title: Combining the Optimized Yeast Cytosine Deaminase Protein Fragment Complementation Assay and an In Vitro Cdk1 Targeting Assay to Study the Regulation of the γ-Tubulin Complex.
abstract: Cdk1 is the essential cyclin-dependent kinase in the budding yeast Saccharomyces cerevisiae. Cdk1 orchestrates cell cycle control by phosphorylating target proteins with extraordinary temporal and spatial specificity by complexing with one of the nine cyclin regulatory subunits. The identification of the cyclin required for targeting Cdk1 to a substrate can help to place the regulation of that protein at a specific time point during the cell cycle and reveal information needed to elucidate the biological significance of the regulation. Here, we describe a combination of strategies to identify interaction partners of Cdk1, and associate these complexes to the appropriate cyclins using a cell-based protein-fragment complementation assay. Validation of the specific reliance of the OyCD interaction between Cdk1 and budding yeast γ-tubulin on the Clb3 cyclin, relative to the mitotic Clb2 cyclin, was performed by an in vitro kinase assay using the γ-tubulin complex as a substrate.
authors.affiliation: Département de Biochimie, Université de Montréal, C.P. 6128, Succursale Centre-Ville, Montréal, QC, Canada, H3C 3J7.
authors.forename: Po Hien
authors.initials: PH
authors.lastname: Ear
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Kowarzyk
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Booth
authors.forename: Diala
authors.initials: D
authors.lastname: Abd-Rabbo
authors.forename: Kristian
authors.initials: K
authors.lastname: Shulist
authors.forename: Conrad
authors.initials: C
authors.lastname: Hall
authors.forename: Jackie
authors.initials: J
authors.lastname: Vogel
authors.forename: Stephen W
authors.initials: SW
authors.lastname: Michnick
mesh_terms: D000818:Animals; D016203:CDC2 Protein Kinase / Q000302:isolation & purification / Q000378:metabolism*; D043525:Cytosine Deaminase / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D017353:Gene Deletion; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D061987:Sf9 Cells; D018411:Spodoptera; D014404:Tubulin / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D014404:Tubulin; D016203:CDC2 Protein Kinase; D043525:Cytosine Deaminase