Cdk1 and its activating subunit cyclin B are required for entry into and maintenance of the mitotic state. The prevailing model of mitotic induction by Cdk1 is that the key substrates must be activated by Cdk1 and in turn drive downstream events that create and sustain mitosis. After exhaustive analysis on the part of many laboratories, the identities of key substrates that Cdk1 activates to create the mitotic state remain unknown. Our recent work suggests that Cdk1 does not function by activating key substrates, but rather that it phosphorylates and suppresses one or more isoforms of protein phosphatase 1 (PP1), and that the suppression of PP1 activity initiates and drives mitosis. Thus, we reason that, upon Cdk1 activation at mitotic entry, Cdk1 phosphorylation inactivates PP1, causing a profound change in the metabolic state of the cell. Further, at the end of mitosis, Cdk1 inactivation permits PP1 reactivation and restoration of the interphase state. If our hypothesis is correct that PP1 sustains mitosis, then both mitotic entry and exit will depend on the state of PP1 activity. We will thus perform assays to address both the role of PP1 suppression in mitotic entry and the role of PP1 reactivation in mitotic exit. The intention of assaying PP1 function both in mitotic entry and exit is to establish a role for PP1 suppression throughout mitosis. We propose several independent lines of inquiry to determine the roles of the PP1 isoforms in mitotic entry and exit. As a first approach, we will use shRNA to knock down each of the PP1 isoforms in turn, to determine if absence of a PP1 isoform permits cells to enter mitosis following drug induced suppression of Cdk1 activity, or if ablation of a PP1 isoform prevents mitotic exit. Rescue of shRNA ablated cells with shRNA resistant PP1 will confirm PP1 function. Calyculin A suppresses serine/threonine phosphatases including PP1. Thus, as a second approach, we will use calyculin A resistant mutants of each of the PP1 isoforms, in combination with calyculin A suppression of PP1, to test if the activity of a specific PP1 isoform permits mitotic exit, in contast to control cells where calyculin A imposes mitotic arrest following Cdk1 inactivation. As a third approach, we will express PP1 isoform mutants that cannot be phosphorylated and inactivated by Cdk1, to determine whether a specific constitutively active PP1 isoform prevents mitotic entry following Cdk1 activation. These experiments should cleanly distinguish whether our hypothesis is correct. If it is correct, our work will lead to a completely new understanding of the mitotic process, and a clear understanding of the principal role of Cdk1 activity in mitosis. This will be a paradigm shift, with the potential of broad implications for understanding the underlying nature of cell cycle mechanisms throughout the cell cycle.

Public Health Relevance

Relevance to public health: Mitosis is the stage of the cell cycle when chromosomes correctly segregate and cell cleavage creates two daughter cells. The mitotic process is driven by Cdk1 protein kinase activity, but the targets of Cdk1 that create the mitotic state remain undefined. We propose that the essential target of Cdk1 is phosphorylation dependent suppression of protein phosphatase 1. If our hypothesis is true, our discovery will profoundly change understanding of mitotic control, and thus reveal new targets for cancer chemotherapy.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZGM1-CBB-7 (EU))
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Hamlet, Michelle R
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Sanford-Burnham Medical Research Institute
La Jolla
United States
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Panopoulos, Andreas; Pacios-Bras, Cristina; Choi, Justin et al. (2014) Failure of cell cleavage induces senescence in tetraploid primary cells. Mol Biol Cell 25:3105-18
Earnshaw, W C; Allshire, R C; Black, B E et al. (2013) Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant. Chromosome Res 21:101-6
Panopoulos, Andreas; Howell, Michael; Fotedar, Rati et al. (2011) Glioblastoma motility occurs in the absence of actin polymer. Mol Biol Cell 22:2212-20
Kim, J A; Lee, J; Margolis, R L et al. (2010) SP600125 suppresses Cdk1 and induces endoreplication directly from G2 phase, independent of JNK inhibition. Oncogene 29:1702-16