Home LiteratureArticle Details
PMID: 13679510 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Importin alpha/beta and Ran-GTP regulate XCTK2 microtubule binding through a bipartite nuclear localization signal.

Molecular biology of the cell ·Vol. 15 ·No. 1 ·2004-01-00 ·Pages 46-57

Ems-McClung SC, Zheng Y, Walczak CE

Abstract

The small GTPase Ran is essential for spindle assembly. Ran is proposed to act through its nuclear import receptors importin alpha and/or importin beta to control the sequestration of proteins necessary for spindle assembly. To date, the molecular mechanisms by which the Ran pathway functions remain unclear. Using purified proteins, we have reconstituted Ran-regulated microtubule binding of the C-terminal kinesin XCTK2, a kinesin important for spindle assembly. We show that the tail of XCTK2 binds to microtubules and that this binding is inhibited in the presence of importin alpha and beta (alpha/beta) and restored by addition of Ran-GTP. The bipartite nuclear localization signal (NLS) in the tail of XCTK2 is essential to this process, because mutation of the NLS abolishes importin alpha/beta-mediated regulation of XCTK2 microtubule binding. Our data show that importin alpha/beta directly regulates the activity of XCTK2 and that one of the molecular mechanisms of Ran-regulated spindle assembly is identical to that used in classical NLS-driven nuclear transport.

MeSH Terms
Animals Cells, Cultured Escherichia coli Kinesins/metabolism Microtubules/metabolism Models, Molecular Mutagenesis, Site-Directed Nuclear Localization Signals/metabolism Protein Binding Protein Structure, Tertiary/physiology Recombinant Proteins/metabolism Spindle Apparatus/metabolism Xenopus Proteins/metabolism Xenopus laevis/metabolism alpha Karyopherins/metabolism beta Karyopherins/metabolism ran GTP-Binding Protein/metabolism
Chemicals
KIFC1 protein, Xenopus Nuclear Localization Signals Recombinant Proteins Xenopus Proteins alpha Karyopherins beta Karyopherins Kinesins ran GTP-Binding Protein
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Ems-McClung Stephanie C
Medical Sciences Program, Indiana University, Bloomington, Indiana 47405, USA.
Zheng Yixian
Walczak Claire E
References (68)
68 references, click to expand
  1. C-terminal kinesin motor KIFC1 participates in acrosome biogenesis and vesicle transport.
    Biol Reprod. 2003 Nov;69(5):1719-29 PMID: 12826589
  2. Structural and functional domains of the Drosophila ncd microtubule motor protein.
    J Biol Chem. 1993 Apr 25;268(12):9005-13 PMID: 8473343
  3. A mutant form of the Ran/TC4 protein disrupts nuclear function in Xenopus laevis egg extracts by inhibiting the RCC1 protein, a regulator of chromosome condensation.
    EMBO J. 1994 Dec 1;13(23):5732-44 PMID: 7988569
  4. Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1.
    Biochemistry. 1995 Jan 17;34(2):639-47 PMID: 7819259
  5. Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1.
    J Virol. 1996 Feb;70(2):1317-22 PMID: 8551602
  6. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts.
    Nature. 1996 Aug 1;382(6590):420-5 PMID: 8684481
  7. Opposing motor activities are required for the organization of the mammalian mitotic spindle pole.
    J Cell Biol. 1996 Oct;135(2):399-414 PMID: 8896597
  8. A complex of NuMA and cytoplasmic dynein is essential for mitotic spindle assembly.
    Cell. 1996 Nov 1;87(3):447-58 PMID: 8898198
  9. Centrosome and spindle function of the Drosophila Ncd microtubule motor visualized in live embryos using Ncd-GFP fusion proteins.
    J Cell Sci. 1996 Oct;109 ( Pt 10):2429-42 PMID: 8923204
  10. Mutations within the Ran/TC4 GTPase. Effects on regulatory factor interactions and subcellular localization.
    J Biol Chem. 1996 Dec 20;271(51):32834-41 PMID: 8955121
  11. Different binding domains for Ran-GTP and Ran-GDP/RanBP1 on nuclear import factor p97.
    J Biol Chem. 1997 Mar 7;272(10):6818-22 PMID: 9045717
  12. XCTK2: a kinesin-related protein that promotes mitotic spindle assembly in Xenopus laevis egg extracts.
    J Cell Biol. 1997 Feb 24;136(4):859-70 PMID: 9049251
  13. Microtubule interaction site of the kinesin motor.
    Cell. 1997 Jul 25;90(2):207-16 PMID: 9244295
  14. Kinetic characterization of the human retinoblastoma protein bipartite nuclear localization sequence (NLS) in vivo and in vitro. A comparison with the SV40 large T-antigen NLS.
    J Biol Chem. 1997 Aug 29;272(35):22134-9 PMID: 9268357
  15. Pathways of spindle pole formation: different mechanisms; conserved components.
    J Cell Biol. 1997 Sep 8;138(5):953-6 PMID: 9281574
  16. Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha.
    Cell. 1998 Jul 24;94(2):193-204 PMID: 9695948
  17. A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity.
    Curr Biol. 1998 Jul 30-Aug 13;8(16):903-13 PMID: 9707401
  18. Equilibrium binding studies of non-claret disjunctional protein (Ncd) reveal cooperative interactions between the motor domains.
    J Biol Chem. 1998 Dec 25;273(52):35307-18 PMID: 9857072
  19. The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.
    Methods Cell Biol. 1999;61:385-412 PMID: 9891325
  20. Kin I kinesins are microtubule-destabilizing enzymes.
    Cell. 1999 Jan 8;96(1):69-78 PMID: 9989498
  21. Identification of microtubule binding sites in the Ncd tail domain.
    Biochemistry. 1999 Feb 9;38(6):1838-49 PMID: 10026264
  22. Autoinhibition by an internal nuclear localization signal revealed by the crystal structure of mammalian importin alpha.
    Nat Struct Biol. 1999 Apr;6(4):388-97 PMID: 10201409
  23. The ran GTPase regulates mitotic spindle assembly.
    Curr Biol. 1999 May 6;9(9):481-4 PMID: 10322113
  24. Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import. Differential role of NTF2.
    J Biol Chem. 1999 May 28;274(22):15820-7 PMID: 10336485
  25. Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran.
    Science. 1999 May 21;284(5418):1356-8 PMID: 10334990
  26. Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran.
    Science. 1999 May 21;284(5418):1359-62 PMID: 10334991
  27. Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation.
    Nature. 1999 Jul 8;400(6740):178-81 PMID: 10408446
  28. Beyond nuclear transport. Ran-GTP as a determinant of spindle assembly.
    J Cell Biol. 1999 Sep 20;146(6):1205-10 PMID: 10491385
  29. The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle.
    J Cell Biol. 1999 Oct 18;147(2):351-66 PMID: 10525540
  30. The Ran GTPase as a marker of chromosome position in spindle formation and nuclear envelope assembly.
    Nat Cell Biol. 2002 Jul;4(7):E177-84 PMID: 12105431
  31. Ran localizes around the microtubule spindle in vivo during mitosis in Drosophila embryos.
    Curr Biol. 2002 Jul 9;12(13):1124-9 PMID: 12121620
  32. Ran binds to chromatin by two distinct mechanisms.
    Curr Biol. 2002 Jul 9;12(13):1151-6 PMID: 12121625
  33. The Ran GTPase: theme and variations.
    Curr Biol. 2002 Jul 23;12(14):R502-8 PMID: 12176353
  34. Targeting of RCC1 to chromosomes is required for proper mitotic spindle assembly in human cells.
    Curr Biol. 2002 Aug 20;12(16):1442-7 PMID: 12194828
  35. Function of a minus-end-directed kinesin-like motor protein in mammalian cells.
    J Cell Sci. 1999 Nov;112 ( Pt 22):4041-50 PMID: 10547364
  36. 15 A resolution model of the monomeric kinesin motor, KIF1A.
    Cell. 2000 Jan 21;100(2):241-52 PMID: 10660047
  37. Identification of isoforms of a mitotic motor in mammalian spermatogenesis.
    Biol Reprod. 2000 May;62(5):1360-9 PMID: 10775188
  38. Nuclear targeting signal recognition: a key control point in nuclear transport?
    Bioessays. 2000 Jun;22(6):532-44 PMID: 10842307
  39. Quantitative analysis of nuclear localization signal (NLS)-importin alpha interaction through fluorescence depolarization. Evidence for auto-inhibitory regulation of NLS binding.
    J Biol Chem. 2000 Jul 14;275(28):21218-23 PMID: 10806202
  40. Spindle assembly in animal cells.
    Annu Rev Biochem. 2000;69:95-114 PMID: 10966454
  41. Assays for microtubule-destabilizing kinesins.
    Methods Mol Biol. 2001;164:109-21 PMID: 11217601
  42. Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity.
    Cell. 2001 Jan 12;104(1):83-93 PMID: 11163242
  43. Importin beta is a mitotic target of the small GTPase Ran in spindle assembly.
    Cell. 2001 Jan 12;104(1):95-106 PMID: 11163243
  44. Role of importin-beta in coupling Ran to downstream targets in microtubule assembly.
    Science. 2001 Jan 26;291(5504):653-6 PMID: 11229403
  45. Dissection of a nuclear localization signal.
    J Biol Chem. 2001 Jan 12;276(2):1317-25 PMID: 11038364
  46. Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities.
    Nat Cell Biol. 2001 Mar;3(3):221-7 PMID: 11231570
  47. Ran-GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly.
    Nat Cell Biol. 2001 Mar;3(3):228-34 PMID: 11231571
  48. Ran hits the ground running.
    Nat Cell Biol. 2001 Mar;3(3):E69-70 PMID: 11231589
  49. Biophysical characterization of interactions involving importin-alpha during nuclear import.
    J Biol Chem. 2001 Sep 7;276(36):34189-98 PMID: 11448961
  50. Nucleocytoplasmic transport: Ran, beta and beyond.
    Trends Cell Biol. 2001 Dec;11(12):497-503 PMID: 11719056
  51. Karyopherins and nuclear import.
    Curr Opin Struct Biol. 2001 Dec;11(6):703-15 PMID: 11751052
  52. Role of importin-beta in the control of nuclear envelope assembly by Ran.
    Curr Biol. 2002 Mar 19;12(6):498-502 PMID: 11909537
  53. The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo.
    Curr Biol. 2002 Mar 19;12(6):503-7 PMID: 11909538
  54. Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts.
    Science. 2002 Mar 29;295(5564):2452-6 PMID: 11923538
  55. Microscopic evidence for a minus-end-directed power stroke in the kinesin motor ncd.
    EMBO J. 2002 Nov 15;21(22):5969-78 PMID: 12426369
  56. LGN blocks the ability of NuMA to bind and stabilize microtubules. A mechanism for mitotic spindle assembly regulation.
    Curr Biol. 2002 Nov 19;12(22):1928-33 PMID: 12445386
  57. Nuclear translocation of plk1 mediated by its bipartite nuclear localization signal.
    J Biol Chem. 2002 Dec 13;277(50):48884-8 PMID: 12364337
  58. Ran GTPase cycle and importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos.
    Mol Biol Cell. 2002 Dec;13(12):4355-70 PMID: 12475958
  59. The auto-inhibitory function of importin alpha is essential in vivo.
    J Biol Chem. 2003 Feb 21;278(8):5854-63 PMID: 12486120
  60. Characterization of Ran-driven cargo transport and the RanGTPase system by kinetic measurements and computer simulation.
    EMBO J. 2003 Mar 3;22(5):1088-100 PMID: 12606574
  61. A mechanism of coupling RCC1 mobility to RanGTP production on the chromatin in vivo.
    J Cell Biol. 2003 Mar 3;160(5):635-44 PMID: 12604592
  62. A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly.
    Nat Cell Biol. 2003 Mar;5(3):242-8 PMID: 12577065
  63. A novel mechanism for activation of the protein kinase Aurora A.
    Curr Biol. 2003 Apr 15;13(8):691-7 PMID: 12699628
  64. Importin alpha-regulated nucleation of microtubules by TPX2.
    EMBO J. 2003 May 1;22(9):2060-70 PMID: 12727873
  65. Unconventional motoring: an overview of the Kin C and Kin I kinesins.
    Traffic. 2003 Jun;4(6):367-75 PMID: 12753646
  66. Identification of Ncd tail domain-binding sites on the tubulin dimer.
    Biochem Biophys Res Commun. 2003 Jun 6;305(3):523-8 PMID: 12763024
  67. The small GTPase Ran: interpreting the signs.
    Curr Opin Cell Biol. 2003 Jun;15(3):338-44 PMID: 12787777
  68. Part of Ran is associated with AKAP450 at the centrosome: involvement in microtubule-organizing activity.
    Mol Biol Cell. 2003 Oct;14(10):4260-71 PMID: 14517334
Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.e03-07-0454
Published
2004-01-00
Epub
2003-00-17
Pages
46-57
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC307526
Subset
IM
Grants
NIGMS NIH HHS · R01 GM059618 · United States
External Links
PubMed source DOI: 10.1091/mbc.e03-07-0454 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