Skip to main content
PMC home page
Molecular Biology of the Cell logoLink to Molecular Biology of the Cell
. 1992 Nov;3(11):1279–1294. doi: 10.1091/mbc.3.11.1279

Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits.

H Kobayashi 1, E Stewart 1, R Poon 1, J P Adamczewski 1, J Gannon 1, T Hunt 1
PMCID: PMC275694  PMID: 1333843

Abstract

The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.

Full text

PDF
1279

Images in this article

1285Image
on p.1285
1286Image
on p.1286
1286Image
on p.1286
1287Image
on p.1287
1287Image
on p.1287
1288Image
on p.1288
1289Image
on p.1289
1290Image
on p.1290

Selected References

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

  1. 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]
  2. Cicirelli M. F., Pelech S. L., Krebs E. G. Activation of multiple protein kinases during the burst in protein phosphorylation that precedes the first meiotic cell division in Xenopus oocytes. J Biol Chem. 1988 Feb 5;263(4):2009–2019. [PubMed] [Google Scholar]
  3. Dasso M. C., Jackson R. J. Efficient initiation of mammalian mRNA translation at a CUG codon. Nucleic Acids Res. 1989 Aug 25;17(16):6485–6497. doi: 10.1093/nar/17.16.6485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Desai D., Gu Y., Morgan D. O. Activation of human cyclin-dependent kinases in vitro. Mol Biol Cell. 1992 May;3(5):571–582. doi: 10.1091/mbc.3.5.571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Devault A., Fesquet D., Cavadore J. C., Garrigues A. M., Labbé J. C., Lorca T., Picard A., Philippe M., Dorée M. Cyclin A potentiates maturation-promoting factor activation in the early Xenopus embryo via inhibition of the tyrosine kinase that phosphorylates cdc2. J Cell Biol. 1992 Sep;118(5):1109–1120. doi: 10.1083/jcb.118.5.1109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Ducommun B., Brambilla P., Félix M. A., Franza B. R., Jr, Karsenti E., Draetta G. cdc2 phosphorylation is required for its interaction with cyclin. EMBO J. 1991 Nov;10(11):3311–3319. doi: 10.1002/j.1460-2075.1991.tb04895.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. 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]
  11. Elledge S. J., Spottswood M. R. A new human p34 protein kinase, CDK2, identified by complementation of a cdc28 mutation in Saccharomyces cerevisiae, is a homolog of Xenopus Eg1. EMBO J. 1991 Sep;10(9):2653–2659. doi: 10.1002/j.1460-2075.1991.tb07808.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Evan G. I., Hancock D. C., Littlewood T., Pauza C. D. Characterization of the human c-myc protein using antibodies prepared against synthetic peptides. Ciba Found Symp. 1986;119:245–263. doi: 10.1002/9780470513286.ch14. [DOI] [PubMed] [Google Scholar]
  13. Fang F., Newport J. W. Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes. Cell. 1991 Aug 23;66(4):731–742. doi: 10.1016/0092-8674(91)90117-h. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Fleig U. N., Gould K. L. Regulation of cdc2 activity in Schizosaccharomyces pombe: the role of phosphorylation. Semin Cell Biol. 1991 Aug;2(4):195–204. [PubMed] [Google Scholar]
  16. Gabrielli B. G., Roy L. M., Gautier J., Philippe M., Maller J. L. A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2. J Biol Chem. 1992 Jan 25;267(3):1969–1975. [PubMed] [Google Scholar]
  17. 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]
  18. Gibbs C. S., Zoller M. J. Rational scanning mutagenesis of a protein kinase identifies functional regions involved in catalysis and substrate interactions. J Biol Chem. 1991 May 15;266(14):8923–8931. [PubMed] [Google Scholar]
  19. Girard F., Strausfeld U., Fernandez A., Lamb N. J. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts. Cell. 1991 Dec 20;67(6):1169–1179. doi: 10.1016/0092-8674(91)90293-8. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Gould K. L., Moreno S., Owen D. J., Sazer S., Nurse P. Phosphorylation at Thr167 is required for Schizosaccharomyces pombe p34cdc2 function. EMBO J. 1991 Nov;10(11):3297–3309. doi: 10.1002/j.1460-2075.1991.tb04894.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Gu Y., Rosenblatt J., Morgan D. O. Cell cycle regulation of CDK2 activity by phosphorylation of Thr160 and Tyr15. EMBO J. 1992 Nov;11(11):3995–4005. doi: 10.1002/j.1460-2075.1992.tb05493.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hadwiger J. A., Wittenberg C., Richardson H. E., de Barros Lopes M., Reed S. I. A family of cyclin homologs that control the G1 phase in yeast. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6255–6259. doi: 10.1073/pnas.86.16.6255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kobayashi H., Golsteyn R., Poon R., Stewart E., Gannon J., Minshull J., Smith R., Hunt T. Cyclins and their partners during Xenopus oocyte maturation. Cold Spring Harb Symp Quant Biol. 1991;56:437–447. doi: 10.1101/sqb.1991.056.01.051. [DOI] [PubMed] [Google Scholar]
  26. Kobayashi H., Minshull J., Ford C., Golsteyn R., Poon R., Hunt T. On the synthesis and destruction of A- and B-type cyclins during oogenesis and meiotic maturation in Xenopus laevis. J Cell Biol. 1991 Aug;114(4):755–765. doi: 10.1083/jcb.114.4.755. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. Lehner C. F., O'Farrell P. H. Drosophila cdc2 homologs: a functional homolog is coexpressed with a cognate variant. EMBO J. 1990 Nov;9(11):3573–3581. doi: 10.1002/j.1460-2075.1990.tb07568.x. [DOI] [PMC free article] [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. Lew D. J., Dulić V., Reed S. I. Isolation of three novel human cyclins by rescue of G1 cyclin (Cln) function in yeast. Cell. 1991 Sep 20;66(6):1197–1206. doi: 10.1016/0092-8674(91)90042-w. [DOI] [PubMed] [Google Scholar]
  32. Lohka M. J., Maller J. L. Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts. J Cell Biol. 1985 Aug;101(2):518–523. doi: 10.1083/jcb.101.2.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lorca T., Devault A., Colas P., Van Loon A., Fesquet D., Lazaro J. B., Dorée M. Cyclin A-Cys41 does not undergo cell cycle-dependent degradation in Xenopus extracts. FEBS Lett. 1992 Jul 13;306(1):90–93. doi: 10.1016/0014-5793(92)80844-7. [DOI] [PubMed] [Google Scholar]
  34. Luca F. C., Ruderman J. V. Control of programmed cyclin destruction in a cell-free system. J Cell Biol. 1989 Nov;109(5):1895–1909. doi: 10.1083/jcb.109.5.1895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Luca F. C., Shibuya E. K., Dohrmann C. E., Ruderman J. V. Both cyclin A delta 60 and B delta 97 are stable and arrest cells in M-phase, but only cyclin B delta 97 turns on cyclin destruction. EMBO J. 1991 Dec;10(13):4311–4320. doi: 10.1002/j.1460-2075.1991.tb05009.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Léopold P., O'Farrell P. H. An evolutionarily conserved cyclin homolog from Drosophila rescues yeast deficient in G1 cyclins. Cell. 1991 Sep 20;66(6):1207–1216. doi: 10.1016/0092-8674(91)90043-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Maller J. L. Mitotic control. Curr Opin Cell Biol. 1991 Apr;3(2):269–275. doi: 10.1016/0955-0674(91)90151-n. [DOI] [PubMed] [Google Scholar]
  38. Matsushime H., Roussel M. F., Ashmun R. A., Sherr C. J. Colony-stimulating factor 1 regulates novel cyclins during the G1 phase of the cell cycle. Cell. 1991 May 17;65(4):701–713. doi: 10.1016/0092-8674(91)90101-4. [DOI] [PubMed] [Google Scholar]
  39. Meyerson M., Enders G. H., Wu C. L., Su L. K., Gorka C., Nelson C., Harlow E., Tsai L. H. A family of human cdc2-related protein kinases. EMBO J. 1992 Aug;11(8):2909–2917. doi: 10.1002/j.1460-2075.1992.tb05360.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Milarski K. L., Dunphy W. G., Russell P., Gould S. J., Newport J. W. Cloning and characterization of Xenopus cdc2, a component of MPF. Cold Spring Harb Symp Quant Biol. 1991;56:377–384. doi: 10.1101/sqb.1991.056.01.045. [DOI] [PubMed] [Google Scholar]
  41. Millar J. B., Russell P. The cdc25 M-phase inducer: an unconventional protein phosphatase. Cell. 1992 Feb 7;68(3):407–410. doi: 10.1016/0092-8674(92)90177-e. [DOI] [PubMed] [Google Scholar]
  42. Minshull J., Golsteyn R., Hill C. S., Hunt T. The A- and B-type cyclin associated cdc2 kinases in Xenopus turn on and off at different times in the cell cycle. EMBO J. 1990 Sep;9(9):2865–2875. doi: 10.1002/j.1460-2075.1990.tb07476.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Minshull J., Murray A., Colman A., Hunt T. Xenopus oocyte maturation does not require new cyclin synthesis. J Cell Biol. 1991 Aug;114(4):767–772. doi: 10.1083/jcb.114.4.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. 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]
  46. 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]
  47. Nielsen D. A., Shapiro D. J. Preparation of capped RNA transcripts using T7 RNA polymerase. Nucleic Acids Res. 1986 Jul 25;14(14):5936–5936. doi: 10.1093/nar/14.14.5936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Nugent J. H., Alfa C. E., Young T., Hyams J. S. Conserved structural motifs in cyclins identified by sequence analysis. J Cell Sci. 1991 Jul;99(Pt 3):669–674. doi: 10.1242/jcs.99.3.669. [DOI] [PubMed] [Google Scholar]
  49. Nurse P. Universal control mechanism regulating onset of M-phase. Nature. 1990 Apr 5;344(6266):503–508. doi: 10.1038/344503a0. [DOI] [PubMed] [Google Scholar]
  50. Pagano M., Pepperkok R., Verde F., Ansorge W., Draetta G. Cyclin A is required at two points in the human cell cycle. EMBO J. 1992 Mar;11(3):961–971. doi: 10.1002/j.1460-2075.1992.tb05135.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Paris J., Le Guellec R., Couturier A., Le Guellec K., Omilli F., Camonis J., MacNeill S., Philippe M. Cloning by differential screening of a Xenopus cDNA coding for a protein highly homologous to cdc2. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):1039–1043. doi: 10.1073/pnas.88.3.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. 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]
  54. Pines J., Hunter T. p34cdc2: the S and M kinase? New Biol. 1990 May;2(5):389–401. [PubMed] [Google Scholar]
  55. Pondaven P., Meijer L., Beach D. Activation of M-phase-specific histone H1 kinase by modification of the phosphorylation of its p34cdc2 and cyclin components. Genes Dev. 1990 Jan;4(1):9–17. doi: 10.1101/gad.4.1.9. [DOI] [PubMed] [Google Scholar]
  56. Roy L. M., Swenson K. I., Walker D. H., Gabrielli B. G., Li R. S., Piwnica-Worms H., Maller J. L. Activation of p34cdc2 kinase by cyclin A. J Cell Biol. 1991 May;113(3):507–514. doi: 10.1083/jcb.113.3.507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Shamu C. E., Murray A. W. Sister chromatid separation in frog egg extracts requires DNA topoisomerase II activity during anaphase. J Cell Biol. 1992 Jun;117(5):921–934. doi: 10.1083/jcb.117.5.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  60. 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]
  61. Tsai L. H., Harlow E., Meyerson M. Isolation of the human cdk2 gene that encodes the cyclin A- and adenovirus E1A-associated p33 kinase. Nature. 1991 Sep 12;353(6340):174–177. doi: 10.1038/353174a0. [DOI] [PubMed] [Google Scholar]
  62. Wang J., Chenivesse X., Henglein B., Bréchot C. Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma. Nature. 1990 Feb 8;343(6258):555–557. doi: 10.1038/343555a0. [DOI] [PubMed] [Google Scholar]
  63. Xiong Y., Connolly T., Futcher B., Beach D. Human D-type cyclin. Cell. 1991 May 17;65(4):691–699. doi: 10.1016/0092-8674(91)90100-d. [DOI] [PubMed] [Google Scholar]
  64. Yamashita M., Fukada S., Yoshikuni M., Bulet P., Hirai T., Yamaguchi A., Lou Y. H., Zhao Z., Nagahama Y. Purification and characterization of maturation-promoting factor in fish. Dev Biol. 1992 Jan;149(1):8–15. doi: 10.1016/0012-1606(92)90259-j. [DOI] [PubMed] [Google Scholar]
  65. van Loon A. E., Colas P., Goedemans H. J., Néant I., Dalbon P., Guerrier P. The role of cyclins in the maturation of Patella vulgata oocytes. EMBO J. 1991 Nov;10(11):3343–3349. doi: 10.1002/j.1460-2075.1991.tb04898.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular Biology of the Cell are provided here courtesy of American Society for Cell Biology

RESOURCES