title: Measuring Activity and Specificity of Protein Phosphatases.
abstract: Reversible protein phosphorylation plays essential roles in coordinating cell division and many other biological processes. Cell cycle regulation by opposing kinase and protein phosphatase activities is often complex and major challenges exist in identifying the direct substrates of these enzymes and the specific sites at which they act. While cell cycle kinases are known to exhibit strict substrate specificities important for coordinating the complex events of cell division, phosphatases have only recently been recognized to exert similarly precise regulatory control over cell cycle events through timely dephosphorylation of specific substrates. The molecular determinants for substrate recognition by many phosphatases that function in cell division are still poorly delineated. To understand phosphatase specificity, it is critical to employ methods that monitor the dephosphorylation of individual phosphorylation sites on physiologically relevant substrates. Here, using the cell cycle phosphatase Cdc14 as an example, we describe two methods for studying phosphatase specificity, one using synthetic phosphopeptide substrates and the other using intact phosphoprotein substrates. These methods are useful for targeted characterization of small substrate sets and are also adaptable to large-scale applications for global specificity studies.
authors.affiliation: Department of Biochemistry and Center for Cancer Research, Purdue University, West Lafayette, IN, 47907, USA.
authors.forename: Brendan L
authors.initials: BL
authors.lastname: Powers
authors.forename: Michael
authors.initials: M
authors.lastname: Melesse
authors.forename: Christie L
authors.initials: CL
authors.lastname: Eissler
authors.forename: Harry
authors.initials: H
authors.lastname: Charbonneau
authors.forename: Mark C
authors.initials: MC
authors.lastname: Hall
mesh_terms: D002853:Chromatography, Liquid; D057075:Enzyme Assays / Q000379:methods*; D013058:Mass Spectrometry; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D013379:Substrate Specificity
chemical_list: D010748:Phosphopeptides; D010749:Phosphoprotein Phosphatases