title: Dissection of Cdk1-cyclin complexes in vivo.
abstract: Cyclin-dependent kinases (Cdks) are regulatory enzymes with temporal and spatial selectivity for their protein substrates that are governed by cell cycle-regulated cyclin subunits. Specific cyclin-Cdk complexes bind to and phosphorylate target proteins, coupling their activity to cell cycle states. The identification of specific cyclin-Cdk substrates is challenging and so far, has largely been achieved through indirect correlation or use of in vitro techniques. Here, we use a protein-fragment complementation assay based on the optimized yeast cytosine deaminase to systematically identify candidate substrates of budding yeast Saccharomyces cerevisiae Cdk1 and show dependency on one or more regulatory cyclins. We identified known and candidate cyclin dependencies for many predicted protein kinase Cdk1 targets and showed elusory Clb3-Cdk1-specific phosphorylation of γ-tubulin, thus establishing the timing of this event in controlling assembly of the mitotic spindle. Our strategy can be generally applied to identify substrates and accessory subunits of multisubunit protein complexes.
authors.affiliation: Département de Biochimie and Centre Robert-Cedergren, Bio-Informatique et Génomique, Université de Montréal, Montréal, QC, Canada H3C 3J7.
authors.forename: Po Hien
authors.initials: PH
authors.lastname: Ear
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Booth
authors.forename: Diala
authors.initials: D
authors.lastname: Abd-Rabbo
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Kowarzyk Moreno
authors.forename: Conrad
authors.initials: C
authors.lastname: Hall
authors.forename: Daici
authors.initials: D
authors.lastname: Chen
authors.forename: Jackie
authors.initials: J
authors.lastname: Vogel
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Michnick
mesh_terms: D016213:Cyclins / Q000378:metabolism*; D004258:DNA Polymerase III / Q000378:metabolism*; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014404:Tubulin / Q000378:metabolism
chemical_list: D016213:Cyclins; D046912:Multiprotein Complexes; C491232:POL3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D004258:DNA Polymerase III
keywords: cyclin specificity; in vivo enzyme complexes screen
references.citation: Microbiol Mol Biol Rev. 1998 Dec;62(4):1191-243
references.pmid: 9841670
references.citation: Science. 2009 Sep 25;325(5948):1682-6
references.pmid: 19779198
references.citation: Science. 2005 Feb 4;307(5710):724-7
references.pmid: 15692050
references.citation: Curr Genet. 1999 Sep;36(3):130-6
references.pmid: 10501935
references.citation: Trends Biochem Sci. 2008 Mar;33(3):113-21
references.pmid: 18291659
references.citation: Cell. 2008 Nov 28;135(5):894-906
references.pmid: 19041752
references.citation: Nat Rev Mol Cell Biol. 2011 Oct 12;12(11):709-21
references.pmid: 21993292
references.citation: Nature. 2005 Mar 3;434(7029):104-8
references.pmid: 15744308
references.citation: Nature. 2005 Dec 1;438(7068):679-84
references.pmid: 16319894
references.citation: Mol Cell. 2011 Jun 10;42(5):610-23
references.pmid: 21658602
references.citation: Annu Rev Cell Dev Biol. 1997;13:261-91
references.pmid: 9442875
references.citation: Cell. 2003 Feb 21;112(4):561-74
references.pmid: 12600318
references.citation: J Cell Biol. 2010 Dec 13;191(6):1097-112
references.pmid: 21149565
references.citation: Nature. 2003 Oct 23;425(6960):859-64
references.pmid: 14574415
references.citation: Mol Biol Cell. 2007 Apr;18(4):1187-202
references.pmid: 17251549
references.citation: Mol Cell Biol. 1993 Sep;13(9):5659-69
references.pmid: 8395009
references.citation: Protein Expr Purif. 2009 Jun;65(2):122-32
references.pmid: 19174191
references.citation: Nature. 2003 Oct 16;425(6959):737-41
references.pmid: 14562106
references.citation: Cell. 1994 Jul 15;78(1):67-74
references.pmid: 8033213
references.citation: Mol Cell. 2004 Jun 18;14(6):699-711
references.pmid: 15200949
references.citation: Cell. 2000 Jul 7;102(1):21-31
references.pmid: 10929710
references.citation: Nature. 2010 Aug 12;466(7308):879-82
references.pmid: 20631709
references.citation: Science. 2011 Jun 24;332(6037):1557-61
references.pmid: 21700874
references.citation: Genome Biol. 2005;6(13):R115
references.pmid: 16420674
references.citation: Nature. 1995 Mar 9;374(6518):131-4
references.pmid: 7877684
references.citation: Genetics. 1998 Apr;148(4):1701-13
references.pmid: 9560388
references.citation: Mol Biol Cell. 2002 Apr;13(4):1144-57
references.pmid: 11950928
references.citation: Nat Methods. 2009 Nov;6(11):813-6
references.pmid: 19820714
references.citation: Yeast. 2007 Oct;24(10):913-9
references.pmid: 17583893
references.citation: Genetics. 2012 Apr;190(4):1197-224
references.pmid: 22491889
references.citation: Nature. 2002 Jul 25;418(6896):387-91
references.pmid: 12140549
references.citation: Science. 1993 Jan 8;259(5092):216-9
references.pmid: 8421781
references.citation: Annu Rev Biochem. 1989;58:453-508
references.pmid: 2549856
references.citation: Genome Biol. 2011;12(4):R39
references.pmid: 21492431
references.citation: EMBO J. 2000 Nov 1;19(21):5824-34
references.pmid: 11060033
references.citation: PLoS One. 2011 May 05;6(5):e19700
references.pmid: 21573187
references.citation: Genes Dev. 2008 Jun 1;22(11):1534-48
references.pmid: 18519644
references.citation: Genetics. 1993 Jan;133(1):67-77
references.pmid: 8417990
references.citation: Nat Rev Mol Cell Biol. 2007 Feb;8(2):149-60
references.pmid: 17245415
references.citation: Cell. 1994 Oct 21;79(2):233-44
references.pmid: 7954792
references.citation: Mol Biol Cell. 2013 Oct;24(20):3238-50
references.pmid: 23966467
references.citation: Dev Cell. 2006 Apr;10(4):425-39
references.pmid: 16580990
references.citation: Genes Dev. 2005 Dec 1;19(23):2816-26
references.pmid: 16322557
references.citation: Nature. 1996 Jul 25;382(6589):325-31
references.pmid: 8684460
references.citation: Genes Dev. 2006 Nov 15;20(22):3069-78
references.pmid: 17114580