title: Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited.
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.
authors.affiliation: Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA. tpo@stowers.org
authors.forename: Tamara A
authors.initials: TA
authors.lastname: Potapova
authors.forename: Sushama
authors.initials: S
authors.lastname: Sivakumar
authors.forename: Jennifer N
authors.initials: JN
authors.lastname: Flynn
authors.forename: Rong
authors.initials: R
authors.lastname: Li
authors.forename: Gary J
authors.initials: GJ
authors.lastname: Gorbsky
mesh_terms: D000818:Animals; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D025461:Feedback, Physiological / Q000187:drug effects; D005260:Female; D016195:G2 Phase / Q000187:drug effects; D015870:Gene Expression / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins; D008938:Mitosis; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D049468:Prometaphase / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000187:drug effects; D029867:Xenopus Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis; D020687:cdc25 Phosphatases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D019926:Cyclin B; D008565:Membrane Proteins; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D029867:Xenopus Proteins; C095970:MYT1 kinase, Xenopus; C530005:WEE1 protein, Xenopus; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C490679:PKMYT1 protein, human; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D020687:cdc25 Phosphatases