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.
