Analog-sensitive Cdk1 As a Tool to Study Mitotic Exit: Protein Phosphatase 1 is Required Downstream from Cdk1 Inactivation in Budding Yeast

Overview

Journal Chromosome Res

Date 2023 Sep 10

PMID 37690059

Authors

Jason M Keaton

Benjamin G Workman

Linfeng Xie

James R Paulson

Affiliations

Soon will be listed here.

Abstract

We show that specific inactivation of the protein kinase Cdk1/cyclin B (Cdc28/Clb2) triggers exit from mitosis in the budding yeast Saccharomyces cerevisiae. Cells carrying the allele cdc28-as1, which makes Cdk1 (Cdc28) uniquely sensitive to the ATP analog 1NM-PP1, were arrested with spindle poisons and then treated with 1NM-PP1 to inhibit Cdk1. This caused the cells to leave mitosis and enter G1-phase as shown by initiation of rebudding (without cytokinesis), induction of mating projections ("shmoos") by α-factor, stabilization of Sic1, and degradation of Clb2. It is known that Cdk1 must be inactivated for cells to exit mitosis, but our results show that inactivation of Cdk1 is not only necessary but also sufficient to initiate the transition from mitosis to G1-phase. This result suggests a system in which to test requirements for particular gene products downstream from Cdk1 inactivation, for example, by combining cdc28-as1 with conditional mutations in the genes of interest. Using this approach, we demonstrate that protein phosphatase 1 (PPase1; Glc7 in S. cerevisiae) is required for mitotic exit and reestablishment of interphase following Cdk1 inactivation. This system could be used to test the need for other protein phosphatases downstream from Cdk1 inactivation, such as PPase 2A and Cdc14, and it could be combined with phosphoproteomics to gain information about the substrates that the various phosphatases act upon during mitotic exit.

References

Ajiro K, Yasuda H, Tsuji H . Vanadate triggers the transition from chromosome condensation to decondensation in a mitotic mutant (tsTM13) inactivation of p34cdc2/H1 kinase and dephosphorylation of mitosis-specific histone H3. Eur J Biochem. 1996; 241(3):923-30. DOI: 10.1111/j.1432-1033.1996.00923.x. View

Akiyoshi B, Biggins S . Cdc14-dependent dephosphorylation of a kinetochore protein prior to anaphase in Saccharomyces cerevisiae. Genetics. 2010; 186(4):1487-91. PMC: 2998326. DOI: 10.1534/genetics.110.123653. View

Alberghina L, Coccetti P, Orlandi I . Systems biology of the cell cycle of Saccharomyces cerevisiae: From network mining to system-level properties. Biotechnol Adv. 2009; 27(6):960-978. DOI: 10.1016/j.biotechadv.2009.05.021. View

Al-Ghabkari A, Moffat L, Walsh M, MacDonald J . Validation of chemical genetics for the study of zipper-interacting protein kinase signaling. Proteins. 2018; 86(11):1211-1217. DOI: 10.1002/prot.25607. View

Amon A . Regulation of B-type cyclin proteolysis by Cdc28-associated kinases in budding yeast. EMBO J. 1997; 16(10):2693-702. PMC: 1169880. DOI: 10.1093/emboj/16.10.2693. View

Bembenek J, Yu H . Regulation of CDC14: pathways and checkpoints of mitotic exit. Front Biosci. 2003; 8:d1275-87. DOI: 10.2741/1128. View

Bishop A, Ubersax J, Petsch D, Matheos D, Gray N, Blethrow J . A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature. 2000; 407(6802):395-401. DOI: 10.1038/35030148. View

Black S, Andrews P, Sneddon A, Stark M . A regulated MET3-GLC7 gene fusion provides evidence of a mitotic role for Saccharomyces cerevisiae protein phosphatase 1. Yeast. 1995; 11(8):747-59. DOI: 10.1002/yea.320110806. View

Bloecher A, Tatchell K . Defects in Saccharomyces cerevisiae protein phosphatase type I activate the spindle/kinetochore checkpoint. Genes Dev. 1999; 13(5):517-22. PMC: 316501. DOI: 10.1101/gad.13.5.517. View

10.

Bloecher A, Tatchell K . Dynamic localization of protein phosphatase type 1 in the mitotic cell cycle of Saccharomyces cerevisiae. J Cell Biol. 2000; 149(1):125-40. PMC: 2175104. DOI: 10.1083/jcb.149.1.125. View

11.

Bloom J, Cross F . Novel role for Cdc14 sequestration: Cdc14 dephosphorylates factors that promote DNA replication. Mol Cell Biol. 2006; 27(3):842-53. PMC: 1800703. DOI: 10.1128/MCB.01069-06. View

12.

Bloom J, Cristea I, Procko A, Lubkov V, Chait B, Snyder M . Global analysis of Cdc14 phosphatase reveals diverse roles in mitotic processes. J Biol Chem. 2010; 286(7):5434-45. PMC: 3037656. DOI: 10.1074/jbc.M110.205054. View

13.

Bohm S, Buchberger A . The budding yeast Cdc48(Shp1) complex promotes cell cycle progression by positive regulation of protein phosphatase 1 (Glc7). PLoS One. 2013; 8(2):e56486. PMC: 3572051. DOI: 10.1371/journal.pone.0056486. View

14.

de Castro I, Gokhan E, Vagnarelli P . Resetting a functional G1 nucleus after mitosis. Chromosoma. 2016; 125(4):607-19. PMC: 5023730. DOI: 10.1007/s00412-015-0561-6. View

15.

de Castro I, Sales Gil R, Ligammari L, Di Giacinto M, Vagnarelli P . CDK1 and PLK1 coordinate the disassembly and reassembly of the nuclear envelope in vertebrate mitosis. Oncotarget. 2018; 9(8):7763-7773. PMC: 5814256. DOI: 10.18632/oncotarget.23666. View

16.

Dischinger S, Krapp A, Xie L, Paulson J, Simanis V . Chemical genetic analysis of the regulatory role of Cdc2p in the S. pombe septation initiation network. J Cell Sci. 2008; 121(Pt 6):843-53. DOI: 10.1242/jcs.021584. View

17.

Dunphy W, Kumagai A . The cdc25 protein contains an intrinsic phosphatase activity. Cell. 1991; 67(1):189-96. DOI: 10.1016/0092-8674(91)90582-j. View

18.

Emanuele M, Lan W, Jwa M, Miller S, Chan C, Stukenberg P . Aurora B kinase and protein phosphatase 1 have opposing roles in modulating kinetochore assembly. J Cell Biol. 2008; 181(2):241-54. PMC: 2315672. DOI: 10.1083/jcb.200710019. View

19.

Feldman R, Correll C, Kaplan K, Deshaies R . A complex of Cdc4p, Skp1p, and Cdc53p/cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p. Cell. 1997; 91(2):221-30. DOI: 10.1016/s0092-8674(00)80404-3. View

20.

Feng Z, Wilson S, Peng Z, Schlender K, Reimann E, Trumbly R . The yeast GLC7 gene required for glycogen accumulation encodes a type 1 protein phosphatase. J Biol Chem. 1991; 266(35):23796-801. View