A second question relates to what the signal transduction pathway of the antephase checkpoint is that prevents mitotic entry. One of the key downstream factors needed for mitotic entry is the cyclin B1-Cdk1 complex (reviewed recently in reference 68
). As alluded to in the review in reference 68
, the cyclin B1-Cdk1 complex is under tight regulation and it is conceivable that the cyclin B1-Cdk1 complex is targeted by the antephase checkpoint at specific regulatory points to prevent activation of the complex and hence entry into mitosis. Cdk1 is inhibited by phosphorylation at threonine-14 (T-14) and tyrosine-15 (Y-15) by Myt1 and Wee1, respectively (88
). Reversal of phosphorylation on T-14 and Y-15 depends upon the Cdc25 phosphatase, of which there are three isoforms, Cdc25A, Cdc25B, and Cdc25C (11
). The antephase checkpoint possibly impinges upon Wee1 and Myt1, as well as Cdc25, so as to control activation of the cyclin B1-Cdk1 complex and hence entry into mitosis.
Indeed, CHFR has been implicated in the ubiquitination and destruction of polo-like kinase 1 (Plk1) (59
). Plk1 is needed for the destruction of Wee1, as well as inhibition of Myt1 activity (reviewed in reference 133
). In addition, the activity of the Cdc25C phosphatase appears to be activated by Plk1 (133
). Destruction of Plk1 prior to mitosis will lead to persistence of the inhibitory phosphorylation on T-14 and Y-15 of Cdk1, resulting in failure of mitotic entry. In Xenopus
extracts, as well as in in vitro reconstitution assays, Plx1, Xenopus
Plk1, was ubiquitinated and degraded in a CHFR-dependent manner (59
). As a result, both the inactivation of Wee1 and the activation of Cdc25C were delayed, with the consequence that Cdk1 was kept inactive and mitotic entry was delayed (59
In CHFR-defective HCT116 cells transfected with a CHFR construct, it was proposed that the cells were able to delay progression to metaphase when exposed to microtubule poisons, likely by preventing the nuclear localization of cyclin B1 and keeping cyclin B1-Cdk1 inactive (116
). As the forced expression of cyclin B1 with a mutated nuclear export sequence was able to override the delay in early prophase upon activation of CHFR, the authors proposed that in the presence of microtubule poisons, CHFR acts to inhibit the cyclin B1-Cdk1 complex to prevent progression through prophase to metaphase (116
However, several lines of evidence support the idea that cyclin A-Cdk2 may, in fact, be the initial target of the antephase checkpoint. Cyclin A was shown to function in human cells not just during S phase but in mitosis as well (95
). Also, cyclin A localizes to the nuclei of HeLa cells from S phase until prophase (96
), after which cyclin A is destroyed in prometaphase in a manner dependent on the anaphase-promoting complex/cyclosome (34
) and 26S proteasome (27
). Unlike cyclin A, cyclin B1 only localizes to the nucleus during prophase (96
In addition, downregulation of cyclin A by short hairpin RNA leads to a G2
arrest in HeLa cells (32
) while a dominant-negative form of Cdk2 when expressed in U2OS cells can delay cells in G2
). Furthermore, cyclin A-Cdk2 activity can promote entry into mitosis (26
), specifically for mitotic progression until late prophase (33
). The notion that cyclin A-Cdk2 is important for the antephase checkpoint is also supported by the finding that the overexpression of a constitutively active cyclin A-Cdk2AF
complex can bypass the antephase checkpoint in the presence of stress inducers (71
). It is therefore likely that cyclin A-Cdk2 activity is normally downregulated by the antephase checkpoint via the p38-Cdc25 pathway in the presence of stress factors.
The three isoforms of Cdc25 phosphatase are thought to perform overlapping functions during the different cell cycle stages (11
). For instance, Cdc25A, previously implicated in the G1
/S transition (87
), can cause an increase in cyclin A-Cdk activities at G2
/M when overexpressed in U2OS cells (124
). Of note is the authors' finding that the activity of Cdc25A occurs prior to Cdc25B activity during mitosis. Given that cyclin A-Cdk2 likely plays an important role in the activation of cyclin B-Cdk1 (32
), presumably via activation of Cdc25B and Cdc25C at G2
/M in U2OS cells (76
), it is conceivable that upon activation, the antephase checkpoint targets Cdc25A for export from the nuclei and away from cyclin A-Cdk2 (11
). In so doing, it tips the balance toward the inhibition of Cdk2 by phosphorylation of Y-15 by the Wee1 kinase, leading to a downregulation of cyclin A-Cdk2 activity. Without active cyclin A-Cdk2, cyclin B1-Cdk1, whose activation requires cyclin A-Cdk2, will be kept inactive and the cells will be prevented from entering mitosis. However, a functional interaction between p38 and Cdc25A has yet to be demonstrated.
The idea that cyclin A-Cdk2 is the target of the antephase checkpoint is disputed by Summers and coworkers, who proposed that cyclin B1-Cdk1 is the target (116
; see above). In their study, they noted that during activation of the antephase, the cyclin A-Cdk2 complex was active, as judged by the mobility shift of Cdk2 in Western blot analysis. They contended that the block in mitotic entry upon antephase checkpoint activation was not likely due to inactivation of the cyclin A-Cdk2 complex. Rather, they suggested that cyclin B1-Cdk1 was inactive because cyclin B1 was sequestered in the cytoplasm.
It should be noted, however, that Cdk2 mobility is affected not by T-14 or Y-15 but by phosphorylation on T-160 (106
) and therefore does not serve well as an indication of cyclin A-Cdk2 activity. The inactive cyclin B1-Cdk1 noted by Summers and colleagues (116
) could therefore be a consequence of the antephase checkpoint acting to downregulate cyclin A-Cdk2 activity. Their observation that the CHFR-mediated delay in mitotic entry led to a failure to import cyclin B1 into the nuclei may not be a direct effect of the antephase checkpoint. In fact, cyclin B1 is normally localized to the cytoplasm during interphase due to a higher rate of nuclear export compared to nuclear import (43
). In prophase, it is the activation of cyclin B1-Cdk1 in the cytoplasm (42
) that results in the reduced rate of nuclear export and accumulation of cyclin B in the nucleus (43
). The lack of nuclear accumulation of cyclin B1 could again be a result of the antephase checkpoint acting to downregulate cyclin A-Cdk2 activity. Furthermore, the finding that overexpression of a cyclin B1 export mutant was able to override the antephase checkpoint could simply mean that cyclin B1-Cdk1 has a function that overlaps that of cyclin A-Cdk2 in promoting the early stages of mitosis.
While it remains a speculation, the likelihood that cyclin A-Cdk2 is a target of the antephase checkpoint could account for the reversibility of antephase in cells exposed to stress factors given that cyclin A-Cdk2 is an upstream effector of mitosis. Once the cells pass antephase, cyclin B1-Cdk1 would be activated. At such a point, the cells are committed to mitosis and would pass the point of no return and proceed to mitosis even in the presence of stress factors. Further studies to resolve the issue of whether cyclin A-Cdk2 or cyclin B1-Cdk1 is relevant in the antephase checkpoint pathway will help provide a clearer picture of how cells respond to insults as they approach mitosis.
However, the pathway from CHFR to the cyclin-Cdk complexes remains unclear. As alluded to above, p38 appears to function downstream of CHFR (71
), though the exact links between CHFR and p38 have yet to be established. Interestingly, Matsusaka and Pines isolated in a yeast two-hybrid screen an interactor of CHFR known as TRAF6-binding protein (T6BP) (71
and references therein). TRAF6 is a ubiquitin ligase that activates C-TAK1 (a MAP kinase kinase kinase). C-TAK is a MAP kinase kinase kinase that functions in the p38 pathway in response to interferon. The authors proposed that CHFR, in combination with T6BP, could activate C-TAK1. C-TAK1 is known to be able to phosphorylate Cdc25 and, as a result, creates a 14-3-3 binding site on Cdc25 (11
). The binding of 14-3-3 sequesters Cdc25 away from cyclin-Cdk complexes, thereby abolishing the ability of the phosphatase to dephosphorylate cyclin-Cdk complexes on T-14 and Y-15. Without active cyclin-Cdk complexes, cells fail to trigger mitotic events and are delayed at antephase. Much of this scheme requires experimental verification at this stage.
CHFR also has been shown to downregulate HDAC1 (89
). More significantly, HDAC1 normally represses the expression of p21, a CDK inhibitor (134
). The downregulation of HDAC1 by CHFR increased p21 mRNA and protein levels (89
). Interestingly, microinjection of cells with the N-terminal fragment of p21 inhibited cyclin A-Cdk2 and caused a reversal of chromosome condensation in cells at prophase (33
). It was previously proposed that p21 binds to and inhibits the cyclin A-Cdk 2 complex (29
), likely by preventing the binding of the Cdk-activating kinase to Cdk2 (3
). It is possible that this constitutes a slow-response arm of the antephase checkpoint (74
) that can cause a sustained antephase delay via p21-mediated inactivation of cyclin-Cdk complexes.