title: Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase.
abstract: We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.
authors.affiliation: Cell Biology Unit, Institut de Genetique Humaine, UPR 1142 CNRS, 141 rue de la Cardonille, F34396 Montpellier Cedex 05, France.
authors.forename: Celine
authors.initials: C
authors.lastname: Franckhauser
authors.forename: Anne
authors.initials: A
authors.lastname: Fernandez
authors.forename: Ned J C
authors.initials: NJ
authors.lastname: Lamb
mesh_terms: D000814:Aniline Compounds / Q000737:chemistry / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002384:Catalysis; D056744:Cyclin B1; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D000814:Aniline Compounds; D056744:Cyclin B1; D009943:Organophosphorus Compounds; C068798:4-aminophenylphosphate; D016203:CDC2 Protein Kinase; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D020687:cdc25 Phosphatases