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Vol. 20, Issue 6, 1737-1748, March 15, 2009
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Cell Cycle and Cancer Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104
Submitted July 29, 2008;
Revised December 10, 2008;
Accepted January 12, 2009
Monitoring Editor: Orna Cohen-Fix
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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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), and inactivation drives exit from mitosis (Murray et al., 1989). Cdk1 kinase requires an activating partner—a cyclin. Cyclin B is a pivotal activator of Cdk1 in mitosis. During mitotic exit, cyclin B is degraded by the ubiquitin-proteasome pathway (Glotzer et al., 1991). Cyclin B degradation causes irreversible inactivation of Cdk1 and therefore provides directionality for the cell cycle during the M phase to G1 transition. In addition to the activation by cyclins, Cdk1 activity can be negatively regulated by phosphorylation on two inhibitory residues—T14 and Y15. Wee1 is a tyrosine kinase that phosphorylates Y15 (Parker and Piwnica-Worms, 1992), and Myt1 is a dual-specificity kinase that can phosphorylate both sites (Kornbluth et al., 1994; Mueller et al., 1995), with a propensity toward the T14 (Liu et al., 1997). These inhibitory phosphorylations are removed by Cdc25 phosphatases. The functions of Wee1, Myt1, and Cdc25, in turn, are regulated by Cdk activity itself. During the G2-to-M transition, Wee1 and Myt1 kinases become phosphorylated and inactive when Cdk1 activity rises (Mueller et al., 1995; Watanabe et al., 2005), and the Cdc25 becomes phosphorylated and active (Hoffmann et al., 1993). Therefore, Cdk activity feeds back on itself by activating its activator Cdc25 and inhibiting its inhibitors Wee1 and Myt1. These two mechanisms serve as positive feedback that induces a robust autoamplification of Cdk1 activity (O'Farrell, 2001), and they are responsible for the switch-like activation of Cdk1 (Pomerening et al., 2005). Here, we show that after mitotic exit, Wee1 and Myt1 kinases become activated and can lock Cdk1 in the inactive state, thus serving to inhibit Cdk1 in the presence of its activators during G1. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemical Inhibitors
The Cdk inhibitor flavopiridol, provided by the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (Bethesda, MD) was used at 5–10 µM except for cells overexpressing Cdk1-GFP constructs in which it was used at 25 µM. The proteasome inhibitor MG132 (Calbiochem, San Diego, CA) was used at 25 µM. The Wee1/Myt1 inhibitor PD0166285 (Pfizer, New York, NY) was used at 0.5 µM. The Aurora kinase inhibitor ZM447439 (AstraZeneca Pharmaceuticals LP, Wilmington, DE) was used at 25 µM. The Cdc25 phosphatase inhibitor NSC663284 (Sigma-Aldrich, St. Louis, MO) was used at 25 µM. The general caspase inhibitor Z-VAD-FMK (Calbiochem) was used at 100 µM. Nocodazole (Sigma-Aldrich) was used at 100 ng/ml.
Drug Treatments and Western Blotting
HeLa cells were collected by shake-off after being blocked in mitosis as detailed in figure legends. Nocodazole or MG132 block was used for nontransfected cells. For R42A cyclin B transfection experiments in Figures 2–5, mitotic cells were collected without nocodazole addition because the cells transfected with the nondegradable cyclin B arrest in mitosis spontaneously. The mitotic cells were split into several experimental subgroups and treated with specified drugs for indicated periods (detailed in figure legends). For the flavopiridol washout, cells were pelleted by centrifugation, the flavopiridol-containing medium was aspirated, and cells were resuspended in the medium without flavopiridol. Cells treated with MG132 were maintained in media containing MG132 for the duration of the experiment. For cells collected in nocodazole, it was also maintained in the media throughout experiments except for the nocodazole release studies. At the time of collection, cells were pelleted by centrifugation and subsequently lysed in NuPAGE protein sample buffer (Invitrogen) containing 50 mM dithiothreitol (DTT). Protein samples were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 4–12% Bis-Tris gels (Invitrogen), transferred to polyvinylidene difluoride (PVDF) (Millipore, Billerica, MA), and blocked in 5% nonfat dry milk or 5% bovine serum albumin (BSA). Primary antibodies used were as follows: anti-pNucleolin (gift from P. Davies, Albert Einstein College of Medicine, Bronx NY), anti-pT14Cdk1 (Abcam, Cambridge, MA), anti-pY15Cdk1 (BD Biosciences, San Jose, CA or Cell Signaling Technology, Danvers, MA), anti-cyclin B1 (BD Biosciences), anti-Plk1 (Zymed Laboratories, South San Francisco, CA), anti-Wee1 (Cell Signaling Technology), anti-Myt1 (Cell Signaling Technology), anti-Cdc25C (Cell Signaling Technology), anti-pS10 histone H3 (Cell Signaling Technology), anti-nucleolin (Abcam), and anti-Cdk1 (BIOMOL Research Laboratories, Plymouth Meeting, PA). Primary antibodies were detected using horseradish-peroxidase-conjugated immunoglobulin G (Jackson ImmunoResearch Laboratories, West Grove, PA) and visualized using West Pico Chemiluminescent kit (Pierce Chemical, Rockford, IL).
Flow Cytometry
For pS10 histone H3 analysis, cells were collected by centrifugation, fixed in 2% formaldehyde in PHEM [60 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 25 mM HEPES, pH 6.8, 10 mM EDTA, and 4 mM MgCl2] for 15 min and permeabilized with 90%, –20°C methanol. Later cells were washed three times with phosphate-buffered saline (PBS), pH 7.2, blocked with 5% BSA in PBS, and labeled with anti-pS10 histone H3 antibody conjugated to Alexa Fluor 647 (Cell Signaling Technology). For cell cycle analysis, cells were fixed in 90% ethanol for at least 24 h and stained with 50 µg/ml propidium iodide in PBS in the presence of 100 µg/ml RNAse A (Sigma-Aldrich). Analysis was carried out on a FACSCalibur flow cytometer (BD Biosciences).
Live Imaging
Cells were grown on 25-mm glass coverslips that were inserted in an Attofluor culture chamber (Invitrogen) before the experiment. When applicable, cells were preincubated for 30–60 min with MG132. Xenopus S3 cells were imaged at room temperature in their normal growth medium. HeLa cells were imaged in L-15 medium with 10% FBS at 37°C. Temperature was maintained with an air curtain incubator (Nevtek, Burnsville, VA) and an objective heater (Bioptechs, Butler, PA). Time-lapse phase contrast and fluorescent images were collected using Axiovert 200M microscopes (Carl Zeiss, Thornwood, NY), either wide-field fluorescent or high-speed Yokogawa spinning disk confocal. Both microscopes were equipped with ORCA-ERG digital cameras (Hamamatsu, Bridgewater, NJ). PlanApochromat oil immersion objectives (63x) were used for all live imaging. Drugs were substituted by exchange of the media after a brief rinse. Time-lapse videos were assembled using MetaMorph software (Molecular Devices, Sunnyvale, CA).
Myt1 Kinase Assays
HeLa cells were blocked in mitosis with nocodazole for 2–3 h, collected by shake-off, washed in nocodazole-free medium, and incubated without nocodazole for 1, 2, 3, and 4 h. Interphase cells were obtained by trypsinizing adherent cells left after shake-off. Equal numbers of cells (typically between 1.6 and 2 x 106) were collected by centrifugation and flash-frozen in liquid nitrogen. For the assay, cell pellets were thawed in 250–300 µl of kinase lysis buffer (50 mM Tris, 50 mM NaCl, 5 mM MgCl2, 1% Triton X-100, 1 mM DTT supplemented with protease inhibitor cocktail, 1 mM NaVO4, and 400 nM Microcystin LR) and cleared by centrifugation. A portion (
5%) of each lysate was spared for Myt1 immunoblotting.
Recombinant 6His-Cdk1(K33R)-
90cyclin B1, a generous gift from P. Todd Stukenberg (University of Virginia, Charlottesville, VA), was conjugated to nickel-nitrilotriacetic acid magnetic beads (QIAGEN, Valencia, CA) and incubated in kinase lysates supplemented with 2 mM ATP for 30 min at 37°C. Beads were subsequently washed, and the bound proteins were released by boiling in NuPAGE protein sample buffer (Invitrogen) for 10 min. Protein samples were run on SDS-PAGE, transferred to PVDF, blocked in 5% BSA, and probed with anti-pT14Cdk1 antibody (Abcam). The membrane was subsequently stained with Coomassie Blue to control for protein loading.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Cyclin B was preserved by treatment with the proteasome inhibitor MG132 or by expressing a nondegradable mutant of the cyclin B protein. Preserving cyclin B1 causes cells to arrest in mitosis with high Cdk1 activity levels. This mitotic arrest could be overcome by treatments with Cdk inhibitors, such as flavopiridol (Losiewicz et al., 1994), which induces cytokinesis and full mitotic exit in the absence of cyclin proteolysis. This ability to induce mitotic exit by application of chemical Cdk inhibitors to cells arrested in M phase in the presence of proteasome inhibitor was recently disputed (Skoufias et al., 2007). However, in our hands, Cdk inhibitors applied to mitotic cells drives complete mitotic exit in both the absence or presence of proteasome inhibitor by all morphological criteria including cytokinesis, midbody formation, chromosome decondensation, reassembly of the nuclear envelope, and rearrangement of the mitotic spindle microtubules into the interphase array (Potapova et al., 2006; Supplemental Videos 3 and 4). As we reported, chromosome segregation fails in the presence of proteasome inhibitor, presumably due to the necessity for degradation of the securin protein. In cells arrested in M phase with microtubule drugs, chemical Cdk1 inhibition induces loss of mitotic phosphorylations again in both the absence and presence of proteasome inhibitors (see Supplemental Figure 1). In our previous work, we found that mitotic exit in the presence of proteasome inhibitors could be driven by several different Cdk inhibitors in addition to flavopiridol (Potapova et al., 2006).
Flavopiridol is a reversible Cdk inhibitor. Thus, when flavopiridol was removed after the induced mitotic exit, the two daughter cells could reverse their cell cycle progression and reenter mitosis. However, the longer cells were incubated with flavopiridol, the less likely they were to reenter mitosis. Thus, the ability of cells to reverse mitotic exit declined with time spent in G1, even though cyclin B was preserved. To understand the loss of reversibility in later G1, we carried out live imaging studies with human HeLa cells and with an immortalized Xenopus cell line S3, which expresses GFP-
tubulin. Cyclin B was preserved by addition of the proteasome inhibitor MG132 to the medium, or by expressing a nondegradable mutant CyclinB1 (R42A) tagged with GFP. Flavopiridol was added to the medium to induce mitotic exit and subsequently washed out. When flavopiridol was removed 30 min after its addition, most cells reentered mitosis (Figure 1A). However, after treatment with flavopiridol for 60 min or more, only a small percentage of cells were capable of reversing mitotic exit.
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), by synthesis of small protein kinase inhibitors (Sherr and Roberts, 2004), or by phosphorylation on inhibitory sites T14 and Y15 (Atherton-Fessler et al., 1994). The loss of reversibility accompanying prolonged flavopiridol treatment was not affected by protein synthesis inhibitors in Xenopus S3 cells (data not shown), which suggests the de novo synthesis of small protein kinase inhibitors was not involved in the late G1 block. Dephosphorylation of Cdk1 on the activating site T161 did not occur when cyclin was preserved, indicating that the lack of reversibility was not due to loss of the activating T161 phosphorylation (Figure 1B, lanes 5–8).
We next tested the status of Cdk phosphorylation on its inhibitory sites, T14 and Y15, during chemically induced mitotic exit and reversal. Cells were accumulated in mitosis by treatment with nocodazole for 3 h. Mitotic cells were collected by shake-off and treated with flavopiridol with or without MG132 for 30 or 60 min. Flavopiridol was subsequently washed out, and cells were allowed to reenter mitosis for another 60 min. Cdk1 inhibitory phosphorylation was examined by Western blotting using phospho-specific Cdk1 T14 and Y15 antibodies. Cdk1-specific phosphorylation of nucleolin (Dranovsky et al., 2001) served as a readout for Cdk1 activity. Without the proteasome inhibitor, addition of flavopiridol caused degradation of cyclin B (Figure 1B, lanes 1–4). As expected, removal of flavopiridol did not result in reactivation of Cdk1 when cyclin B was not preserved (Figure 1B, lanes 2 and 4). Consistent with the reports that Cdk1 phosphorylation on the inhibitory residues occurs solely when it is bound to cyclin (Solomon et al., 1990), Cdk1 was not phosphorylated on T14 and Y15 when cyclin B was degraded. When cyclin B was preserved by the proteasome inhibitor, flavopiridol treatment resulted in only weak phosphorylation of the inhibitory sites T14 and Y15 at 30 min but strong phosphorylation at 60 min (Figure 1B, lanes 7 and 8). Thus, Cdk1 activity (phospho-nucleolin) returned to high levels in cells reversed after 30 min (Figure 1B, lane 6) but increased only modestly in cells reversed after 60 min (Figure 1B, lane 8).
The activities of Wee1 and Myt1 kinases as well as Cdc25 phosphatase are reflected in their electrophoretic mobilities caused by phosphorylation changes. Wee1 and Myt1 are hyperphosphorylated and inactive in mitosis (Mueller et al., 1995; Watanabe et al., 2005), whereas Cdc25C, is hyperphosphorylated and active (Hoffmann et al., 1993). Chemical inhibition of Cdk1 resulted in increased mobility of all of three, indicating loss of phosphorylation and consequently activation of the Wee1/Myt1 kinases and inactivation of Cdc25C phosphatase. The changes in mobility increased progressively from 30 to 60 min of treatment with flavopiridol, particularly in the presence of MG132 (Figure 1B, lanes 5 and 7). This suggests that the kinases become progressively dephosphorylated and active as cells exit mitosis, whereas Cdc25 becomes inactive, providing a window of time for Cdk1 reactivation when cyclin B is preserved.
We conducted the same type of analysis in HeLa cells expressing the wild-type or the nondegradable (R42A) cyclin B1 (Figure 1D). For this experiment, cells were transfected with indicated constructs for 24 h; mitotic cells were collected in nocodazole and treated with Cdk inhibitor for 30 or 60 min. Cells expressing the nondegradable (R42A) cyclin B1 lose the ability to rephosphorylate nucleolin and histone H3 with time in G1 similarly to MG132-treated cells (Figure 1B, lanes 7 vs. 9). The Cdk1 phosphorylation on inhibitory T14 and Y15 also increased (Figure 1B, lanes 8 and 9). The wild-type cyclin B was degraded upon Cdk inhibition, and the cells that expressed it did not reenter mitosis after flavopiridol washout (lanes 2 and 4) in spite of similar initial cyclin B1 levels. The differences in phosphorylation shifts of Wee1, Myt1, and Cdc25 at 30 versus 60 min of flavopiridol treatment were less sharp than in the MG132 experiment in Figure 1B. This was likely because not all of the nocodazole-blocked mitotic cells expressed the nondegradable cyclin—the typical transfection efficiency was 70%, and also because expression levels vary among the transfected cells. To get a higher proportion of cells expressing the nondegradable cyclin B for the subsequent experiments, the R42A cyclin B-expressing mitotic cells were collected by shake-off without nocodazole, because they arrest in mitosis spontaneously.
To assess whether the Cdk1 reactivation indicated by phospho-nucleolin labeling correlated with other markers of cells reentering mitosis, cells from each treatment in Figure 1, B and D, were fixed and stained with antibody against the mitotic marker phospho-S10 on histone H3. The cells were analyzed by flow cytometry to determine mitotic indices (Figure 1, C and E). The samples with high Cdk1 activity as delineated by strong blotting signals to phospho-nucleolin correlated with high mitotic indices in the fixed cells. The decreased ability to reactivate Cdk1 after 60 min of flavopiridol treatment was consistent with the loss of reversibility observed by live imaging. Together, these data indicate that despite presence of cyclin B, cells lose the ability to reenter mitosis with longer time in G1, and this loss correlates with phosphorylation of Cdk1 inhibitory sites T14 and Y15.
Chemical Inhibition of Wee1 and Myt1 Kinases Allows Cdk1 Reactivation and Mitotic Reentry from Late G1
To address the functional significance of Cdk inhibitory phosphorylations in G1, we inhibited the Wee1 and Myt1 kinases with a potent and specific Wee1 and Myt1 inhibitor PD0166285 (Wang et al., 2001; Hashimoto et al., 2003; Hashimoto et al., 2006). Judged against mitotic exit of an untreated cell (Supplemental Video 1), PD0166285 had no obvious effect on normal mitotic exit, although it seemed to inhibit complete extension of the cortex in telophase (Supplemental Video 2). We used live videomicroscopy to study the consequences of Wee1 and Myt1 inhibition on the reversibility of mitotic exit by adding this inhibitor during flavopiridol-induced exit. Cells were arrested at metaphase by treatment with the proteasome inhibitor MG132. Flavopiridol and PD0166285 were added for 60 min. Then, the flavopiridol was removed, and the cells were maintained in PD0166285. Remarkably, under these conditions, 100% of the MG132-treated cells reentered mitosis (Figure 2A). A time-lapse sequence of mitotic exit and reversal of a Xenopus S3 cell after 60 min of Cdk inhibition in the presence of PD0166285 is shown in Figure 2B and Supplemental Video 3. In contrast, in the absence of PD0166285, only a small percentage of MG132-treated Xenopus S3 cells and HeLa cells were able to reenter mitosis after 60 min of treatment with flavopiridol (Figure 2A). Shown in Figure 2B and Supplemental Video 4 is a typical example of Xenopus S3 cell that remained in G1 after 60 min of flavopiridol treatment in the absence of PD0166285.
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). In the three cells that failed to reverse in these experiments the expression of the nondegradable cyclin was relatively low (data not shown). In the absence of PD0166285, very few HeLa cells expressing R42A cyclin B1 were able to reenter mitosis after 60 min of Cdk inhibition (Figure 2A). An example of R42A cyclin B1-GFP–expressing cell in the absence of PD0166285 that did not reverse from G1 after 60 min of flavopiridol treatment is shown in Figure 2E and Supplemental Video 6. The persistence of the GFP fluorescence in the cell demonstrates that the mutant cyclin B1 protein is stable. However, the cell remained in G1 after flavopiridol washout, and eventually the R42A cyclin B1 was transported out of the newly formed nuclei.
To ensure that the reversibility seen in the presence of the Wee1/Myt1 inhibitor PD0166285 occurs only when cyclin B is preserved, we also expressed wild-type cyclin B1-GFP in HeLa cells. Proteolysis of wild-type cyclin B1 is initiated before anaphase onset and continues during anaphase and mitotic exit (Clute and Pines, 1999). For this experiment, cells were treated with flavopiridol before initiation of spontaneous degradation. The fluorescent intensity of the cyclin B1-GFP dropped rapidly upon Cdk1 inhibition, indicating degradation of the cyclin. None of the 13 observed cells cultured in the presence of PD0166285 and expressing wild-type cyclin B1-GFP were able to reenter mitosis after 60 min of flavopiridol treatment. A representative cell expressing wild-type cyclin B1-GFP blocked in G1 after 60 min of flavopiridol treatment in the presence of the PD0166285 is shown in Supplemental Video 7.
We analyzed directly the effect of Wee1/Myt1 inhibition by blotting Cdk1 inhibitory phosphorylations in extracts of mitotic HeLa cells treated with flavopiridol for 60 min. Cyclin B was preserved with MG132 (Figure 3A) or by expression of the nondegradable mutant (Figure 3B). PD0166285 prevented Cdk1 phosphorylation on T14 and Y15 and allowed Cdk1 to regain activity when Cdk inhibitor was removed (Figure 3, A and B, lanes 4). In contrast to the results obtained with PD0166285, the inhibition of another class of mitotic kinases, the Aurora kinases, with the drug ZM447439 did not affect reversibility of mitotic exit when tested with Xenopus S3 cells (data not shown).
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). We asked whether Cdc25 phosphatases were active during mitotic exit reversal. HeLa cells treated with MG132 were induced to exit mitosis for 60 min in flavopiridol. Flavopiridol treatment induced high levels of phosphorylated T14 and Y15 on Cdk1 (Figure 3C, lane 1). These levels remained high when flavopiridol was removed (Figure 3C, lane 2). When PD0166285 was added to inhibit Wee1 and Myt1 kinases at the time of flavopiridol removal the Cdk1 inhibitory phosphorylations diminished and Cdk1 activity returned, indicating that Cdc25 phosphatases were active (Figure 3C, lane 3). To test whether Cdk1 reactivation was due to the activity of Cdc25 phosphatases, we added a specific Cdc25 inhibitor NSC663284 (Pu et al., 2002) together with PD0166285 after the flavopiridol washout (Figure 3C, lane 4). When Cdc25 activity was inhibited, Cdk1 remained highly phosphorylated on T14 and Y15, and Cdk1 kinase activity was not fully restored. Thus, at least for 1 h after the induced mitotic exit, Cdc25 phosphatase(s) have the potential to dephosphorylate and activate Cdk1, but eventually the Wee1/Myt1 kinases outbalance this potential.
Mutation of Inhibitory Phosphorylation Sites on Cdk1 Allows Cdk1 Reactivation and Mitotic Reentry from Late G1
To confirm the results obtained with the chemical Wee1/Myt1 inhibitor PD0166285, we ectopically expressed the phosphorylation site mutants of human Cdk1 (T14A, Y15F, and the double mutant T14A/Y15F) tagged with GFP in HeLa cells. All populations showed similar transfection efficiencies. Cells expressing each of the phosphorylation mutants, but not the wild-type Cdk1, were able to reactivate Cdk1 to differing degrees after 1 h of flavopiridol treatment in the presence of MG132 (Figure 4A, lanes 6, 9, and 12). The Y15F mutant and the double mutant showed stronger recovery of Cdk1 activity than did the T14A mutant. This is consistent with a report that Y15, but not T14, is essential to prevent mitotic entry after the radiation damage (Fletcher et al., 2002). Live imaging of HeLa cells expressing Cdk1 mutants further supported the idea that inhibitory phosphorylation blocks cells in G1 when cyclin B is preserved. Time-lapse sequences of mitotic exit and reversal after 60 min of Cdk inhibition in HeLa cell expressing Cdk1-Y15F in the presence of MG132 are shown in Figure 4C and Supplemental Video 8. Cells expressing Cdk1 mutants reentered mitosis more readily after 1 h of Cdk inhibition than cells expressing wild-type Cdk1 (Figure 4B). Interestingly, a few of the reversed cells expressing the single T14A or Y15F Cdk1 mutants exhibited a prophase-like morphology with condensed chromosomes surrounded by an intact nuclear envelope, perhaps indicating that such cells had intermediate levels of Cdk1 activity. Notably, many of the mitotic cells expressing the double mutant demonstrated aberrant metaphase morphology and reduced viability, consistent with reports that the expression T14A/Y15F Cdk1 causes cells to enter mitosis prematurely and aberrantly (Krek and Nigg, 1991). Overall, the data obtained with Cdk1 mutants were consistent with our results using the Wee1/Myt1 inhibitor PD0166285 and highlighted a more potent contribution of Y15 in comparison with T14 in regulation of Cdk1 activity. Together, these results support the hypothesis that blocking Wee1 and Myt1 kinase action on Cdk1 extends the period of reversibility into G1 when cyclin B is preserved.
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Because cell death was not observed in cells treated with proteasome inhibitor, we reasoned that in cells expressing nondegradable cyclin B and forced into G1, cell death during reversal might result from reactivation of Cdk1 in the absence of other mitotic proteins that are normally degraded by late G1. To test this hypothesis, we added the proteasome inhibitor MG132 with the flavopiridol to mitotic cells expressing nondegradable cyclin B1. Incubation with MG132 showed substantial rescue of cells during reversal after 120 min of flavopiridol treatment (Figure 5D). One mitotic protein degraded during G1 is polo-like kinase 1 (Plk1) (Lindon and Pines, 2004). Plk1 was substantially degraded upon treatment of cells with flavopiridol for 120 min in the absence of the proteasome inhibitor MG132 (compare lanes 0 and 1, Figure 5C). The presence of MG132 preserved Plk1 and restored the levels of phosphorylated nucleolin after flavopiridol washout (Figure 5C, lane 5).
To determine whether the loss of viability involved the action of caspases, the general caspase inhibitor Z-VAD-FMK was added to the cells at the time of flavopiridol washout. The caspase inhibitor restored phospho-nucleolin in the reversing cells to levels similar to those of cells incubated with MG132 (Figure 5C, lane 6). Plk1 levels were low in these cells compared with the MG132-treated cells, indicating that the proteasome-dependent protein degradation of Plk1 during G1 was not inhibited. By live imaging, caspase inhibitor also restored the viability of the reversing cells to 90% (Figure 5D). A time-lapse sequence of mitotic exit for 120 min and reentry in the presence of Z-VAD-FMK is shown in Figure 5E and Supplemental Video 12. Together, these results indicate that the Cdk activation in late G1 may lead to cell death in a caspase-dependent manner. Many essential mitotic regulators, such as Plk1, are substrates for the anaphase-promoting complex and are degraded in late G1. The cell death that we observed may stem from a lack of one or more of the proteins that are essential for viability of mitotic cells when Cdk1 becomes reactivated in late G1. This mechanism of cell death may be relevant to the aberrant Cdk1 activation and apoptosis occurring in quiescent neurons, a feature of certain neurodegenerative diseases such as Alzheimer's disease (Vincent et al., 1997). Our ability to regulate Cdk1 reactivation in G1 cells may provide a model to study the signaling pathways of unscheduled Cdk1 activity under controlled conditions.
Myt1 and Wee1 Activation in Normal G1
Although cyclin degradation plays a key role in inducing exit from mitosis, we have shown that the inhibitory phosphorylations of Cdk1 can lock the kinase in the low activity state in G1. Therefore, the inhibitory kinases Wee1 and Myt1 must be reactivated in G1 after the Cdk1 activity drops. However, cyclin B1 in our experiments was preserved, providing a detectable substrate for their activity. We next asked the question whether the Wee1 and Myt1 were active during the normal physiological G1. For these experiments, cells were synchronized in G1 by release of mitotic cells from the nocodazole block. Under these conditions, most cells are in G1 by 4 h after nocodazole washout as demonstrated by DNA content (Figure 6A). As cells were exiting mitosis, Wee1, Myt1, and Cdc25C underwent electrophoretic mobility shifts corresponding to the active forms of Wee1 and Myt1 and inactive form of Cdc25C (Figure 6B), suggesting that these changes in enzyme activities are normal aspects of mitotic exit. The presence of the Wee1/Myt1 inhibitor did not severely affect these processes, but there were some detectable differences. For example, the antibodies against phospho-Cdk1 T14 and phospho-Cdk1 Y15 show some weak immunoreactivity 3–4 h after nocodazole release, a the time when cyclin B levels are very low (Figure 6B). Importantly, phospho-T14 and phospho-Y15 bands were absent in the Wee1/Myt1 inhibitor-treated cells. We next directly assayed the Myt1 activity of G1 cells in vitro. Technical difficulties precluded us from directly assaying the activity of Wee1. The activity of Myt1 toward a specific substrate was tested in lysates obtained from cells undergoing the M-to-G1 transition. For the substrate, we used a recombinant His-tagged kinase-dead (K33R) Cdk1 protein complexed with nondegradable 90 cyclin B. The substrate was incubated in lysates in the presence of ATP and analyzed for phosphorylation on T14 by using the phospho-specific antibody. The substrate phosphorylation on T14 begun in lysates of cells released from mitotic nocodazole block for 2 to 3 h (Figure 6C). By 4 h, when most of the cells have exited mitosis (Figure 6A), the activity of Myt1 was nearly equal the level detected in interphase lysates from cycling cells. The specific inhibitor PD0166285 blocked the activity of Myt1 nearly as well as the lack of ATP. The activation of the kinase corresponded with its electrophoretic mobility shift in lysates (Figure 6C, bottom). Together, these results suggest that the Myt1 kinase, and likely the Wee1 kinase, is activated shortly after mitotic exit. Specific in vivo targets for these kinases in G1 are yet to be established and are currently under investigation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, previously there was no evidence that the kinases that catalyze these phosphorylations had the capability to negatively regulate Cdk1 activity in G1. Here, we suggest that the reactivation of this pathway is initiated by the drop in Cdk1 activity levels and works to progressively activate the Wee1 and Myt1 kinases and inhibit the Cdc25 phosphatases. At early times, 30 min after induction of mitotic exit with the reversible Cdk inhibitor flavopiridol, cells could be driven back into mitosis simply by removing the flavopiridol. After that, the Wee1 and Myt1 activities begin to outbalance the Cdc25 activity and flavopiridol removal fails to reactivate Cdk1 even when cyclin B is preserved. By inhibiting the Wee1 or Myt1 kinases, or by expressing mutant Cdk1 protein that was not susceptible to inhibition by phosphorylation, we could greatly extend the time of reversibility.
Although the steps in the activation and inactivation of Cdk1 during the transition into and out of M phase have been the subject of considerable investigation, much less is understood about potential alternative controls that may function in later G1. Our evidence suggests that inhibitory phosphorylation kinases Wee1 and Myt1 are active during this time. The lack of known substrates for them in G1 does not mean the absence of substrates per se. We speculate that these kinases may participate in less explored processes involving Cdk inhibition in by phosphorylation. For example, there exists a poorly explored family of RINGO/Speedy proteins some of which are noncyclin Cdk1 activators. These proteins are expressed in G1 and can bind and activate Cdk1 and Cdk2 (Cheng et al., 2005). Importantly, at least the Myt1 kinase is able to phosphorylate inhibitory sites leading to inhibition of Cdk1 when Cdk1 is bound to RINGO/Speedy protein (Karaiskou et al., 2001). In mice, the knockout data for Cdks and cyclins reveal that Cdk1 can in principle bind many cyclins in vivo, including the G1 cyclins (see review by Sherr and Roberts, 2004). Moreover, mouse embryos expressing only a single cyclin dependent kinase, Cdk1, develop to midgestation and cells from these embryos can undergo continuous growth where Cdk1 forms functional complexes with a G1, S and G2-M cyclins (Santamaria et al., 2007). Therefore, the back-up mechanism must exist to dampen the activation of Cdk1 molecules that happened to find themselves an activating partner in G1. Last, virally encoded cyclins, provide another situation where cell cycle control may be co-opted by noncanonical pathways (see review by Nebreda, 2006). The activity of Wee1 and Myt1 kinases in G1 may serve other physiological purposes than simply reinforcing the G1. For example, a recent siRNA study indicated that Myt1 kinase has a specific role in G1, stimulating proper postmitotic reassembly of the ER and Golgi networks (Nakajima et al., 2008). Using the inhibitor PD0166285, we observed similar effects on Golgi reassembly (data not shown).
As we have shown here, reactivation of Cdk1 activity in late G1 leads to cell death. The relevant pathological condition may be illustrated by the apoptosis of postmitotic neurons in neurodegenerative diseases such as Alzheimer's disease. Terminally differentiated neurons permanently exit the cell cycle and stay at resting (G1/G0) phase (reviewed by Yoshikawa, 2000), but in Alzheimer's disease, they display aberrant Cdk1 activity (Vincent et al., 1997). Cdk1 activation has been shown to alter gene expression and precede the formation of the neurofibrillary tangles and apoptosis in neurons (Pei et al., 2002; reviewed by Becker and Bonni, 2004). Thus, it is not surprising that backup mechanisms to restrain Cdk1 activity during the G1 stage of the cell cycle have evolved.
In summary, although cyclin proteolysis is the dominant control for down-regulation of Cdk1 activity during mitotic exit, additional backup mechanisms may contribute to its fine-tuning and to the maintenance of the G1 state. To fully comprehend cell cycle regulation at the systems level, it is critical that all latent networks be identified and their potential roles be investigated. Elucidation of these accessory controls may also contribute to pathologies that involve aberrations in cell cycle control mechanisms, for example in hyperproliferative diseases such as cancer and in neurodegenerative diseases such as Alzheimer's.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Gary J. Gorbsky (gary-gorbsky{at}omrf.ouhsc.edu)
Abbreviations used: Cdk1, cyclin-dependent kinase 1; GFP, green fluorescent protein.
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REFERENCES |
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