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B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast


Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.


Vyšlo v časopise: B-Cyclin/CDKs Regulate Mitotic Spindle Assembly by Phosphorylating Kinesins-5 in Budding Yeast. PLoS Genet 6(5): e32767. doi:10.1371/journal.pgen.1000935
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1000935

Souhrn

Although it has been known for many years that B-cyclin/CDK complexes regulate the assembly of the mitotic spindle and entry into mitosis, the full complement of relevant CDK targets has not been identified. It has previously been shown in a variety of model systems that B-type cyclin/CDK complexes, kinesin-5 motors, and the SCFCdc4 ubiquitin ligase are required for the separation of spindle poles and assembly of a bipolar spindle. It has been suggested that, in budding yeast, B-type cyclin/CDK (Clb/Cdc28) complexes promote spindle pole separation by inhibiting the degradation of the kinesins-5 Kip1 and Cin8 by the anaphase-promoting complex (APCCdh1). We have determined, however, that the Kip1 and Cin8 proteins are present at wild-type levels in the absence of Clb/Cdc28 kinase activity. Here, we show that Kip1 and Cin8 are in vitro targets of Clb2/Cdc28 and that the mutation of conserved CDK phosphorylation sites on Kip1 inhibits spindle pole separation without affecting the protein's in vivo localization or abundance. Mass spectrometry analysis confirms that two CDK sites in the tail domain of Kip1 are phosphorylated in vivo. In addition, we have determined that Sic1, a Clb/Cdc28-specific inhibitor, is the SCFCdc4 target that inhibits spindle pole separation in cells lacking functional Cdc4. Based on these findings, we propose that Clb/Cdc28 drives spindle pole separation by direct phosphorylation of kinesin-5 motors.


Zdroje

1. MorganDO

1997 Cyclin-dependent kinases: Engines, clocks, and microprocessors. Annual Review of Cell and Developmental Biology 13 261 291

2. MurrayAW

2004 Recycling the cell cycle: Cyclins revisited. Cell 116 221 234

3. LaceyKR

JacksonPK

StearnsT

1999 Cyclin-dependent kinase control of centrosome duplication. Proceedings of the National Academy of Science USA 96 2817 2822

4. HaaseSB

WineyM

ReedSI

2001 Multi-step control of spindle pole body duplication by cyclin-dependent kinase. Nature Cell Biology 3 38 42

5. AdamsIR

KilmartinJV

2000 Spindle pole body duplication: a model for centrosome duplication? Trends in Cell Biology 10 329 335

6. Simmons KovacsLA

NelsonCL

HaaseSB

2008 Intrinsic and Cyclin-dependent Kinase-dependent Control of Spindle Pole Body Duplication in Budding Yeast. Molecular Biology of the Cell 19 3243 3253

7. WalczakC

2000 Molecular mechanisms of spindle function. Genome Biology 1 reviews101.101 101.104

8. JaspersenSL

WineyM

2004 The budding yeast spindle pole body: Structure, duplication and function. Annual Review of Cell and Developmental Biology 20 1 28

9. O'TooleMWE

2001 The spindle cycle in budding yeast. Nature Cell Biology 3 E23 E27

10. AmonA

SuranaU

MuroffI

NasmythK

1992 Regulation of p34CDC28 tyrosine phosphorylation is not required for entry into mitosis in S. cerevisiae. Nature 355 368 371

11. KeatonMA

BardesESG

MarquitzAR

FreelCD

ZylaTR

2007 Differential susceptibility of S and M phase cyclin/CDK complexes to inhibitory tyrosine phosphorylation in yeast. Current Biology 17 1181 1189

12. LimHH

GohP-Y

SuranaU

1996 Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28. Molecular and Cellular Biology 16 6385 6397

13. FitchI

DahmannC

SuranaU

AmonA

NasmythK

1992 Characterization of four B-type cyclin genes of the budding yeast Saccharomyces cerevisiae. Molecular Biology of the Cell 3 805 818

14. RichardsonH

LewDJ

HenzeM

SugimotoK

ReedSI

1992 Cyclin-B homologs in Saccharomyces cerevisiae function in S phase and in G2. Genes & Development 6 2021 2034

15. BaiC

SenP

HofmannK

MaL

GoeblM

1996 SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell 86 263 274

16. MathiasN

JohnsonSL

WineyM

AdamsAE

GoetschL

1996 Cdc53p acts in concert with Cdc4p and Cdc34p to control G1-to-S-Phase transition and identifies a conserved family of proteins. Molecular and Cellular Biology 16 6634 6643

17. ByersB

GoetschL

1975 Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. Journal of Bacteriology 124 511 523

18. GoeblMG

YochemJ

JentschS

McGrathJP

VarshavskyA

1988 The yeast cell cycle gene CDC34 encodes a ubiquitin-conjugating enzyme. Science 241 1331 1335

19. ByersB

GoetschL

1974 Duplication of spindle plaques and integration of the yeast cell cycle. Cold Spring Harbor Symposium on Quantitative Biology 38 123 131

20. SchwobE

BohmT

MendenhallMD

NasmythK

1994 The B-type cyclin kinase inhibitor p40SIC1 controls the G1 to S transition in S. cerevisiae. Cell 79 233 244

21. VermaR

FeldmanRR

DeshaiesRJ

1997 SIC1 is ubiquitinated in vitro by a pathway that requires CDC4, CDC34, and cyclin/CDK activities. Molecular Biology of the Cell 8 1427 1437

22. FeldmanRR

CorrellCC

KaplanKB

DeshaiesRJ

1997 A complex of Cdc4p, Skp1p, and Cdc53p/Cullin catalyzes ubiquitination of the phosphorylated CDK inhibitor Sic1p. Cell 91 221 230

23. VodermaierHC

2004 APC/C and SCF: Controlling Each Other and the Cell Cycle. Current Biology 14 R787 R796

24. RoofDM

MeluhPB

RoseMD

1992 Kinesin-related proteins required for assembly of the mitotic spindle. The Journal of Cell Biology 118 95 108

25. HoytMA

HeL

LooKK

SaundersWS

1992 Two Saccharomyces cerevisiae kinesin-related gene products required for mitotic spindle assembly. Journal of Cell Biology 118 109 120

26. MikiH

OkadaY

HirokawaN

2005 Analysis of the kinesin superfamily: insights into structure and function. Trends in Cell Biology 15 467 476

27. EnosAP

MorrisNR

1990 Mutation of a gene that encodes a kinesin-like protein that blocks nuclear division in A. nidulans. Cell 60 1019 1027

28. HeckMS

PereiraA

PesaventoP

YannoniY

SpradlingAC

1993 The kinesin-like protein KLP61F is essential for mitosis in Drosophila. The Journal of Cell Biology 123 665 679

29. HaganIM

YanagidaM

1990 Novel potential mitotic motor protein encoded by the fission yeast cut7+ gene. Nature 347 563 566

30. SawinKE

LeGuellecK

PhilippeM

MitchisonTJ

1992 Mitotic spindle organization by a plus-end-directed microtubule motor. Nature 359 540 543

31. JacobsCW

AdamsAE

SzaniszloPJ

PringleJR

1988 Functions of microtubules in the Saccharomyces cerevisiae cell cycle. Journal of Cell Biology 107 1409 1426

32. ReijoRA

CooperEM

BeagleGJ

HuffakerTC

1994 Systematic mutational analysis of the yeast ß-tubulin gene. Molecular Biology of the Cell 5 29 43

33. AmosL

2008 Spindle assembly: Kinesin-5 is in control. Current Biology 18 R1146 R1149

34. SchwobE

NasmythK

1993 CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae. Genes & Development 7 1160 1175

35. BlangyA

LaneHA

d'HérinP

HarperM

KressM

1995 Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell 83 1159 1169

36. BlangyA

ArnaudL

NiggEA

1997 Phosphorylation by p34cdc2 protein kinase regulates binding of the kinesin-related motor HsEg5 to the dynactin subunit p150Glued. Journal of Biological Chemistry 272 19418 19424

37. SawinKE

MitchisonTJ

1995 Mutations in the kinesin-like protein Eg5 disrupt localization to the mitotic spindle. Proceedings of the National Academy of Science USA 92 4289 4293

38. GietR

UzbekovR

CubizollesF

Le GuellecK

PrigentC

1999 The Xenopus laevis Aurora-related protein kinase pEg2 associates with and phosphorylates the kinesin-related protein XlEg5. Journal of Biological Chemistry 274 15005 15013

39. SharpDJ

McDonaldKL

BrownHM

MatthiesHJ

WalczakC

1999 The bipolar kinesin KLP61F, cross-links microtubules within interpolar microtubule bundles of Drosophila embryonic mitotic spindles. Journal of Cell Biology 144 125 138

40. GoshimaG

ValeRD

2005 Cell cycle-dependent dynamics and regulation of mitotic kinesins in Drosophila S2 cells. Molecular Biology of the Cell 16 3896 3907

41. CrastaK

HuangP

MorganG

WineyM

SuranaU

2006 Cdk1 regulates centrosome separation by restraining proteolysis of microtubule-associated proteins. EMBO Journal 1 13

42. CrastaK

LimHH

GiddingsTHJr

WineyM

SuranaU

2008 Inactivation of Cdh1 by synergistic action of Cdk1 and polo kinase is necessary for proper assembly of the mitotic spindle. Nature Cell Biology 10 665 675

43. VisintinR

PrinzS

AmonA

1997 CDC20 and CDH1: A family of substrate-specific activators of APC-dependent proteolysis. Science 278 460 463

44. HuangJN

ParkI

EllingsonE

LittlepageLE

PellmanD

2001 Activity of the APCCdh1 form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p. Journal of Cell Biology 154 85 94

45. van LeukenR

CligstersL

WothuisR

2008 To cell cycle, swing the APC/C. Biochimica et Biophysica Acta 1786 49 59

46. GordonDM

RoofDM

2001 Degradation of the kinesin Kip1p at anaphase onset if mediated by the anaphase-promoting complex and Cdc20p. Proceedings of the National Academy of Science USA 98 12515 12520

47. HildebrandtER

HoytMA

2001 Cell cycle-dependent degradation of the Saccharomyces cerevisiae spindle motor Cin8p requires APCCdh1 and a bipartite destruction sequence. Molecular Biology of the Cell 12 3402 3416

48. BishopAC

UbersaxJA

PetschDT

MatheosDP

GrayNS

2000 A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407 395 401

49. VermaR

AnnanRS

HuddlestonMJ

CarrSA

ReynardG

1997 Phosphorylation of Sic1p by G1 Cdk is required for its degradation and entry into S phase. Science 278 455 460

50. HartwellLH

MortimerRK

CulottiJ

CulottiM

1973 Genetic control of the cell division cycle in yeast: V. Genetic analysis of cdc mutants. Genetics 74 267 286

51. HerefordLM

HartwellLH

1974 Sequential gene function in the initiation of Saccharomyces cerevisiae DNA synthesis. Journal of Molecular Biology 84 445 461

52. KnappD

BhoiteL

StillmanDJ

NasmythK

1996 The transcription factor Swi5 regulates expression of the cyclin kinase inhibitor p40SIC1. Molecular and Cellular Biology 16 5701 5707

53. GohP-Y

SuranaU

1999 Cdc4, a protein required for the onset of S phase, serves an essential function during G2/M transition in Saccharomyces cerevisiae. Molecular and Cellular Biology 5512 5522

54. UbersaxJA

WoodburyEA

QuangPN

ParazM

BlethrowJD

2003 Targets of the cyclin-dependent kinase Cdk1. Nature 425 859 864

55. CrastaK

SuranaU

2006 Disjunction of conjoined twins: Cdk1, Cdh1 and separation of centrosomes. Cell Division 1 12 20

56. OrlandoDA

LinCY

BernardA

WangJY

ESJ

2008 Global control of cell-cycle transcription by coupled CDK and network oscillators. Nature 453 944 947

57. HildebrandtER

GheberL

HoytMA

2006 Homotetrameric form of Cin8p, a Saccharomyces cerevisiae kinesin-5 motor, is essential for its in vivo function. Journal of Biological Chemistry 281 26004 26013

58. ShanerNC

CampbellRE

SteinbachPA

NG.GB

PalmerAE

2004 Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnology 22 1567 1572

59. HarveySL

CharletA

HaasW

GygiSP

KelloggDR

2005 Cdk1-dependent regulation of the mitotic inhibitor Wee1. Cell 122 407 420

60. NashP

TangX

OrlickyS

ChenQ

GertierFB

2001 Multisite phosphorylation of a CDK inhibitor sets a threshold for the onset of DNA replication. Nature 414 514 521

61. EndowSA

2000 Molecular motors - a paradigm for mutant analysis. Journal of Cell Science 113 1311 1318

62. BlackburnK

GosheMB

2009 Challenges and strategies for targeted phosphorylation site identification and quantification using mass spectrometry analysis. Briefings in Functional Genomics and Proteomics 8 90 103

63. GarciaBA

ShabanowitzJ

HuntDF

2005 Analysis of protein phosphorylation by mass spectrometry. Methods 35 256 264

64. GuexN

PeitschMC

1997 SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18 2714 2723

65. SchwedeT

KoppJ

GuexN

PeitschMC

2003 SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Research 31 3381 3385

66. ArnoldK

BordoliL

KoppJ

SchwedeT

2006 The SWISS-MODEL Workspace: A web-based environment for protein structure homology modelling. Bioinformatics 22 195 201

67. TurnerJ

AndersonR

GuoJ

BeraudC

FletterickR

2001 Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck linker. Journal of Biological Chemistry 276 25496 25502

68. GulickAM

SongH

EndowSA

RaymentI

1998 X-ray Crystal Structure of the Yeast Kar3 Motor Domain Complexed with Mg·ADP to 2.3 Å Resolution. Biochemistry 37 1769 1776

69. ValeRD

FletterickRJ

1997 The Design Plan of Kinesin Motors. Annual Review of Cell and Developmental Biology 13 745 777

70. WoehlkeG

RubyAK

HartCL

LyB

Hom-BooherN

1997 Microtubule Interaction Site of the Kinesin Motor. Cell 90 207 216

71. AlonsoM

van DammeJ

VandekerchkhoveJ

CrossRA

1998 Proteolytic mapping of kinesin/ncd-microtubule interface: nucleotide-dependent conformational changes in the loops L8 and L12. Embo J 17 949 951

72. CochranJC

GilbertSP

2005 ATPase Mechanism of Eg5 in the Absence of Microtubules: Insight into Microtubule Activation and Allosteric Inhibition by Monastrol. Biochemistry 44 16633 16648

73. KorolyevE

Steinberg-NeifachO

EshelD

2005 Mutations in the yeast kinesin-like Cin8p are alleviated by osmotic support. FEMS Microbiology Letters 244 379 383

74. GeiserJR

SchottEJ

KingsburyTJ

ColeNB

TotisLJ

1997 Saccharomyces cerevisiae genes required in the absence of the CIN8-encoded spindle motor act in functionally diverse mitotic pathways. Molecular Biology of the Cell 8 1035 1050

75. BasmajiF

Martin-YkenH

DurandF

DagkessamanskaiaA

PichereauxC

2006 The ‘interactome’ of the Knr4/Smi1, a protein implicated in coordinating cell wall synthesis with bud emergence in Saccharomyces cerevisiae. Molecular Genetics and Genomics 275 217 230

76. CollinsSR

MillerKM

MaasNL

RoguevA

FillinghamJ

2007 Functional dissection of protein complexes involved in yeast chromosome biology using a genetic interaction map. Nature 446 806 810

77. TongAH

LesageG

BaderGD

DingH

XuH

2004 Global mapping of the yeast genetic interaction network. Science 303 808 813

78. PanX

YuanDS

XiangD

WangX

Sookhai-MahadeoS

2004 A robust toolkit for functional profiling of the yeast genome. Mol Cell 16 487 496

79. RobbinsJA

CrossFR

2010 Requirements and reasons for effective inhibition of the Anaphase Promoting Complex activator Cdh1. Molecular Biology of the Cell In Press: published online ahead of print

80. CahuJ

OlichonA

HentrichC

SchekH

DrinjakovicJ

2008 Phosphorylation by Cdk1 increases the binding of Eg5 to microtubules in vitro and in Xenopus egg extract spindles. PLoS ONE 3 e3936 doi:10.1371/journal.pone.0003936

81. van den WildenbergSM

TaoL

KapiteinLC

SchmidtCF

ScholeyJM

2008 The homotetrameric kinesin-5 KLP61F preferentially crosslinks microtubules in antiparallel orientations. Current Biology 18 1860 1864

82. GheberL

KuoSC

HoytMA

1999 Motile properties of the kinesin-related Cin8p spindle motor extracted from Saccharomyces cerevisiae cells. The Journal of Biological Chemistry 274 9564 9572

83. KapiteinLC

PetermanEJG

KwokBH

KimJH

KapoorTM

2005 The bipolar mitotic kinesin Eg5 moves on both microtubules that it crosslinks. Nature 435 114 118

84. GardnerMK

BouckDC

PaliulisLV

MeehlJB

O'TooleET

2008 Chromosome congression by Kinesin-5 motor-mediated disassembly of longer kinetochore microtubules. Cell 135 894 906

85. MennellaV

TanD-Y

BusterDW

AsenjoAB

RathU

2009 Motor domain phosphorylation and regulation of the Drosophila kinesin 13, KLP10A. Journal of Cell Biology 186 481 490

86. MishimaM

PavicicV

GrünebergU

NiggEA

GlotzerM

2004 Cell cycle regulation of central spindle assembly. Nature 430 908 913

87. EspeutJ

GaussenA

BielingP

MorinV

PrietoS

2008 Phosphorylation Relieves Autoinhibition of the Kinetochore Motor Cenp-E. Molecular Cell 29 637 643

88. JangC-Y

CoppingerJA

SekiA

YatesJRI

FangG

2009 Plk1 and Aurora A regulate the depolymerase activity and the cellular localization of Kif2a. Journal of Cell Science 122 1334 1341

89. MondésertG

ClarkeDJ

ReedSI

1997 Identification of genes controlling growth polarity in the budding yeast Saccharomyces cerevisiae: a possible role of N-glycosylation and involvement of the exocyst complex. Genetics 147 421 434

90. PuigO

CasparyF

RigautG

RutzB

BouveretE

2001 The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24 218 229

91. HaaseSB

ReedSI

2002 Improved flow cytometric analysis of the budding yeast cell cycle. Cell Cycle 1 132 136

92. ShevchenkoA

WilmM

VormO

MannM

1996 Mass Spectrometric Sequencing of Proteins from Silver-Stained Polyacrylamide Gels. Analytical Chemistry 68 850 858

93. PengJ

GygiSP

2001 Proteomics: the move to mixtures. Journal of Mass Spectrometry 36 1083 1091

94. EngJK

McCormackAL

YatesJRI

1994 An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. Journal of the American Society for Mass Spectrometry 5 976 989

95. HooftRW

VriendG

SanderC

AbolaEE

1996 Errors in protein structure. Nature 381 272

96. LovellSC

WordJM

RichardsonJS

RichardsonDC

2000 The penultimate rotamer library. Proteins: Structure, Function and Genetics 40 389 408

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