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. 1994 Sep;5(9):989–1001. doi: 10.1091/mbc.5.9.989

Cell cycle regulation of the p34cdc2 inhibitory kinases.

S Atherton-Fessler 1, F Liu 1, B Gabrielli 1, M S Lee 1, C Y Peng 1, H Piwnica-Worms 1
PMCID: PMC301122  PMID: 7841526

Abstract

In cells of higher eukaryotic organisms the activity of the p34cdc2/cyclin B complex is inhibited by phosphorylation of p34cdc2 at two sites within its amino-terminus (threonine 14 and tyrosine 15). In this study, the cell cycle regulation of the kinases responsible for phosphorylating p34cdc2 on Thr14 and Tyr15 was examined in extracts prepared from both HeLa cells and Xenopus eggs. Both Thr14- and Tyr15- specific kinase activities were regulated in a cell cycle-dependent manner. The kinase activities were high throughout interphase and diminished coincident with entry of cells into mitosis. In HeLa cells delayed in G2 by the DNA-binding dye Hoechst 33342, Thr14- and Tyr15-specific kinase activities remained high, suggesting that a decrease in Thr14- and Tyr15- kinase activities may be required for entry of cells into mitosis. Similar cell cycle regulation was observed for the Thr14/Tyr15 kinase(s) in Xenopus egg extracts. These results indicate that activation of CDC2 and entry of cells into mitosis is not triggered solely by activation of the Cdc25 phosphatase but by the balance between Thr14/Tyr15 kinase and phosphatase activities. Finally, we have detected two activities capable of phosphorylating p34cdc2 on Thr14 and/or Tyr15 in interphase extracts prepared from Xenopus eggs. An activity capable of phosphorylating Tyr15 remained soluble after ultracentrifugation of interphase extracts whereas a second activity capable of phosphorylating both Thr14 and Tyr15 pelleted. The pelleted fraction contained activities that were detergent extractable and that phosphorylated p34cdc2 on both Thr14 and Tyr15. The Thr14- and Tyr15-specific kinase activities co-purified through three successive chromatographic steps indicating the presence of a dual-specificity protein kinase capable of acting on p34cdc2.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Atherton-Fessler S., Parker L. L., Geahlen R. L., Piwnica-Worms H. Mechanisms of p34cdc2 regulation. Mol Cell Biol. 1993 Mar;13(3):1675–1685. doi: 10.1128/mcb.13.3.1675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Clarke P. R., Hoffmann I., Draetta G., Karsenti E. Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific regulation during the cell cycle in Xenopus egg extracts. Mol Biol Cell. 1993 Apr;4(4):397–411. doi: 10.1091/mbc.4.4.397. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Coleman T. R., Tang Z., Dunphy W. G. Negative regulation of the wee1 protein kinase by direct action of the nim1/cdr1 mitotic inducer. Cell. 1993 Mar 26;72(6):919–929. doi: 10.1016/0092-8674(93)90580-j. [DOI] [PubMed] [Google Scholar]
  4. Crissman H. A., Hofland M. H., Stevenson A. P., Wilder M. E., Tobey R. A. Supravital cell staining with Hoechst 33342 and DiOC5(3). Methods Cell Biol. 1990;33:89–95. doi: 10.1016/s0091-679x(08)60514-2. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Draetta G., Piwnica-Worms H., Morrison D., Druker B., Roberts T., Beach D. Human cdc2 protein kinase is a major cell-cycle regulated tyrosine kinase substrate. Nature. 1988 Dec 22;336(6201):738–744. doi: 10.1038/336738a0. [DOI] [PubMed] [Google Scholar]
  7. Ducommun B., Draetta G., Young P., Beach D. Fission yeast cdc25 is a cell-cycle regulated protein. Biochem Biophys Res Commun. 1990 Feb 28;167(1):301–309. doi: 10.1016/0006-291x(90)91765-k. [DOI] [PubMed] [Google Scholar]
  8. Dunphy W. G., Kumagai A. The cdc25 protein contains an intrinsic phosphatase activity. Cell. 1991 Oct 4;67(1):189–196. doi: 10.1016/0092-8674(91)90582-j. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Edgar B. A., O'Farrell P. H. Genetic control of cell division patterns in the Drosophila embryo. Cell. 1989 Apr 7;57(1):177–187. doi: 10.1016/0092-8674(89)90183-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Edgar B. A., O'Farrell P. H. The three postblastoderm cell cycles of Drosophila embryogenesis are regulated in G2 by string. Cell. 1990 Aug 10;62(3):469–480. doi: 10.1016/0092-8674(90)90012-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Featherstone C., Russell P. Fission yeast p107wee1 mitotic inhibitor is a tyrosine/serine kinase. Nature. 1991 Feb 28;349(6312):808–811. doi: 10.1038/349808a0. [DOI] [PubMed] [Google Scholar]
  13. Galaktionov K., Beach D. Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins. Cell. 1991 Dec 20;67(6):1181–1194. doi: 10.1016/0092-8674(91)90294-9. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Gautier J., Solomon M. J., Booher R. N., Bazan J. F., Kirschner M. W. cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2. Cell. 1991 Oct 4;67(1):197–211. doi: 10.1016/0092-8674(91)90583-k. [DOI] [PubMed] [Google Scholar]
  16. Glotzer M., Murray A. W., Kirschner M. W. Cyclin is degraded by the ubiquitin pathway. Nature. 1991 Jan 10;349(6305):132–138. doi: 10.1038/349132a0. [DOI] [PubMed] [Google Scholar]
  17. Hannig G., Ottilie S., Schievella A. R., Erikson R. L. Comparison of the biochemical and biological functions of tyrosine phosphatases from fission yeast, budding yeast and animal cells. Yeast. 1993 Oct;9(10):1039–1052. doi: 10.1002/yea.320091002. [DOI] [PubMed] [Google Scholar]
  18. Hoffmann I., Clarke P. R., Marcote M. J., Karsenti E., Draetta G. Phosphorylation and activation of human cdc25-C by cdc2--cyclin B and its involvement in the self-amplification of MPF at mitosis. EMBO J. 1993 Jan;12(1):53–63. doi: 10.1002/j.1460-2075.1993.tb05631.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Honda R., Ohba Y., Yasuda H. The cell cycle regulator, human p50weel, is a tyrosine kinase and not a serine/tyrosine kinase. Biochem Biophys Res Commun. 1992 Aug 14;186(3):1333–1338. doi: 10.1016/s0006-291x(05)81552-9. [DOI] [PubMed] [Google Scholar]
  20. Igarashi M., Nagata A., Jinno S., Suto K., Okayama H. Wee1(+)-like gene in human cells. Nature. 1991 Sep 5;353(6339):80–83. doi: 10.1038/353080a0. [DOI] [PubMed] [Google Scholar]
  21. Izumi T., Walker D. H., Maller J. L. Periodic changes in phosphorylation of the Xenopus cdc25 phosphatase regulate its activity. Mol Biol Cell. 1992 Aug;3(8):927–939. doi: 10.1091/mbc.3.8.927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jessus C., Beach D. Oscillation of MPF is accompanied by periodic association between cdc25 and cdc2-cyclin B. Cell. 1992 Jan 24;68(2):323–332. doi: 10.1016/0092-8674(92)90473-p. [DOI] [PubMed] [Google Scholar]
  23. Kornbluth S., Sebastian B., Hunter T., Newport J. Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14. Mol Biol Cell. 1994 Mar;5(3):273–282. doi: 10.1091/mbc.5.3.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Krek W., Nigg E. A. Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites. EMBO J. 1991 Feb;10(2):305–316. doi: 10.1002/j.1460-2075.1991.tb07951.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kumagai A., Dunphy W. G. Regulation of the cdc25 protein during the cell cycle in Xenopus extracts. Cell. 1992 Jul 10;70(1):139–151. doi: 10.1016/0092-8674(92)90540-s. [DOI] [PubMed] [Google Scholar]
  26. Lee M. S., Ogg S., Xu M., Parker L. L., Donoghue D. J., Maller J. L., Piwnica-Worms H. cdc25+ encodes a protein phosphatase that dephosphorylates p34cdc2. Mol Biol Cell. 1992 Jan;3(1):73–84. doi: 10.1091/mbc.3.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lorca T., Labbé J. C., Devault A., Fesquet D., Capony J. P., Cavadore J. C., Le Bouffant F., Dorée M. Dephosphorylation of cdc2 on threonine 161 is required for cdc2 kinase inactivation and normal anaphase. EMBO J. 1992 Jul;11(7):2381–2390. doi: 10.1002/j.1460-2075.1992.tb05302.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lundgren K., Walworth N., Booher R., Dembski M., Kirschner M., Beach D. mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell. 1991 Mar 22;64(6):1111–1122. doi: 10.1016/0092-8674(91)90266-2. [DOI] [PubMed] [Google Scholar]
  29. McGowan C. H., Russell P. Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. EMBO J. 1993 Jan;12(1):75–85. doi: 10.1002/j.1460-2075.1993.tb05633.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Meijer L., Azzi L., Wang J. Y. Cyclin B targets p34cdc2 for tyrosine phosphorylation. EMBO J. 1991 Jun;10(6):1545–1554. doi: 10.1002/j.1460-2075.1991.tb07674.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Millar J. B., Blevitt J., Gerace L., Sadhu K., Featherstone C., Russell P. p55CDC25 is a nuclear protein required for the initiation of mitosis in human cells. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10500–10504. doi: 10.1073/pnas.88.23.10500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Millar J. B., Lenaers G., Russell P. Pyp3 PTPase acts as a mitotic inducer in fission yeast. EMBO J. 1992 Dec;11(13):4933–4941. doi: 10.1002/j.1460-2075.1992.tb05600.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Millar J. B., McGowan C. H., Lenaers G., Jones R., Russell P. p80cdc25 mitotic inducer is the tyrosine phosphatase that activates p34cdc2 kinase in fission yeast. EMBO J. 1991 Dec;10(13):4301–4309. doi: 10.1002/j.1460-2075.1991.tb05008.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Moreno S., Nurse P., Russell P. Regulation of mitosis by cyclic accumulation of p80cdc25 mitotic inducer in fission yeast. Nature. 1990 Apr 5;344(6266):549–552. doi: 10.1038/344549a0. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Motokura T., Bloom T., Kim H. G., Jüppner H., Ruderman J. V., Kronenberg H. M., Arnold A. A novel cyclin encoded by a bcl1-linked candidate oncogene. Nature. 1991 Apr 11;350(6318):512–515. doi: 10.1038/350512a0. [DOI] [PubMed] [Google Scholar]
  37. 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]
  38. Norbury C., Blow J., Nurse P. Regulatory phosphorylation of the p34cdc2 protein kinase in vertebrates. EMBO J. 1991 Nov;10(11):3321–3329. doi: 10.1002/j.1460-2075.1991.tb04896.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Parker L. L., Atherton-Fessler S., Lee M. S., Ogg S., Falk J. L., Swenson K. I., Piwnica-Worms H. Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee1+ dependent manner. EMBO J. 1991 May;10(5):1255–1263. doi: 10.1002/j.1460-2075.1991.tb08067.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Parker L. L., Atherton-Fessler S., Piwnica-Worms H. p107wee1 is a dual-specificity kinase that phosphorylates p34cdc2 on tyrosine 15. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2917–2921. doi: 10.1073/pnas.89.7.2917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Parker L. L., Piwnica-Worms H. Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science. 1992 Sep 25;257(5078):1955–1957. doi: 10.1126/science.1384126. [DOI] [PubMed] [Google Scholar]
  42. Parker L. L., Walter S. A., Young P. G., Piwnica-Worms H. Phosphorylation and inactivation of the mitotic inhibitor Wee1 by the nim1/cdr1 kinase. Nature. 1993 Jun 24;363(6431):736–738. doi: 10.1038/363736a0. [DOI] [PubMed] [Google Scholar]
  43. Russell P., Nurse P. Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog. Cell. 1987 May 22;49(4):559–567. doi: 10.1016/0092-8674(87)90458-2. [DOI] [PubMed] [Google Scholar]
  44. Russell P., Nurse P. cdc25+ functions as an inducer in the mitotic control of fission yeast. Cell. 1986 Apr 11;45(1):145–153. doi: 10.1016/0092-8674(86)90546-5. [DOI] [PubMed] [Google Scholar]
  45. Sebastian B., Kakizuka A., Hunter T. Cdc25M2 activation of cyclin-dependent kinases by dephosphorylation of threonine-14 and tyrosine-15. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3521–3524. doi: 10.1073/pnas.90.8.3521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Smythe C., Newport J. W. Coupling of mitosis to the completion of S phase in Xenopus occurs via modulation of the tyrosine kinase that phosphorylates p34cdc2. Cell. 1992 Feb 21;68(4):787–797. doi: 10.1016/0092-8674(92)90153-4. [DOI] [PubMed] [Google Scholar]
  47. Solomon M. J., Glotzer M., Lee T. H., Philippe M., Kirschner M. W. Cyclin activation of p34cdc2. Cell. 1990 Nov 30;63(5):1013–1024. doi: 10.1016/0092-8674(90)90504-8. [DOI] [PubMed] [Google Scholar]
  48. Solomon M. J., Lee T., Kirschner M. W. Role of phosphorylation in p34cdc2 activation: identification of an activating kinase. Mol Biol Cell. 1992 Jan;3(1):13–27. doi: 10.1091/mbc.3.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Strausfeld U., Labbé J. C., Fesquet D., Cavadore J. C., Picard A., Sadhu K., Russell P., Dorée M. Dephosphorylation and activation of a p34cdc2/cyclin B complex in vitro by human CDC25 protein. Nature. 1991 May 16;351(6323):242–245. doi: 10.1038/351242a0. [DOI] [PubMed] [Google Scholar]
  50. Tang Z., Coleman T. R., Dunphy W. G. Two distinct mechanisms for negative regulation of the Wee1 protein kinase. EMBO J. 1993 Sep;12(9):3427–3436. doi: 10.1002/j.1460-2075.1993.tb06017.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wu L., Russell P. Nim1 kinase promotes mitosis by inactivating Wee1 tyrosine kinase. Nature. 1993 Jun 24;363(6431):738–741. doi: 10.1038/363738a0. [DOI] [PubMed] [Google Scholar]
  52. Zheng X. F., Ruderman J. V. Functional analysis of the P box, a domain in cyclin B required for the activation of Cdc25. Cell. 1993 Oct 8;75(1):155–164. [PubMed] [Google Scholar]

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