Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 111, Issue 31, Pages 11323-11328Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1409666111
Keywords
kinase substrate identification; NESKA
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Funding
- National Institutes of Health [2P41GM103314, GM103362, GM47238, GM78153]
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In eukaryotes, cell cycle progression is controlled by cyclin/cyclin-dependent kinase (CDK) pairs. To better understand the details of this process, it is necessary to dissect the CDK's substrate pool in a cyclin-and cell cycle stage-specific way. Here, we report a mass spectrometry-based method that couples rapid isolation of native kinase-substrate complexes to on-bead phosphorylation with heavy-labeled ATP (ATP-gamma-O-18(4)). This combined in vivo/in vitro method was developed for identifying cyclin/CDK substrates together with their sites of phosphorylation. We used the method to identify Clb5 (S-cyclin)/Cdc28 and Cln2 (G1/S-cyclin)/Cdc28 substrates during S phase in Saccharomyces cerevisiae (Cdc28 is the master CDK in budding yeast). During the work, we discovered that Clb5/Cdc28 specifically phosphorylates S429 in the disordered tail of Cdc14, an essential phosphatase antagonist of Cdc28. This phosphorylation severely decreases the activity of Cdc14, providing a means for modulating the balance of CDK and phosphatase activity.
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