Home LiteratureArticle Details
PMID: 12475960 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Identification of the nuclear localization signal in Xenopus cyclin E and analysis of its role in replication and mitosis.

Molecular biology of the cell ·Vol. 13 ·No. 12 ·2002-12-00 ·Pages 4388-400

Moore JD, Kornbluth S, Hunt T

Abstract

Cyclin-dependent kinase (Cdk)2/cyclin E is imported into nuclei assembled in Xenopus egg extracts by a pathway that requires importin-alpha and -beta. Here, we identify a basic nuclear localization sequence (NLS) in the N-terminus of Xenopus cyclin E. Mutation of the NLS eliminated nuclear accumulation of both cyclin E and Cdk2, and such versions of cyclin E were unable to trigger DNA replication. Addition of a heterologous NLS from SV40 large T antigen restored both nuclear targeting of Cdk2/cyclin E and DNA replication. We present evidence indicating that Cdk2/cyclin E complexes must become highly concentrated within nuclei to support replication and find that cyclin A can trigger replication at much lower intranuclear concentrations. We confirmed that depletion of endogenous cyclin E increases the concentration of cyclin B necessary to promote entry into mitosis. In contrast to its inability to promote DNA replication, cyclin E lacking its NLS was able to cooperate with cyclin B in promoting mitotic entry.

MeSH Terms
Active Transport, Cell Nucleus Amino Acid Sequence Animals CDC2-CDC28 Kinases Cell Nucleus/metabolism Cyclin E/genetics,metabolism Cyclin-Dependent Kinase 2 Cyclin-Dependent Kinases/metabolism Cytoplasm/metabolism DNA Replication Dose-Response Relationship, Drug Glutathione Transferase/metabolism Mitosis Molecular Sequence Data Mutation Nuclear Localization Signals/biosynthesis Plasmids/metabolism Protein Serine-Threonine Kinases/metabolism Protein Structure, Tertiary Sequence Homology, Amino Acid Xenopus/metabolism Xenopus Proteins
Chemicals
Cyclin E Nuclear Localization Signals Xenopus Proteins Glutathione Transferase Protein Serine-Threonine Kinases CDC2-CDC28 Kinases Cdk2 protein, Xenopus Cyclin-Dependent Kinase 2 Cyclin-Dependent Kinases
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Moore Jonathan D
Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD, United Kingdom.
Kornbluth Sally
Hunt Tim
References (55)
55 references, click to expand
  1. Four-dimensional control of the cell cycle.
    Nat Cell Biol. 1999 Jul;1(3):E73-9 PMID: 10559915
  2. Human cyclin A is required for mitosis until mid prophase.
    J Cell Biol. 1999 Oct 18;147(2):295-306 PMID: 10525536
  3. Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation.
    Genes Dev. 2000 Dec 15;14(24):3102-14 PMID: 11124803
  4. The localization of human cyclins B1 and B2 determines CDK1 substrate specificity and neither enzyme requires MEK to disassemble the Golgi apparatus.
    J Cell Biol. 2001 Mar 5;152(5):945-58 PMID: 11238451
  5. S and G2 phase roles for Cdk2 revealed by inducible expression of a dominant-negative mutant in human cells.
    Mol Cell Biol. 2001 Apr;21(8):2755-66 PMID: 11283255
  6. Cyclin A is destroyed in prometaphase and can delay chromosome alignment and anaphase.
    J Cell Biol. 2001 Apr 2;153(1):121-36 PMID: 11285279
  7. Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint.
    J Cell Biol. 2001 Apr 2;153(1):137-48 PMID: 11285280
  8. Cyclin-dependent kinase 2 nucleocytoplasmic translocation is regulated by extracellular regulated kinase.
    J Biol Chem. 2001 Jun 22;276(25):22404-9 PMID: 11304535
  9. Mitotic kinases as regulators of cell division and its checkpoints.
    Nat Rev Mol Cell Biol. 2001 Jan;2(1):21-32 PMID: 11413462
  10. Tumor-specific proteolytic processing of cyclin E generates hyperactive lower-molecular-weight forms.
    Mol Cell Biol. 2001 Sep;21(18):6254-69 PMID: 11509668
  11. Role for cyclin-dependent kinase 2 in mitosis exit.
    Curr Biol. 2001 Aug 7;11(15):1221-6 PMID: 11516956
  12. Human F-box protein hCdc4 targets cyclin E for proteolysis and is mutated in a breast cancer cell line.
    Nature. 2001 Sep 20;413(6853):316-22 PMID: 11565034
  13. Cyclin A- and cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm.
    Mol Biol Cell. 2002 Mar;13(3):1030-45 PMID: 11907280
  14. Distinct roles for cyclins E and A during DNA replication complex assembly and activation.
    Nat Cell Biol. 2002 Jul;4(7):523-8 PMID: 12080347
  15. The spindle checkpoint kinase bub1 and cyclin e/cdk2 both contribute to the establishment of meiotic metaphase arrest by cytostatic factor.
    Curr Biol. 2002 Jun 25;12(12):1027-33 PMID: 12123578
  16. A short amino acid sequence able to specify nuclear location.
    Cell. 1984 Dec;39(3 Pt 2):499-509 PMID: 6096007
  17. Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: studies in Xenopus.
    Cell. 1990 Jun 1;61(5):811-23 PMID: 2160859
  18. 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):2865-75 PMID: 2143983
  19. Cyclin B in Xenopus oocytes: implications for the mechanism of pre-MPF activation.
    EMBO J. 1991 Jan;10(1):177-82 PMID: 1824935
  20. Identification of cellular proteins that can interact specifically with the T/E1A-binding region of the retinoblastoma gene product.
    Cell. 1991 Feb 8;64(3):521-32 PMID: 1825028
  21. Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport.
    J Cell Biol. 1991 Oct;115(1):1-17 PMID: 1717476
  22. Cyclin A is required for the onset of DNA replication in mammalian fibroblasts.
    Cell. 1991 Dec 20;67(6):1169-79 PMID: 1836977
  23. A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2.
    J Biol Chem. 1992 Jan 25;267(3):1969-75 PMID: 1309805
  24. Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts.
    Methods Cell Biol. 1991;35:449-68 PMID: 1664032
  25. Cyclin A is required at two points in the human cell cycle.
    EMBO J. 1992 Mar;11(3):961-71 PMID: 1312467
  26. Cell cycle extracts.
    Methods Cell Biol. 1991;36:581-605 PMID: 1839804
  27. Nuclear translocation of viral Jun but not of cellular Jun is cell cycle dependent.
    Proc Natl Acad Sci U S A. 1992 May 15;89(10):4290-4 PMID: 1584763
  28. Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation.
    Cell. 1994 Apr 8;77(1):107-20 PMID: 8156587
  29. Maternal Xenopus Cdk2-cyclin E complexes function during meiotic and early embryonic cell cycles that lack a G1 phase.
    J Biol Chem. 1995 Mar 24;270(12):6843-55 PMID: 7896832
  30. Human cyclin E, a nuclear protein essential for the G1-to-S phase transition.
    Mol Cell Biol. 1995 May;15(5):2612-24 PMID: 7739542
  31. Control of the Cdc2/cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors.
    Mol Biol Cell. 1995 Feb;6(2):199-213 PMID: 7787246
  32. Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.
    Mol Biol Cell. 1995 Feb;6(2):215-26 PMID: 7787247
  33. Early events in DNA replication require cyclin E and are blocked by p21CIP1.
    J Cell Biol. 1995 Aug;130(4):755-69 PMID: 7642695
  34. Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins.
    Cell. 1995 Dec 1;83(5):683-92 PMID: 8521485
  35. Cdk2 kinase is required for entry into mitosis as a positive regulator of Cdc2-cyclin B kinase activity.
    Cell. 1996 Jan 12;84(1):73-82 PMID: 8548828
  36. S-phase-promoting cyclin-dependent kinases prevent re-replication by inhibiting the transition of replication origins to a pre-replicative state.
    Curr Biol. 1995 Nov 1;5(11):1257-69 PMID: 8574583
  37. The crystal structure of cyclin A.
    Structure. 1995 Nov 15;3(11):1235-47 PMID: 8591034
  38. Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.
    J Cell Sci. 1996 Jun;109 ( Pt 6):1555-63 PMID: 8799842
  39. Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells.
    Genes Dev. 1996 Nov 15;10(22):2819-30 PMID: 8918884
  40. A role for Cdk2 kinase in negatively regulating DNA replication during S phase of the cell cycle.
    J Cell Biol. 1997 Apr 7;137(1):183-92 PMID: 9105046
  41. S-phase function of Drosophila cyclin A and its downregulation in G1 phase.
    Curr Biol. 1997 Jul 1;7(7):488-99 PMID: 9210381
  42. CDK1 inactivation regulates anaphase spindle dynamics and cytokinesis in vivo.
    J Cell Biol. 1997 Jul 28;138(2):385-93 PMID: 9230080
  43. Cyclin-dependent kinases: engines, clocks, and microprocessors.
    Annu Rev Cell Dev Biol. 1997;13:261-91 PMID: 9442875
  44. Identification of a substrate-targeting domain in cyclin E necessary for phosphorylation of the retinoblastoma protein.
    Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2535-40 PMID: 9482921
  45. Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint.
    EMBO J. 1998 May 15;17(10):2728-35 PMID: 9582266
  46. Nuclear localization of cyclin B1 controls mitotic entry after DNA damage.
    J Cell Biol. 1998 May 18;141(4):875-85 PMID: 9585407
  47. Regulated chromosomal DNA replication in the absence of a nucleus.
    Mol Cell. 1998 Mar;1(4):519-29 PMID: 9660936
  48. MPF localization is controlled by nuclear export.
    EMBO J. 1998 Jul 15;17(14):4127-38 PMID: 9670027
  49. Control of cyclin B1 localization through regulated binding of the nuclear export factor CRM1.
    Genes Dev. 1998 Jul 15;12(14):2131-43 PMID: 9679058
  50. Nuclear import of Cdk/cyclin complexes: identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1.
    J Cell Biol. 1999 Jan 25;144(2):213-24 PMID: 9922449
  51. Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts.
    Science. 1999 Feb 5;283(5403):851-4 PMID: 9933170
  52. Cyclin-dependent kinase control of centrosome duplication.
    Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2817-22 PMID: 10077594
  53. Ran-independent nuclear import of cyclin B1-Cdc2 by importin beta.
    Proc Natl Acad Sci U S A. 1999 Jul 6;96(14):7938-43 PMID: 10393926
  54. Translocation of cyclin B1 to the nucleus at prophase requires a phosphorylation-dependent nuclear import signal.
    Curr Biol. 1999 Jul 1;9(13):680-9 PMID: 10395539
  55. Nuclei and microtubule asters stimulate maturation/M phase promoting factor (MPF) activation in Xenopus eggs and egg cytoplasmic extracts.
    J Cell Biol. 2000 Sep 4;150(5):963-74 PMID: 10973988
Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.e02-07-0449
Published
2002-12-00
Pages
4388-400
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC138641
Subset
IM
Grants
NIGMS NIH HHS · R01 GM060500 · United States
NIGMS NIH HHS · R01 GM60500 · United States
External Links
PubMed source DOI: 10.1091/mbc.e02-07-0449 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: product@genelibs.com

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2