Dissection of Cdk1–cyclin complexes in vivo

Dissection of Cdk1–cyclin complexes in vivo

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....

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS Vol. 110; no. 39; pp. 15716 - 15721
Main Authors: Ear, Po Hien, Booth, Michael J., Abd-Rabbo, Diala, Moreno, Jacqueline Kowarzyk, Hall, Conrad, Chen, Daici, Vogel, Jackie, Michnick, Stephen W.
Format: Journal Article
Language:English
Published: United States National Academy of Sciences 24-09-2013
National Acad Sciences
Subjects:
Biochemistry
Biological Sciences
Cell cycle
Cell growth
Cyclin-dependent kinases
Cyclins
Cyclins - metabolism
DNA Polymerase III - metabolism
Enzymes
Microtubules
Mitotic spindle apparatus
Models, Biological
Multiprotein Complexes - metabolism
Phosphorylation
Principal components analysis
Protein Binding
Proteins
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - metabolism
Substrate Specificity
Substrates
Tubulin - metabolism
Yeasts
in vivo enzyme complexes screen
cyclin specificity
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Summary: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.
Bibliography:http://dx.doi.org/10.1073/pnas.1305420110
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2Present address: Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, United Kingdom.
1Present address: Department of Genetics, Harvard Medical School, Boston, MA 02115.
3Present address: Institute for Research in Immunology and Cancer, University of Montreal, Montreal, QC, Canada H3C 3J7.
Edited by James A. Wells, University of California, San Francisco, CA, and approved August 13, 2013 (received for review March 20, 2013)
Author contributions: P.H.E., M.J.B., J.K.M., C.H., J.V., and S.W.M. designed research; P.H.E., M.J.B., J.K.M., C.H., and D.C. performed research; P.H.E., M.J.B., D.A.-R., J.K.M., D.C., J.V., and S.W.M. analyzed data; and P.H.E., M.J.B., D.A.-R., J.K.M., C.H., D.C., J.V., and S.W.M. wrote the paper.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1305420110