Oncogene (2008) 27, 4986–4997
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ORIGINAL ARTICLE
The regulatory b-subunit of protein kinase CK2 regulates cell-cycle progression at the onset of mitosis CW Yde1, BB Olsen1, D Meek2, N Watanabe3 and B Guerra1 1 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark; 2Biomedical Research Centre, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK and 3Antibiotics Laboratory, Discovery Research Institute, RIKEN, Wako, Saitama, Japan
Cell-cycle transition from the G2 phase into mitosis is regulated by the cyclin-dependent protein kinase 1 (CDK1) in complex with cyclin B. CDK1 activity is controlled by both inhibitory phosphorylation, catalysed by the Myt1 and Wee1 kinases, and activating dephosphorylation, mediated by the CDC25 dual-specificity phosphatase family members. In somatic cells, Wee1 is downregulated by phosphorylation and ubiquitin-mediated degradation to ensure rapid activation of CDK1 at the beginning of M phase. Here, we show that downregulation of the regulatory b-subunit of protein kinase CK2 by RNA interference results in delayed cell-cycle progression at the onset of mitosis. Knockdown of CK2b causes stabilization of Wee1 and increased phosphorylation of CDK1 at the inhibitory Tyr15. PLK1–Wee1 association is an essential event in the degradation of Wee1 in unperturbed cell cycle. We have found that CK2b participates in PLK1–Wee1 complex formation whereas its cellular depletion leads to disruption of PLK1–Wee1 interaction and reduced Wee1 phosphorylation at Ser53 and 121. The data reported here reinforce the notion that CK2b has functions that are independent of its role as the CK2 regulatory subunit, identifying it as a new component of signaling pathways that regulate cell-cycle progression at the entry of mitosis. Oncogene (2008) 27, 4986–4997; doi:10.1038/onc.2008.146; published online 12 May 2008 Keywords: CK2b; Wee1; PLK1; CDC25A; cell cycle
Introduction Protein kinase CK2 is a serine/threonine kinase highly conserved throughout evolution and is ubiquitously expressed in eukaryotic cells. Traditionally, CK2 is described as a constitutively active tetrameric enzyme composed of two catalytic a and/or a0 subunits and two Correspondence: Dr B Guerra, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, Odense 5230, Denmark. E-mail:
[email protected] Received 21 November 2007; revised 14 March 2008; accepted 1 April 2008; published online 12 May 2008
regulatory b subunits. More recently, the classical view of CK2 as a stable tetrameric complex has been modified, suggesting that the individual subunits may have independent functions and they may be asymmetrically distributed inside the cell (reviewed in Litchfield, 2003; Bibby and Litchfield 2005). CK2 is involved in various intracellular processes ranging from regulation of transcription and DNA replication to proliferation, survival and differentiation. The involvement of CK2 in the regulation of cell-cycle progression has been explored in lower as well as higher eukaryotes. Genetic studies in yeast indicated that CK2 regulates both the G1/S and G2/M transitions (reviewed in Glover, 1998). In this respect, Toczyski et al. (1997), by exploring the yeast DNA damage checkpoint regulation, demonstrated the involvement of CK2b and Cdc5 in the adaptation to DNA damage in budding yeast. Evidence that CK2 plays a role in cell-cycle regulation in mammalian cells comes from findings that CK2 is associated with the mitotic spindle and centrosomes and interacts with and/or phosphorylates numerous cellcycle regulatory proteins including Pin1, topoisomerase II, cdc34 and cyclin-dependent kinase 1 (CDK1; reviewed in Faust and Montenarh, 2000; Litchfield, 2003). Recent results have also indicated the involvement of CK2 in the cellular decision of life and death in response to ionizing radiation damage (Yamane and Kinsella, 2005). Cell-cycle progression is tightly regulated by a plethora of proteins that act in order to preserve the integrity of the genetic information to be transferred to daughter cells. Entry into mitosis requires the involvement of CDK1 that becomes active following its association with cyclin B and phosphorylation on a threonine residue located in the conserved T-loop domain. During interphase, CDK1 is maintained in an inactive state by phosphorylation at two amino-acid residues (that is, Thr14 and Tyr15) located within the socalled ATP-binding loop. Myt1 and Wee1 protein kinases mediate the phosphorylation of CDK1 at the aforementioned residues, whereas the CDC25 dualspecificity protein phosphatase family members catalyse the activating dephosphorylation at the same sites (reviewed in Pines, 1999). Entry into mitosis involves a remarkably high number of kinases and phosphatases and it is regulated by both positive and negative feed
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back loops. Interestingly, Wee1 and CDC25 proteins are similarly regulated despite their inhibiting and activating roles on mitotic entry, respectively. Their activities oscillate in opposition to one another but their turnover is regulated by phosphorylation at specific amino acids during cell-cycle progression. CDC25A is an unstable protein that is thought to play a role in both G1/S and G2/M cell-cycle transitions (reviewed in Busino et al., 2004). In interphase, Chk1 phosphorylates CDC25A on four different sites (Ser124, 178, 279 and 293; S rensen et al., 2003; reviewed in Busino et al., 2004) contributing to its rapid turnover in unperturbed cells. Upon DNA damage, both Chk1 and Chk2 kinases contribute to further phosphorylate CDC25A at Ser124, 178 and 293 inducing a rapid proteasome-mediated degradation of the latter thus activating the cell-cycle G2/M checkpoint (S rensen et al., 2003). Wee1 becomes inactive at the onset of mitosis when active CDK phosphorylates it at Ser123 (Van Vugt et al., 2004; Watanabe et al., 2004). Phosphorylation of the aforementioned residue contributes to the formation of a polo box domain (PBD)binding site that allows the binding of PLK1 to Wee1 with the outcome of promoting phosphorylation at Ser53 and subsequent proteasome-mediated degradation of Wee1 (Watanabe et al., 2005). We have recently shown that ectopic expression of CK2b during unperturbed cell-cycle progression enhances the degradation of CDC25A. This effect is mediated by Chk1 as the pharmacological inhibition of Chk1 kinase activity and the cellular depletion of Chk1 by RNA interference, respectively, counteract the downregulation of CDC25A induced by CK2b overexpression. Interestingly, we also showed that the sole depletion of CK2b by siRNA is able to enhance the stability of the phosphatase (Kreutzer and Guerra, 2007). In the present study, we aimed to closely investigate the role of CK2b with respect to cell-cycle regulation at the G2/M transition. Surprisingly, during our studies we found that the cellular depletion of CK2b by siRNA leads to enhanced levels of Wee1 resulting in delayed progression into mitosis. Our data suggest an undisclosed role of CK2b in regulating the stability of Wee1, revealing a novel component of the complex machinery that operates at the G2/M transition.
Results Depletion of CK2b stabilizes CDC25A Previously, we showed that the cellular depletion of CK2b by short RNA duplexes (siRNA) led to a significant higher level of CDC25A protein (Kreutzer and Guerra, 2007). In order to verify whether this effect was exclusively mediated by CK2b we transfected H1299 lung cancer cells with siRNAs directed against the individual CK2 subunits (that is, -a, -a0 and -b) and analysed the endogenous CDC25A protein level (Figure 1a). Interestingly, CDC25A levels increased not only in cells depleted of CK2b (lane 5) but also after
treatment with siRNAs against CK2a or a combination of CK2a and CK2a0 (lanes 2 and 4) but not CK2a0 (lane 3). As CDC25C contributes to the regulation of the G2/ M cell-cycle transition, we also analyzed its expression under the aforementioned conditions. As shown in Figure 1a, the downregulation of the individual CK2 subunits did not affect CDC25C expression levels. The notion that CK2a also contributes to the stability of CDC25A could not be excluded at this point although we noticed that the depletion of CK2a, but not CK2a0 , led to downregulation of CK2b expression (lanes 2 and 4), an effect that underlines the functional specialization of the CK2 catalytic subunits and that we previously attributed to the ability of CK2b to assemble with the catalytic subunits in stable heterotetramers for preserving its stability (Seeber et al., 2005). In this respect, it has been shown that if CK2b fails to form complexes with the catalytic subunit it undergoes rapid degradation (Lu¨scher and Litchfield, 1994). To clarify the contribution of CK2a in the stability of CDC25A, we analysed the levels of endogenous CDC25A in cells treated with a panel of CK2 kinase inhibitors, that is, 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), emodin and apigenin with respect to control cells incubated with dimethyl sulfoxide. CK2 kinase activity in whole cell lysates from H1299 cells treated as indicated in Figure 1b showed that all treatments were effective in inhibiting the kinase activity of CK2. Next, western blot analysis of protein extracts from cells treated as described above revealed that the inhibition of CK2 did not influence the expression level of endogenous CDC25A protein (Figure 1c), thus excluding a possible contribution of the CK2 catalytic activity in the regulation of CDC25A stability. On the contrary, cell treatment with emodin lowered the expression of CDC25A (Figure 1c, lane 3), an effect which was not further investigated because beyond the scope of the present paper. As a positive control, cells were also treated with Go¨6976, a known inhibitor of Chk1 (Kohn et al. 2003; Figure 1c, lane 5), but not of CK2 (Figure 1b), and we verified that inhibition of Chk1 kinase activity enhanced the expression of endogenous CDC25A. Enhanced level of CDC25A in cells depleted of CK2b does not lead to accelerated G2/M cell-cycle transition Although originally considered a regulator of the cellcycle G1/S transition, CDC25A has also been shown to be required for cell-cycle progression from G2 to M phase (reviewed in Boutros et al., 2006; KarlssonRosenthal and Millar, 2006). Cellular exposure to a wide variety of DNA insults leads to Chk1-mediated CDC25A degradation triggering the G2/M checkpoint, whereas the cellular depletion of Chk1 or overexpression of CDC25A abrogates checkpoint arrest (Mailand et al., 2000; Xiao et al., 2003). As treatment of cells with CK2b siRNA led to higher levels of CDC25A, we next investigated how CK2b might influence the G2/M transition. As indicated in Figure 2a, cells were treated with siRNAs against the indicated proteins and inOncogene
CK2b regulates the G2/M cell-cycle transition CW Yde et al
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Figure 1 Contribution of the CK2 subunits to the stability of the CDC25A protein phosphatase. (a) H1299 cells were transfected with siRNAs for downregulating CK2a (lane 2), CK2a0 (lane 3), CK2a and CK2a0 (lane 4) and CK2b (lane 5), respectively. Lane 1 refers to cells transfected with a scramble siRNA (Scramble). Cell lysates were subjected to western blot analysis with the indicated antibodies. The blot was probed with anti-b-actin antibody in order to verify equal protein loading. The values expressed in percentages below the lane numbers refer to the densitometric analysis of the protein bands detected with anti-CDC25A antibody. The quantification was performed by assigning a value of 100% to the band in lane 1. (b) H1299 cells were treated for 5 h with dimethyl sulfoxide (DMSO; Control), 25 mM TBB, 40 mM emodin, 50 mM apigenin and for 24 h with 300 nM Go¨6976, respectively. Total lysates (20 mg) were subjected to CK2 kinase activity with a specific CK2 peptide substrate as described in the materials and methods. The average and standard deviation of three independent experiments are shown. (c) Cells were treated as in (a). Total lysates were subjected to SDS– polyacrylamide gel electrophoresis (PAGE). Separated proteins were transferred to polyvinylidene difluoride (PVDF) membrane by western blot. Proteins were visualized by probing the membranes with the indicated monoclonal antibodies. Data shown are representative of three independent experiments.
cubated with doxorubicin to induce DNA damage. As expected, the cellular depletion of Chk1 (lane 2) led to stabilization of CDC25A as compared to the control experiment (lane 1). Expression of CDC25A also increased in cells treated with CK2b and CK2a siRNAs (lanes 3 and 4, respectively) although not to the same extent. Given the lower expression of CDC25A in cells depleted of CK2b and treated with doxorubicin (as compared with cells treated with Chk1 siRNA), we investigated whether the CDC25A level was enough to disrupt the G2/M checkpoint induced by drug treatOncogene
ment. As shown in Figures 2b and c, cells treated with scramble siRNA and exposed to doxorubicin for 48 h arrested at G2 phase whereas cells treated with Chk1specific siRNA (positive control) continued to proliferate showing a cell-cycle distribution similar to that of cells treated with scramble siRNA in the absence of doxorubicin as reported earlier (Xiao et al., 2003). When cells transfected with CK2b siRNA were exposed to doxorubicin, we did not observe a significant abrogation of G2/M checkpoint, indicating that the moderate stabilization of CDC25A (in Figure 2a, the level of
CK2b regulates the G2/M cell-cycle transition CW Yde et al
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Relative DNA content Figure 2 The regulatory b-subunit of CK2 is dispensable for G2/M checkpoint activation. (a) H1299 cells were transfected with scramble (lane 1), Chk1 (lane 2), CK2b (lane 3) and CK2a (lane 4) siRNAs, respectively. After 24 h from transfection, cells were incubated with 0.25 mM doxorubicin (Dox) for additional 48 h. The analysis of the indicated proteins and the densitometric analysis of CDC25A protein bands were done as described in the legend to Figure 1. (b) Cells were treated as indicated in (a). After 48 h of incubation with doxorubicin cells were harvested for flow cytometry analysis of cell-cycle distribution. The distribution of cells in the cell-cycle phases is indicated in the upper-left panel. Sub-G1 cell populations are indicated with an asterisk. (c) Cells from the experiment shown in (b) were gated and their percentage was calculated according to the phases of the cell cycle. Three separate experiments were performed and data from one representative experiment are shown.
CDC25A in lane 3 is about 50% less than that in lane 2) was not sufficient to abrogate the G2 checkpoint as in the case of Chk1 siRNA treatment. Consistent with the notion that overexpression of CDC25A accelerates entry into mitosis (Molinari et al., 2000; Zhao et al., 2002), we tested whether the stabilization of CDC25A in cells treated with CK2b siRNA affected the G2/M transition in the absence of DNA damage (Figure 3). Cells transfected with scramble or CK2b siRNAs were shortly exposed to the mitotic inhibitor nocodazole to trap cells exiting G2 and were analysed afterwards by flow cytometry. Unexpectedly, we observed delayed cell-cycle
progression (Figures 3b and c) despite stabilization of CDC25A in CK2b-reduced cells (Figure 3a). Whereas about 17% of the control cell population was phosphohistone H3 (a marker of entry into mitosis) positive after 8 h of incubation with nocodazole, only 10% of the CK2b-depleted cells were found arrested in mitosis at the aforementioned time point (Figures 3b and c). While suggesting a possible role of CK2b in G2/M transition, the data, however, were not consistent with the observation that CDC25A becomes stabilized in cells transfected with CK2b siRNA (Figures 1 and 3a). Oncogene
CK2b regulates the G2/M cell-cycle transition CW Yde et al
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Figure 3 The cellular depletion of CK2b stabilizes CDC25A but leads to delayed cell-cycle progression at the onset of mitosis. (a) H1299 cells were transfected with the indicated siRNAs for 72 h. Cell lysates were analysed by western blot probing the membranes with the indicated antibodies. The intensity of the protein bands detected with anti-CDC25A antibody was quantified in percentage by densitometric analysis by assigning a value of 100% to the band in lane 1. (b) Cells were transfected with scramble and CK2b siRNAs, respectively, for 48 h prior to treatment with 200 ng/ml nocodazole for the indicated time points. Next, cells were fixed and subsequently stained with propidium iodide and anti-phospho-histone H3 (Ser10) antibody conjugated with Alexa Fluor 488 for flow cytometry analysis. The percentage of mitotic cells from the total amount of cells was quantified by Cell Quest Pro analysis software gating the appropriate cell populations. (c) The percentage of mitotic cells from the experiment described in (b) was plotted against incubation time in the presence of 200 ng/ml nocodazole. Experiments were performed three times obtaining similar results.
Knockdown of CK2b destabilizes the complex between Wee1 and PLK1 kinases and upregulates Wee1 protein As mentioned previously, both activating and inhibiting phosphorylations are required for modulating CDK1 activity at the onset of mitosis. Wee1 is considered the major kinase phosphorylating CDK1 at the Tyr15 site (reviewed in Perry and Kornbluth, 2007). As cellular depletion of CK2b led to delayed cell-cycle progression at the G2/M transition, we analysed the protein expression of Wee1 in cells transfected with various siRNAs (Figure 4). The cellular depletion of CK2b stabilized Wee1 (lane 5) as indicated also by the densitometric analysis of Wee1 protein bands. Additionally, we noticed stabilization of Wee1 in cells Oncogene
transfected with CK2a siRNA (lane 3) but, as mentioned earlier, we believe that this is an indirect effect due to the CK2a knockdown-dependent destabilization of CK2b. The stabilization of Wee1 was also accompanied by a higher phosphorylation of CDK1 at Tyr15 as also indicated by the densitometric analysis of phospho-CDK1 protein bands (lanes 3 and 5). A kinase assay revealed a 40% decrease CDK1 activity in CK2bdepleted cells as compared to cells transfected with scramble siRNA (results not shown). The aforementioned data suggest that the higher level of Wee1 observed in cells transfected with CK2b siRNA, might be responsible for the observed delayed cell-cycle G2/M transition. Additionally, these data raise
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questions as to how Wee1 stabilization is achieved in cells depleted of CK2b. Wee1 is inactivated at the onset of mitosis by both phosphorylation and degradation in human somatic cells whereas in lower eukaryotes it is regulated either at the protein level as in the case of budding yeast, or mainly by phosphorylation as in fission yeast (Coleman et al., 1993; Parker et al., 1993; Tang et al., 1993; Wu and Russell, 1993; Sia et al., 1998). Evidence indicates that although Wee1 lacks a canonical b-TrCP binding consensus sequence for ubiquitinmediated degradation, phosphorylation at three serine residues, namely Ser53, 121 and 123, were found to be required for b-TrCP recognition (Watanabe et al., 2004, 2005). Interestingly, it has been reported that CDK, PLK1 and CK2 kinases create the two b-TrCPphosphodegrons that are responsible of Wee1 degradation at the onset of mitosis (Watanabe et al., 2005). Therefore, to explore the possible involvement of these enzymes we analysed whether the higher expression level of Wee1 reflected a downregulation of PLK1 at the protein and/or at the activity levels. We synchronized cells and looked at the expression- and activity levels of PLK1 at the different phases of the cell cycle (Figures 5a, b and c). As shown in Figure 5, PLK1 expression and activity increase at the onset of mitosis that occurs 8 h after release from the aphidicolin-induced G1/S arrest and remain high during mitosis. Next, we analysed whether CK2b knockdown affected the abunanti-Wee1
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Figure 4 Reduced level of CK2b stabilizes Wee1 kinase. H1299 cells were transfected with the indicated siRNAs for 72 h. Total cell lysates were analysed by western blot using the indicated antibodies. The densitometric analysis of Wee1 and phosphoCDK1 protein bands is expressed in percentage assigning a value of 100% to the Wee1 and phospho-CDK1 protein bands in lane 1.
dance of PLK1 (Figure 5d) in synchronized cells harvested at 10 h after release from the G1/S block and arrested in mitosis, respectively. Additionally, we immunoprecipitated PLK1 from cells treated, as indicated above, and tested the kinase activity (Figure 5e). Based on the obtained results, we excluded the possibility that the accumulation of Wee1 observed in cells transfected with CK2b siRNA, reflected decreased expression and/or activity of PLK1. A possible direct CK2b-mediated effect on PLK1 was also excluded by in vitro experiments, where the activity of PLK1 was tested in the presence of increasing amounts of recombinant CK2b, showing that CK2b does not modulate the activity of PLK1 (Figure 5f). Next, a plausible hypothesis was to assume a direct involvement of CK2 in the observed stabilization of Wee1 in cells depleted of CK2b. We reasoned that cellular depletion of CK2b would lower the kinase activity of endogenous CK2 required for Wee1 phosphorylation at Ser121, which promotes the b-TrCPdependent destabilization of Wee1 (Watanabe et al., 2005). Indeed, we observed approximately 50% reduction in CK2 kinase activity in both asynchronous and synchronized cells transfected with CK2b siRNA (Figure 6a). Cells, treated as described in the legend to Figure 6b, were analysed for the expression of the indicated proteins and subjected to co-immunoprecipitation employing anti-PLK1 antibody. Transfection of cells with CK2b siRNA resulted in a shift in Wee1 electrophoretic mobility on SDS–polyacylamide gels (Figure 6b, lanes 4–6). In Figure 6c, in asynchronous cells (lanes 2 and 5) and in cells arrested in mitosis (lanes 4 and 7), we detected a slight interaction between PLK1 and Wee1. In contrast, in synchronized cells harvested at 10 h after release from the G1/S block and transfected with scramble siRNA, we observed a strong interaction among CK2b, PLK1 and Wee1 (lane 3) indicating that CK2b participates in the Wee1–PLK1 complex formation at the onset of mitosis. Strikingly, we did not observe any protein–protein association in synchronized cells transfected with CK2b siRNA (lane 6). We, then, examined whether CK2b affects the binding of PBD motif to Wee1 and thus, Wee1 phosphorylation at Ser123. Results shown in Figure 7a demonstrated that the PBD-mediated binding of PLK1 to Wee1 is not regulated by CK2b. The phosphorylation of Wee1 in lysates from cells treated as described in Figure 6b was analysed with respect to phosphorylation at Ser53 and 121 (Figure 7b). While the Ser53 phosphorylation was slightly lowered, the Ser121 phosphorylation was dramatically reduced by the CK2b knockdown when compared with the total Wee1 level (lanes 4–6). As both these phosphorylation events are important for the binding of b-TrCP to Wee1, we conclude that the reduction in phosphorylation by the CK2b depletion seems to be responsible for the stabilization of Wee1 in vivo. Discussion Cell-cycle progression is spatially and timely regulated by cyclin-dependent kinases, which are subjected to a Oncogene
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Cell cycle distribution (%)
complex post-translational regulation in a stepwise fashion. While surveillance pathways provide information on the integrity of the genome and/or the general well being of the cells, the influence exerted on CDKs by positive and negative effectors drives cellular decisions, such as normal cell-cycle progression or activation of checkpoints in response to DNA damage. At the G2/M
transition, inhibition of CDK1 is mediated by Wee1and Myt1-dependent phosphorylations, whereas its activation is made possible by the involvement of CDC25 phosphatases such as CDC25A. Thus, the delicate balance between the expression and activity of CDC25s, Wee1 and Myt1 is crucial in the modulation of CDK1 activity at the onset of mitosis.
100
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100 anti-Wee1
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The data presented in this study show that in an unperturbed cell cycle, alteration in the level of CK2b influences the expression of proteins that play key roles in the modulation of the G2/M transition. Stabilization of CDC25A is observed in cells transfected with CK2b siRNA but it appears that CK2 kinase activity is not directly involved in the upregulation of the aforementioned phosphatase. Based on previous studies on cellular effects induced by CDC25A overexpression, we anticipated that stabilization of CDC25A would lead to unscheduled mitosis in cells depleted of CK2b. Unexpectedly, one of the most remarkable results in this study is the fact that upregulation of CDC25A does not lead to accelerated entry into mitosis. Following CK2b siRNA treatment, the observed delayed cell-cycle progression may be attributed to the unchanged expression of CDC25C, which contributes to the regulation of the G2/M transition, and also to the stabilization of Wee1 that preserves the inhibitory phosphorylation of CDK1. In this respect, it has been reported that the activity of Wee1 exceeds that of CDC25s in G2 phase and that AKT promotes the G2/M transition by phosphorylation-dependent 14-3-3y binding and cytoplasmic localization of Wee1 (Katayama et al., 2005). The sum of these effects might explain why in cells transfected with CK2b siRNA the stabilization of CDC25A does not accelerate the entry into mitosis as initially postulated. Stabilization of CDC25A was also seen in cells treated with doxorubicin and transfected with CK2b siRNA although not to the same extent as in cells depleted of Chk1. Downregulation of Chk1 was found to abrogate the doxorubicin induced G2 checkpoint as a consequence of CDC25A stabilization, whereas the same effect was not observed in cells treated with CK2b siRNA. This suggests that a certain CDC25A threshold level might be required for the G2 checkpoint abrogation, which can be reached in Chk1 siRNA-treated cells but not in CK2b-deficient cells. An intriguing question that we addressed during our study is how Wee1 stability is achieved. Based on the current knowledge on the regulation of Wee1 (Watanabe et al., 2005), we speculated that knockdown of
CK2b would negatively affect the activity and/or expression levels of PLK1 contributing to the observed stabilization of Wee1. Based on our results, we excluded a direct involvement of PLK1 in the observed phenotype and we, instead, came up with a model showing that components of distinct pathways contribute to stabilize Wee1 in cells depleted of CK2b (Figure 8). Expression of Wee1 mutated at Ser123 leads to disruption of PLK1–Wee1 association (Watanabe et al., 2005). As a similar effect is observed in cells depleted of CK2b, this indicates that other Wee1–protein domains, targeted by CK2b, might contribute to PLK1 binding. Previous observations implicated CK2 kinase activity in the regulation of Wee1 stability at the onset of mitosis (Watanabe et al., 2005). Our data add new insights into the mechanism of Wee1 regulation uncovering an unprecedented role of the CK2 regulatory b-subunit by affecting the phosphorylation of Wee1 at Ser53 and 121. Specifically, whereas reduction of Wee1 phosphorylation at Ser121 can be attributed to the lower CK2 kinase activity seen in cells with reduced CK2b expression, decreased phosphorylation at Ser53 may be the result of attenuated Wee1–PLK1 interaction seen under the aforementioned treatment conditions. Numerous studies have reported evidence for the unbalanced expression of the catalytic and regulatory CK2 subunits in a variety of tissues (reviewed in Bibby and Litchfield, 2005). The fact that aberrant high expression levels of CK2b relative to CK2a have been observed in tumors (Stalter et al., 1994; Timofeeva et al., 2006) and isolated CK2b has been shown in cells (Lu¨scher and Litchfield, 1994; Guerra et al., 1999) supports the notion that CK2b may be able to modulate the expression of critical regulators of CDK1/cyclin B activity thereby controlling mitotic entry in a CK2-independent fashion. At the onset of mitosis, CDK phosphorylates Wee1 at Ser123 and this event promotes the subsequent CK2catalysed phosphorylation of Wee1 at Ser121 creating a phosphodegron. Phosphorylation at Ser123 creates a PBD-binding domain that favors the interaction of PLK1 with Wee1 and the subsequent PLK1-catalysed phosphorylation of Wee1 at Ser53. This latter event
Figure 5 CK2b does not influence the activity of PLK1 kinase. (a) H1299 cells were synchronized as described in materials and methods. They were released from the G1/S block and then harvested at the indicated time points. Next, cells were fixed and stained with propidium iodide for cell-cycle analysis by flow cytometry. The cell-cycle profiles of asynchronous (As) or nocodazole-treated cells (Noc) are also shown. (b) Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Equal protein loading was verified by probing the western blot membranes with monoclonal anti-b-actin antibody. (c) Cell lysates from synchronized cells harvested after release from the G1/S block at the indicated time were subjected to immunoprecipitation with rabbit monoclonal antiPLK1 antibody. The immunoprecipitates were used for the determination of PLK1 kinase activity using casein as substrate. Bar 1 (), refers to the activity of PLK1 tested in the absence of substrate. The numbers are expressed in percentage by assigning a value of 100% to the activity of PLK1 in cells treated with nocodazole. (d) Cell lysates from asynchronous (lanes 1 and 4) and synchronized cells harvested after 10 h (lanes 2 and 5) or 12 h in the presence of nocodazole (lanes 3 and 6) after release from the G1/S block, respectively, were subjected to western blot and probed with the indicated antibodies. The cells were transfected with scramble siRNA (lanes 1–3) or CK2b siRNA (lanes 4–6). (e) Cell lysates from cells treated as in (d) were subjected to immunoprecipitation with rabbit monoclonal anti-PLK1 antibody. Lane 1 shows a negative control () where cell extract was subjected to immunoprecipitation with a control serum. The immunoprecipitates were used for the determination of PLK1 kinase activity using GST-tagged MDM2(203282) as substrate. The activity is expressed in percentage by assigning a value of 100% to the PLK1 activity in cells treated with nocodazole (lane 4). Lane 8* refers to the kinase activity of immunoprecipitated PLK1 in the presence of mutant GST-tagged MDM2(203282) where the PLK1 major phosphorylation site (that is, serine 260) has been mutated to alanine. (f) In vitro PLK1 kinase assay was performed in the presence of 1 pmol recombinant PLK1 (lane 1) or 1 pmol recombinant PLK1 and increasing amounts of recombinant CK2b (lane 2, 0.5 pmol; lane 3, 1 pmol; lane 4, 2 pmol; lane 5, 5 pmol; lane 6, 10 pmol). Oncogene
CK2b regulates the G2/M cell-cycle transition CW Yde et al
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Figure 6 Cellular depletion of CK2b disrupts the complex between PLK1 and Wee1 at the G2/M transition. (a) Whole lysates (10 mg) from H1299 cells left asynchronous (bars 1 and 4) or released from the G1/S block and harvested after 10 h (bars 2 and 5) or 12 h in the presence of nocodazole (bars 3 and 6), respectively, were subjected to CK2 kinase activity with a specific CK2 peptide substrate as described in Materials and methods. The average and standard deviation of three independent experiments are shown. (b) Whole lysates from cells treated as in (a) were analysed by western blot probing the membranes with the indicated antibodies. The cells were transfected with scramble siRNA (lanes 1–3) or CK2b siRNA (lanes 4–6) 12 h prior to synchronization. (c) Whole lysates from cells treated as in (a) were subjected to immunoprecipitation with rabbit monoclonal anti-PLK1 antibody (lanes 2–7). Lane 1 is a control experiment () were lysate from synchronized cells harvested after 10 h from the G1/S block was subjected to immunoprecipitation in the presence of control serum. Immunoprecipitates were then analysed by western blot probing the membranes with the indicated monoclonal antibodies.
promotes the formation of a second phosphodegron promoting the ubiquitin-mediated degradation of Wee1. At present, we cannot exclude the possibility that downregulation of CK2b might also negatively influence components of the ubiquitylation machinery resulting in stabilization of Wee1 kinase. Oncogene
-- P-Wee1 -- P-Wee1 -- CK2β 1
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Figure 7 CK2b influences the stability of Wee1 through Wee1 phosphorylation at Ser53 and 121. (a) Wild-type (Wt) GST-PBD and its mutant (Mt) form (H538A/K540M) were expressed in bacteria as reported in (Watanabe et al., 2005). Bacteria lysates were combined with whole extracts from synchronized H1299 cells overexpressing wild-type Wee1 harvested at 10 h after release from G1/S arrest. 12 h before transfection with plasmid DNA expressing wild-type Wee1, cells were transfected with either scramble siRNA (lanes 2, 4 and 7) or CK2b siRNA (lanes 5 and 8). PBDwt and -mt were precipitated by adding glutathione sepharose beads to the mixture as described in (Watanabe et al., 2005). Lanes 1 and 2 are control experiments where whole cell extracts were either mixed with recombinant purified GST (lane 1) or directly added to the sonication buffer containing the beads (lane 2). Lanes 3 and 6 are also control experiments where GST-PBDwt and GST-PBDmt were added to the sonication buffer containing the beads in the absence of cell lysates, respectively. (b) Level of Wee1 and its phosphorylation at Ser53 and 121, respectively, were analysed with the indicated antibodies by western blot of total lysates from cells treated as described in Figure 6b.
Stabilization of Wee1 and delayed cell-cycle progression may have important implications for the pathogenesis of cancer. Promoting Wee1 stability by CK2b downregulation might be important for maintaining an interphase state as long as cell-cycle checkpoints, which operate to control the state of CDK1 tyrosine phosphorylation, are activated. The present study establishes a previously unrecognized unique role of CK2b in contributing to the timing of entry into mitosis in mammalian cells. Future studies will certainly provide further insights into the impact of CK2b downregulation on the pathways that regulate the G2/M transition in normal cell-cycle progression.
Materials and methods Cell culture conditions and reagents H1299 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in Roswell
CK2b regulates the G2/M cell-cycle transition CW Yde et al
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Figure 8 Model of the proposed influence of CK2b on G2/M transition. Active cyclin B-CDK1 kinase promotes G2/M transition. The activity of CDK1 is negatively controlled by Wee1-dependent phosphorylation and positively regulated by CDC25A-dependent dephosphorylation. The stability of Wee1 is controlled by a series of phosphorylations, including phosphorylation by CK2, which creates the first phosphodegron (pSer121), responsible for Wee1 instability during interphase (Watanabe et al., 2004, 2005). At the onset of M phase, stabilized PLK1 binds to CDK-mediated phosphorylated Wee1. Subsequently, PLK1 phosphorylates Wee1 at Ser53 (that is, pSer53 creates the second phosphodegron) leading to ubiquitination and proteasome-dependent degradation of Wee1. The CDC25A phosphatase is also a target of CDK1 albeit the CDK1-dependent phosphorylation leads to CDC25A stabilization. Upon DNA damage, CDC25A is subjected to multiple phosphorylations from Chk1 and other protein kinases (‘PK’), including Chk2 and p38 MAPK, leading to destabilization of the phosphatase. Disruption of CK2b leads to stabilization of both Wee1 and CDC25A. At present, it is unclear exactly how CK2b regulates CDC25A stability but CK2b presumably enhances CDC25A phosphorylation via Chk1 and/or other protein kinases. Lack of CK2b, however, leads to partial inhibition of CDK1 and this is explained by the requirement of CK2b for PLK1–Wee1 interaction. Without CK2b the CK2- and PLK1-dependent Wee1 degradation is compromised, resulting in stabilization of Wee1 and increased phosphorylation of CDK1.
Park Memorial Institute medium (RPMI; Gibco, Taastrup, Denmark) supplemented with 10% fetal bovine serum in a humidified incubator at 37 1C and in 5% CO2. Cell-cycle synchronization was achieved by starving the cells in the absence of serum for 24 h. Subsequently, fresh medium was added and cells were cultured in the presence of 3 mg/ml aphidicolin (Calbiochem, Nottingham, UK). After 24 h, cells were extensively washed with phosphate buffered saline (PBS) and released from G1/S arrest by adding fresh culture medium. Cells were harvested at various times as indicated in the figure legends. Where indicated, 200 ng/ml nocodazole (Calbiochem) was added 1 h after release from the block at the G1/S interphase and mitotic cells were collected as indicated. For inhibition of endogenous CK2, cells were incubated for 5 h with 25 mM TBB, 40 mM emodin and 50 mM apigenin (all from Calbiochem), respectively. Pharmacological inhibition of Chk1 kinase was performed by incubating the cells with 300 nM Go¨6976 (Calbiochem) for 24 h. Induction of DNA damage was induced by incubating the cells with 250 nM doxorubicin (Calbiochem) for 48 h.
siRNA experiments The silencing of CK2a expression was achieved by transfecting cells with the following oligonucleotide sequence: 50 -GAU GACUACCAGCUGGUUCdTdT-30 . The expression of CK2a0 was silenced by transfecting cells with the following sequence: 50 -CAGUCUGAGGAGCCGCGAGdTdT-30 . A set of four siRNA duplexes directed against CK2b and Chk1 (ON-TARGET plus SMARTpools), respectively, were employed for downregulating the expression of the aforementioned proteins. Control experiments were performed employing a scramble oligonucleotide sequence: 50 -GAAGC AGUCGCAGUGAAGAdTdT-30 . All siRNAs were purchased from Dharmacon (Lafayette, CO, USA). Cells were transfected with Lipofectamine 2000 (Invitrogen, Paisley, UK) for up to 72 h following the manufacturer’s recommendations.
Cell-cycle analysis H1299 cells were collected after various treatments by trypsinization, washed with PBS, fixed overnight in 70% ethanol at 20 1C, incubated for 30 min with 20 mg/ml Oncogene
CK2b regulates the G2/M cell-cycle transition CW Yde et al
4996 propidium iodide (Sigma, Br ndby, Denmark) and 40 mg/ml RNaseA (Roche, Penzberg, Germany) in PBS prior to analysis on a FACS-Calibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). Staining of cells arrested in mitosis with anti-phospho-histone H3 (Ser10) antibody (Alexa Fluor 488 conjugate; Cell Signaling Technology, Beverly, MA, USA) was performed according to the manufacturer’s recommendations. Collected data were analysed by Cell Quest Pro Analysis software. Antibodies and immunological methods Cells were lysed in lysis buffer (50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM DTT, 1 mM Na3VO4, 30 mM b-glycerophosphate, 10 mM NaF, 100 nM okadaic acid) containing a protease inhibitor cocktail (Roche). After sonication and centrifugation, whole cell extracts were subjected to SDS–polyacrylamide gel electrophoresis (PAGE), western blot, protein kinase assay or immunoprecipitation experiments. Proteins were detected by probing western blot membranes with the following antibodies: mouse monoclonal anti-CK2a, mouse monoclonal anti-CK2b (both from Calbiochem); rabbit polyclonal antiCK2a0 obtained by immunizing rabbits with specific peptide sequence of human CK2a0 , SQPCADNAVLSSGTAAR; mouse monoclonal anti-CDC25A, rabbit polyclonal antiCDC25C, mouse monoclonal anti-Chk1, mouse monoclonal anti-CDK1, mouse monoclonal anti-Wee1, rabbit polyclonal anti-cyclin E, rabbit polyclonal anti-cyclin A, mouse monoclonal anti-PLK1 antibody (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal anti-phospho-CDK1 (Tyr15), rabbit monoclonal anti-PLK1 (both from Cell Signaling Technology); mouse monoclonal anti-b-actin (Sigma). Anti-phospho-Wee1 (Ser121) antibody was raised against phospho-Ser121 peptides, which were also employed for affinity purification; anti-phospho-Wee1 (Ser53) antibody was as described in Watanabe et al. (2005). Protein– antibody complexes were visualized by a chemiluminescence detection system following the manufacturer’s guidelines (CDP-Star; Applied Biosytems, Foster City, CA, USA). Unless otherwise indicated, the antibodies employed in the study detect endogenous levels of total proteins according to the manufacturers’ specifications. Densitometric analysis of the protein bands was performed with Gelwork 1D Intermediate software. Cell lysates were employed for immunoprecipitation of PLK1 using rabbit monoclonal anti-PLK1 antibody (Cell Signaling Technology) essentially as previously described (Guerra et al., 1999).
Protein kinase assays CK2 kinase assay was performed as previously described (Guerra et al., 1999) in a reaction mixture containing the CK2specific synthetic peptide RRRDDDSDDD and 10 or 20 mg cell lysate. Immunoprecipitated PLK1 was employed as kinase source in enzyme assays performed for 20 min at 30 1C in a 20 ml reaction mixture containing 50 mM 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (pH 7.5), 10 mM MgCl2, 1 mM EDTA, 2 mM DTT, 0.01% NP-40, 150 mM ATP, 10 mCi[g-32P] ATP and 1 mg recombinant purified GST-MDM2(203282) or 1 mg dephosphorylated casein (Sigma). Reactions were terminated by adding sample buffer and boiling for 5 min. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes by western blot. Radioactive substrates were revealed by autoradiography and radioactivity was quantified by scintillation counting (Hewlett-Packard, Birker d, Denmark) of the excised radioactive bands. Kinase assays performed employing 1 pmol recombinant PLK1 (Carna Biosciences, Kobe, Japan) and increasing amounts of recombinant CK2b were performed as described above. Purification of recombinant proteins CK2b was expressed and purified as described in (Guerra et al., 2003). GST-MDM2(203282) and GSTMDM2(203282)(S260A) constructs (the latter contains a mutation in the target serine phosphorylated by PLK1) were generated and expressed in bacteria essentially as described in (Kurki et al., 2004). Pellets from bacteria expressing the aforementioned proteins were resuspended in sonication buffer containing 50 mM Tris/HCl (pH 8), 300 mM NaCl and protease inhibitor cocktail (Roche). Disruption of the bacteria was followed by sonication and centrifugation at 4 1C for 30 min at 10 000 g. The supernatant was loaded onto a GSH-agarose column chromatography (Amersham-Biosciences, Hiller d, Denmark) equilibrated in sonication buffer. After extensive wash, proteins were eluted in buffer containing 50 mM Tris/ HCl (pH 8) and 10 mM reduced glutathione (Sigma). Collected fractions were dialysed overnight in a buffer containing 50 mM Tris/HCl (pH 8), 1 mM DTT, 300 mM NaCl and 50% glycerol and stored in aliquots at 70 1C. Acknowledgements We thank T Holm for technical assistance. This study was supported by the Novo Nordisk Foundation, grant no. 5373 and the Danish Medical Research Council, grant no. 271-070464 to BG.
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4997 Kreutzer J, Guerra B. (2007). The regulatory beta-subunit of protein kinase CK2 accelerates the degradation of CDC25A phosphatase through the checkpoint kinase Chk1. Int J Oncol 31: 1251–1259. Kurki S, Peltonen K, Latonen L, Kiviharju TM, Ojala PM, Meek D et al. (2004). Nucleolar protein NPM interacts with HDM2 and protects tumor suppressor protein p53 from HDM2-mediated degradation. Cancer Cell 5: 465–475. Litchfield DW. (2003). Protein kinase CK2: structure, regulation and role in cellular decisions of life and death. Biochem J 369: 1–15. Lu¨scher B, Litchfield DW. (1994). Biosynthesis of casein kinase II in lymphoid cell lines. Eur J Biochem 220: 521–526. Mailand N, Falck J, Lukas C, Syljua˚sen RG, Welcker M, Bartek J et al. (2000). Rapid destruction of human Cdc25A in response to DNA damage. Science 288: 1425–1429. Molinari M, Mercurio C, Dominguez J, Goubin F, Draetta GF. (2000). Human Cdc25A inactivation in response to S phase inhibition and its role in preventing premature mitosis. EMBO Rep 1: 71–79. Parker LL, Walter SA, Young PG, Piwnica-Worms H. (1993). Phosphorylation and inactivation of the mitotic inhibitor Wee1 by the nim1/cdr1 kinase. Nature 363: 736–738. Perry JA, Kornbluth S. (2007). Cdc25 and Wee1: analogous opposites? Cell Div 2: 12. Pines J. (1999). Four-dimensional control of the cell cycle. Nat Cell Biol 1: E73–E79. Seeber S, Issinger O-G, Holm T, Kristensen LP, Guerra B. (2005). Validation of protein kinase CK2 as oncological target. Apoptosis 10: 875–885. Sia RA, Bardes ESG, Lew DJ. (1998). Control of Swe1p degradation by the morphogenesis checkpoint. EMBO J 17: 6678–6688. Stalter G, Siemer S, Becht E, Ziegler M, Remberger K, Issinger OG. (1994). Asymmetric expression of protein kinase CK2 subunits in human kidney tumors. Biochem Biophys Res Commun 202: 141–147. S rensen CS, Syljua˚sen RG, Falck J, Schroeder T, Ronnstrand L, Khanna KK et al. (2003). Chk1 regulates the S phase checkpoint by
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