Expression, purification, and enzymatic characterization of the dual specificity mitogen-activated protein kinase phosphatase, MKP-4 |
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Authors: | Hong Suk-Bong Lubben Thomas H Dolliver Christine M Petrolonis Anthony J Roy Rebecca A Li Zhi Parsons Thomas F Li Ping Xu Haiyan Reilly Regina M Trevillyan James M Nichols Andrew J Tummino Peter J Gant Thomas G |
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Affiliation: | Metabolic Disease Biology, Millennium Pharmaceuticals, Inc., 75 Sidney Street, Cambridge, MA 02139, USA. vhong@wyeth.com |
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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. |
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Keywords: | Dual specificity phosphatase Protein tyrosine phosphatase superfamily Type 2 diabetes MAP kinase Insulin signaling Diabetes mellitus |
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