Maintenance of the mitotic state depends on Cdk1 activity. Loss of Cdk1 activity normally occurs at the metaphase to anaphase transition once the spindle assembly checkpoint and other mitotic checkpoints have been satisfied (Kops et al., 2005
). Mitotic exit normally occurs through the cdc20-activated APC/C-dependent degradation of two key substrates, cyclin B1 and securin.
We have demonstrated that mitotic cells will remain mitotic for several hours in the absence of Cdk1 activity provided that APC/C-dependent protease activity is suppressed. Continued mitotic status in the absence of Cdk1 activity has been verified by several independent criteria: the continued presence of condensed chromosomes, the presence of a mitotic spindle, the presence of the mitosis-specific phosphoantigen markers MPM-2 and S10 of histone H3, and the presence on chromosomes of mitotic passenger proteins. Finally, and most importantly, we show that Cdk1 substrates remain phosphorylated in the absence of Cdk1 activity.
Thus, our results require a revision of the prevailing paradigm, which holds that destruction of cyclin B1, which inactivates Cdk1, is itself necessary and sufficient to induce mitotic exit. This paradigm requires that suppression of Cdk1 activity should therefore induce mitotic exit even in the absence of cyclin B1 destruction. Instead, our results show there is a pathway downstream of Cdk1 inactivation that requires both further proteolysis and phosphatase activation to complete the mitotic exit pathway.
Our results contrast with a recent study that proposed that cells exposed to both a Cdk1 inhibitor and an inhibitor of proteolysis undergo cell cleavage but remain competent to revert to mitosis when the Cdk1 inhibitor is removed (Potapova et al., 2006
). The interpretation of these results as evidence for a reversible exit from mitosis dependent on the continued presence of cyclin B1 rested on the reversibility of cell cleavage and the reappearance of a rounded mitotic cell. Other markers that would confirm that mitotic exit had indeed occurred in the absence of Cdk1 activity combined with the suppression of proteolysis were not examined except phosphorylated nucleolin.
Potapova et al. (2006)
suggested that mitotic exit occurred with some Cdk1 inhibitors such as purvalanol A but not with roscovitine. Importantly, our results have been obtained with three different Cdk inhibitors and with two different proteasome inhibitors, eliminating the possibility that the capacity to remain in mitosis in the absence of Cdk1 activity is dependent on a particular drug combination.
In accord with Potapova et al. (2006)
, we find that a portion of blocked monoastral cells undergo transient furrowing. This furrowing is accompanied by the relocation of proteins important to cytokinesis, such as the passenger proteins Aurora B, survivin, and TD60, and of PRC1 to the cell cortex along a bundle of microtubules. As a result, these cells form a bud that contains no chromatin. Such furrowing is quite similar to that previously shown to occur in cells with monoastral spindles in which the metaphase checkpoint had been suppressed by the introduction of mutant Mad2 (Canman et al., 2003
). These results and previous work (Martineau et al., 1995
; Wheatley et al., 1997
; Stemmann et al., 2001
; Niiya et al., 2005
) indicate that cytokinesis is an event that is independent of, and neither synchronous nor synonymous with, mitotic exit. Furrowing can occur well after mitotic exit has occurred or, as in the case presented here, can occur in the absence of mitotic exit. However, induction to undergo furrowing may depend on the absence of Cdk1 activity.
Cells released from STLC arrest in the presence of MG132 proceed to metaphase and maintain a bipolar metaphase spindle for at least an additional 2 h after the suppression of Cdk1 activity by roscovitine. The absence of Cdk1 activity clearly does not, of itself, drive cells forward from metaphase to anaphase. It is of interest that a substantial percentage of cells revert from metaphase to prometaphase when Cdk1 activity is suppressed in MG132-treated cells (Fig. S2), whereas none are driven forward to anaphase. When chromosomes are lost from the metaphase plate, they appear to have a merotelic (both kinetochores associated with one spindle pole) kinetochore alignment (unpublished data). Therefore, it appears that continuous Cdk1 activity is required to maintain proper metaphase chromosome alignment. This apparently unique role for Cdk1 activity in maintaining bipolar kinetochore attachment has not been noted before and deserves attention.
Retention of cells in mitosis is not caused by the continued presence of either cyclin B1 or securin in the absence of Cdk1 activity, as nondegradable mutants of these proteins do not prevent mitotic exit in the absence of Cdk1 activity. As MG132 nonetheless prevents mitotic exit, the reasonable conclusion is that a protease substrate other than cyclin B1 or securin must be degraded to permit mitotic exit in the absence of Cdk1 activity. Two proteins other than cyclin B1 and securin must be degraded for mitosis to progress, but these proteins, cyclin A (den Elzen and Pines, 2001
; Geley et al., 2001
) and Emi1 (Guardavaccaro et al., 2003
; Margottin-Goguet et al., 2003
), are both eliminated very early in mitosis and are unlikely to play a role in mitotic exit.
The putative protease substrate must be involved in phosphatase activation. Cdk1 substrates remain substantially phosphorylated for hours in the combined presence of different Cdk and proteasome inhibitors, and we obtain parallel results on exposing cells to the combination of roscovitine with protein phosphatase inhibitors (okadaic acid or calyculin A).
Suppression of the PP1 and PP2A protein phosphatase families is required for entry into mitosis (Cyert and Thorner, 1989
; Dohadwala et al., 1994
; Wera and Hemmings, 1995
; Kwon et al., 1997
; Puntoni and Villa-Moruzzi, 1997
), and both okadaic acid and calyculin A specifically inhibit the PP1 and PP2A protein phosphatase families (Ishihara et al., 1989
). Reasonable candidates for control of mitotic exit are members of the PP1 family, as they remain suppressed in mammalian cells by phosphorylation until metaphase and can be prolonged in this suppressed state by exposure of mitotic cells to either okadaic acid or calyculin A (Kwon et al., 1997
). Furthermore, microinjection of anti-PP1 antibody arrests mammalian cells at metaphase (Fernandez et al., 1992
). Similarly, the two PP1 proteins in Schizosaccharomyces pombe
are suppressed by cdc2 phosphorylation in mitosis, and their reactivation is required to proceed past metaphase (Ishii et al., 1996
). PP1 activity is also required for mitotic exit in Aspergillus
(Doonan and Morris, 1989
) and Drosophila
(Axton et al., 1990
; Chen et al., 2007
Our results with the combination of roscovitine and the phosphatase inhibitors okadaic acid or calyculin A indicate that phosphatase activity is required for exit from the mitotic state, presumably by the dephosphorylation of mitotic Cdk1 substrates, and, importantly, that there is little phosphatase activity evident on Cdk1 substrates in the presence of roscovitine and MG132. The continuing mitotic state, which is characterized by condensed chromosomes and by the presence of MPM-2 and S10-phosphorylated histone H3 markers, indicates that the phosphatase activity required for mitotic exit is minimal when APC/C-dependent proteolysis has been suppressed by MG132. The potential role of protease inhibition in the suppression of phosphatase-dependent mitotic exit is of substantial interest.
The key phosphatase activity must be downstream of the cdc20-driven APC/C-dependent protease activity, as cells treated with phosphatase inhibitors in the presence of roscovitine have lost both cyclin B1 and securin () but remain metabolically mitotic through the absence of the phosphatase-dependent destruction of Cdk1 substrates.
In light of a potential key role for protein phosphatases in mitotic exit, it is important to note that both budding and fission yeast contain mitotic exit networks that are dependent on unique protein phosphatases of the cdc14 family (Visintin et al., 1998
; Trautmann and McCollum, 2002
; Stegmeier and Amon, 2004
). Although mammalian cdc14A and B are functional homologues of cdc14 phosphatases in the yeast system (Vazquez-Novelle et al., 2005
), suppression of cdc14A in mammalian cells does not prevent mitotic exit (Mailand et al., 2002
). We do not believe that the cdc14 mechanism is likely to play an equivalent role in the pathway we describe here, as S. pombe
cdc14 (Clp1/Flp1) directly inactivates cdc2 (the S. pombe
homologue of Cdk1) by regulating its phosphorylation status. In this scenario, phosphatase inhibitors would not be expected to retain mitotic status in the absence of Cdk1 activity. In light of our results with combined Cdk and phosphatase inhibitors, it is interesting that in Saccharomyces cerevisiae
, Cdk activity is required to activate Net1 and, thus, cdc14 in the mitotic exit pathway and that this overrides PP2A-dependent metaphase arrest (Queralt et al., 2006
). It will be of substantial interest if a parallel exists between our results and the yeast pathway that uses Cdk activity and phosphatase activation for mitotic exit.
Our results strongly implicate phosphatase activity in key events in mitotic exit and protease in activating this pathway. Although it remains to be seen whether these effects directly or indirectly involve cdc14, it is nonetheless of great interest that there appears to be a convergence between the yeast systems and the mammalian system in the functional requirement for activation of protein phosphatase activity to enable mitotic exit.
In summary, we find that cells without Cdk1 activity remain mitotic by several criteria as long as there is no APC/C-dependent protease activity and that this effect is dependent on mitotic suppression of a key protein phosphatase activity. It will now be of great interest to elucidate this important pathway, to determine the protease substrate that is retaining these cells in mitosis, and to identify the protein phosphatase whose activation is apparently required for mitotic exit.