also tested whether the Cig2 cyclin was capable of inhibiting rereplication. For this, cig2+ was expressed from the nmt1 promoter to uncouple cig2+ expression from cell cycle effects and to provide high levels of Cig2 that might overcome negative regulation by inhibitors or proteolysis. Selective spore germination was used to obtain cells lacking the essential B-cyclin Cdc13. As previously reported, Δcdc13 spores having only the wild-type allele of cig2+ are unable control replication and rereplicate their DNA without entering mitosis. Overexpression of cig2+ prevented the rereplication but was not sufficient to drive mitosis (Figure 6B). Germination experiments of Δcdc13 nmt1:cig2+ spores in presence of B1 gave an intermediate result, thus the level of cig2+ expression is responsible for the activity to inhibit rereplication. The observed effects are unlikely to be caused by cig2+ toxicity because overexpression of cig2+ has little effect on wild-type cells. | |||||||||||||
Genetic studies of fission yeast have established that the cyclin-dependent kinase Cdc2 is required for both positive and negative regulation of DNA replication. Put simply, these studies have shown that elimination of Cdc2 activity in G1 phase prevents the onset of S phase, whereas elimination of Cdc2 kinase in G2 phase causes the reinitiation of S phase without an intervening M phase (Broek et al., 1991 Herein we have described a series of physical and genetic interactions between Cdc2 and Cdc18. Foremost among these findings is the fact that functional GST-Cdc18 produced in fission yeast is associated with Cdc2 and two B-type cyclins, Cdc13 and Cig2. The GST-Cdc18 is isolated in association with an active protein kinases that phosphorylate Cdc18 and which are strongly inhibited by GST-Rum1. Rum1 is a highly specific inhibitor of Cdc2-Cdc13, as shown both by in vitro protein kinase assays and by the remarkably similar phenotypes which arise from high expression of rum1+ or deletion of cdc13+ (Hayles et al., 1994 Having discovered a close physical relationship involving Cdc18 and Cdc2, the questions remains as whether the interaction is important for regulating DNA replication and if so, is it concerned with the positive or negative regulation of S phase, or perhaps both. One approach to this problem is to begin with the hypothesis that Cdc2-Cdc18 association exists because Cdc18 is phosphorylated by Cdc13, hence mapping and mutating phosphorylation sites may reveal the role of Cdc2 in regulating Cdc18. As a first step in this investigative process we have mutated the six threonine residues in Cdc18 that are most likely to be phosphorylated by Cdc2. High expression of all six mutant forms of GST-Cdc18 induces continuous DNA replication at rate that is undiminished from that caused by high expression of wild-type GST-Cdc18. Therefore, in this type of assay, individual phosphorylation of the six sites is not required for Cdc18 activity. None of the mutant GST-Cdc18 proteins are deficient at inducing overreplication, but one of the mutants, T104A, consistently causes a moderate increase in the rate of DNA replication relative to wild-type. Importantly, T104 appears to be one of the major sites that is phosphorylated by Cdc2. Thus, at least in the Cdc18-induced DNA rereplication assay (Nishitani and Nurse, 1995 The biochemical data described above, which suggest that Cdc2 may catalyze the negative regulation of Cdc18, are consistent with the genetic data showing that Cdc2-Cdc13 represses the reinitiation of S phase in cells that are in the G2 phase. These data are also consistent with the observation that a moderate increase in Rum1 abundance will suppress, at least partially, the temperature-sensitive phenotype of a cdc18-K9 mutant (Jallepalli and Kelly, 1996 The genetic data indicating that Cdc2-Cig2 contributes to the inhibition of Cdc18 is derived from two experiments. The first experiment showed that elimination of the cig2+ gene rescues cdc18-K9, whereas the second experiment revealed that constitutive overproduction of Cig2 suppressed inappropriate DNA replication in Δcdc13 cells. The rescue of cdc18-K9 by Δcig2 is reminiscent of the rescue of cdc18-K9 by Rum1 overproduction (Jallepalli and Kelly, 1996 As mentioned above, a limitation of the phosphorylation site mutation studies presented in this report is that they have relied on high expression of GST-Cdc18 to assay changes in Cdc18 activity. Overexpression studies have been used in the past to illuminate the role of Cdc18 in regulating DNA replication (Nishitani and Nurse, 1995 In budding yeast Cdc6p associates with Cdc28p, a Cdc2 homologue, and Cdc28p is able to phosphorylate Cdc6p in vitro (Elsasser et al., 1996 The strong physical association between Cdc18 and Cdc2 is unusual for a kinase-substrate pair and is remarkably similar to the interaction of the Sic1p with Saccharomyces cerevisiae Cdc28p (Mendenhall, 1993 | |||||||||||||
ACKNOWLEDGMENTS We thank G. Degols, C. McGowan, N. Rhind, and K. Shiozaki for technical advice; K. Gould and S. Reed for antibodies; the Scripps Cell Cycle Labs for general support; T. Kelly for communicating results before publication; Ministerio de Educación y Ciencia (Spain) (A.L.-G.), American Cancer Society (J.L.), and the National Institutes of Health (P.R.). | |||||||||||||
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