Inhibition of DUSP13B Phosphatase Activity by PTP Inhibitor V

Abstract

We next confirmed the inhibition mechanism of PTP inhibitor V on DUSP13B by kinetic measurements based on Michaelis-Menten equation. The Lineweaver-Burk plots show that the maximum reaction velocity (Vmax) is constant regardless of the presence of the inhibitor. This result indicates that PTP inhibitor V binds to the active site of DUSP13B and functions as a competitive inhibitor of DUSP13B (Fig. 2(b)). The calculated inhibition constant (Ki) value was 6.36 μM. To clarify the inhibitory effect of PTP inhibitor V on intact DUSP13B expressed in the mammalian cells, human embryonic kidney 293 (HEK 293) cells were transfected with FLAG-tagged DUSP13B expression plasmid and incubated for 48 h. Then the cells were harvested and lysed with PTP lysis buffer. FLAG-DUSP13B was immunoprecipitated from cell lysates by anti-FLAG M2 affinity gel. Immunoprecipitated DUSP13B was incubated with or without PTP inhibitor V and the phosphatase activities of DUSP13B were measured by using 3-O-methylfluorescein phosphate (OMFP) as a substrate. Since PTP inhibitor V is hardly soluble in Dulbecco's modified Eagle's medium (DMEM), we decided to treat the inhibitor to the immunoprecipitated DUSP13B. The phosphatase activity of DUSP13B decreased as the