Chk1 is a wee1 kinase in the G2 DNA damage checkpoint inhibiting cdc2 by Y15 phosphorylation
The EMBO Journal
(1997)
16: 545 - 554
The G2 DNA damage checkpoint ensures maintenance of cell viability by delaying progression into mitosis in cells which have suffered genomic damage. It is controlled by a number of proteins which are hypothesized to transduce signals through cell cycle regulators to delay activation of p34cdc2. Studies in mammalian cells have correlated induction of inhibitory tyrosine 15 (Y15) phosphorylation on p34cdc2 with the response to DNA damage. However, genetic studies in fission yeast have suggested that the major Y15 kinase, p107wee1, is not required for the cell cycle delay in response to DNA damage, although it is required for survival after irradiation. Thus, the target of the checkpoint, and hence the mechanism of cell cycle delay, remains unknown. We show here that Y15 phosphorylation is maintained in checkpoint‐arrested fission yeast cells. Further, wee1 is required for cell cycle arrest induced by up‐regulation of an essential component of this checkpoint, chk1. We observed that p107wee1 is hyperphosphorylated in cells delayed by chk1 overexpression or UV irradiation, and that p56chk1 can phosphorylate p107wee1 directly in vitro. These observations suggest that in response to DNA damage p107wee1 is phosphorylated by p56chk1 in vivo, and this results in maintenance of Y15 phosphorylation and hence G2 delay. In the absence of wee1, other Y15 kinases, such as p66mik1, may partially substitute for p107wee1 to induce cell cycle delay, but this wee1‐independent delay is insufficient to maintain full viability. This study establishes a link between a G2 DNA damage checkpoint function and a core cell cycle regulator.
Introduction
Controls over cell cycle transitions influence the activities of cyclin‐dependent protein kinases (cdks). The cdks required for each transition are activated and inactivated sequentially as cells cycle between alternating S‐phases and mitoses. There is an interdependency between these cell cycle transitions that must be maintained to ensure genomic integrity and prevent changes in ploidy; i.e. S‐phase onset is dependent on the completion of the previous mitosis, and the onset of mitosis is dependent on the completion of the previous S‐phase (Nurse, 1994). Further, as the fidelity of these events is absolutely crucial, a cell will not continue progression through the cell cycle when defects in its division programme are detected. The surveillance mechanisms which monitor the state of the cell, and are believed ultimately to influence cell cycle transitions either directly or indirectly through the timing of cdk activation, are known as checkpoints (Hartwell and Weinert, 1989).
The transition from G2 into mitosis requires activation of an archetypal cdk, p34cdc2 (Nurse, 1990). In the fission yeast Schizosaccharomyces pombe, as in all eukaryotes, this requires association of p34cdc2 with its regulatory B‐type cyclin subunit and phosphorylation on threonine 167 of p34cdc2 by a constitutive activity known as cdk‐activating kinase (CAK). This potentially active complex is maintained in an inactive state by inhibitory phosphorylation in the active site of p34cdc2 on tyrosine 15 (Y15) by wee1‐ and mik1‐encoded tyrosine kinases throughout the G2 period. When conditions are appropriate for entry into mitosis, the inhibitory phosphorylation on Y15 is removed by the cdc25‐encoded Y15 phosphatase, and mitosis is initiated rapidly. This activating modification is the rate‐limiting step for entry into mitosis.
During G2, there are checkpoints which monitor completion of S‐phase and prevent mitosis in its absence, and which also monitor the integrity of the genome and act to prevent the onset of mitosis when damage is detected. Genetic studies in fission yeast have identified a number of checkpoint proteins which are required for either, or in most cases both, of these checkpoints (Humphrey and Enoch, 1995). These proteins are non‐essential, and so act to alter cell cycle transitions only under conditions of stress. In some cases, the predicted amino acid sequences have given some clue as to their function, but in general we have little idea as to how these proteins function to elicit cell cycle arrest. Genetic studies so far have not been particularly illuminating, as the non‐essential nature of these genes has led in most part to the isolation of null alleles, which then fail to show interactions with each other. Similarly, it has also been difficult to build direct links between the checkpoint proteins and the core cell cycle regulators in response to G2 checkpoint‐mediated signals. In the case of the DNA replication checkpoint, inhibitory Y15 phosphorylation has been shown to be the end‐point of this checkpoint, but what lies directly upstream is unknown (Enoch and Nurse, 1990). With the G2 DNA damage checkpoint, the involvement of p34cdc2 Y15 phosphorylation is less clear. Cells lacking the major Y15 kinase encoded by wee1, p107wee1, delay cell cycle progression following DNA damage and yet are still hypersensitive to DNA‐damaging agents (Barbet and Carr, 1993). Therefore, this delay may not represent a true checkpoint in that cell viability is not maintained. This is compounded further by the observation that wee1− mutants are synthetically lethal with G2 checkpoint mutants, indicating that these cells require a constitutively active checkpoint to maintain viability (Al‐Khodairy et al., 1994). Although there have been no reports as to the effects of evoking the G2 DNA damage checkpoint on Y15 phosphorylation in fission yeast, in mammalian cells it has been shown to correlate with cell cycle arrest following DNA damage in G2 (Kharbanda et al., 1994a). However, the kinase responsible for this is unknown, although possible candidates are the wee1 kinase and the src‐related kinase p53/p56lyn (Kharbanda et al., 1994b). Furthermore, in the G1 DNA damage checkpoint, it has been shown through overexpression of a mutant cdk4 allele (Cdk4F17) that cell cycle progression may in part be delayed through tyrosine phosphorylation of cdk4 (Tereda et al., 1995), in addition to the well established mechanism acting through cdk2 inhibition by binding of the inhibitory protein p21CIP1/WAF1 (Dulic et al., 1994; Macleod et al., 1995).
Given the confusion surrounding these issues, we chose to address the role of p34cdc2 Y15 phosphorylation in the fission yeast G2 DNA damage checkpoint. Our studies described here show that Y15 remains phosphorylated after cells sustain DNA damage in G2 even though they have passed beyond the point at which Y15 normally would be dephosphorylated. This suggests that evoking the checkpoint leads to a maintenance of G2 through maintaining p34cdc2 in its Y15‐phosphorylated state. To confirm that maintenance of Y15 phosphorylation is linked to activation of the checkpoint, we have used the gain‐of‐function phenotype caused by overexpression of one component of the G2 DNA damage checkpoint, chk1. This gene encodes a protein kinase, p56chk1, which appears to function downstream of the other identified checkpoint functions. We show here that p56chk1 elicits a cell cycle arrest in G2 that acts through p107wee1‐dependent Y15 phosphorylation of p34cdc2. Further, we show that p107wee1 is phosphorylated in cells arrested by chk1 overexpression or UV irradiation. Finally, we show that p56chk1 can phosphorylate p107wee1 directly in vitro, thus establishing a potentially direct link between the signal transduction cascade which makes up the G2 DNA damage checkpoint and a core cell cycle regulator.
Results
DNA damage‐induced cell cycle arrest results in maintenance of p34cdc2 Y15 phosphorylation
In fission yeast, it has been established that activation of p34cdc2 through dephosphorylation of Y15 is the rate‐limiting step for entry into mitosis (Millar et al., 1991). During the cell cycle, there is a checkpoint which prevents the onset of mitosis until the completion of S‐phase. This checkpoint has been shown, at least in part, to act through inhibitory Y15 phosphorylation of p34cdc2 (Enoch and Nurse, 1990). Many proteins involved in this checkpoint have been identified by mutational studies, and most of these are also required for the checkpoint which prevents mitosis from occurring when the cell has suffered DNA damage (Humphrey and Enoch, 1995). Despite this, no effect of DNA damage‐induced arrest in G2 on Y15 phosphorylation has yet been demonstrated, and so we sought to establish whether evoking this checkpoint influenced the phosphorylation of p34cdc2 on Y15.
In order to investigate this, we prepared small fission yeast cells synchronized in early G2 by centrifugal elutriation. These cells were split into two populations: one was irradiated with 100 J/m2 UV‐C to evoke the DNA damage checkpoint, and the other was left unirradiated as a control. These cells were then re‐inoculated into media, and samples were taken over a timecourse to assay for cell cycle progression, and for p34cdc2 Y15 phosphorylation (Figure 1). In the irradiated cells, we observed a delay in cell cycle progression and dephosphorylation of Y15 compared with unirradiated controls. Dephosphorylation of Y15 was evident after 60 min in the control cells, but not until 120 min in the irradiated cells. By the end of the timecourse (180 min), Y15 phosphorylation re‐appeared in the unirradiated controls as they had passed START of the subsequent cell cycle. A similar delay was also observed for activation of p34cdc2 kinase activity (data not shown). These data show that in fission yeast, as in mammalian cells, p34cdc2 is maintained in its Y15‐phosphorylated state during a G2 cell cycle delay in response to DNA damage. Although there was not an increase in Y15 phosphorylation, this was already at high levels as the cells used for the experiment had been synchronized in G2. However, this G2 level of Y15 phosphorylation is maintained past the point at which p34cdc2 normally would be activated by Y15 dephosphorylation due to the cells passing both the size and completion of S‐phase requirements for mitosis. This finding suggests that the maintenance of G2 as a consequence of the DNA damage checkpoint control in fission yeast may involve inhibition of p34cdc2 by Y15 phosphorylation.
Chk1 promotes cell cycle arrest associated with inhibition of p34cdc2 by Y15 phosphorylation
One problem in studying the G2 DNA damage checkpoint in fission yeast has been that the majority of identified mutations represent null alleles, and so it has been difficult to establish genetic interactions between the checkpoint functions themselves, or with core cell cycle regulators. One exception to this is chk1, which encodes a protein kinase, p56chk1, that is essential for the G2 DNA damage checkpoint. There is a gain‐of‐function caused by overexpression of chk1 which can, by itself, elicit a cell cycle arrest in G2 in both wild‐type and checkpoint mutant cells (Walworth et al., 1993; Al‐Khodairy et al., 1994; Ford et al., 1994). In keeping with this, moderate increases in the level of chk1 can rescue the radiation sensitivity caused by lack of some checkpoint functions. Furthermore, p56chk1 is phosphorylated in response to DNA damage, but this response is blocked in cells carrying mutations in the other checkpoint genes (Walworth and Bernards, 1996). It would appear, therefore, that p56chk1 acts near the end of the G2 DNA damage checkpoint signal transduction pathway, downstream of the other identified components, and possibly close to p34cdc2.
We wished to determine whether the cell cycle arrest caused by chk1 overexpression was equivalent to that involving activation of p56chk1 by upstream components of the checkpoint following DNA damage, and whether this would result in our observed maintenance of Y15 phosphorylation. To this end, we overexpressed chk1 from the nmt1 promoter in wild‐type cells. Between 10 and 12 h growth in the absence of thiamine, which is required for derepression of the promoter (Maundrell, 1993), we observed a cessation of cell division and loss of p34cdc2 kinase activity, which was confirmed to be concomitant with induction of p56chk1 by Western blotting (Figure 2). Unlike the more transient effects of evoking the G2 DNA damage checkpoint by UV irradiation, continued overexpression of chk1 led to cells becoming highly elongated, as they were maintained in a G2 arrest with inactive p34cdc2. Consistent with this, strains arrested by chk1 overexpression accumulated significantly higher levels of Y15‐phosphorylated p34cdc2 than did unarrested controls (Figure 2). Thus chk1 overexpression has a similar effect to evoking the G2 DNA damage checkpoint: cells are arrested in G2 because p34cdc2 is maintained inactive by Y15 phosphorylation, and continuation of chk1 overexpression is, therefore, analogous to a constitutively acting checkpoint arrest.
Chk1 overexpression elicits a cell cycle arrest through wee1
wee1 encodes the major p34cdc2 Y15 kinase in fission yeast, p107wee1 (Russell and Nurse 1987b; Lundgren et al., 1991). We therefore asked whether chk1 mediates its arrest on cell cycle progression via p107wee1. If this were the case, then a wee1Δ mutant should be defective in its response to chk1 overexpression. We tested this hypothesis by overexpressing chk1 in a strain (wee1Δ) lacking the p107wee1 kinase. Under the same conditions as the wild‐type controls, wee1Δ strains overexpressing chk1 continued to divide at the same rate (Figure 3), and maintained the cell size and cell cycle distribution of vector‐only controls (Figure 3), i.e. in the absence of wee1, cells are completely non‐responsive to overexpression of chk1. These data show that the chk1‐mediated cell cycle arrest acts through wee1. If p56chk1 were alternatively acting by inhibiting the p80cdc25 phosphatase, then we would have observed a suppression of the ‘wee’ phenotype in these cells, which was clearly not the case. This is in keeping with the biochemical studies showing p80cdc25 levels to be low in G2 and activated on entry into mitosis (Moreno et al., 1990; Dunphy, 1994).
The p107wee1 protein kinase phosphorylates p34cdc2 on Y15, and levels of this accumulate during a wee1 dependent cell cycle arrest induced by chk1 overexpression. We wished, therefore, to confirm that under these conditions p107wee1 and p56chk1 were acting through this mechanism. To this end, we compared the effects of chk1 overexpression in strains carrying alleles of cdc2 which are altered in their sensitivity to regulators of Y15 phosphorylation, and confer a wee phenotype due to a shortening of the G2 period. As predicted, cdc2‐1w mutants, which have a reduced sensitivity to wee1 (Russell and Nurse, 1987b), were less sensitive to chk1 overexpression than cdc2‐3w mutants, which have a reduced requirement for the cdc25‐encoded phosphatase (Nurse, 1975) (Figure 3). Furthermore, the chk1‐induced arrest was suppressed by co‐overexpression of the cdc25 phosphatase, and was not dependent on the presence of the nim1/cdr1 kinase (Russell and Nurse, 1987a), a negative regulator of p107wee1 (data not shown). These data confirm that chk1 overexpression arrests cells due to maintaining wee1‐mediated Y15 phosphorylation of p34cdc2.
p107wee1 is hyperphosphorylated in cells arrested by DNA damage or chk1 overexpression
Given the requirement for p107wee1 for chk1‐mediated cell cycle arrest, we decided to assay p107wee1 for modifications consistent with a direct interaction between p107wee1 and p56chk1. Previous studies have shown that p107wee1 is present at concentrations too low to be detected by conventional antibodies (Aligue et al., 1994). Detection of p107wee1 cannot be aided by overexpression of the protein as this induces a strong cell cycle arrest. In order to alleviate this problem, we constructed strains expressing a fully functional wee1 from its endogenous promoter, which was engineered to allow detection and isolation of p107wee1: a triplicated influenza virus haemagglutinin (HA) tag, which has been shown to be effective in detecting low abundance proteins (Tyers et al., 1993), was added to allow immunodetection of the protein with the 12CA5 monoclonal antibody. Six consecutive histidines were also added to allow isolation of the protein on Ni–agarose under conditions which would preserve any post‐translational modifications (Figure 4). That the tagged form of wee1 was functional was confirmed by complementation of mutations in wee1 (wee1‐50 and wee1Δ) and the ability of this construct to induce G2 arrest when overexpressed from the nmt1 promoter (data not shown).
Using Western blotting, we showed that p107wee1 migrated predominantly as a single band with a smearing of higher molecular weight species. However, when chk1 was overexpressed, p107wee1 now migrated as a doublet, and the amount of the higher molecular weight band was dependent on the level of chk1 expression (Figure 4). In cells containing a single integrated copy of nmt1::chk1, the higher molecular weight form of p107wee1 was dependent on the derepression of the promoter. A similar distribution of p107wee1 species was seen in cells arrested by chk1 overexpression from a multi‐copy plasmid. Even under repressing conditions, the nmt1 promoter directs substantial levels of gene expression (Forsburg, 1993). Therefore, cells carrying multi‐copy nmt1::chk1 have increased levels of p56chk1 (data not shown). In these strains, a substantial proportion of p107wee1 was retarded in mobility, but this was to a lesser extent than when the promoter was derepressed and was insufficient to result in cell cycle arrest. Thus, there is a chk1 dose‐dependent modification on p107wee1 which is associated with cell cycle arrest and maintenance of p34cdc2 Y15 phosphorylation. In no case did we observe a mobility shift for all the p107wee1, even though cells were first boiled and extracted under denaturing conditions in the presence of phosphatase inhibitors (Materials and methods). This observation suggests that a proportion of this protein may be inaccessible to p56chk1 in vivo, and may reside in a separate cellular compartment.
Overexpression of chk1 has a similar effect on p34cdc2 Y15 phosphorylation as did DNA damage. We therefore assayed the effect of DNA damage on p107wee1, and observed that it was similarly retarded in mobility when prepared from irradiated cells, although to a lesser extent (Figure 4). This may be due to the more transient effects of radiation‐induced DNA damage (Figure 1), as compared with the sustained cell cycle arrest elicited by continued chk1 overexpression (Figure 2). The latter would allow a more pronounced accumulation of the modified form of p107wee1 and Y15‐phosphorylated p34cdc2. The mobility shift was not, however, a consequence of G2 arrest in these cells, as it was not observed in elutriated G2 cells, or in cells arrested in G2 by mutation in cdc25 (data not shown).
As would be predicted, the modification to p107wee1 was shown to be phosphorylation. We isolated the tagged p107wee1 on Ni–agarose from control cells and cells arrested by chk1 overexpression. The mobility shift on p107wee1 prepared from cells overexpressing chk1 was reversed in vitro by treatment with phosphatase (Figure 4). The higher molecular weight species in the control extracts were also shown by phosphatase treatment to represent phosphorylated forms of p107wee1. Thus, chk1‐mediated cell cycle arrest is both wee1 dependent and associated with hyperphosphorylation of p107wee1.
Chk1 is a wee1 kinase
We next asked whether p56chk1 could directly phosphorylate p107wee1. For this purpose, we constructed a recombinant baculovirus expressing a (HIS)6‐tagged p56chk1, which allowed purification from insect cell lysates using cobalt–Sepharose affinity chromatography (Figure 5). A previously characterized baculovirus expressing catalytically inactive p107wee1 as a GST fusion protein (GST–wee1K596L) (Parker et al., 1993) was used to prepare a substrate for in vitro kinase assays on glutathione–Sepharose. These assays showed that p56chk1 could directly phosphorylate p107wee1, and phosphoamino acid analysis showed that this occurred exclusively on serine residues (Figure 5). Control experiments confirmed that p56chk1 was unable to phosphorylate GST. We consistently observed low level labelling of GST–wee1K596L incubated either alone or with cobalt–Sepharose preparations of extracts infected with wild‐type baculovirus. This may be due either to residual activity of the mutant kinase or to a low level of contamination.
The in vitro phosphorylated p107wee1 did not undergo the mobility shift which was seen in the fission yeast extracts, which may be because in vitro we are observing phosphorylation by a single kinase (p56chk1). In the fission yeast extracts, we were observing phosphorylation of p107wee1 in the background of other potential phosphorylation events such as those described for phosphorylation by p67nim1/cdr1, a mitosis‐specific kinase, and autophosphorylation of p107wee1 itself (Featherstone and Russell, 1991; Coleman et al., 1993; Parker et al., 1993; Tang et al., 1993). The lack of the mobility shift caused by p56chk1 phosphorylation of p107wee1 may also be due to the effects of the GST portion of the fusion protein used in these experiments.
The observations from these in vitro kinase assays are fully consistent with the genetic observations showing p56chk1 functions through p107wee1, and that up‐regulation of p56chk1 by either overexpression or DNA damage results in hyperphosphorylation of p107wee1 in vivo. They confirm that p107wee1 and p56chk1 can interact directly, and thus establish a link between a member of the signal transduction pathway controlling the DNA damage checkpoint and the core cell cycle machinery.
Phosphorylation of p107wee1 by p56chk1 does not alter its Y15 kinase activity
Evoking a checkpoint which functions through p107wee1‐dependent Y15 phosphorylation of p34cdc2 could have either of two effects on p107wee1 activity: either it could maintain G2 levels of this kinase in cells that, in the absence of DNA damage, would have entered mitosis, or it may stimulate its activity to enhance cell cycle arrest further. Our observation of maintenance of Y15 phosphorylation during a G2 DNA damage checkpoint arrest (Figure 1) suggests the former may result. In order to assess whether in vitro phosphorylation of p107wee1 by p56chk1 altered its activity as a Y15 kinase, we performed similar assays to those described above, but used baculovirus‐expressed active GST–wee1 as the substrate (Parker et al., 1992). We observed that p56chk1 also phosphorylated active GST–wee1, resulting in [32P]ATP labelling above that due to autophosphorylation (Figure 6). When we assayed the Y15 kinase activity of these two forms of active GST–wee1, we found no change in p107wee1 activity caused by p56chk1 phosphorylation when assayed by Western blotting for the presence of phosphotyrosine on p34cdc2 (Figure 6). As expected, the mutant p107wee1 (GST–wee1K596L) had no detectable Y15 kinase activity. These data were confirmed in other experiments by 32P labelling of p34cdc2, and also by assays of p34cdc2 histone H1 kinase subsequent to phosphorylation by p107wee1 (data not shown). Therefore, although p107wee1 is phosphorylated by p56chk1, this does not affect p107wee1 kinase activity in vitro.
Discussion
Although many proteins involved in eliciting a G2 cell cycle arrest in response to DNA damage have been identified, little is known about how they interact to send signals to the core cell cycle regulators. Further, we have not known what the ultimate effect of evoking this checkpoint is on the p34cdc2–cyclin B complexes. In this study we have described experiments which address both of these issues.
By Western blot analysis of extracts prepared from irradiated G2 cells, we showed that p34cdc2 is maintained in its inactive, Y15‐phosphorylated form. This was maintained in cells despite the fact that they had fulfilled the cell size and completion of S‐phase requirements for entry into mitosis. These experiments were performed at a radiation dose which causes little effect in terms of viability on wild‐type cells, which recover due to the transient cell cycle delay induced by the checkpoint. This delay is, however, not long enough to see a significant accumulation of high levels of the Y15‐phosphorylated form of p34cdc2. These data are consistent with those described for mammalian cells which have correlated p34cdc2 Y15 phosphorylation with DNA damage‐induced G2 delay (Kharbanda et al., 1994a). Furthermore, other groups have demonstrated recently that Y15 phosphorylation is crucial in the G2 DNA checkpoints of Aspergillus nidulans (Ye et al., 1997) and fission yeast (N.Rhind and P.Russell, personal communication), and in a G2 checkpoint resulting from irradiation of human cells in S–phase (Jin et al., 1996).
In our experiments, the cells are already in G2 when they suffer DNA damage, and so our data would indicate that, under these conditions, the checkpoint functions to hold the cells in G2 and prevent the onset of mitosis whilst the DNA is repaired. This maintenance of p34cdc2 inhibition by Y15 phosphorylation rather than induction of an additional arrest mechanism is in contrast to that described for vertebrate cells in G1: here, the potentially active cdk2–cyclin E complexes are inhibited by induction of the p21 cdk inhibitor (Harper et al., 1993; Macleod et al., 1995). A role for inhibition of G1 cdks by tyrosine phosphorylation in G1 checkpoints has also been shown by mutation in cdk4 (Tereda et al., 1995). Furthermore, cdc25 phosphatases acting in G1 have the ability, when overproduced, to override G1 checkpoints in cellular transformation (Galaktionov et al., 1995). Thus, inhibition of cdks by tyrosine phosphorylation appears to be generally important in checkpoints responding to DNA damage.
Although we had shown that p34cdc2 Y15 phosphorylation is associated with G2 checkpoint arrest, we asked whether this was a result of activation of the checkpoint. To answer this question, we chose to investigate the effects of up‐regulation of the p56chk1 kinase by overexpression. This line of investigation was particularly useful as it allowed investigation of the cell cycle arrest phenotype as a constitutive event in the absence of DNA damage. From these experiments, we established that overexpression of chk1 also resulted in G2 cell cycle arrest with p34cdc2 inactivated by Y15 phosphorylation. Importantly, we established that this arrest mechanism functioned directly through wee1 rather than through another p34cdc2 inhibitory mechanism: in the absence of wee1, cells were completely non‐responsive to the effects of chk1 overexpression. The fact that these cells retained their wee phenotype rules out the possibility that p56chk1 could be acting alternatively through inhibition of p80cdc25, as this would suppress the effects of lacking p107wee1. Furthermore, interactions with wee alleles of cdc2 confirmed the requirement for wee1 in this arrest, and that this was at the level of p107wee1‐mediated p34cdc2 Y15 phosphorylation.
Through Western blot analysis, we showed that p107wee1 became hyperphosphorylated when cells were arrested by chk1 overexpression or UV‐induced DNA damage. Together with the genetic observations, these data strongly implicated p107wee1 as a direct target of p56chk1. Evidence for this model was gained by in vitro kinase assays using baculovirus‐expressed proteins, which showed that p56chk1 can phosphorylate p107wee1 directly on serine residues. Although these data strongly suggest that p56chk1 is phosphorylating p107wee1 directly in vivo, we do not have direct evidence for this. Due to the complexity of phosphorylation events on p107wee1, this issue can only be resolved by mapping the in vitro phosphorylation sites, and assessing the function of these by mutation studies in vivo. Such experiments are underway.
The phosphorylation of p107wee1 by p56chk1 did not affect its in vitro Y15 kinase activity, despite the fact that p56chk1 clearly affects both p107wee1 and Y15 phosphorylation. These data should be compared with published work concerning the checkpoint function associated with fission yeast rad24, which encodes a 14‐3‐3 protein (Ford et al., 1994). Analysis of chk1 overexpression has shown it to act downstream of all checkpoint mutants with the exception of rad24. Strains deleted for rad24 are only partially responsive to chk1, becoming delayed but not arrested in cell cycle progression. Importantly, however, rad24Δ cells exhibit a wee phenotype, and thus also have a defect in cell cycle progression accelerating them through G2. These phenotypes could be related if rad24Δ strains are defective in some aspect of p107wee1 control, which in turn makes them less responsive to p56chk1‐mediated signals. In view of these data, it is noteworthy that a physical interaction between murine homologues of p107wee1 and 14‐3‐3 proteins have been observed in vitro and in vivo, without an effect on p107wee1 activity (R.Honda and H.Yasuda, personal communication). Further, 14‐3‐3 proteins have been implicated in subcellular localization and protein–protein interactions (Burbelo and Hall, 1995) through association with phosphoserine residues, which were the sites of phosphorylation of p107wee1 by p56chk1. Both the wee phenotype and insensitivity to chk1 of rad24Δ could, therefore, be a result of inappropriate localization or protein–protein interactions of p107wee1 when the rad24‐encoded 14‐3‐3 protein is not present, and not a result of alteration of its intrinsic kinase activity.
Based on the genetic and biochemical experiments performed in fission yeast, and the in vitro phosphorylation experiments with recombinant protein, we propose a model for cell cycle arrest following DNA damage in G2: as a consequence of DNA damage, p56chk1 is activated and maintains p34cdc2 Y15 phosphorylation by phosphorylating p107wee1 (Figure 7). This phosphorylation does not affect the in vitro p107wee1 kinase activity, but does affect the in vivo function of p107wee1, perhaps by affecting its intracellular localization, interaction with p34cdc2 or interaction with negative regulatory proteins functioning at the G2–M transition. This leads to the activity of p107wee1 being maintained after genome damage beyond the point when an appropriate cell size is achieved and cells would normally enter mitosis. In vertebrates, a similar mechanism has been proposed for the DNA replication checkpoint (McGowan and Russell, 1995). This checkpoint clearly functions through Y15 phosphorylation (Enoch et al., 1992), but is not associated with an increase in p107wee1 activity above that observed in normal G2 cells (McGowan and Russell, 1995; Mueller et al., 1995). Although the DNA replication and DNA damage checkpoints have overlapping components, p56chk1 is not involved in the replication checkpoint, suggesting that separable but overlapping pathways are involved in G2 checkpoint controls. A good candidate for a protein carrying out an analogous function to p56chk1 in the fission yeast DNA replication checkpoint is p52cds1 (Murakami and Okayama, 1995). This protein kinase is required for cell cycle delay following a block to DNA replication, but not following DNA damage. At this stage, we do not know the mechanism by which DNA damage is detected, or how this results in activation of p56chk1. However, is has been shown that p56chk1 becomes phosphorylated after DNA damage (Walworth and Bernards, 1996), and this may play some role in its activation.
If p107wee1 is a target of the G2 DNA damage checkpoint, how can strains completely lacking wee1 arrest in response to irradiation (Barbet and Carr, 1993)? The answer to this is likely to lie in the redundancy of Y15 kinases in fission yeast (Lundgren et al., 1991). We suggest that p107wee1 is a target of the checkpoint, but not the only target. At least one other Y15 kinase, p66mik1, is likely to play some role. The radiation sensitivity of wee1Δ cells indicates that a wee1‐independent arrest involving mik1 is not sufficient to maintain wild‐type levels of viability after DNA damage. When both kinases are absent, all G2 controls are lost and cells enter mitosis constitutively, leading to a lethal mitotic catastrophe phenotype. Although wee1 and mik1 have overlapping functions, mik1Δ mutants do not exhibit a wee phenotype, suggesting that p66mik1 plays a minor role in G2 control in the presence of p107wee1. It is also important to note that the viability of wee1Δ cells, even when unirradiated, requires all known G2 checkpoint functions, including chk1 (Al‐Khodairy et al., 1994), which may also act through mik1 under these conditions. This indicates that checkpoint functions are constitutively active in these cells, and may act to up‐regulate p66mik1 so as to prevent premature entry into mitosis. Our data show that Y15 phosphorylation plays a role in the G2 DNA damage checkpoint functioning through p56chk1, but we cannot rule out additional regulatory mechanisms.
The data presented here provide a direct link between a G2 checkpoint function and a core cell cycle regulator. This link establishes a firm basis on which to build this pathway, which although it has many identified members, still lacks the biochemical links required to understand this important biological phenomenon.
Materials and methods
Fission yeast methods
All strains are derivatives of wild‐type S.pombe 972h−. Standard procedures for propagation, transformation and extract preparation were used. Small G2 cells prepared from synchronous cultures were derived by centrifugal elutriation. For UV irradiation, cells were re‐innoculated into pre‐warmed (30°C) medium for a recovery period of 20 min, and then filtered in aliquots onto PVDF membranes. Half the membranes were then irradiated with 100 J/m2 UV‐C (254 nm), following which both populations of cells were washed into pre‐warmed medium. Cell cycle progression was followed by septation indices, which were estimated by phase contrast microscopy every 10 min, and increases in cell number in samples fixed in 3.7% formaldehyde for cell counting in a Sysmex K‐1000 cell counter.
Detection and dephosphorylation of p107wee1 from fission yeast
In order to detect p107wee1 by Western blotting, a tagging fragment corresponding to six consecutive histidines and three copies of the influenza virus HA epitope (RSHHHHHHGGRIFYPYDVPDYAGYPYDVPDYAGSYPYDVPDYAAQCGRS) was inserted into the BglII site of pWee1‐10 (Russell and Nurse, 1987b). This construct rescues the temperature‐sensitive allele wee1‐50, and was carried extrachromosomally under ura4 selection. For detection of p107wee1, proteins were resolved on a 6% SDS–PAGE gel, transferred to nitrocellulose and probed with the monoclonal antibody 12CA5. For dephosphorylation of p107wee1, protein extracts were prepared from boiled cells in 8 M urea, 100 mM NaH2PO4, 10 mM Tris, 60 mM β‐glycerophosphate, 100 μM Na orthovanadate, 15 mM p‐Nitrophenyl Phosphate (PNPP), 1% Triton X‐100, pH 8, and p107wee1 was affinity isolated on Ni‐NTA–agarose (Qiagen). Beads were washed extensively in extraction buffer, and urea removed by dilution batch washing. Precipitates were washed three times in CIP buffer (50 mM Tris, 5 mM MgCl2, pH 8), treated with the indicated units of calf intestinal phosphatase (Gibco‐BRL) for 30 min at 37°C and processed for Western blotting.
Baculovirus expression
The chk1 open reading frame was amplified by PCR to include BglII sites at codon 2 and directly after the termination codon, and cloned into pBlueBacHisB (Invitrogen). DNA sequencing was performed (ABI) to confirm the integrity of the amplified sequence. A recombinant virus was then constructed using the linear transfection module (Invitrogen) as recommended, and was propagated at an m.o.i. of 5, with extracts prepared 2–3 days post‐infection. Recombinant p56chk1 was isolated from Sf21 lysates prepared in 250 mM NaCl, 50 mM Tris pH 8, 10% glycerol, 0.1% NP‐40, 50 mM NaF, 10 mM Na pyrophosphate, 100 μM Na orthovanadate, 10 mM β‐mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μg/ml aprotinin, leupeptin and pepstatin, on cobalt–Sepharose (Talon, Clontech) as recommended. The other recombinant viruses used here for expression of wee1, cyclin B and cdc2 have been described (Parker et al., 1993).
Protein kinase assays
Fission yeast p34cdc2 kinase assays using histone H1 as a substrate were performed on immunoprecipitates using a monoclonal antibody directed against the cdc13‐encoded B‐type cyclin. Assays were quantified by PhosphoImager analysis (Molecular Dynamics). For phosphorylation of p107wee1 by p56chk1, baculovirus‐expressed proteins were selected on affinity reagents (cobalt–Sepharose or glutathione–Sepharose), washed extensively in extraction buffer, and finally in kinase assay buffers. Assays were performed in 50 mM Tris, 10 mM MgCl2, 1 mM dithiothreitol (DTT), 10 μM ATP, with 10 μCi of 3000 Ci/mmol [32P]ATP. Reactions were incubated for 30 min at 30°C, and terminated by boiling in SDS–PAGE buffer. Samples were run on an 8% SDS–PAGE gel, stained, dried and exposed to film. Assays were quantified by PhosphorImager analysis (Molecular Dynamics). Control experiments confirmed that p56chk1 was unable to phosphorylate GST. For phosphoamino acid analysis, 32P‐labelled GST–wee1K596L was transferred to PVDF and incubated in 5.7 M HCl at 100°C for 2 h. Reactions were lyophilized, resuspended with phosphoamino acid standards and electrophoresed at pH 3.5 on a thin‐layer cellulose plate. For assays of p107wee1 activity, p34cdc2(K33R) was isolated in complexes with GST–cyclin B on glutathione–Sepharose, and GST–wee1 kinase assays were performed as described (Coleman et al., 1993; Parker et al., 1993). Reactions were electrophoresed on a 10% SDS–PAGE gel, blotted to nitrocellulose, probed with antibodies to phosphotyrosine and subsequently reprobed with anti‐p34cdc2 (PSTAIRE) to control for loading. Western blots were quantified by densitometry (Molecular Dynamics).
Antibodies
Polyclonal antisera against p56chk1 were raised in rabbits immunized with full‐length bacterially expressed, histidine‐tagged p56chk1. Procedures for protein expression, isolation, immunization and affinity purification were as described (O'Connell et al., 1994). The affinity‐purified sera were then used at a concentration of 1:100 on crude extracts. That this specifically detected p56chk1 was confirmed by comparing extracts of wild‐type‐, chk1Δ‐ and chk1‐overexpressing cells. For detection of Y15‐phosphorylated p34cdc2, a phospho‐specific rabbit polyclonal antibody (New England Biolabs) was used at a concentration of 1:250. For detection of epitope‐tagged p107wee1, the 12CA5 monoclonal antibody was used at a concentration of 40 μg/ml. For detection of total p34cdc2, a rabbit polyclonal antiserum directed against the PSTAIR region (a kind gift of Dr Hideo Nishitani) was used at a concentration of 1:10 000.
Acknowledgements
The authors wish to thank Dr H.Piwnica‐Worms for providing baculoviruses, and Drs R.Honda and H.Yasuda; and N.Rhind and P.Russell for communication of results prior to publication. We are also grateful to Dr D.Germain (PMCI) for critical reading of the manuscript and many useful discussions, and to Drs J.Hayles (ICRF), J.Correa‐Bordes (ICRF), R.Pearson (PMCI) and R.Ramsey (PMCI) for much helpful advice. We are also most grateful to Mrs Liz Eaton for much assistance in trans‐global communications. H.V. is a recipient of an Australian Postgraduate Award, and M.O. is a Special Fellow of the Leukemia Society of America.
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Received: 21 October 1996
Revision received: 19 November 1996
Published online: 1 February 1997
Published in issue: 1 February 1997
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