title: B-cyclin/CDKs regulate mitotic spindle assembly by phosphorylating kinesins-5 in budding yeast.
abstract: 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 SCF(Cdc4) 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 (APC(Cdh1)). 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 SCF(Cdc4) 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.
authors.affiliation: Department of Biology, Duke University, Durham, North Carolina, United States of America.
authors.forename: Mark K
authors.initials: MK
authors.lastname: Chee
authors.forename: Steven B
authors.initials: SB
authors.lastname: Haase
mesh_terms: D017489:CDC28 Protein Kinase, S cerevisiae / Q000235:genetics / Q000378:metabolism*; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism*; D016547:Kinesins; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D008941:Spindle Apparatus / Q000235:genetics / Q000378:metabolism*
chemical_list: C075342:CIN8 protein, S cerevisiae; C093749:CLB2 protein, S cerevisiae; D019926:Cyclin B; C431747:KIP1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D029701:Saccharomyces cerevisiae Proteins; D017489:CDC28 Protein Kinase, S cerevisiae; D016547:Kinesins
references.citation: Cell Div. 2006 Jun 22;1:12
references.pmid: 16792804
references.citation: Science. 1997 Oct 17;278(5337):455-60
references.pmid: 9334303
references.citation: Science. 1988 Sep 9;241(4871):1331-5
references.pmid: 2842867
references.citation: Nature. 1996 May 23;381(6580):272
references.pmid: 8692262
references.citation: Mol Cell Biol. 1999 Aug;19(8):5512-22
references.pmid: 10409741
references.citation: Mol Cell Biol. 1996 Dec;16(12):6634-43
references.pmid: 8943317
references.citation: J Biol Chem. 2006 Sep 8;281(36):26004-13
references.pmid: 16829678
references.citation: Proc Natl Acad Sci U S A. 2001 Oct 23;98(22):12515-20
references.pmid: 11606759
references.citation: Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2817-22
references.pmid: 10077594
references.citation: J Cell Biol. 1992 Jul;118(1):109-20
references.pmid: 1618897
references.citation: PLoS One. 2008;3(12):e3936
references.pmid: 19079595
references.citation: J Bacteriol. 1975 Oct;124(1):511-23
references.pmid: 1100612
references.citation: Methods. 2001 Jul;24(3):218-29
references.pmid: 11403571
references.citation: Electrophoresis. 1997 Dec;18(15):2714-23
references.pmid: 9504803
references.citation: Nature. 2004 Aug 19;430(7002):908-13
references.pmid: 15282614
references.citation: Mol Cell Biol. 1996 Nov;16(11):6385-97
references.pmid: 8887667
references.citation: Trends Cell Biol. 2005 Sep;15(9):467-76
references.pmid: 16084724
references.citation: J Am Soc Mass Spectrom. 1994 Nov;5(11):976-89
references.pmid: 24226387
references.citation: J Biol Chem. 1999 May 21;274(21):15005-13
references.pmid: 10329703
references.citation: Curr Biol. 2008 Dec 23;18(24):R1146-9
references.pmid: 19108775
references.citation: Nature. 2003 Oct 23;425(6960):859-64
references.pmid: 14574415
references.citation: Cell. 1997 Jul 25;90(2):207-16
references.pmid: 9244295
references.citation: Mol Cell Biol. 1996 Oct;16(10):5701-7
references.pmid: 8816483
references.citation: Cell. 2004 Jan 23;116(2):221-34
references.pmid: 14744433
references.citation: J Biol Chem. 1999 Apr 2;274(14):9564-72
references.pmid: 10092642
references.citation: Cell Cycle. 2002 Mar-Apr;1(2):132-6
references.pmid: 12429922
references.citation: Nat Cell Biol. 2008 Jun;10(6):665-75
references.pmid: 18500339
references.citation: Annu Rev Cell Dev Biol. 2004;20:1-28
references.pmid: 15473833
references.citation: Proteins. 2000 Aug 15;40(3):389-408
references.pmid: 10861930
references.citation: EMBO J. 1998 Feb 16;17(4):945-51
references.pmid: 9463373
references.citation: Mol Cell. 2008 Mar 14;29(5):637-43
references.pmid: 18342609
references.citation: Biochim Biophys Acta. 2008 Sep;1786(1):49-59
references.pmid: 18544349
references.citation: J Cell Biol. 1993 Nov;123(3):665-79
references.pmid: 8227131
references.citation: Curr Biol. 2004 Sep 21;14(18):R787-96
references.pmid: 15380093
references.citation: Cell. 2008 Nov 28;135(5):894-906
references.pmid: 19041752
references.citation: Cell. 1990 Mar 23;60(6):1019-27
references.pmid: 2138511
references.citation: Mol Cell. 2004 Nov 5;16(3):487-96
references.pmid: 15525520
references.citation: Brief Funct Genomic Proteomic. 2009 Mar;8(2):90-103
references.pmid: 19109306
references.citation: Nature. 2001 Nov 29;414(6863):514-21
references.pmid: 11734846
references.citation: J Biol Chem. 2001 Jul 6;276(27):25496-502
references.pmid: 11328809
references.citation: Curr Biol. 2008 Dec 9;18(23):1860-4
references.pmid: 19062285
references.citation: Mol Biol Cell. 1994 Jan;5(1):29-43
references.pmid: 8186463
references.citation: Genes Dev. 1992 Nov;6(11):2021-34
references.pmid: 1427070
references.citation: Mol Biol Cell. 2005 Aug;16(8):3896-907
references.pmid: 15958489
references.citation: Mol Biol Cell. 2001 Nov;12(11):3402-16
references.pmid: 11694576
references.citation: Nucleic Acids Res. 2003 Jul 1;31(13):3381-5
references.pmid: 12824332
references.citation: J Cell Biol. 1992 Jul;118(1):95-108
references.pmid: 1618910
references.citation: J Cell Biol. 2009 Aug 24;186(4):481-90
references.pmid: 19687256
references.citation: Mol Biol Cell. 2008 Aug;19(8):3243-53
references.pmid: 18480404
references.citation: Cell. 1995 Dec 29;83(7):1159-69
references.pmid: 8548803
references.citation: Mol Genet Genomics. 2006 Mar;275(3):217-30
references.pmid: 16362369
references.citation: Nat Cell Biol. 2001 Jan;3(1):E23-7
references.pmid: 11146646
references.citation: Curr Biol. 2007 Jul 17;17(14):1181-9
references.pmid: 17614281
references.citation: FEMS Microbiol Lett. 2005 Mar 15;244(2):379-83
references.pmid: 15766794
references.citation: J Mass Spectrom. 2001 Oct;36(10):1083-91
references.pmid: 11747101
references.citation: Nature. 1992 Jan 23;355(6358):368-71
references.pmid: 1731251
references.citation: Cell. 1996 Jul 26;86(2):263-74
references.pmid: 8706131
references.citation: Annu Rev Cell Dev Biol. 1997;13:745-77
references.pmid: 9442886
references.citation: Bioinformatics. 2006 Jan 15;22(2):195-201
references.pmid: 16301204
references.citation: Nature. 2000 Sep 21;407(6802):395-401
references.pmid: 11014197
references.citation: Science. 2004 Feb 6;303(5659):808-13
references.pmid: 14764870
references.citation: Mol Biol Cell. 2010 Mar 15;21(6):914-25
references.pmid: 20089834
references.citation: Genetics. 1973 Jun;74(2):267-86
references.pmid: 17248617
references.citation: J Cell Sci. 2009 May 1;122(Pt 9):1334-41
references.pmid: 19351716
references.citation: Mol Biol Cell. 1992 Jul;3(7):805-18
references.pmid: 1387566
references.citation: J Biol Chem. 1997 Aug 1;272(31):19418-24
references.pmid: 9235942
references.citation: J Cell Biol. 1999 Jan 11;144(1):125-38
references.pmid: 9885249
references.citation: Nature. 1990 Oct 11;347(6293):563-6
references.pmid: 2145514
references.citation: Anal Chem. 1996 Mar 1;68(5):850-8
references.pmid: 8779443
references.citation: Nat Biotechnol. 2004 Dec;22(12):1567-72
references.pmid: 15558047
references.citation: Nature. 2008 Jun 12;453(7197):944-7
references.pmid: 18463633
references.citation: Genome Biol. 2000;1(1):REVIEWS101
references.pmid: 11104520
references.citation: Methods. 2005 Mar;35(3):256-64
references.pmid: 15722222
references.citation: EMBO J. 2006 Jun 7;25(11):2551-63
references.pmid: 16688214
references.citation: Biochemistry. 1998 Feb 17;37(7):1769-76
references.pmid: 9485302
references.citation: Nature. 2005 May 5;435(7038):114-8
references.pmid: 15875026
references.citation: Genetics. 1997 Oct;147(2):421-34
references.pmid: 9335583
references.citation: Mol Biol Cell. 1997 Jun;8(6):1035-50
references.pmid: 9201714
references.citation: J Cell Sci. 2000 Apr;113 ( Pt 8):1311-8
references.pmid: 10725215
references.citation: Annu Rev Cell Dev Biol. 1997;13:261-91
references.pmid: 9442875
references.citation: Nature. 1992 Oct 8;359(6395):540-3
references.pmid: 1406972
references.citation: J Cell Biol. 2001 Jul 9;154(1):85-94
references.pmid: 11448992
references.citation: Cold Spring Harb Symp Quant Biol. 1974;38:123-31
references.pmid: 4598635
references.citation: Genes Dev. 1993 Jul;7(7A):1160-75
references.pmid: 8319908
references.citation: Proc Natl Acad Sci U S A. 1995 May 9;92(10):4289-93
references.pmid: 7753799
references.citation: Trends Cell Biol. 2000 Aug;10(8):329-35
references.pmid: 10884685
references.citation: Nat Cell Biol. 2001 Jan;3(1):38-42
references.pmid: 11146624
references.citation: Mol Biol Cell. 1997 Aug;8(8):1427-37
references.pmid: 9285816
references.citation: Cell. 2005 Aug 12;122(3):407-20
references.pmid: 16096060
references.citation: Science. 1997 Oct 17;278(5337):460-3
references.pmid: 9334304
references.citation: J Cell Biol. 1988 Oct;107(4):1409-26
references.pmid: 3049620
references.citation: Biochemistry. 2005 Dec 20;44(50):16633-48
references.pmid: 16342954
references.citation: Cell. 1994 Oct 21;79(2):233-44
references.pmid: 7954792
references.citation: Nature. 2007 Apr 12;446(7137):806-10
references.pmid: 17314980
references.citation: Cell. 1997 Oct 17;91(2):221-30
references.pmid: 9346239
references.citation: J Mol Biol. 1974 Apr 15;84(3):445-61
references.pmid: 4618856