title: Expression, purification, and enzymatic characterization of the dual specificity mitogen-activated protein kinase phosphatase, MKP-4.
abstract: Mitogen-activated protein kinase phosphatase-4 (MKP-4) is a dual specificity phosphatase, which acts as a negative regulator of insulin-stimulated pathways. Here, we describe expression, purification, and biochemical characterization of MKP-4. We used the Baculovirus expression system and purification with a combination of affinity and gel filtration chromatography to generate pure MKP-4 and MKP-4/p38 complex. Both MKP-4 and the MKP-4/p38 complex exhibited moderate activity toward the surrogate substrates p-nitrophenyl phosphate, 6, 8-difluoro-4-methylumbelliferyl phosphate, and 3-O-methylfluorescein phosphate. The phosphatase activity could be inhibited by peroxovanate, a potent inhibitor of protein tyrosine phosphatases. We further determined kinetic parameters for the MKP-4 and the MKP-4/p38 by using spectrophotometric and fluorescence intensity methods. The MKP-4/p38 complex was found to provide substantially higher phosphatase activity than MKP-4 alone, similar to what has been shown for MKP-3. Our data allow the configuration of screens for modulators of MKP-4 activity.
authors.affiliation: Metabolic Disease Biology, Millennium Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, MA 02139, USA. vhong@wyeth.com
authors.forename: Suk-Bong
authors.initials: SB
authors.lastname: Hong
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Lubben
authors.forename: Christine M
authors.initials: CM
authors.lastname: Dolliver
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Petrolonis
authors.forename: Rebecca A
authors.initials: RA
authors.lastname: Roy
authors.forename: Zhi
authors.initials: Z
authors.lastname: Li
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Parsons
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Haiyan
authors.initials: H
authors.lastname: Xu
authors.forename: Regina M
authors.initials: RM
authors.lastname: Reilly
authors.forename: James M
authors.initials: JM
authors.lastname: Trevillyan
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Nichols
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Tummino
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Gant
mesh_terms: D002478:Cells, Cultured; D054637:Dual-Specificity Phosphatases; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D054639:Mitogen-Activated Protein Kinase Phosphatases; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D007328:Insulin; D048052:Mitogen-Activated Protein Kinase 3; D048051:p38 Mitogen-Activated Protein Kinases; D054639:Mitogen-Activated Protein Kinase Phosphatases; D010749:Phosphoprotein Phosphatases; C104821:DUSP9 protein, human; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases