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Vol. 20, Issue 16, 3671-3679, August 15, 2009
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Department of Biomedical Sciences, College of Medicine, Florida State University, Tallahassee, FL 32306
Submitted October 22, 2008;
Revised May 27, 2009;
Accepted June 19, 2009
Monitoring Editor: Fred Chang
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The activity of Cdc14 is regulated through its subcellular localization. During most of the cell cycle stages, Cdc14 is sequestered within the nucleolus to prevent the dephosphorylation of its substrates. Two signaling cascades, Cdc fourteen early anaphase release (FEAR) and mitotic exit network (MEN), are responsible for the regulation of Cdc14 release. The FEAR network promotes a brief Cdc14 release from the nucleolus during early anaphase, and this pathway consists of Polo-like kinase Cdc5, separase Esp1, Slk19, and Spo12 (Stegmeier et al., 2002). Our recent work indicates that FEAR-induced Cdc14 release during early anaphase is responsible for the reversal of protein phosphorylation imposed by S phase cyclin-associated Cdk1 (Clb5-Cdk1), which facilitates spindle stabilization and elongation (Jin et al., 2008). The activation of the MEN further promotes the dissociation of Cdc14 from its inhibitor during late anaphase and telophase (Shou et al., 1999; Visintin et al., 1999). In addition, the phosphorylation of Cdc14 by Dbf2, a component of MEN, promotes transfer of Cdc14 to cytoplasm (Mohl et al., 2009). Released Cdc14 inactivates mitotic Cdk by destroying mitotic cyclin Clb2 and stabilizing the Cdk inhibitor Sic1 (Visintin et al., 1998). The complete inactivation of Cdk makes it possible for the assembly of the prereplication complex for the next round of cell cycle (Noton and Diffley, 2000).
FEAR-dependent Cdc14 release may be facilitated by the phosphorylation of Net1 that binds to and sequesters Cdc14 within the nucleolus (Shou et al., 1999; Visintin et al., 1999; Azzam et al., 2004). Before anaphase entry, the presence of phosphatase PP2ACdc55 keeps Net1 in its hypophosphorylated form, which prevents the dissociation of Cdc14 from the nucleolar localized Net1 (Queralt et al., 2006; Wang and Ng, 2006; Yellman and Burke, 2006). After anaphase entry, separase Esp1 inactivates PP2ACdc55 with the assistance of Slk19, Spo12, Zds1, and Zds2 (Queralt et al., 2006; Queralt and Uhlmann, 2008), allowing the phosphorylation of Net1 by Clb2-Cdk1 (Azzam et al., 2004). This modification may favor Cdc14 release.
Cdc5 is a conserved Polo-like kinase that plays multiple roles during the cell cycle. As a component of the MEN, Cdc5 promotes mitotic exit by phosphorylating Bfa1, the negative regulator of the MEN (Hu et al., 2001). It is interesting that Cdc5 also functions in the FEAR pathway because Cdc5 is required for the release of Cdc14 from the nucleolus during early anaphase (Stegmeier et al., 2002). Some evidence indicates that the direct phosphorylation of Cdc14 by Cdc5 may facilitate Cdc14 release (Visintin et al., 2003; Rahal and Amon, 2008). Also, the degradation of Cdc5 protein is essential for the return of Cdc14 to the nucleolus after exit mitosis (Visintin et al., 2008). Previous data indicate that Cdc5 kinase phosphorylates Swe1 to promote its degradation (Park et al., 2004; Sakchaisri et al., 2004; Asano et al., 2005). In vitro and in vivo evidence indicates that Swe1 inhibits mitotic cyclin-associated Cdk activity by phosphorylating Cdk1 at tyrosine 19 (Booher et al., 1993). Recent evidence suggests that Swe1 has little effect on S phase cyclin-associated Cdk1 (Hu and Aparicio, 2005; Liu and Wang, 2006; Keaton et al., 2007). In this study, we present evidence indicating that Cdc5 regulates the FEAR network in part by inducing Swe1 degradation, which enables Clb2-Cdk1 to phosphorylate Net1.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-factor was added into cell cultures (YPD, pH 3.9). After 2 h of incubation, the G1-arrested cells were washed twice with water and then released into YPD medium to start cell cycle.
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Protein Techniques
The pellets from 1.5 ml of cell cultures were resuspended in 200 µl of 0.1 N NaOH and incubated at room temperature for 5min. After centrifugation, the cells were resuspended in equal volume (30 µl) of double distilled H2O and SDS protein-loading buffer. The samples were then boiled for 5 min and resolved with 8% SDS-polyacrylamide gel. Proteins were detected with enhanced chemiluminescence (PerkinElmer Life and Analytical Sciences, Boston, MA) after probing with anti-myc antibody (Covance Research Products, Princeton, NJ) and horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Previous studies have shown that Cdc5 phosphorylates Swe1 and promotes its degradation (Asano et al., 2005), and cdc5-2 mutant cells exhibit Swe1 degradation defects when incubated at 37°C (Liu and Wang, 2006). These observations raise the possibility that the failure of Swe1 degradation might contribute to the FEAR defects in cdc5 mutant cells. Because cdc5-1 mutants show FEAR defects, we first examined Swe1 degradation defects in this mutant. Compared with wild-type (WT) cells, cdc5-1 mutant cells exhibited persistent Swe1 protein levels when incubated at 37°C, but Swe1 protein degradation was not compromised in other MEN (cdc15-2) or FEAR (cdc15-2 spo12
) mutants (data not shown). Therefore, we believe that the Swe1 degradation defect is specific to cdc5 mutants but not to other FEAR or MEN mutant cells.
Given that Swe1 accumulates in both cdc5-1 and cdc5-2 mutants, it is possible that the Swe1 accumulation in these mutants contributes to FEAR defects. To test this possibility, we compared cell cycle progression and Cdc14 release in cdc5-1 and cdc5-1 swe1 mutants incubated at 37°C. Very few cdc5-1 mutant cells exhibited Cdc14 release as reported previously (Stegmeier et al., 2002), but 26% of cdc5-1 swe1 double mutant cells showed released Cdc14 after G1 release for 100 min, similar to cdc15-2 mutant, where the FEAR, but not MEN, is active (Figure 1, A and B). We also compared the spindle elongation kinetics in these mutant cells, and the spindles with one end that has elongated into daughter cells are counted as anaphase spindle. Delayed spindle elongation was noticed in cdc5-1 mutants at 80 and 100 min after G1 release compared with cdc15-2, consistent with the role of the FEAR pathway in spindle morphogenesis (Jin et al., 2008). The spindle elongation defect was partially suppressed by the deletion of SWE1 (Figure 1A). Similarly, we observed Cdc14 release defect in cdc5-2 mutant cells and deletion of SWE1 suppressed this defect (data not shown). These observations indicate that Swe1 accumulation contributes to the Cdc14 release defect in both cdc5-1 and cdc5-2 mutants.
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; Queralt et al., 2006). Because Swe1 exhibits specific inhibition on Clb2-Cdk1 (Booher et al., 1993), and Cdc5 promotes Swe1 degradation, we reason that Cdc5 induces Net1 phosphorylation by alleviating Swe1-imposed inhibition on Clb2-Cdk1. If that is the case, the impediment of Net1 phosphorylation by Cdk1 should abolish the transient Cdc14 release in cdc5 swe1 double mutants. Consistent with our prediction, net1-6Cdk mutants, wherein the six Cdk1 phosphorylation sites are mutated to alanine, largely suppressed Cdc14 release in cdc5-1 swe1 mutants (Figure 1A). However, a few cdc5-1 swe1 net1-6Cdk cells still showed released Cdc14 when grown at the restrictive temperature. One possible explanation is that net1-6Cdk mutant is not a mutant allele that loses FEAR function completely (Azzam et al., 2004) or that some mechanism other than Net1 phosphorylation also controls Cdc14 release during early anaphase.
Swe1 Degradation Is Required for Efficient rDNA Separation
FEAR-dependent Cdc14 release is important for efficient rDNA separation (D'Amours et al., 2004; Sullivan et al., 2004). Our data indicate that Cdc5-dependent Swe1 degradation promotes Cdc14 release during early anaphase, and we speculate that the failure of Swe1 degradation would lead to the rDNA separation defects in cdc5 mutants. To test this speculation, rDNA separation in cdc15-2, cdc5-1, and cdc5-1 swe1 mutants with a tetO array integrated adjacent to the rDNA locus (rDNA-GFP) was analyzed (D'Amours et al., 2004). After G1 release for 2 h at 37°C, 74% of cdc15-2 cells showed separated rDNA-GFP dots, but only 26% of cdc5-1 mutant cells exhibited rDNA separation. In contrast, 47% of cdc5-1 swe1 double mutant cells exhibited separated rDNA (Figure 2, A and B), indicating that swe1 deletion could partially suppress the rDNA separation defect in cdc5-1 mutants. Similar results were obtained using cdc5-2 and cdc5-2 swe1 cells (data not shown). To further confirm the suppression of the rDNA separation defect in cdc5-1 mutants by swe1, we also analyzed the distribution of Net1-GFP, which localizes within the nucleolus throughout the cell cycle (Shou et al., 1999; Visintin et al., 1999; Machin et al., 2006). swe1 deletion suppressed the Net1 separation defects in both cdc5-1 and cdc5-2 mutants (data not shown), suggesting that the FEAR defects in cdc5 mutants are attributable to the failure of Swe1 protein degradation.
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). If the compromised FEAR function in cdc5 mutants is a result of Swe1 accumulation, we expect defective Sld2 dephosphorylation in cdc5 mutants and deletion of SWE1 should suppress this defect. Therefore, we compared the dephosphorylation kinetics of Sld2 in cdc5 and cdc5 swe1 mutants. G1-arrested WT, cdc15-2, cdc5-1, cdc5-1 swe1, cdc5-2, and cdc5-2 swe1 mutants carrying SLD2-myc were released into YPD medium at 37°C. After G1 release for 40 min, Sld2 became phosphorylated in all the tested cells as indicated by the appearance of slow migrating bands. Although cdc15-2 cells showed Sld2 dephosphorylation after G1 release for 80 min, both cdc5-1 and cdc5-2 mutant cells exhibited dramatically delayed Sld2 dephosphorylation, supporting the role of Cdc5 in both FEAR and MEN pathways. However, swe1 deletion alleviated this delay in both cdc5-1 and cdc5-2 mutants, although the suppression is not complete (Figure 3B). Together, the defects in Cdc14 release, rDNA separation, and Sld2 dephosphorylation observed in cdc5 mutants can be partially suppressed by the deletion of SWE1, supporting the conclusion that Cdc5 kinase promotes FEAR function in part by stimulating Swe1 degradation.
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; Sorger and Murray, 1992; Booher et al., 1993). To test whether increased Cdk1 phosphorylation by Swe1 is responsible the FEAR defects in cdc5 mutants, we compared Cdc14 release and rDNA separation in cdc5 single and cdc5 cdc28F19 double mutants, where the Swe1 phosphorylation site in Cdk1 is mutated. It was obvious that cdc28F19 mutation suppressed the defects of Cdc14 release and rDNA separation in cdc5-1 mutants, although the suppression is not complete (Figure 4, A and B), suggesting that the FEAR defects in cdc5 mutants is due to the increased inhibitory phosphorylation of Cdk1 by Swe1 kinase.
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). The localization of Cdc5 at the spindle pole bodies is essential for Bfa1 phosphorylation, whereas the bud-neck localization of Cdc5 is responsible for the phosphorylation of Swe1 (Park et al., 2004). cdc5 mutant cells are defective for both FEAR and MEN. If the FEAR defect in cdc5 is a result of Swe1 accumulation, the expression of CDC5 that specifically localized at the bud neck will suppress the defects of cdc5 mutants in FEAR but not in MEN. Therefore, we introduced a plasmid pKL2438 containing CDC5C-CDC12 into cdc5-1 mutant. This Cdc5C-Cdc12 fusion protein has been shown to exclusively localize at the bud neck and is competent for Swe1 phosphorylation and degradation (Park et al., 2004). The introduction of this plasmid partially suppressed the Swe1 degradation defects in cdc5-1 mutants, but the transformants were still arrested as large-budded cells when incubated at 37°C, indicating the defective MEN (Figure 5A). The expression of Cdc5C-Cdc12 protein also caused Cdc14 release from the nucleolus in some cdc5-1 mutant cells, but the suppression by Cdc5C-Cdc12 was not as efficient as swe1 (Figure 5B). We reason that the slower Swe1 degradation kinetics in cdc5-1 mutant cells containing Cdc5C-Cdc12 contributes to the less efficient suppression of the FEAR defects in cdc5-1 mutants. Thus, the bud-neck localization of Cdc5 and the subsequent Swe1 phosphorylation and degradation are required for FEAR activation.
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), Swe1 overexpression is expected to block the FEAR-dependent Cdc14 release. Indeed, we found that overexpression of Swe1 blocked the Cdc14 release in WT cells but not in cdc28F19 mutant cells (data not shown), suggesting a negative role of Swe1 in Cdc14 release by inhibiting Clb2-Cdk1. However, we could not exclude the possibility that the failure of Cdc14 release in cells overexpressing SWE1 is a result of anaphase entry block. Hsl1 is required for the recruitment of Swe1 to the bud-neck and subsequent Swe1 degradation (Shulewitz et al., 1999; Asano et al., 2005). Although hsl1 mutant cells exhibit elevated Swe1 protein levels, the mutant cells are viable. To further define the negative role of Swe1 in FEAR-dependent Cdc14 release, we examined Cdc14 localization in cdc15-2 hsl1 mutant cells. cdc15-2 hsl1 and cdc15-2 hsl1 swe1 cells with 5GFP-tagged Cdc14 were arrested in G1 phase at 25°C and then released into cell cycle at 37°C. After G1 release for 100 min, 22% cdc15-2 cells showed released Cdc14, indicating that FEAR pathway is active. In contrast, cdc15-2 hsl1 mutant cells showed delayed and decreased Cdc14 release, but the defect was suppressed by introducing swe1 deletion (Figure 6A). The results suggest that high levels of Swe1 in cdc15-2 hsl1 cells contribute to the Cdc14 release defects. We also examined the kinetics of Sld2 dephosphorylation in cdc15-2 and cdc15-2 hsl1 cells incubated at 37°C. In cdc15-2 cells, Sld2 became dephosphorylated after G1 release for 80 min, whereas a delayed Sld2 dephosphorylation was observed in cdc15-2 hsl1 cells (Figure 3C). Together, these observations demonstrate that low level of Swe1 protein is needed for Cdc14 release during early anaphase as well as the subsequent dephosphorylation of Clb5-Cdk1 substrates.
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mutants in the presence of DNA damage (Liang and Wang, 2007). If Swe1 plays a negative role in FEAR activation, high levels of Swe1 would block the mitotic exit in chk1 mutant cells with damaged DNA. To test this possibility, we examined the microcolony formation in chk1 and chk1 hsl1 mutants arrested with cdc13-1, which activates DNA damage checkpoint because of the unprotected telomeres when incubated at the restrictive temperature. Consistent with previous data, 61% of cdc13-1 chk1 cells formed microcolonies when incubated at 37°C for 8 h, indicating cells exit mitosis, but only 11% cdc13-1 chk1 hsl1 cells did, presumably due to the Swe1 degradation defects in hsl1. Deletion of SWE1 allowed cdc13-1 chk1 hsl1 cells to regain the ability for microcolony formation (Figure 6B), confirming that high levels of Swe1 compromise the FEAR-dependent mitotic exit.
FEAR and MEN promote Cdc14 release from the nucleolus and deletion of LTE1, a MEN component, is synthetically lethal with FEAR mutants slk19 and spo12 (Stegmeier et al., 2002). To further determine the FEAR defects in hsl1 mutants, double mutants between hsl1 and MEN mutants, mob1-77, cdc15-2 and lte1, were generated. When incubated at 30°C, hsl1 mob1-77 double mutants failed to grow, whereas mob1-77 single mutant cells grew well. Similarly, the growth of cdc15-2 hsl1 mutant cells at 30°C was not as well as each single mutant. Deletion of SWE1 suppressed the growth defects of the double mutants (Figure 6C). Also, hsl1 lte1 double mutants exhibited poor growth phenotype when incubated at 25°C (data not shown). All these observations support the notion that Swe1 plays a negative role in FEAR activation.
Swe1 Accumulation Is Responsible for the Decreased Net1 Phosphorylation in cdc5 Mutants
The phosphorylation of Net1 by Clb2-Cdk1 promotes FEAR activation (Azzam et al., 2004; Queralt and Uhlmann, 2008). Given that Swe1 negatively regulates Clb2-Cdk1 and that cdc5 mutants exhibit failure in Swe1 degradation, it is likely that the FEAR defect in cdc5 mutants is due to the inability of Net1 phosphorylation. To confirm this speculation, we examined the phosphorylation status of Net1 protein in synchronous cdc5-1 and cdc5-1 swe1 mutants incubated at 37°C. After G1 release for 80 min, both WT and cdc15-2 mutants showed slow migrating forms of Net1, presumably due to phosphorylation (Azzam et al., 2004; Queralt and Uhlmann, 2008). In contrast, only hypophosphorylated Net1 was observed in cdc5-1 mutant cells and deletion of SWE1 suppressed the Net1 phosphorylation defects in cdc5-1 mutants (Figure 7A). This is a strong indication that Cdc5 regulates Net1 phosphorylation through Swe1. In cdc15-2 mutant cells, we noticed persistent Net1 phosphorylation even after G1 release for 2 h, but fewer cdc15-2 mutant cells exhibited released Cdc14 after G1 release for 2 h (Figure 1A). This observation indicates that Net1 phosphorylation might be essential, but not sufficient, for Cdc14 release during early anaphase.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We believe that Cdc5 executes its FEAR function in part by promoting Swe1 degradation on the basis of the following observations. First, we found that both cdc5-1 and cdc5-2 mutants exhibited Swe1 degradation defects. Deletion of SWE1 partially alleviated the defects in Cdc14 release and rDNA separation in cdc5 mutants. In contrast, hsl1 mutant strains, wherein Swe1 degradation is compromised, exhibited FEAR defects, as indicated by the delayed Cdc14 release and the synthetic growth defects with MEN mutants. More importantly, cdc5-1 mutants exhibited Swe1-dependent defect in Net1 phosphorylation.
Our evidence suggests that Swe1 acts as a negative regulator of FEAR. S phase-expressed Swe1 is likely to prevent the premature activation of Clb2-Cdk1, whereas the activity of Clb5-Cdk1 remains unaffected. Because we have shown that the activation of FEAR facilitates spindle stabilization and elongation (Jin et al., 2008), Swe1 accumulation in S phase cells could be an important mechanism to prevent mitotic activities when DNA replication is underway. Consistent with this speculation, we have demonstrated previously that the block of DNA replication stabilizes Swe1 (Liu and Wang, 2006). Surprisingly, no premature mitosis is observed in swe1 mutant cells when DNA synthesis is blocked (Amon et al., 1992; Sorger and Murray, 1992), indicating that other mechanisms might be present to prevent premature activation of Clb2-Cdk1. For example, the low transcription levels of CLB2 gene during S phase could avoid premature mitosis induced by Clb2-Cdk1.
Although swe1 deletion can suppress the FEAR defects in cdc5 mutants, the kinetics of rDNA separation and Sld2 dephosphorylation in cdc5 swe1 double mutant cells is a little slower than that in cdc15-2 mutants, indicating that additional defects in cdc5 mutants may also contribute to the failure of FEAR activation. Previous studies suggest that overexpression of Cdc5 leads to hyperphosphorylated Cdc14 and that this modification may play a positive role in Cdc14 release (Visintin et al., 2003). Recently, Cdc5 has been shown to interact with Cdc14 directly, but the significance of this interaction remains to be defined (Rahal and Amon, 2008). Thus, Cdc5 may regulate Cdc14 release in multiple ways and Cdc5-dependent Swe1 degradation could be one of the functions of Cdc5 in mitotic exit.
We and others have demonstrated that FEAR components Esp1, Slk19, and Spo12 promote FEAR function by inhibiting PP2ACdc55-dependent Net1 dephosphorylation (Queralt et al., 2006; Wang and Ng, 2006; Yellman and Burke, 2006). Unlike other FEAR components, the Polo-like kinase Cdc5 regulates Net1 phosphorylation through Swe1. Cdc5-induced Swe1 degradation enables the activation of mitotic cyclin-associated Cdk1, which phosphorylates Net1. Therefore, two different mechanisms control FEAR activation through the regulation of Net1 phosphorylation. The activation of Cdc5 kinase before metaphase results in the degradation of Swe1, which allows the activation of Clb2-Cdk1. However, the presence of the phosphatase PP2ACdc55 keeps Net1 protein from being phosphorylated by Clb2-Cdk1. On anaphase onset, the degradation of the anaphase inhibitor Pds1 frees the separase Esp1 that inactivates PP2ACdc55 and shifts the equilibrium toward Net1 phosphorylation. Recent works from the Amon laboratory indicate that Spo12 is also a substrate of Clb2-Cdk1 (Tomson et al., 2009), raising the possibility that Cdc5-dependent Swe1 degradation may also lead to Spo12 phosphorylation and further activate FEAR. In conclusion, both Cdc5-dependent Clb2-Cdk1 activation and Esp1-induced inactivation of PP2ACdc55 promote FEAR activation by stimulating the phosphorylation of Net1 (Figure 7B).
Mammalian cells express Cdc14A and Cdc14B, which are the functional homologues of budding yeast Cdc14 (Trinkle-Mulcahy and Lamond, 2006). Cdc14A is located at centrosomes, whereas Cdc14B resides in the nucleolus during interphase but not during mitosis (Bembenek and Yu, 2001; Kaiser et al., 2002). We found that S phase cyclin substrates, including Ase1, a spindle midzone component, are subjected to FEAR-dependent dephosphorylation after anaphase entry (Jin et al., 2008). PRC1, the mammalian homologue of Ase1, also becomes dephosphorylated during metaphase-to-anaphase transition, and this dephosphorylation is essential for the spindle midzone formation (Zhu et al., 2006), raising the possibility that the dephosphorylation of some Cdk substrates occurs after anaphase entry in mammalian cells. However, further experiments are needed to determine whether PRC1 is a substrate of Cdc14A or B. So far, there is no solid evidence indicating the presence of FEAR and MEN networks that regulate Cdc14 activity in mammalian cells. Much more research work is needed to determine whether mammalian cells dephosphorylate some Cdk substrates after anaphase entry through the activation of phosphatase Cdc14.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Present address: Department of Pharmacology, University of Texas Southwestern Medical Center, 6001 Forest Park Rd., Dallas, TX 75390. 
Address correspondence to: Yanchang Wang (yanchang.wang{at}med.fsu.edu).
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