Mitotic progression requires a wave of Cdk1 activity that phosphorylates a large number of substrates. However, the details of how this wave of phosphorylation coordinates the precisely ordered physiological processes of mitosis are incompletely understood. A particularly important issue that awaits explanation is the relationship between mitotic kinases and their antagonistic phosphatases. Here, we show that cells become capable of the “forward” M-to-G1 cell cycle transition only after Cdk1 is fully activated. Under normal circumstances, positive feedback-mediated Cdk1 activation may function to overcome the activity of Cdk1-opposing phosphatases. This mode of Cdk activation appears to be essential for maintaining the mitotic state and for the proper ordering of mitotic events.
By chemically inhibiting Cdk1 at different stages of mitosis from prophase to metaphase, we demonstrated that Cdk1 inhibition results in complete cyclin B breakdown and irreversible cell division (“forward” mitotic transition) only if the Cdk inhibitor was applied after prophase. Application of Cdk inhibitor in prophase caused return to interphase without substantial cyclin B breakdown, and cells could re-enter mitosis when the Cdk inhibitor was removed. Thus, Cdk inhibition in prophase induces cells to retreat back to G2. Estimation of the Cdk1 activity at different stages of mitotic progression by immunofluorescence analysis of the phosphorylation of three mitotic substrates revealed that the rapid rise of Cdk1-mediated phosphorylation occurs primarily during the short transition from prophase to prometaphase. This is generally consistent with previous immunofluorescence measurements by Lindqvist et al.
, where Cdk activation was assessed by measuring the dephosphorylation of the inhibitory Y15 on Cdk1 and phosphorylation of the Cdk1 substrate APC/C subunit Cdc27 (Lindqvist et al.
). More recently, Gavet and Pines were able to measure the activity of Cdk1/cyclin B complex in individual cells directly, by using a FRET biosensor designed specifically for Cdk1/cyclin B1 kinase (Gavet and Pines 2010a
). This elegant molecular tool used a short fragment of human cyclinB1 harboring an autophosphorylation site. This biosensor exhibited a steep increase in FRET signal during prophase and early prometaphase. Overall, this trend was similar to the one observed in our immunofluorescence experiments. Taken together, these data point toward the conclusion that the rapid increase of Cdk1 activity in prometaphase determines the moment when cells become committed to “forward” mitotic progression.
The primary indicator for “forward” mitotic progression in our studies was proteolysis of cyclin B, which depends on the activation of APC/C-Cdc20. APC/C-Cdc20 is itself a Cdk substrate that is heavily phosphorylated in mitosis (Kraft et al.
; Herzog et al.
; Steen et al.
). Even though we did not assess APC/C phosphorylation directly due to the lack of suitable phosphoepitope antibodies, we anticipate the kinetics of APC/C phosphorylation to be similar to that of the other mitotic substrates we did assess. Lindqvist et al.
performed quantitative analysis of mitotic phosphorylation of specific Cdk1 target residues on one of the subunits of the APC/C–Cdc27/APC3–T446 and S426. Their study showed that the bulk of these residues became phosphorylated during prophase and prometaphase (Lindqvist et al.
). In our study, live imaging analysis of fluorescent cyclin B breakdown induced by Cdk inhibition showed that, functionally, APC/C-Cdc20 becomes progressively more efficient at targeting cyclin B for degradation with advancing stages of mitosis. Therefore activation of Cdk1 is likely to be a determining factor for the ability of the APC/C-Cdc20 to process mitotic substrates.
Our immunofluorescence analysis showed that there is considerable variability in final (metaphase) levels of Cdk1 activity from cell to cell. However, this variability did not seem to impact mitotic progression. The final level of Cdk1/cyclin B activity in the cell is likely determined by the amount of cyclin B because Cdk1 was reported to be in vast excess over cyclins in cells (Arooz et al.
). Several cyclin B knockdown studies reported a variety of relatively minor mitotic perturbation in different cell lines, suggesting that overall mitotic progression has room to be remarkably tolerant to reduction of cyclin B levels by siRNA or shRNA (Yuan et al.
; Bellanger et al.
; Gong et al.
; Androic et al.
; Chen et al.
). Although the efficiency of knockdown may partially explain the weak phenotype, this observation is also consistent with the idea that the total level of Cdk1/cyclin B activity is less important than the positive feedback-mediated rapidity of Cdk activation. For instance, overexpression of the Cdk1-AF mutant, which lacks inhibitory phosphorylation sites, causes a profound effect on cell cycle progression, manifested by premature chromatin condensation, aberrant mitosis, and abbreviated cell cycles (Jin et al.
; Pomerening et al.
; Gavet and Pines, 2010b
). This phenotype was somewhat distinct from the mitotic collapse phenotype, particularly in the aspect of persistent oscillations between mitotic and interphase state that were not observed in our experiments. However, in the above studies, Cdk1-AF mutant was overexpressed above the endogenous wild-type Cdk1. Therefore a portion of Cdk1/cyclin B complex in these studies may have been assembled with endogenous, wild-type Cdk1 that retained the ability to be regulated by phosphorylation.
In this study, we used fast-acting chemical inhibitors to analyze the importance of the switch-like activation of endogenous Cdk1 for the proper order of mitotic progression. Inhibition of the Wee1 and Myt1 kinases in cells induced a relatively normal mitosis in cells synchronized at the end of S phase, without requiring a G2 stage. Ordinarily, during G2, cells grow and accumulate various proteins, including mitotic cyclins. In cells pushed into mitosis by the Wee1/Myt1 inhibitor, cyclin B1 did not accumulate to the level characteristic of cells that entered mitosis without the inhibitor. Surprisingly, the amount of cyclin B present by the end of the S phase in synchronized cells was sufficient for entry into mitosis. Because inhibition of Wee1 and Myt1 kinases resulted in rapid dephosphorylation of Cdk1 on inhibitory T14 and Y15, Cdk1 activation in these cells was still rapid, even though their cyclin B levels were lower than in cells that entered mitosis spontaneously. Nevertheless, these cells were able to progress through mitosis, supporting the idea that, for the proper order of mitotic events, the final Cdk1 activity levels may be less critical than the feedback-mediated dynamics of its activation.
Simultaneous inhibition of Wee1/Myt1 kinases and Cdc25 phosphatases prevented both phosphorylation and dampened dephosphorylation of Cdk1 on inhibitory T14 and Y15. Unexpectedly, this led to a sluggish mitotic entry followed by dephosphorylation of mitotic substrates without cyclin B breakdown—a phenotype that we termed “mitotic collapse.” The failure to degrade cyclin B likely reflects insufficient activation of APC/C-Cdc20 by low levels of Cdk1 activity, similar to the situation in prophase cells. The substrate dephosphorylation was prevented by 1 μM okadaic acid, indicating that the Cdk1 was actively antagonized by phosphatase(s).
The possibility that the combination of Wee1 and Cdc25 inhibitors could have some off-target effect that can influence phenotypic changes observed in cells undergoing mitotic collapse cannot be completely excluded. This caveat is intrinsic to any chemical inhibitor studies. However, it is highly unlikely that these inhibitors can trigger the nonspecific phosphatase activation, because phosphorylation of nucleolin and histone H3 was not lost in cells that were already in mitosis at the time of drug addition ().
Historically, mitosis research has highlighted the mitotic kinases as key regulators of cell division, whereas phosphatases have received much less attention. However, it is becoming clear that the normal progression of mitosis is not only a consequence of the change in activity of mitotic kinases, primarily Cdk1, but requires balanced actions of counteracting phosphatases (Trinkle-Mulcahy and Lamond, 2006
In budding yeast, the primary phosphatase opposing Cdk1 is Cdc14 (Visintin et al.
; Jaspersen et al.
; reviewed in Stegmeier and Amon, 2004
; Bosl and Li, 2005
; Amon, 2008
). However, in metazoans, neither of the two Cdc14 homologues, Cdc14A or Cdc14B, has been shown to counteract Cdk1 kinase during mitotic exit (Vazquez-Novelle et al.
; Berdougo et al.
). Instead, in higher eukaryotes, the PP1 and PP2A families of protein phosphatases, enzymes that can be inhibited by okadaic acid, appear to play more important roles in mitotic entry and exit. In Xenopus
egg extracts, depletion studies have implicated both PP1 and PP2A in the dephosphorylation of Cdk1 substrates (Mochida et al.
; Wu et al.
). Interestingly, both PP1 and PP2A phosphatases appear to be inhibited by high Cdk1 activity, constituting another feedback mechanism where the Cdk1 kinase inactivates its antagonists, shifting the balance toward mitotic phosphorylation ()
FIGURE 7: (A) Cdk substrate phosphorylation regulatory network. The phosphorylation of mitotic substrates (enzymes and structural proteins) by Cdk1/cyclin B complex underlies mitotic entry. Cdk1/cyclin B is antagonized by phosphatases PP1 and PP2A that dephosphorylate (more ...)
PP1 is phosphorylated by Cdk1 on the inhibitory T320 residue (Dohadwala et al.
; Kwon et al.
; Wu et al.
). When Cdk1 is inactivated during mitotic exit, PP1 activates itself by dephosphorylating this T320 residue and another residue, T35 (likely a MAP kinase site), responsible for the binding of the inhibitory protein I-1 (Wu et al.
). Another small protein inhibitor of PP1 is the inhibitory protein 2 (I-2), which is also heavily phosphorylated in mitosis (Li et al.
) and may be a Cdk1 substrate. Therefore the activation of Cdk1 may switch PP1 off, and inactivation of Cdk1 may switch PP1 on. Further experimental and modeling studies are needed to evaluate the dynamics and robustness of this switch.
A similar mechanism of Cdk-dependent inhibition may exist for PP2A. The activity of PP2A–B55 delta is low when Cdk1 is fully active in mitosis (Mochida and Hunt, 2007
). Unlike PP1, PP2A has not yet been shown to be inhibited by Cdk1 phosphorylation directly. However, a kinase called Greatwall (human MastL) has been shown to inhibit anti-mitotic phosphatases in the Xenopus
egg extract system (Castilho et al.
; Vigneron et al.
). Greatwall kinase is a Cdk1/cyclin B substrate. Active Cdk1/cyclin B complex phosphorylates and activates Greatwall, which then inhibits PP2A and perhaps other phosphatases, constituting another feedback loop that promotes mitotic phosphorylation ().
Because the substrate of the human MastL kinase is not yet identified, we were not able to assay its activity directly. By Western blotting, we observed a phosphorylation shift during mitotic entry that was absent in mitotic collapse, suggesting that MastL may be inactive in collapsed cells (). This may partially explain the elevated phosphatase activity in these cells. MastL knockdown was shown to cause defects in chromosome alignment and segregation and also incomplete cyclin B breakdown upon mitotic exit (Burgess et al.
; Voets and Wolthuis, 2010
). However, strong MastL knockdown as well as the Greatwall depletion in Xenopus
egg extracts were reported to block entry in mitosis. We attempted to override this block in MastL siRNA-treated HeLa cells synchronized at the S/G2 border by treating them with the Wee1/Myt1 inhibitor PD0166285. The mitotic entry in this case was comparable in both MastL siRNA and negative control siRNA-treated cells. The phenotype of MastL knockdown cells that entered mitosis in Wee1 inhibitor was generally similar to what has been reported previously (Supplemental Figure 6 and Video 11), although there was an increased incidence of mitotic cell death. We did not observe defects reminiscent of mitotic collapse, which suggests that MastL may be responsible for inhibition of some, but not all Cdk-opposing phosphatases involved in generating mitotic collapse phenotype. Alternatively, the depletion of MastL by siRNA may have been insufficient to fully release phosphatase activities.
The phosphatase(s) responsible for the mitotic collapse phenotype in our studies likely belonged to the PP2A family because the dephosphorylation of mitotic substrates was prevented by 1 μM okadaic acid. At this concentration, PP1 is only partially inhibited (Bialojan and Takai, 1988
). Okadaic acid not only prevented the dephosphorylation of Cdk1 substrates but also markedly increased their phosphorylation (). Without okadaic acid, mitotic phosphatases eventually overcame Cdk activity when it was not fueled by positive feedback, resulting in mitotic collapse. One possible mechanism that may aid somatic cells in countering phosphatase activity during mitotic entry is spatial concentration of Cdk1 activity within the nucleus in early mitosis. Cdk1/cyclin B complex translocates into the nucleus in prophase and then disperses throughout the cytoplasm after nuclear envelope breakdown (Pines and Hunter, 1991
; Hagting et al.
). It was recently confirmed that translocation of Cdk1/cyclin B complex into the nucleus coincides with its activation (Gavet and Pines, 2010a
). Consistent with this, our immunolabeling experiments show that the Cdk activity is concentrated in the nucleus in prophase, and after nuclear envelope breakdown, the cytoplasm fills with phosphorylated Cdk1 substrates (Supplemental Figure 2, A–C). Overall, it appears that Cdk1 activity spikes around the time of the nuclear envelope disassembly, when the activated Cdk/cyclin B complex spreads through the cytoplasm. Therefore it is possible that in the absence of the positive feedback, active Cdk1 became too dilute in the cytoplasm when the nuclear envelope disassembled or became permeable enough to permit the diffusion of Cdk1/cyclin complexes out of the nucleus (). Under these circumstances, the concentration of the active kinase per unit of cytosol may have fallen below the level that is needed to efficiently counteract Cdk-opposing phosphatases and maintain mitosis.
The mitotic collapse phenotype that we observed was accompanied by substrate dephosphorylation, but morphologically it was far from normal mitotic exit. Mitotic exit, like mitotic entry, is a well-ordered sequence of events: chromatid segregation is followed by cytokinesis, nuclear envelope reassembly, cytosceletal rearrangements, etc. Whether this orderly progression requires a particular sequence of dephosphorylation reactions is not known. However, our results suggest that the proper interplay of kinase and phosphatase activities, where feedback-mediated activation of Cdk first overcomes the activity of phosphatases then is rapidly turned off, is essential for the normal mitotic entry and exit.