Mitosis persists in the absence of Cdk1 activity when proteolysis or protein phosphatase activity is suppressed

Abstract

Cellular transition to anaphase and mitotic exit has been linked to the loss of cyclin-dependent kinase 1 (Cdk1) kinase activity as a result of anaphase-promoting complex/cyclosome (APC/C)-dependent specific degradation of its cyclin B1 subunit. Cdk1 inhibition by roscovitine is known to induce premature mitotic exit, whereas inhibition of the APC/C-dependent degradation of cyclin B1 by MG132 induces mitotic arrest. In this study, we find that combining both drugs causes prolonged mitotic arrest in the absence of Cdk1 activity. Different Cdk1 and proteasome inhibitors produce similar results, indicating that the effect is not drug specific. We verify mitotic status by the retention of mitosis-specific markers and Cdk1 phosphorylation substrates, although cells can undergo late mitotic furrowing while still in mitosis. Overall, we conclude that continuous Cdk1 activity is not essential to maintain the mitotic state and that phosphatase activity directed at Cdk1 substrates is largely quiescent during mitosis. Furthermore, the degradation of a protein other than cyclin B1 is essential to activate a phosphatase that, in turn, enables mitotic exit.

Figure 1.

Loss of Cdk1 kinase activity in the absence of proteasome activity does not lead to mitotic exit. (A) Mitotic HeLa cells were collected by selective detachment after being blocked in mitosis with 7.5 μM STLC (left) or with 0.1 μg/ml nocodazole (right) for 16 h. Cells in the continuous presence of the mitotic inhibitors were exposed to 100 μM roscovitine (ROS) or 20 μM MG132 or to both roscovitine and MG132, and samples for 2D FACScan analysis were taken 2 h after drug addition. In contrast to roscovitine treatment, cells in the presence of MG132 retained their mitotic state, as indicated by MPM2 signal. Similarly, the majority of cells coincubated with roscovitine and MG132 remained mitotic. The percentages shown indicate the cell subpopulation that was positive for MPM-2. (B) Quantitation of the percentage of cells positive for MPM-2 calculated relative to the mitotic value of cells at time 0, which was defined as the time of shake-off and addition of the various drug combinations. (C) Immunoblot analysis of various mitotic markers assayed at 2 h after shake-off of mitotic cells and addition of various drug combinations. Mitotic exit induced by roscovitine leads to the degradation of Aurora A, cyclin B1, and securin as well as loss of the mitosis-specific phosphorylation of residue S10 of H3 ((PS10)H3). In contrast, the addition of MG132, even in the presence of roscovitine, leads to stabilization of all of the proteins tested as well as retention of the phosphorylated status of H3. Release in medium alone (DME) served as a mitotic exit control.

Figure 2.

Absence of mitotic exit after loss of Cdk1 kinase activity is independent of specific drugs. (A) Mitotic HeLa cells were collected by selective detachment after being blocked in mitosis with 0.1 μg/ml nocodazole for 16 h. Cells in the continuous presence of nocodazole were exposed to 20 μM MG132 (MG) for 1 h before the addition of Cdk inhibitors roscovitine (ROS; 100 μM) or CGP74514A (CGP; 7.5 μM), or cells were exposed to 25 μM purvalanol A (PURVA) for 2 h in the continuous presence of MG132. Samples for 2D FACScan analysis were taken after drug addition to determine DNA and MPM-2 content (as in Fig. 1). The percentage of 4N cells positive for MPM-2 was quantitated from FACScan and plotted relative to the mitotic value of cells at time 0, which was defined as the time of shake-off and addition of the various drug combinations. In contrast to Cdk inhibitor treatments, cells in the presence of MG132 retained their mitotic state, as indicated by MPM2 signal. Similarly, the majority of cells coincubated with Cdk inhibitors and MG132 remained mitotic. Error bars represent SD. (B) Cells were treated and analyzed as in A, but MG132 was substituted with 20 μM of the proteasome inhibitor AM114. Quantitation of the percentage of 4N cells positive for MPM-2 was calculated as in A. (C) Immunoblot analysis of various mitotic markers assayed at 2 h after shake-off of mitotic cells and addition of various drug combinations. Mitotic exit induced by Cdk inhibitors leads to loss of the mitosis-specific phosphorylation of Cdk substrates as well as the following mitotic markers: residue S10 of H3 ((PS10)H3) and residue T244 of cdc27. In contrast, the addition of MG132, even in the presence of Cdk inhibitors, leads to retention of the phosphorylated status of the proteins. Similar results were obtained for purvalanol A in place of roscovitine or CGP74514A and AM114 in place of MG132 (not depicted). (D) Chromosome spreads of representative cells in CGP74514A + MG132 or CGP74514A + AM114 show mitotic status. Results compared with MG132 or AM114 alone. DNA stain is propidium iodide. Bar, 13 μm.

Figure 3.

Loss of Cdk1 activity in the absence of proteasome activity does not lead to mitotic exit. (A) After STLC treatment and mitotic shake-off, cells were treated with roscovitine in the absence or presence of MG132 for 2 h. Fixed cells were then stained for immunofluorescence microscopy with antitubulin, phosphorylated histone H3 ((PS10)H3), MPM-2, or anti–lamin B antibodies (green) and were counterstained with propidium iodide (PI; red). Roscovitine induced rapid mitotic exit, as evidenced by loss of the monoastral spindles, (PS10)H3, MPM-2 staining, and the assembly of nuclear lamina surrounding decondensed chromatin. However, cells treated with STLC plus roscovitine and MG132 retained monoastral spindles, (PS10)H3, and MPM-2 staining, whereas nuclear lamina were absent. These results were parallel to control cells blocked in mitosis with STLC. Microscope settings were held constant for all image acquisitions. (B and C) The percentage of cells with monoastral spindles (B) or positive for (PS10)H3 (C) were quantitated. The data represent the mean of three counts of >60 cells per count. Gray bar, cells at time 0, the time of mitotic cell selection by shake-off; white bars, cells treated with roscovitine alone; black bars, cells treated with roscovitine plus MG132. In all cases, cells were in the continuous presence of STLC. Error bars represent SD. Bar, 16.5 μm.

Figure 4.

Cdk1 inhibition by purvalanol A and roscovitine but not MG132. Immunoprecipitates pulled down with Cdk1 antibody from nocodazole-arrested HeLa cell lysates were used for in vitro kinase assays. (top) 25 μM purvalanol A or 100 μM roscovitine or the combination of purvalanol A or roscovitine with MG132 was added to the kinase assay cocktail and incubated with the immunoprecipitates for 30 min at 30°C in the presence of [³²P]ATP. (bottom) Immunoblot of Cdk1 immunoprecipitates (∼10% input) cross-blotted using anti-Cdk1 antibody to demonstrate equal lane loading.

Figure 5.

Degradation of cyclin B1 and securin is not necessary for mitotic exit in the absence of Cdk1 activity. (A and B) HeLa cells were transiently transfected with cDNA coding nondegradable cyclin B1 (R4A2GFP) alone (A) or both cDNA coding nondegradable cyclin B1 (R4A2GFP) and nondegradable securin (KEN-box mutant myc-securin-KAA-DM; B). At 30 h after transfection, cells were synchronized in mitosis by 16-h exposure to nocodazole. After shake-off, mitotic cells were maintained in the continuous presence of nocodazole with either roscovitine (ROS), MG132, or both roscovitine + MG132. (A) Immunofluorescence shows that nondegradable cyclin B is not sufficient to maintain mitotic status in the presence of roscovitine because GFP-positive cells are negative for MPM-2 staining. Both GFP-positive and -negative cells are MPM-2 positive after mitotic shake-off in MG132 and in roscovitine + MG132. (B) Immunoblot analysis of cellular extracts from double-transfected cells shows that in the presence of roscovitine, endogenous cyclin B1 and securin were degraded, whereas the ectopically expressed nondegradable proteins were not. Despite the continued presence of cyclin B or securin, exposure to roscovitine induced mitotic exit followed by the loss of (PS10)H3, (PT244)cdc27, and Cdk phosphoprotein substrates. In contrast, in cells treated with MG132 alone or with MG132 plus roscovitine, there was no loss of these markers, confirming the continued mitotic status of these cells. Bar, 44.5 μm.

Figure 6.

Cells with monoastral spindles cleave without mitotic exit after loss of Cdk1 activity in the presence of proteasome inhibitor. (A and B) HeLa cells collected in mitosis by 16-h exposure to STLC followed by mitotic shake-off were maintained in STLC and treated with roscovitine in the presence or absence of MG132 for 2 h. Cells were then fixed and stained for microscopy with antibodies to tubulin (A) or to the cleavage furrow–associated proteins survivin, Aurora B, TD60, PRC1, or anillin (B). Exposure of STLC-treated cells to roscovitine induced a furrowing event, characteristically with formation of a bud devoid of chromatin. The passenger proteins survivin, Aurora B, and TD60 associated with the furrow, as did PRC1 and anillin. (C) The percentage of cells with a bud was quantitated after 1 or 2 h of exposure to drugs. The data represent the mean of three counts of >60 cells per count. White bars, cells treated with roscovitine; black bars, cells treated with both roscovitine and MG132. All cells were in the continuous presence of STLC. Error bars represent SD. Bar, 16.5 μm.

Figure 7.

The absence of Cdk1 activity does not prevent bipolar spindle formation and metaphase chromosome alignment on release from STLC. (A) After 16-h STLC block and mitotic shake-off, cells were released from STLC by three washes in drug-free medium and were treated with MG132 for 2 h followed by 2-h coincubation with both roscovitine and MG132. Samples were analyzed by 2D FACScan as in Fig 1. Controls were released from STLC for 4 h with no further drug treatment. Percentages shown indicate the subpopulations that were positive for MPM-2. (B) Confocal microscopy was performed on identically treated cells, which were stained for tubulin (green) and DNA with propidium iodide (PI; red). The combined FACScan and microscopy data indicate that controls released from STLC form normal bipolar spindles and exit normally from mitosis. In contrast, cells released from STLC in MG132 remain mitotic but within 2 h have normal mitotic spindles with chromosomes aligned to a normal metaphase plate. After an additional 2 h without Cdk1 activity (MG132 plus roscovitine), cells remained mitotic with normal bipolar spindles and metaphase chromosome alignment. Bar, 16.5 μm.

Figure 8.

Phosphatase activity is required for mitotic exit in the absence of Cdk1 activity. (A) Western blot analysis of mitotic markers in extracts. Cells were collected in mitosis by STLC block (16 h) and shake-off and were exposed to different conditions for 2 h as indicated. All cells were maintained in STLC during this time course. Cells were exposed to 100 μM roscovitine (ROS), 20 μM MG132, both roscovitine and MG132, both roscovitine and 0.5 μM okadaic acid (OKAD), or to both roscovitine and 30 nM calyculin A (CALYC). Controls were cells in the continuous presence of STLC (2 h) or STLC + MG132 (MG132). S/O indicates the time 0 shake-off sample. Cell extracts were probed with a polyclonal antibody specific for Cdk substrates phosphorylated on serine (anti P-Ser Cdk substrates), with antibody specific for histone H3 phosphorylated on Ser10 ((PS10)H3), and were also probed with securin, cyclin B1, and aurora A antibodies. Anti-actin was used as loading control. (B) FACScan analysis of mitotic HeLa cells collected by shake-off after being blocked in mitosis with 7.5 μM STLC for 16 h. Cells in the continuous presence of STLC were exposed to 100 μM roscovitine (ROS), 20 μM MG132, both roscovitine and MG132, both roscovitine and 0.5 μM okadaic acid (OKAD), or both roscovitine and 30 nM calyculin A (CALYC). Samples for 2D FACScan analysis were taken 2 h after the collection of mitotic cells by shake-off. Percentages shown indicate the subpopulations that were positive for MPM-2. Parallel results were obtained with nocodazole-treated cells. (C) Comparison of results with nocodazole or with STLC block. Quantitation of the percentage of cells positive for MPM2 includes results as shown in B plus a parallel set of data from nocodazole-arrested cells that were otherwise treated identically. Data demonstrate that parallel results were obtained with nocodazole or STLC-treated cells. (D) Chromosome spreads 2 h after collection by arrest with nocodazole followed by mitotic shake-off as described in A. Cells were maintained in nocodazole and exposed to various conditions after shake-off as indicated. Bar, 19.36 μm.