Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA.
Molecular biology of the cell (Impact Factor: 5.98). 02/2011; 22(8):1191-206. DOI: 10.1091/mbc.E10-07-0599
Source: PubMed

ABSTRACT Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.


Available from: Tamara A Potapova, Jun 15, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Entry into mitosis is driven by the coordinated phosphorylation of thousands of proteins. For the cell to complete mitosis and divide into two identical daughter cells it must regulate dephosphorylation of these proteins in a highly ordered, temporal manner. There is currently a lack of a complete understanding of the phosphorylation changes that occur during the initial stages of mitotic exit in human cells. Therefore, we performed a large unbiased, global analysis to map the very first dephosphorylation events that occur as cells exit mitosis. We identified and quantified the modification of >16,000 phosphosites on >3,300 unique proteins during early mitotic exit, providing up to 8-fold greater resolution than previous studies. The data have been deposited to the ProteomeXchange with identifier PXD001559. Only a small fraction (~10%) of phosphorylation sites were dephosphorylated during early mitotic exit and these occurred on proteins involved in critical early exit events, including organization of the mitotic spindle, the spindle assembly checkpoint, and reformation of the nuclear envelope. Surprisingly this enrichment was observed across all kinase consensus motifs, indicating that it is independent of the upstream phosphorylating kinase. Therefore, dephosphorylation of these sites is likely determined by the specificity of phosphatase/s rather than the activity of kinase/s. Dephosphorylation was significantly affected by the amino acids at and surrounding the phosphorylation site, with several unique evolutionarily conserved amino acids correlating strongly with phosphorylation status. These data provide a potential mechanism for the specificity of phosphatases, and how they co-ordinate the ordered events of mitotic exit. In summary, our results provide a global overview of the phosphorylation changes that occur during the very first stages of mitotic exit, providing novel mechanistic insight into how phosphatase/s specifically regulate this critical transition. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Molecular &amp Cellular Proteomics 06/2015; DOI:10.1074/mcp.M114.046938 · 7.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: When cells enter mitosis, the anaphase-promoting complex/cyclosome (APC/C) is activated by phosphorylation and binding of Cdc20. The RXXL destruction box (D-box) of cyclin B1 only binds Cdc20 after release of the spindle checkpoint in metaphase, initiating cyclin B1 ubiquitination upon chromosome bi-orientation. However, we found that cyclin B1, through Cdk1 and Cks, is targeted to the phosphorylated APC/C(Cdc20) at the start of prometaphase, when the spindle checkpoint is still active. Here, we show that MASTL is essential for cyclin B1 recruitment to the mitotic APC/C and that this occurs entirely independently of Cdc20. Importantly, MASTL-directed binding of cyclin B1 to spindle checkpoint-inhibited APC/C(Cdc20) critically supports efficient cyclin B1 destruction after checkpoint release. A high incidence of anaphase bridges observed in response to MASTL RNAi may result from cyclin B1 remaining after securin destruction, which is insufficient to keep MASTL-depleted cells in mitosis but delays the activation of separase. © 2015. Published by The Company of Biologists Ltd.
    03/2015; 4(4). DOI:10.1242/bio.201410793
  • [Show abstract] [Hide abstract]
    ABSTRACT: Progesterone receptor (PR) activation in the ventrolateral division of the hypothalamic ventromedial nucleus (VMNvl) is essential for promoting female sexual behavior. The estrogen receptor alpha (ERα), in contrast with the ERβ, has been largely implicated in the induction of PRs. The simultaneous activation of ERα and ERβ, while not increasing the number of PR-immunoreactive neurons in the VMNvl, facilitates lordosis, which suggests that ERβ and/or the ERα-ERβ interaction might play a role in PR dynamics and/or PR expression by individual neurons. To address this question, we used Western blot and immunohistochemical studies to determine the amount and subcellular distribution of both PR isoforms in VMNvl neurons of ovariectomized rats injected with estradiol benzoate or with the specific agonists of ERα and ERβ, alone or in association. The present data show that ERα activation does not change PR expression in individual neurons, but augments the quantity of PRs in the VMNvl because it increases the number of neurons expressing PRs. Conversely, ERβ activation does not change the total amount of PRs in the VMNvl, but increases the labeling intensity of the perikaryal cytoplasm, which suggests that it promotes the transport of PRs from neurites into cell bodies. In addition, the simultaneous activation of ERα and ERβ increases the expression of PRs by individual neurons and, consequently, augments the total amount of PRs in the VMNvl. Our findings reveal that individual or simultaneous activation of ERα and ERβ promote different outcomes in the levels and subcellular location of PRs in VMNvl neurons. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    FEBS Journal 01/2015; 282(6). DOI:10.1111/febs.13207 · 3.99 Impact Factor