Expression, purification, and enzymatic characterization of the dual specificity mitogen-activated protein kinase phosphatase, MKP-4.
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.
Hong
Suk-Bong
SB
Metabolic Disease Biology, Millennium Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, MA 02139, USA. vhong@wyeth.com
Lubben
Thomas H
TH
Dolliver
Christine M
CM
Petrolonis
Anthony J
AJ
Roy
Rebecca A
RA
Li
Zhi
Z
Parsons
Thomas F
TF
Li
Ping
P
Xu
Haiyan
H
Reilly
Regina M
RM
Trevillyan
James M
JM
Nichols
Andrew J
AJ
Tummino
Peter J
PJ
Gant
Thomas G
TG
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
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