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. 1990 Mar;9(3):675–683. doi: 10.1002/j.1460-2075.1990.tb08159.x

Cdc2 H1 kinase is negatively regulated by a type 2A phosphatase in the Xenopus early embryonic cell cycle: evidence from the effects of okadaic acid.

M A Félix 1, P Cohen 1, E Karsenti 1
PMCID: PMC551720  PMID: 2155777

Abstract

In Xenopus embryos, the cell cycle is abbreviated to a rapid alternation between interphase and mitosis. The onset of each M phase is induced by the periodic activation of the cdc2 kinase which is triggered by a threshold level of cyclins and apparently involves dephosphorylation of p34cdc2. We have prepared post-ribosomal supernatants from eggs sampled during interphase (interphase extracts) and just before the first mitosis of the early embryonic cell cycle (prophase extracts). In 'interphase extracts', the cdc2 kinase never activates spontaneously upon incubation at room temperature whereas in 'prophase extracts' it does. We show here that in 'interphase extracts', specific inhibition of type 2A phosphatase by okadaic acid induces cdc2 kinase activation. This requires a subthreshold level of cyclin and the presence of a particulate factor in the extract. Inhibition of type 1 phosphatases by inhibitor 1 and inhibitor 2 never results in cdc2 kinase activation. These results demonstrate that during the period of cyclin accumulation, cdc2 kinase activation is inhibited by a type 2A phosphatase. In 'prophase extracts', spontaneous activation of the cdc2 kinase is inhibited by beta-glycerophosphate and NaF, but not by okadaic acid, inhibitor 1 and inhibitor 2 or divalent cation chelation. This demonstrates that when enough cyclin has accumulated, cdc2 kinase activation involves a protein phosphatase which must be distinct from the type 1 and 2A phosphatases, and from the calcium-dependent (type 2B) and magnesium-dependent (type 2C) phosphatases.

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Selected References

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  1. Arion D., Meijer L., Brizuela L., Beach D. cdc2 is a component of the M phase-specific histone H1 kinase: evidence for identity with MPF. Cell. 1988 Oct 21;55(2):371–378. doi: 10.1016/0092-8674(88)90060-8. [DOI] [PubMed] [Google Scholar]
  2. Beach D., Durkacz B., Nurse P. Functionally homologous cell cycle control genes in budding and fission yeast. Nature. 1982 Dec 23;300(5894):706–709. doi: 10.1038/300706a0. [DOI] [PubMed] [Google Scholar]
  3. Bialojan C., Takai A. Inhibitory effect of a marine-sponge toxin, okadaic acid, on protein phosphatases. Specificity and kinetics. Biochem J. 1988 Nov 15;256(1):283–290. doi: 10.1042/bj2560283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Booher R. N., Alfa C. E., Hyams J. S., Beach D. H. The fission yeast cdc2/cdc13/suc1 protein kinase: regulation of catalytic activity and nuclear localization. Cell. 1989 Aug 11;58(3):485–497. doi: 10.1016/0092-8674(89)90429-7. [DOI] [PubMed] [Google Scholar]
  5. Booher R., Beach D. Involvement of a type 1 protein phosphatase encoded by bws1+ in fission yeast mitotic control. Cell. 1989 Jun 16;57(6):1009–1016. doi: 10.1016/0092-8674(89)90339-5. [DOI] [PubMed] [Google Scholar]
  6. Brizuela L., Draetta G., Beach D. Activation of human CDC2 protein as a histone H1 kinase is associated with complex formation with the p62 subunit. Proc Natl Acad Sci U S A. 1989 Jun;86(12):4362–4366. doi: 10.1073/pnas.86.12.4362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Brizuela L., Draetta G., Beach D. p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase. EMBO J. 1987 Nov;6(11):3507–3514. doi: 10.1002/j.1460-2075.1987.tb02676.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cohen P., Alemany S., Hemmings B. A., Resink T. J., Strålfors P., Tung H. Y. Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol. 1988;159:390–408. doi: 10.1016/0076-6879(88)59039-0. [DOI] [PubMed] [Google Scholar]
  9. Cohen P., Cohen P. T. Protein phosphatases come of age. J Biol Chem. 1989 Dec 25;264(36):21435–21438. [PubMed] [Google Scholar]
  10. Cohen P., Klumpp S., Schelling D. L. An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues. FEBS Lett. 1989 Jul 3;250(2):596–600. doi: 10.1016/0014-5793(89)80803-8. [DOI] [PubMed] [Google Scholar]
  11. Cohen P. The structure and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. doi: 10.1146/annurev.bi.58.070189.002321. [DOI] [PubMed] [Google Scholar]
  12. Cyert M. S., Kirschner M. W. Regulation of MPF activity in vitro. Cell. 1988 Apr 22;53(2):185–195. doi: 10.1016/0092-8674(88)90380-7. [DOI] [PubMed] [Google Scholar]
  13. Doonan J. H., Morris N. R. The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1. Cell. 1989 Jun 16;57(6):987–996. doi: 10.1016/0092-8674(89)90337-1. [DOI] [PubMed] [Google Scholar]
  14. Draetta G., Beach D. Activation of cdc2 protein kinase during mitosis in human cells: cell cycle-dependent phosphorylation and subunit rearrangement. Cell. 1988 Jul 1;54(1):17–26. doi: 10.1016/0092-8674(88)90175-4. [DOI] [PubMed] [Google Scholar]
  15. Dunphy W. G., Brizuela L., Beach D., Newport J. The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis. Cell. 1988 Jul 29;54(3):423–431. doi: 10.1016/0092-8674(88)90205-x. [DOI] [PubMed] [Google Scholar]
  16. Dunphy W. G., Newport J. W. Fission yeast p13 blocks mitotic activation and tyrosine dephosphorylation of the Xenopus cdc2 protein kinase. Cell. 1989 Jul 14;58(1):181–191. doi: 10.1016/0092-8674(89)90414-5. [DOI] [PubMed] [Google Scholar]
  17. Evans T., Rosenthal E. T., Youngblom J., Distel D., Hunt T. Cyclin: a protein specified by maternal mRNA in sea urchin eggs that is destroyed at each cleavage division. Cell. 1983 Jun;33(2):389–396. doi: 10.1016/0092-8674(83)90420-8. [DOI] [PubMed] [Google Scholar]
  18. Felix M. A., Pines J., Hunt T., Karsenti E. A post-ribosomal supernatant from activated Xenopus eggs that displays post-translationally regulated oscillation of its cdc2+ mitotic kinase activity. EMBO J. 1989 Oct;8(10):3059–3069. doi: 10.1002/j.1460-2075.1989.tb08457.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Foulkes J. G., Maller J. L. In vivo actions of protein phosphatase inhibitor-2 in Xenopus oocytes. FEBS Lett. 1982 Dec 13;150(1):155–160. doi: 10.1016/0014-5793(82)81325-2. [DOI] [PubMed] [Google Scholar]
  20. Gautier J., Matsukawa T., Nurse P., Maller J. Dephosphorylation and activation of Xenopus p34cdc2 protein kinase during the cell cycle. Nature. 1989 Jun 22;339(6226):626–629. doi: 10.1038/339626a0. [DOI] [PubMed] [Google Scholar]
  21. Gautier J., Norbury C., Lohka M., Nurse P., Maller J. Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+. Cell. 1988 Jul 29;54(3):433–439. doi: 10.1016/0092-8674(88)90206-1. [DOI] [PubMed] [Google Scholar]
  22. Goris J., Hermann J., Hendrix P., Ozon R., Merlevede W. Okadaic acid, a specific protein phosphatase inhibitor, induces maturation and MPF formation in Xenopus laevis oocytes. FEBS Lett. 1989 Mar 13;245(1-2):91–94. doi: 10.1016/0014-5793(89)80198-x. [DOI] [PubMed] [Google Scholar]
  23. Gould K. L., Nurse P. Tyrosine phosphorylation of the fission yeast cdc2+ protein kinase regulates entry into mitosis. Nature. 1989 Nov 2;342(6245):39–45. doi: 10.1038/342039a0. [DOI] [PubMed] [Google Scholar]
  24. Haystead T. A., Sim A. T., Carling D., Honnor R. C., Tsukitani Y., Cohen P., Hardie D. G. Effects of the tumour promoter okadaic acid on intracellular protein phosphorylation and metabolism. Nature. 1989 Jan 5;337(6202):78–81. doi: 10.1038/337078a0. [DOI] [PubMed] [Google Scholar]
  25. Huchon D., Ozon R., Demaille J. G. Protein phosphatase-1 is involved in Xenopus oocyte maturation. Nature. 1981 Nov 26;294(5839):358–359. doi: 10.1038/294358a0. [DOI] [PubMed] [Google Scholar]
  26. Karsenti E., Bravo R., Kirschner M. Phosphorylation changes associated with the early cell cycle in Xenopus eggs. Dev Biol. 1987 Feb;119(2):442–453. doi: 10.1016/0012-1606(87)90048-0. [DOI] [PubMed] [Google Scholar]
  27. Labbe J. C., Lee M. G., Nurse P., Picard A., Doree M. Activation at M-phase of a protein kinase encoded by a starfish homologue of the cell cycle control gene cdc2+. Nature. 1988 Sep 15;335(6187):251–254. doi: 10.1038/335251a0. [DOI] [PubMed] [Google Scholar]
  28. Labbe J. C., Picard A., Peaucellier G., Cavadore J. C., Nurse P., Doree M. Purification of MPF from starfish: identification as the H1 histone kinase p34cdc2 and a possible mechanism for its periodic activation. Cell. 1989 Apr 21;57(2):253–263. doi: 10.1016/0092-8674(89)90963-x. [DOI] [PubMed] [Google Scholar]
  29. Labbé J. C., Capony J. P., Caput D., Cavadore J. C., Derancourt J., Kaghad M., Lelias J. M., Picard A., Dorée M. MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B. EMBO J. 1989 Oct;8(10):3053–3058. doi: 10.1002/j.1460-2075.1989.tb08456.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Labbé J. C., Picard A., Karsenti E., Dorée M. An M-phase-specific protein kinase of Xenopus oocytes: partial purification and possible mechanism of its periodic activation. Dev Biol. 1988 May;127(1):157–169. doi: 10.1016/0012-1606(88)90197-2. [DOI] [PubMed] [Google Scholar]
  31. Maller J. L. Regulation of amphibian oocyte maturation. Cell Differ. 1985 Jun;16(4):211–221. doi: 10.1016/0045-6039(85)90570-6. [DOI] [PubMed] [Google Scholar]
  32. McGowan C. H., Cohen P. Protein phosphatase-2C from rabbit skeletal muscle and liver: an Mg2+-dependent enzyme. Methods Enzymol. 1988;159:416–426. doi: 10.1016/0076-6879(88)59041-9. [DOI] [PubMed] [Google Scholar]
  33. Meijer L., Arion D., Golsteyn R., Pines J., Brizuela L., Hunt T., Beach D. Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase. EMBO J. 1989 Aug;8(8):2275–2282. doi: 10.1002/j.1460-2075.1989.tb08353.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meijer L., Pondaven P., Tung H. Y., Cohen P., Wallace R. W. Protein phosphorylation and oocyte maturation. II. Inhibition of starfish oocyte maturation by intracellular microinjection of protein phosphatases 1 and 2A and alkaline phosphatase. Exp Cell Res. 1986 Apr;163(2):489–499. doi: 10.1016/0014-4827(86)90079-0. [DOI] [PubMed] [Google Scholar]
  35. Minshull J., Blow J. J., Hunt T. Translation of cyclin mRNA is necessary for extracts of activated xenopus eggs to enter mitosis. Cell. 1989 Mar 24;56(6):947–956. doi: 10.1016/0092-8674(89)90628-4. [DOI] [PubMed] [Google Scholar]
  36. Moreno S., Hayles J., Nurse P. Regulation of p34cdc2 protein kinase during mitosis. Cell. 1989 Jul 28;58(2):361–372. doi: 10.1016/0092-8674(89)90850-7. [DOI] [PubMed] [Google Scholar]
  37. Morla A. O., Draetta G., Beach D., Wang J. Y. Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis. Cell. 1989 Jul 14;58(1):193–203. doi: 10.1016/0092-8674(89)90415-7. [DOI] [PubMed] [Google Scholar]
  38. Murray A. W., Kirschner M. W. Cyclin synthesis drives the early embryonic cell cycle. Nature. 1989 May 25;339(6222):275–280. doi: 10.1038/339275a0. [DOI] [PubMed] [Google Scholar]
  39. Murray A. W., Solomon M. J., Kirschner M. W. The role of cyclin synthesis and degradation in the control of maturation promoting factor activity. Nature. 1989 May 25;339(6222):280–286. doi: 10.1038/339280a0. [DOI] [PubMed] [Google Scholar]
  40. Nurse P., Bissett Y. Gene required in G1 for commitment to cell cycle and in G2 for control of mitosis in fission yeast. Nature. 1981 Aug 6;292(5823):558–560. doi: 10.1038/292558a0. [DOI] [PubMed] [Google Scholar]
  41. Ohkura H., Kinoshita N., Miyatani S., Toda T., Yanagida M. The fission yeast dis2+ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell. 1989 Jun 16;57(6):997–1007. doi: 10.1016/0092-8674(89)90338-3. [DOI] [PubMed] [Google Scholar]
  42. Picard A., Capony J. P., Brautigan D. L., Dorée M. Involvement of protein phosphatases 1 and 2A in the control of M phase-promoting factor activity in starfish. J Cell Biol. 1989 Dec;109(6 Pt 2):3347–3354. doi: 10.1083/jcb.109.6.3347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Picard A., Peaucellier G., le Bouffant F., Le Peuch C., Dorée M. Role of protein synthesis and proteases in production and inactivation of maturation-promoting activity during meiotic maturation of starfish oocytes. Dev Biol. 1985 Jun;109(2):311–320. doi: 10.1016/0012-1606(85)90458-0. [DOI] [PubMed] [Google Scholar]
  44. Pines J., Hunt T. Molecular cloning and characterization of the mRNA for cyclin from sea urchin eggs. EMBO J. 1987 Oct;6(10):2987–2995. doi: 10.1002/j.1460-2075.1987.tb02604.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pines J., Hunter T. Isolation of a human cyclin cDNA: evidence for cyclin mRNA and protein regulation in the cell cycle and for interaction with p34cdc2. Cell. 1989 Sep 8;58(5):833–846. doi: 10.1016/0092-8674(89)90936-7. [DOI] [PubMed] [Google Scholar]
  46. Russell P., Nurse P. The mitotic inducer nim1+ functions in a regulatory network of protein kinase homologs controlling the initiation of mitosis. Cell. 1987 May 22;49(4):569–576. doi: 10.1016/0092-8674(87)90459-4. [DOI] [PubMed] [Google Scholar]
  47. Simanis V., Nurse P. The cell cycle control gene cdc2+ of fission yeast encodes a protein kinase potentially regulated by phosphorylation. Cell. 1986 Apr 25;45(2):261–268. doi: 10.1016/0092-8674(86)90390-9. [DOI] [PubMed] [Google Scholar]
  48. Stewart A. A., Hemmings B. A., Cohen P., Goris J., Merlevede W. The MgATP-dependent protein phosphatase and protein phosphatase 1 have identical substrate specificities. Eur J Biochem. 1981 Mar 16;115(1):197–205. doi: 10.1111/j.1432-1033.1981.tb06217.x. [DOI] [PubMed] [Google Scholar]
  49. da Cruz e Silva O. B., Cohen P. T. A second catalytic subunit of type-2A protein phosphatase from rabbit skeletal muscle. FEBS Lett. 1987 Dec 21;226(1):176–178. doi: 10.1016/0014-5793(87)80574-4. [DOI] [PubMed] [Google Scholar]
  50. da Cruz e Silva O. B., da Cruz e Silva E. F., Cohen P. T. Identification of a novel protein phosphatase catalytic subunit by cDNA cloning. FEBS Lett. 1988 Dec 19;242(1):106–110. doi: 10.1016/0014-5793(88)80995-5. [DOI] [PubMed] [Google Scholar]

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