PP2A/B55 and Fcp1 Regulate Greatwall and Ensa Dephosphorylation during Mitotic Exit


Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, inhibition or depletion of neither Fcp1 nor PP2A appears to block dephosphorylation of the bulk of mitotic Cdk1 substrates during mitotic exit. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit.


Vyšlo v časopise: PP2A/B55 and Fcp1 Regulate Greatwall and Ensa Dephosphorylation during Mitotic Exit. PLoS Genet 10(1): e32767. doi:10.1371/journal.pgen.1004004
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004004

Souhrn

Entry into mitosis is triggered by activation of Cdk1 and inactivation of its counteracting phosphatase PP2A/B55. Greatwall kinase inactivates PP2A/B55 via its substrates Ensa and ARPP19. Both Greatwall and Ensa/ARPP19 are regulated by phosphorylation, but the dynamic regulation of Greatwall activity and the phosphatases that control Greatwall kinase and its substrates are poorly understood. To address these questions we applied a combination of mathematical modelling and experiments using phospho-specific antibodies to monitor Greatwall, Ensa/ARPP19 and Cdk substrate phosphorylation during mitotic entry and exit. We demonstrate that PP2A/B55 is required for Gwl dephosphorylation at the essential Cdk site Thr194. Ensa/ARPP19 dephosphorylation is mediated by the RNA Polymerase II carboxy terminal domain phosphatase Fcp1. Surprisingly, inhibition or depletion of neither Fcp1 nor PP2A appears to block dephosphorylation of the bulk of mitotic Cdk1 substrates during mitotic exit. Taken together our results suggest a hierarchy of phosphatases coordinating Greatwall, Ensa/ARPP19 and Cdk substrate dephosphorylation during mitotic exit.


Zdroje

1. DephoureN, ZhouC, VillenJ, BeausoleilSA, BakalarskiCE, et al. (2008) A quantitative atlas of mitotic phosphorylation. Proc Natl Acad Sci U S A 105: 10762–10767.

2. OlsenJV, VermeulenM, SantamariaA, KumarC, MillerML, et al. (2010) Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 3: ra3.

3. MedemaRH, LindqvistA (2011) Boosting and suppressing mitotic phosphorylation. Trends Biochem Sci 36: 578–584.

4. Domingo-SananesMR, KapuyO, HuntT, NovakB (2011) Switches and latches: a biochemical tug-of-war between the kinases and phosphatases that control mitosis. Philos Trans R Soc Lond B Biol Sci 366: 3584–3594.

5. NovakB, VinodPK, FreireP, KapuyO (2010) Systems-level feedback in cell-cycle control. Biochem Soc Trans 38: 1242–1246.

6. LindqvistA, Rodriguez-BravoV, MedemaRH (2009) The decision to enter mitosis: feedback and redundancy in the mitotic entry network. J Cell Biol 185: 193–202.

7. TangZ, ColemanTR, DunphyWG (1993) Two distinct mechanisms for negative regulation of the Wee1 protein kinase. EMBO J 12: 3427–3436.

8. MuellerPR, ColemanTR, DunphyWG (1995) Cell cycle regulation of a Xenopus Wee1-like kinase. Mol Biol Cell 6: 119–134.

9. HarveySL, CharletA, HaasW, GygiSP, KelloggDR (2005) Cdk1-dependent regulation of the mitotic inhibitor Wee1. Cell 122: 407–420.

10. HoffmannI, ClarkePR, MarcoteMJ, KarsentiE, DraettaG (1993) Phosphorylation and activation of human cdc25-C by cdc2–cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J 12: 53–63.

11. ClarkePR, HoffmannI, DraettaG, KarsentiE (1993) Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts. Mol Biol Cell 4: 397–411.

12. MochidaS, IkeoS, GannonJ, HuntT (2009) Regulated activity of PP2A-B55 delta is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts. EMBO J 28: 2777–2785.

13. YuJ, FlemingSL, WilliamsB, WilliamsEV, LiZ, et al. (2004) Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila. J Cell Biol 164: 487–492.

14. YuJ, ZhaoY, LiZ, GalasS, GoldbergML (2006) Greatwall kinase participates in the Cdc2 autoregulatory loop in Xenopus egg extracts. Mol Cell 22: 83–91.

15. CastilhoPV, WilliamsBC, MochidaS, ZhaoY, GoldbergML (2009) The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/δδelta, a phosphatase directed against CDK phosphosites. Mol Biol Cell 20: 4777–4789.

16. Gharbi-AyachiA, LabbeJC, BurgessA, VigneronS, StrubJM, et al. (2010) The substrate of Greatwall kinase, Arpp19, controls mitosis by inhibiting protein phosphatase 2A. Science 330: 1673–1677.

17. MochidaS, MaslenSL, SkehelM, HuntT (2010) Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis. Science 330: 1670–1673.

18. RangoneH, WegelE, GattMK, YeungE, FlowersA, et al. (2011) Suppression of scant identifies Endos as a substrate of greatwall kinase and a negative regulator of protein phosphatase 2A in mitosis. PLoS Genet 7: e1002225.

19. KimMY, BucciarelliE, MortonDG, WilliamsBC, Blake-HodekK, et al. (2012) Bypassing the Greatwall-Endosulfine pathway: plasticity of a pivotal cell-cycle regulatory module in Drosophila melanogaster and Caenorhabditis elegans. Genetics 191: 1181–1197.

20. VoetsE, WolthuisRM (2010) MASTL is the human orthologue of Greatwall kinase that facilitates mitotic entry, anaphase and cytokinesis. Cell Cycle 9: 3591–3601.

21. BurgessA, VigneronS, BrioudesE, LabbeJC, LorcaT, et al. (2010) Loss of human Greatwall results in G2 arrest and multiple mitotic defects due to deregulation of the cyclin B-Cdc2/PP2A balance. Proc Natl Acad Sci U S A 107: 12564–12569.

22. Blake-HodekKA, WilliamsBC, ZhaoY, CastilhoPV, ChenW, et al. (2012) Determinants for activation of the atypical AGC kinase Greatwall during M phase entry. Mol Cell Biol 32: 1337–1353.

23. LorcaT, CastroA (2013) The Greatwall kinase: a new pathway in the control of the cell cycle. Oncogene 32: 537–543.

24. VigneronS, BrioudesE, BurgessA, LabbeJC, LorcaT, et al. (2009) Greatwall maintains mitosis through regulation of PP2A. EMBO J 28: 2786–2793.

25. WuJQ, GuoJY, TangW, YangCS, FreelCD, et al. (2009) PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation. Nat Cell Biol 11: 644–651.

26. SchmitzMH, HeldM, JanssensV, HutchinsJR, HudeczO, et al. (2010) Live-cell imaging RNAi screen identifies PP2A-B55alpha and importin-beta1 as key mitotic exit regulators in human cells. Nat Cell Biol 12: 886–893.

27. HocheggerH, DejsuphongD, SonodaE, SaberiA, RajendraE, et al. (2007) An essential role for Cdk1 in S phase control is revealed via chemical genetics in vertebrate cells. J Cell Biol 178: 257–268.

28. SkoufiasDA, IndoratoRL, LacroixF, PanopoulosA, MargolisRL (2007) Mitosis persists in the absence of Cdk1 activity when proteolysis or protein phosphatase activity is suppressed. J Cell Biol 179: 671–685.

29. PotapovaTA, DaumJR, PittmanBD, HudsonJR, JonesTN, et al. (2006) The reversibility of mitotic exit in vertebrate cells. Nature 440: 954–958.

30. ManchadoE, GuillamotM, de CarcerG, EgurenM, TrickeyM, et al. (2010) Targeting mitotic exit leads to tumor regression in vivo: Modulation by Cdk1, Mastl, and the PP2A/B55alpha,delta phosphatase. Cancer Cell 18: 641–654.

31. SonS, OsmaniSA (2009) Analysis of all protein phosphatase genes in Aspergillus nidulans identifies a new mitotic regulator, fcp1. Eukaryot Cell 8: 573–585.

32. ViscontiR, PalazzoL, Della MonicaR, GriecoD (2012) Fcp1-dependent dephosphorylation is required for M-phase-promoting factor inactivation at mitosis exit. Nat Commun 3: 894.

33. GuillamotM, ManchadoE, ChiesaM, Gomez-LopezG, PisanoDG, et al. (2011) Cdc14b regulates mammalian RNA polymerase II and represses cell cycle transcription. Sci Rep 1: 189.

34. MocciaroA, BerdougoE, ZengK, BlackE, VagnarelliP, et al. (2010) Vertebrate cells genetically deficient for Cdc14A or Cdc14B retain DNA damage checkpoint proficiency but are impaired in DNA repair. J Cell Biol 189: 631–639.

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2014 Číslo 1
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa