Journal article
Dissection of Cdk1–cyclin complexes in vivo
Proceedings of the National Academy of Sciences - PNAS, Vol.110(39), pp.15716-15721
09/09/2013
DOI: 10.1073/pnas.1305420110
PMCID: PMC3785786
PMID: 24019491
Abstract
A simple strategy is described to discover cyclin dependency of general cell cycle regulatory kinase cyclin-dependent kinase 1 for substrates in vivo. γ-Tubulin is discovered to be a specific B cyclin Clb3–cyclin-dependent kinase 1 substrate. The strategy can link proteins to specific phases of the cell cycle, and it can be used to determine subunit dependencies for other posttranslational modifying enzymes.
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.
Details
- Title: Subtitle
- Dissection of Cdk1–cyclin complexes in vivo
- Creators
- Po Hien Ear - Université de MontréalMichael J. Booth - Agence pour le développement de l'emploiDiala Abd-Rabbo - Université de MontréalJacqueline Kowarzyk Moreno - Département de Biochimie andConrad Hall - McGill UniversityDaici Chen - McGill UniversityJackie Vogel - McGill UniversityStephen W. Michnick - Université de Montréal
- Resource Type
- Journal article
- Publication Details
- Proceedings of the National Academy of Sciences - PNAS, Vol.110(39), pp.15716-15721
- Publisher
- National Academy of Sciences
- DOI
- 10.1073/pnas.1305420110
- PMID
- 24019491
- PMCID
- PMC3785786
- ISSN
- 0027-8424
- eISSN
- 1091-6490
- Alternative title
- Dissection of Cdk1–cyclin complexes in vivo
- Language
- English
- Date published
- 09/09/2013
- Academic Unit
- Surgery
- Record Identifier
- 9984322817502771
Metrics
13 Record Views