Inhibition of DUSP13A Activity by PTP Inhibitor V

Experimental Section

Cell Culture and Transfection. Human embryonic kidney (HEK) 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS, Thermo Scientific) and penicillin/streptomycin in the presence of 5% CO2. For transfection, 4 × 105 cells were seeded before the day of transfection and transfected with DNA using polyethylenimine, linear MW ~25,000 (PEI, Polysciences, Warrington, PA).

Antibodies and Plasmid Constructions. Monoclonal anti-FLAG antibody was from Sigma-Aldrich (St. Louis, MO). 6 x His-tagged DUSP13A was constructed in pET28a plasmid (Novagen, Madison, WI) for protein expression in Escherichia coli (E. coli) and FLAG-tagged DUSP13A was constructed in pcDNA3.1(+) (Invitrogen, Carlsbad, California).

Purification of 6 x His Tagged Proteins. PTP expression plasmids were constructed in pET28a(+) and transformed into BL21(DE3)-RIL E. coli. Recombinant proteins were purified as previously described.12

In vitro PTP Activity Assays and Kinetic Analysis. The activity of protein phosphatases was measured using the substrate 3-O-methylfluorescein phosphate (OMFP; Sigma-Aldrich) in a 96-well microtiter plate based on methods described previously.13 PTP inhibitor V and OMFP were solubilized in DMSO and all reactions were performed at the final concentration of 1% DMSO. The final incubation mixture (100 μL) was optimized for enzyme activity in assay buffer containing 30 mM Tris-HCl (pH 7), 75 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.4 mM dithiothreitol (DTT), 0.033% bovine serum albumin (BSA) and 100 nM of PTPs. Reaction was initiated by the addition of OMFP to the final concentration of 100 μM and the incubation time was 30 min at 37 °C, fluorescence levels of released products were determined using a fluorescence plate reader (an excitation of 485 nm and emission of 535 nm). The half maximal inhibition constant (IC50) was defined as the concentration of an inhibitor that caused a 50% decrease in the PTP activity by using the curve fitting program Prism3.0 (GraphPad Software). The inhibition constant (Ki) to DUSP13A phosphatase was calculated using the equations from the Lineweaver-Burk plots. The initial rates were measured at various OMFP concentrations for each fixed concentration of the inhibitor and the slopes showed the noncompetitive inhibition pattern. The Ki value was obtained from the below equation of noncompetitive inhibition. All experiments were performed in triplicate and repeated at least three times.

1/V = Km (1 + [I]/Ki) Vmax [S] + 1/Vmax (1 + [I]/Ki)

Immunoblotting Analysis. After HEK 293 cells were transiently transfected with or without FLAG-tagged DUSP13A for 48 h, cells were washed twice with phosphate buffered saline (PBS). The cells were lysed in PTP lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 0.5 % NP- 40, 0.5% Triton X -100, 1 mM EDTA, 1% glycerol, 1 mM PMSF and 1 μg/mL aprotinin. Cell lysates were centrifuged at 13,000 rpm for 30 min at 4 °C and the supernatants were transferred to 1.5 mL Eppendorf tube. Samples were boiled at 100 °C for 5 min. Samples were run in SDS−10% polyacrylamide gels and transferred to nitrocellulose membrane (Whatman, Springfield Mill, UK). The membrane was blocked in 5% nonfat skim milk and incubated with an anti-FLAG antibody, followed by incubation with a secondary antibody conjugated to horseradish peroxidase. The immunoreactive bands were visualized using an ECL system (Pierce, Rockford, IL).