Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Nov 1;109(5):1895–1909. doi: 10.1083/jcb.109.5.1895

Control of programmed cyclin destruction in a cell-free system

PMCID: PMC2115844  PMID: 2530237

Abstract

To ask what controls the periodic accumulation and destruction of the mitotic across the cell cycle, we have developed a cell-free system from clam embryos that reproduces several aspects of cyclin behavior. One or more rounds of cyclin proteolysis and resynthesis occur in vitro, and the destruction of the cyclins is highly specific. The onset, duration, and extent of cyclin destruction and the appropriately stagered disappearance of cyclin A and cyclin B are correctly regulated during the first cycle in the cell-free system. Just as in intact cells, lysates made from early interphase cells require further protein synthesis to reach the cyclin destruction point, and lysates made from later stages do not. Using the cell-free system we show that cyclin disappearance requires ATP and Mg2+. By combining lysates from different cell cycle stages, we show that (a) interphase lysates do not contain a dominant inhibitor of cyclin destruction and (b) the timing of cyclin destruction is determined by the cell cycle stage of the cytoplasm rather than the cell cycle stage of the substrate cyclins themselves. Among a large variety of agents tested, only a few affect cyclin destruction. Tosyl-lysine chlormethyl ketone (TLCK, a protease inhibitor), 6-dimethylaminopurine (6-DMAP, a kinase inhibitor), certain sulfhydryl-blocking agents, ZnCl2 and EDTA (but not EGTA) completely block cyclin destruction in vitro. Addition of 1 mM Ca2+ to the cell- free system has no effect on cyclin stability, but 5 mM Ca2+ leads to the rapid destruction of cyclins and a small number of other proteins.

Full Text

The Full Text of this article is available as a PDF (3.2 MB).

Selected References

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

  1. Anderson C. W., Baum P. R., Gesteland R. F. Processing of adenovirus 2-induced proteins. J Virol. 1973 Aug;12(2):241–252. doi: 10.1128/jvi.12.2.241-252.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrett A. J. Alpha 2-macroglobulin. Methods Enzymol. 1981;80(Pt 100):737–754. doi: 10.1016/s0076-6879(81)80056-0. [DOI] [PubMed] [Google Scholar]
  3. Bond J. S., Butler P. E. Intracellular proteases. Annu Rev Biochem. 1987;56:333–364. doi: 10.1146/annurev.bi.56.070187.002001. [DOI] [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. Interaction between cdc13+ and cdc2+ in the control of mitosis in fission yeast; dissociation of the G1 and G2 roles of the cdc2+ protein kinase. EMBO J. 1987 Nov;6(11):3441–3447. doi: 10.1002/j.1460-2075.1987.tb02667.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Booher R., Beach D. Involvement of cdc13+ in mitotic control in Schizosaccharomyces pombe: possible interaction of the gene product with microtubules. EMBO J. 1988 Aug;7(8):2321–2327. doi: 10.1002/j.1460-2075.1988.tb03075.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Borle A. B. Control, Modulation, and regulation of cell calcium. Rev Physiol Biochem Pharmacol. 1981;90:13–153. doi: 10.1007/BFb0034078. [DOI] [PubMed] [Google Scholar]
  8. Burke B., Gerace L. A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell. 1986 Feb 28;44(4):639–652. doi: 10.1016/0092-8674(86)90273-4. [DOI] [PubMed] [Google Scholar]
  9. Chou I. N., Black P. H., Roblin R. O. Non-selective inhibition of transformed cell growth by a protease inhibitor. Proc Natl Acad Sci U S A. 1974 May;71(5):1748–1752. doi: 10.1073/pnas.71.5.1748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crawford C., Mason R. W., Wikstrom P., Shaw E. The design of peptidyldiazomethane inhibitors to distinguish between the cysteine proteinases calpain II, cathepsin L and cathepsin B. Biochem J. 1988 Aug 1;253(3):751–758. doi: 10.1042/bj2530751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cyclin in fission yeast. Cell. 1988 Sep 9;54(6):738–740. doi: 10.1016/s0092-8674(88)90933-6. [DOI] [PubMed] [Google Scholar]
  12. Dessev G., Palazzo R., Rebhun L., Goldman R. Disassembly of the nuclear envelope of spisula oocytes in a cell-free system. Dev Biol. 1989 Feb;131(2):496–504. doi: 10.1016/s0012-1606(89)80020-x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Draetta G., Luca F., Westendorf J., Brizuela L., Ruderman J., Beach D. Cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF. Cell. 1989 Mar 10;56(5):829–838. doi: 10.1016/0092-8674(89)90687-9. [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. Etlinger J. D., Goldberg A. L. Control of protein degradation in reticulocytes and reticulocyte extracts by hemin. J Biol Chem. 1980 May 25;255(10):4563–4568. [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. Featherstone C. The complexities of the cell cycle. Trends Biochem Sci. 1989 Mar;14(3):85–87. doi: 10.1016/0968-0004(89)90125-4. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Haas A. L., Rose I. A. Hemin inhibits ATP-dependent ubiquitin-dependent proteolysis: role of hemin in regulating ubiquitin conjugate degradation. Proc Natl Acad Sci U S A. 1981 Nov;78(11):6845–6848. doi: 10.1073/pnas.78.11.6845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hagan I., Hayles J., Nurse P. Cloning and sequencing of the cyclin-related cdc13+ gene and a cytological study of its role in fission yeast mitosis. J Cell Sci. 1988 Dec;91(Pt 4):587–595. doi: 10.1242/jcs.91.4.587. [DOI] [PubMed] [Google Scholar]
  22. Hepler P. K. Calcium restriction prolongs metaphase in dividing Tradescantia stamen hair cells. J Cell Biol. 1985 May;100(5):1363–1368. doi: 10.1083/jcb.100.5.1363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. James G. T. Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers. Anal Biochem. 1978 Jun 1;86(2):574–579. doi: 10.1016/0003-2697(78)90784-4. [DOI] [PubMed] [Google Scholar]
  24. Keith C. H., Ratan R., Maxfield F. R., Bajer A., Shelanski M. L. Local cytoplasmic calcium gradients in living mitotic cells. 1985 Aug 29-Sep 4Nature. 316(6031):848–850. doi: 10.1038/316848a0. [DOI] [PubMed] [Google Scholar]
  25. Kiehart D. P. Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium-sequestering system. J Cell Biol. 1981 Mar;88(3):604–617. doi: 10.1083/jcb.88.3.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kinzel V., König N. Interaction of protease inhibitors with the catalytic subunit of cAMP-dependent protein kinase. Biochem Biophys Res Commun. 1980 Mar 28;93(2):349–353. doi: 10.1016/0006-291x(80)91083-9. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. Laura R., Robison D. J., Bing D. H. (p-Amidinophenyl)methanesulfonyl fluoride, an irreversible inhibitor of serine proteases. Biochemistry. 1980 Oct 14;19(21):4859–4864. doi: 10.1021/bi00562a024. [DOI] [PubMed] [Google Scholar]
  30. Lehner C. F., O'Farrell P. H. Expression and function of Drosophila cyclin A during embryonic cell cycle progression. Cell. 1989 Mar 24;56(6):957–968. doi: 10.1016/0092-8674(89)90629-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Lodish H. G., Housman D., Jacobsen M. Initiation of hemoglobin synthesis. Specific inhibition by antibiotics and bacteriophage ribonucleic acid. Biochemistry. 1971 Jun 8;10(12):2348–2356. doi: 10.1021/bi00788a027. [DOI] [PubMed] [Google Scholar]
  32. Lohka M. J., Hayes M. K., Maller J. L. Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Proc Natl Acad Sci U S A. 1988 May;85(9):3009–3013. doi: 10.1073/pnas.85.9.3009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mazia D., Paweletz N., Sluder G., Finze E. M. Cooperation of kinetochores and pole in the establishment of monopolar mitotic apparatus. Proc Natl Acad Sci U S A. 1981 Jan;78(1):377–381. doi: 10.1073/pnas.78.1.377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meijer L., Pelech S. L., Krebs E. G. Differential regulation of histone H1 and ribosomal S6 kinases during sea star oocyte maturation. Biochemistry. 1987 Dec 1;26(24):7968–7974. doi: 10.1021/bi00398a063. [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. 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]
  37. 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]
  38. Nash R., Tokiwa G., Anand S., Erickson K., Futcher A. B. The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog. EMBO J. 1988 Dec 20;7(13):4335–4346. doi: 10.1002/j.1460-2075.1988.tb03332.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Néant I., Charbonneau M., Guerrier P. A requirement for protein phosphorylation in regulating the meiotic and mitotic cell cycles in echinoderms. Dev Biol. 1989 Apr;132(2):304–314. doi: 10.1016/0012-1606(89)90227-3. [DOI] [PubMed] [Google Scholar]
  40. Pelech S. L., Meijer L., Krebs E. G. Characterization of maturation-activated histone H1 and ribosomal S6 kinases in sea star oocytes. Biochemistry. 1987 Dec 1;26(24):7960–7968. doi: 10.1021/bi00398a062. [DOI] [PubMed] [Google Scholar]
  41. Penn A., Lake S., Timourian H., Gledhill B. L. Division delay in sea urchin embryos induced by a specific protease inhibitor. Exp Cell Res. 1976 Jan;97:164–174. doi: 10.1016/0014-4827(76)90665-0. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Poenie M., Alderton J., Steinhardt R., Tsien R. Calcium rises abruptly and briefly throughout the cell at the onset of anaphase. Science. 1986 Aug 22;233(4766):886–889. doi: 10.1126/science.3755550. [DOI] [PubMed] [Google Scholar]
  45. Poenie M., Alderton J., Tsien R. Y., Steinhardt R. A. Changes of free calcium levels with stages of the cell division cycle. Nature. 1985 May 9;315(6015):147–149. doi: 10.1038/315147a0. [DOI] [PubMed] [Google Scholar]
  46. Rebhun L. I., White D., Sander G., Ivy N. Cleavage inhibition in marine eggs by puromycin and 6-dimethylaminopurine. Exp Cell Res. 1973 Mar 15;77(1):312–318. doi: 10.1016/0014-4827(73)90582-x. [DOI] [PubMed] [Google Scholar]
  47. Rechsteiner M. Ubiquitin-mediated pathways for intracellular proteolysis. Annu Rev Cell Biol. 1987;3:1–30. doi: 10.1146/annurev.cb.03.110187.000245. [DOI] [PubMed] [Google Scholar]
  48. Reed S. I., Hadwiger J. A., Lörincz A. T. Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4055–4059. doi: 10.1073/pnas.82.12.4055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rime H., Neant I., Guerrier P., Ozon R. 6-Dimethylaminopurine (6-DMAP), a reversible inhibitor of the transition to metaphase during the first meiotic cell division of the mouse oocyte. Dev Biol. 1989 May;133(1):169–179. doi: 10.1016/0012-1606(89)90308-4. [DOI] [PubMed] [Google Scholar]
  50. Rosenthal E. T., Hunt T., Ruderman J. V. Selective translation of mRNA controls the pattern of protein synthesis during early development of the surf clam, Spisula solidissima. Cell. 1980 Jun;20(2):487–494. doi: 10.1016/0092-8674(80)90635-2. [DOI] [PubMed] [Google Scholar]
  51. SAKAI H., DAN K. Studies on sulfhydryl groups during cell division of sea urchin egg. I. Glutatione. Exp Cell Res. 1959 Jan;16(1):24–41. doi: 10.1016/0014-4827(59)90192-2. [DOI] [PubMed] [Google Scholar]
  52. Schollmeyer J. E. Calpain II involvement in mitosis. Science. 1988 May 13;240(4854):911–913. doi: 10.1126/science.2834825. [DOI] [PubMed] [Google Scholar]
  53. Shaw E., Green G. D. Inactivation of thiol proteases with peptidyl diazomethyl ketones. Methods Enzymol. 1981;80(Pt 100):820–826. doi: 10.1016/s0076-6879(81)80064-x. [DOI] [PubMed] [Google Scholar]
  54. Shoji-Kasai Y., Senshu M., Iwashita S., Imahori K. Thiol protease-specific inhibitor E-64 arrests human epidermoid carcinoma A431 cells at mitotic metaphase. Proc Natl Acad Sci U S A. 1988 Jan;85(1):146–150. doi: 10.1073/pnas.85.1.146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Silver R. B., Cole R. D., Cande W. Z. Isolation of mitotic apparatus containing vesicles with calcium sequestration activity. Cell. 1980 Feb;19(2):505–516. doi: 10.1016/0092-8674(80)90525-5. [DOI] [PubMed] [Google Scholar]
  56. Standart N., Minshull J., Pines J., Hunt T. Cyclin synthesis, modification and destruction during meiotic maturation of the starfish oocyte. Dev Biol. 1987 Nov;124(1):248–258. doi: 10.1016/0012-1606(87)90476-3. [DOI] [PubMed] [Google Scholar]
  57. Steinhardt R. A., Alderton J. Intracellular free calcium rise triggers nuclear envelope breakdown in the sea urchin embryo. Nature. 1988 Mar 24;332(6162):364–366. doi: 10.1038/332364a0. [DOI] [PubMed] [Google Scholar]
  58. Sugita H., Ishiura S., Suzuki K., Imahori K. Inhibition of epoxide derivatives on chicken calcium-activated neutral protease (CANP) in vitro and in vivo. J Biochem. 1980 Jan;87(1):339–341. doi: 10.1093/oxfordjournals.jbchem.a132742. [DOI] [PubMed] [Google Scholar]
  59. Swenson K. I., Farrell K. M., Ruderman J. V. The clam embryo protein cyclin A induces entry into M phase and the resumption of meiosis in Xenopus oocytes. Cell. 1986 Dec 26;47(6):861–870. doi: 10.1016/0092-8674(86)90801-9. [DOI] [PubMed] [Google Scholar]
  60. Twigg J., Patel R., Whitaker M. Translational control of InsP3-induced chromatin condensation during the early cell cycles of sea urchin embryos. Nature. 1988 Mar 24;332(6162):366–369. doi: 10.1038/332366a0. [DOI] [PubMed] [Google Scholar]
  61. Umezawa H. Structures and activities of protease inhibitors of microbial origin. Methods Enzymol. 1976;45:678–695. doi: 10.1016/s0076-6879(76)45058-9. [DOI] [PubMed] [Google Scholar]
  62. Weisenberg R. C. Microtubule formation in vitro in solutions containing low calcium concentrations. Science. 1972 Sep 22;177(4054):1104–1105. doi: 10.1126/science.177.4054.1104. [DOI] [PubMed] [Google Scholar]
  63. Westendorf J. M., Swenson K. I., Ruderman J. V. The role of cyclin B in meiosis I. J Cell Biol. 1989 Apr;108(4):1431–1444. doi: 10.1083/jcb.108.4.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Whitfield W. G., González C., Sánchez-Herrero E., Glover D. M. Transcripts of one of two Drosophila cyclin genes become localized in pole cells during embryogenesis. Nature. 1989 Mar 23;338(6213):337–340. doi: 10.1038/338337a0. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES