title: Method for identifying phosphorylated substrates of specific cyclin/cyclin-dependent kinase complexes.
abstract: 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-γ-(18)O4). 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.
authors: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry and.
authors: Yinyin
authors: Y
authors: Li
authors: Laboratory of Cell Cycle Genetics, The Rockefeller University, New York, NY 10065.
authors: Frederick R
authors: FR
authors: Cross
authors: Laboratory of Mass Spectrometry and Gaseous Ion Chemistry and chait@rockefeller.edu.
authors: Brian T
authors: BT
authors: Chait
mesh_terms: D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D013058:Mass Spectrometry; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D016213:Cyclins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D010768:Phosphoserine; D018844:Cyclin-Dependent Kinases; D010749:Phosphoprotein Phosphatases
keywords: NESKA; kinase substrate identification