Home LiteratureArticle Details
PMID: 15247293 Published · ppublish English Journal Article Research Support, U.S. Gov't, P.H.S.

Mechanistic analysis of the mitotic kinesin Eg5.

The Journal of biological chemistry ·Vol. 279 ·No. 37 ·2004-09-10 ·Pages 38861-70

Cochran JC, Sontag CA, Maliga Z, Kapoor TM, Correia JJ, Gilbert SP

Abstract

Eg5 is a slow, plus-end-directed microtubule-based motor of the BimC kinesin family that is essential for bipolar spindle formation during eukaryotic cell division. We have analyzed two human Eg5/KSP motors, Eg5-367 and Eg5-437, and both are monomeric based on results from sedimentation velocity and sedimentation equilibrium centrifugation as well as analytical gel filtration. The steady-state parameters were: for Eg5-367: k(cat) = 5.5 s(-1), K(1/2,Mt) = 0.7 microm, and K(m,ATP) = 25 microm; and for Eg5-437: k(cat) = 2.9 s(-1), K(1/2,Mt) = 4.5 microm, and K(m,ATP) = 19 microm. 2'(3')-O-(N-Methylanthraniloyl)-ATP (mantATP) binding was rapid at 2-3 microm(-1)s(-1), followed immediately by ATP hydrolysis at 15 s(-1). ATP-dependent Mt.Eg5 dissociation was relatively slow and rate-limiting at 8 s(-1) with mantADP release at 40 s(-1). Surprisingly, Eg5-367 binds microtubules more effectively (11 microm(-1)s(-1)) than Eg5-437 (0.7 microm(-1)s(-1)), consistent with the steady-state K(1/2,Mt) and the mantADP release K(1/2,Mt). These results indicate that the ATPase pathway for monomeric Eg5 is more similar to conventional kinesin than the spindle motors Ncd and Kar3, where ADP product release is rate-limiting for steady-state turnover.

MeSH Terms
Adenosine Diphosphate/chemistry Adenosine Triphosphatases/chemistry,metabolism Adenosine Triphosphate/chemistry Binding Sites Cell Division Chromatography, Gel Dose-Response Relationship, Drug Drosophila Proteins/metabolism Humans Hydrolysis Kinesins/chemistry,metabolism Kinetics Microtubule-Associated Proteins Microtubules/metabolism Mitosis Models, Chemical Phosphocreatine/chemistry Protein Binding Protein Structure, Tertiary Saccharomyces cerevisiae Proteins Time Factors Ultracentrifugation
Chemicals
Drosophila Proteins KAR3 protein, S cerevisiae KIF11 protein, human Microtubule-Associated Proteins Saccharomyces cerevisiae Proteins ncd protein, Drosophila Phosphocreatine Adenosine Diphosphate Adenosine Triphosphate Adenosine Triphosphatases Kinesins
Authors & Affiliations
6 authors, click to expand affiliations / ORCID
Cochran Jared C
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Sontag Christopher A
Maliga Zoltan
Kapoor Tarun M
Correia John J
Gilbert Susan P
References (86)
86 references, click to expand
  1. Alternate fast and slow stepping of a heterodimeric kinesin molecule.
    Nat Cell Biol. 2003 Dec;5(12):1079-82 PMID: 14634664
  2. Inhibition of a mitotic motor protein: where, how, and conformational consequences.
    J Mol Biol. 2004 Jan 9;335(2):547-54 PMID: 14672662
  3. Kinesin moves by an asymmetric hand-over-hand mechanism.
    Science. 2003 Dec 19;302(5653):2130-4 PMID: 14657506
  4. What kinesin does at roadblocks: the coordination mechanism for molecular walking.
    EMBO J. 2004 Jan 14;23(1):23-32 PMID: 14685258
  5. Kinesin walks hand-over-hand.
    Science. 2004 Jan 30;303(5658):676-8 PMID: 14684828
  6. Inhibition of kinesin motility by ADP and phosphate supports a hand-over-hand mechanism.
    Proc Natl Acad Sci U S A. 2004 Feb 3;101(5):1183-8 PMID: 14734813
  7. Modulation of kinesin binding by the C-termini of tubulin.
    EMBO J. 2004 Mar 10;23(5):989-99 PMID: 14976555
  8. Kinesin's second step.
    Proc Natl Acad Sci U S A. 2004 Mar 9;101(10):3444-9 PMID: 14985504
  9. Spindle mechanics and dynamics during mitosis in Drosophila.
    Trends Cell Biol. 2004 Apr;14(4):194-205 PMID: 15066637
  10. A mechanistic model for the organization of microtubule asters by motor and non-motor proteins in a mammalian mitotic extract.
    Mol Biol Cell. 2004 May;15(5):2116-32 PMID: 14978218
  11. Determination of molecular weights and frictional ratios of proteins in impure systems by use of gel filtration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductases.
    Biochim Biophys Acta. 1966 Feb 7;112(2):346-62 PMID: 5329026
  12. Kinesin ATPase: rate-limiting ADP release.
    Proc Natl Acad Sci U S A. 1988 Sep;85(17):6314-8 PMID: 2970638
  13. Mutation of a gene that encodes a kinesin-like protein blocks nuclear division in A. nidulans.
    Cell. 1990 Mar 23;60(6):1019-27 PMID: 2138511
  14. Cloning by differential screening of a Xenopus cDNA that encodes a kinesin-related protein.
    Mol Cell Biol. 1991 Jun;11(6):3395-8 PMID: 1710028
  15. Kinesin-related cut7 protein associates with mitotic and meiotic spindles in fission yeast.
    Nature. 1992 Mar 5;356(6364):74-6 PMID: 1538784
  16. Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly.
    J Cell Biol. 1992 Jul;118(1):109-20 PMID: 1618897
  17. Kinesin-related proteins required for assembly of the mitotic spindle.
    J Cell Biol. 1992 Jul;118(1):95-108 PMID: 1618910
  18. Kinesin-related proteins required for structural integrity of the mitotic spindle.
    Cell. 1992 Aug 7;70(3):451-8 PMID: 1643659
  19. Mitotic spindle organization by a plus-end-directed microtubule motor.
    Nature. 1992 Oct 8;359(6395):540-3 PMID: 1406972
  20. Oligomeric structure of bacteriophage T7 DNA primase/helicase proteins.
    J Biol Chem. 1993 May 15;268(14):10668-75 PMID: 8486715
  21. Pre-steady-state kinetics of the microtubule-kinesin ATPase.
    Biochemistry. 1994 Feb 22;33(7):1951-60 PMID: 8110800
  22. Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization.
    J Biol Chem. 1994 Jun 10;269(23):16493-501 PMID: 8206959
  23. The rate-limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule.
    J Biol Chem. 1994 Jun 10;269(23):16508-11 PMID: 8206961
  24. A "slow" homotetrameric kinesin-related motor protein purified from Drosophila embryos.
    J Biol Chem. 1994 Sep 16;269(37):22913-6 PMID: 8083185
  25. Pathway of processive ATP hydrolysis by kinesin.
    Nature. 1995 Feb 23;373(6516):671-6 PMID: 7854446
  26. Sedimentation studies on the kinesin motor domain constructs K401, K366, and K341.
    Biochemistry. 1995 Apr 11;34(14):4898-907 PMID: 7718594
  27. Mutations in the kinesin-like protein Eg5 disrupting localization to the mitotic spindle.
    Proc Natl Acad Sci U S A. 1995 May 9;92(10):4289-93 PMID: 7753799
  28. Expression, purification, ATPase properties, and microtubule-binding properties of the ncd motor domain.
    Biochemistry. 1995 Oct 10;34(40):13259-66 PMID: 7548090
  29. Antagonistic microtubule-sliding motors position mitotic centrosomes in Drosophila early embryos.
    Nat Cell Biol. 1999 May;1(1):51-4 PMID: 10559864
  30. The road less traveled: emerging principles of kinesin motor utilization.
    Annu Rev Cell Dev Biol. 1999;15:141-83 PMID: 10611960
  31. A structural change in the kinesin motor protein that drives motility.
    Nature. 1999 Dec 16;402(6763):778-84 PMID: 10617199
  32. Moving a microtubule may require two heads: a kinetic investigation of monomeric Ncd.
    Biochemistry. 2000 Feb 15;39(6):1346-55 PMID: 10684615
  33. Kinetics: a tool to study molecular motors.
    Methods. 2000 Dec;22(4):337-54 PMID: 11133240
  34. The spindle: a dynamic assembly of microtubules and motors.
    Nat Cell Biol. 2001 Jan;3(1):E28-34 PMID: 11146647
  35. Mitosis: a history of division.
    Nat Cell Biol. 2001 Jan;3(1):E17-21 PMID: 11146645
  36. Switch-based mechanism of kinesin motors.
    Nature. 2001 May 24;411(6836):439-45 PMID: 11373668
  37. A mechanistic model for Ncd directionality.
    J Biol Chem. 2001 Jun 1;276(22):19259-66 PMID: 11278404
  38. A structural pathway for activation of the kinesin motor ATPase.
    EMBO J. 2001 Jun 1;20(11):2611-8 PMID: 11387196
  39. All kinesin superfamily protein, KIF, genes in mouse and human.
    Proc Natl Acad Sci U S A. 2001 Jun 19;98(13):7004-11 PMID: 11416179
  40. Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck-linker.
    J Biol Chem. 2001 Jul 6;276(27):25496-502 PMID: 11328809
  41. Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix.
    J Cell Biol. 2001 Sep 17;154(6):1125-33 PMID: 11564753
  42. The mitotic spindle: a self-made machine.
    Science. 2001 Oct 19;294(5542):543-7 PMID: 11641489
  43. Pathway of ADP-stimulated ADP release and dissociation of tethered kinesin from microtubules. Implications for the extent of processivity.
    Biochemistry. 2002 Apr 2;41(13):4437-46 PMID: 11914091
  44. The role of ATP hydrolysis for kinesin processivity.
    J Biol Chem. 2002 May 10;277(19):17079-87 PMID: 11864969
  45. Measuring kinesin's first step.
    J Biol Chem. 2002 Sep 27;277(39):36731-9 PMID: 12122000
  46. Evidence that monastrol is an allosteric inhibitor of the mitotic kinesin Eg5.
    Chem Biol. 2002 Sep;9(9):989-96 PMID: 12323373
  47. Self-organisation and forces in the microtubule cytoskeleton.
    Curr Opin Cell Biol. 2003 Feb;15(1):118-24 PMID: 12517713
  48. Interaction of the mitotic inhibitor monastrol with human kinesin Eg5.
    Biochemistry. 2003 Jan 21;42(2):338-49 PMID: 12525161
  49. A force balance model of early spindle pole separation in Drosophila embryos.
    Biophys J. 2003 Feb;84(2 Pt 1):757-69 PMID: 12547760
  50. The ATPase cross-bridge cycle of the Kar3 motor domain. Implications for single head motility.
    J Biol Chem. 2003 Feb 7;278(6):3527-35 PMID: 12446697
  51. The molecular motor toolbox for intracellular transport.
    Cell. 2003 Feb 21;112(4):467-80 PMID: 12600311
  52. Loading direction regulates the affinity of ADP for kinesin.
    Nat Struct Biol. 2003 Apr;10(4):308-11 PMID: 12640444
  53. Cell division.
    Nature. 2003 Apr 17;422(6933):746-52 PMID: 12700768
  54. Molecular motors.
    Nature. 2003 Apr 17;422(6933):759-65 PMID: 12700770
  55. Kinesin hydrolyses one ATP per 8-nm step.
    Nature. 1997 Jul 24;388(6640):386-90 PMID: 9237757
  56. Coupling of kinesin steps to ATP hydrolysis.
    Nature. 1997 Jul 24;388(6640):390-3 PMID: 9237758
  57. Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150.
    J Biol Chem. 1997 Aug 1;272(31):19418-24 PMID: 9235942
  58. Kinetic evidence for low chemical processivity in ncd and Eg5.
    J Mol Biol. 1997 Oct 17;273(1):160-70 PMID: 9367754
  59. Kinetic mechanism of monomeric non-claret disjunctional protein (Ncd) ATPase.
    J Biol Chem. 1997 Dec 5;272(49):30735-40 PMID: 9388211
  60. X-ray structure of motor and neck domains from rat brain kinesin.
    Biochemistry. 1997 Dec 23;36(51):16155-65 PMID: 9405049
  61. Alternating site mechanism of the kinesin ATPase.
    Biochemistry. 1998 Jan 20;37(3):792-9 PMID: 9454568
  62. Pathway of ATP hydrolysis by monomeric and dimeric kinesin.
    Biochemistry. 1998 Jan 20;37(3):800-13 PMID: 9454569
  63. X-ray crystal structure of the yeast Kar3 motor domain complexed with Mg.ADP to 2.3 A resolution.
    Biochemistry. 1998 Feb 17;37(7):1769-76 PMID: 9485302
  64. 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
  65. The bipolar kinesin, KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles.
    J Cell Biol. 1999 Jan 11;144(1):125-38 PMID: 9885249
  66. Kinetics processivity and the direction of motion of Ncd.
    Biophys J. 1999 Aug;77(2):1003-16 PMID: 10423445
  67. Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo.
    Cell. 1995 Dec 29;83(7):1159-69 PMID: 8548803
  68. A bipolar kinesin.
    Nature. 1996 Jan 18;379(6562):270-2 PMID: 8538794
  69. Kinetics and motility of the Eg5 microtubule motor.
    Biochemistry. 1996 Feb 20;35(7):2365-73 PMID: 8652578
  70. Crystal structure of the kinesin motor domain reveals a structural similarity to myosin.
    Nature. 1996 Apr 11;380(6574):550-5 PMID: 8606779
  71. Crystal structure of the motor domain of the kinesin-related motor ncd.
    Nature. 1996 Apr 11;380(6574):555-9 PMID: 8606780
  72. Purification and characterization of two monomeric kinesin constructs.
    Biochemistry. 1996 May 21;35(20):6321-9 PMID: 8639576
  73. 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
  74. An improved function for fitting sedimentation velocity data for low-molecular-weight solutes.
    Biophys J. 1997 Jan;72(1):435-44 PMID: 8994630
  75. Kinetic mechanism of a monomeric kinesin construct.
    J Biol Chem. 1997 Jan 10;272(2):717-23 PMID: 8995355
  76. Interacting head mechanism of microtubule-kinesin ATPase.
    J Biol Chem. 1997 Jan 10;272(2):724-30 PMID: 8995356
  77. The role of microtubule assembly dynamics in mitotic force generation and functional organization of living cells.
    J Struct Biol. 1997 Mar;118(2):87-93 PMID: 9126635
  78. Mitotic spindle function in Saccharomyces cerevisiae requires a balance between different types of kinesin-related motors.
    Mol Biol Cell. 1997 Jun;8(6):1025-33 PMID: 9201713
  79. Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen.
    Science. 1999 Oct 29;286(5441):971-4 PMID: 10542155
  80. 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
  81. Stepping and stretching. How kinesin uses internal strain to walk processively.
    J Biol Chem. 2003 May 16;278(20):18550-6 PMID: 12626516
  82. The roles of microtubule-based motor proteins in mitosis: comprehensive RNAi analysis in the Drosophila S2 cell line.
    J Cell Biol. 2003 Sep 15;162(6):1003-16 PMID: 12975346
  83. A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions.
    Anal Biochem. 2000 Mar 15;279(2):151-63 PMID: 10706784
  84. The way things move: looking under the hood of molecular motor proteins.
    Science. 2000 Apr 7;288(5463):88-95 PMID: 10753125
  85. Probing spindle assembly mechanisms with monastrol, a small molecule inhibitor of the mitotic kinesin, Eg5.
    J Cell Biol. 2000 Sep 4;150(5):975-88 PMID: 10973989
  86. Spindle assembly in animal cells.
    Annu Rev Biochem. 2000;69:95-114 PMID: 10966454
Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.M404203200
Published
2004-09-10
Epub
2004-00-06
Pages
38861-70
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC1356567
Subset
IM
Grants
NIGMS NIH HHS · GM54141 · United States
NIGMS NIH HHS · R01 GM054141 · United States
NIAMS NIH HHS · K02-AR47841 · United States
NIAMS NIH HHS · K02 AR047841 · United States
NIGMS NIH HHS · R37 GM054141 · United States
External Links
PubMed source DOI: 10.1074/jbc.M404203200 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: [email protected]

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