Inhibition of PTPN2 by PTP inhibitor V

Experimental Section

Antibodies and Reagents.Anti-FLAG M2 antibody was purchased from Sigma-Aldrich (St. Louis, MO). Linear-formed polyethylenimine (PEI) used for transfection into HEK 293 cell was purchased from Polysciences, Inc. (Warrington, PA). PTP inhibitor V was purchased from Merck KGaA (Darmstadt, Germany).

Cell Culture and Transfection. 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, Waltham, MA) and penicillin/streptomycin in the presence of 5% CO2. For transfection, 1 × 106 cells were seeded in each 60 mm cell culture dish before the day of transfection and transfected with DNA using PEI.

Purification of Recombinant Protein. 6 x His-tagged PTPN2 was constructed in pET28a(+) plasmid (Novagen, Darmstadt, Germany) and transformed into BL21(DE3)-RIL Escherichia coli. Recombinant protein was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 22 °C for overnight. Cells were harvested and then lysed by sonication in 50 mM Tris-HCl (pH 6.8), 300 mM NaCl, 1% IGEPAL CA-630 (NP-40), 1 mM phenylmethanesulfonyl fluoride (PMSF). The lysates were clarified at 10,000 rpm for 20 min at 4 °C. The supernatant was applied by gravity flow to a column of Ni-NTA resin (PEPTRON, Daejon, Korea). The resin was washed with 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 50 mM imidazole and eluted with 20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 200 mM imidazole.

In vitro Phosphatase Assays and Kinetic Analysis.Phosphatase activity was measured using the substrate OMFP (Sigma-Aldrich, St. Louis, MO) in a 96-well microtiter plate assay based on methods described previously.17 PTP inhibitor V and OMFP were solubilized in dimethyl sulfoxide (DMSO). All reactions were performed at the final concentration of 1% DMSO. The final reaction mixture (100 μL) was optimized for enzyme activity and composed of 30 mM Tris-HCl (pH 7.0), 75 mM NaCl, 1 mM ethylenediaminete-traacetic acid (EDTA), 0.4 mM dithiothreitol (DTT), 0.132% bovine serum albumin (BSA), and 100 nM of PTPs. Reactions were started by addition of OMFP and incubated for 30 min at 37 °C. Fluorescence emission from the product was measured with a multi-well plate reader (Biotek, ex-citation filter, 485 nm; emission filter, 535 nm). The reaction was linear over the time period of the experiment and was directly proportional to both enzyme and substrate con-centrations. Half-maximal inhibition constant (IC50) was defined as the concentration of an inhibitor that caused a 50% decrease in the PTP activities. Half-maximal inhibition constants and best curve fit for Lineweaver-Burk plots were determined by using the curve fitting program Prism 3.0 (GraphPad Software, San Diego, CA). All experiments were performed in triplicate and were repeated at least three times.

Immunoblotting Analysis. Cell lysates were run in SDS-10% polyacrylamide gel and transferred to nitrocellulose membrane. The membrane was blocked in 5% skim milk for 1 h and incubated with the appropriate primary antibodies, followed by incubation with the appropriate secondary antibodies conjugated horseradish peroxidase (HRP). The protein bands were visualized by the ECL detection system (Pierce, Rockford, IL).

Immunoprecipitation and in vitro Phosphatase Activity Assay.For immunoprecipitation, HEK 293 cells were transi-ently transfected with FLAG-PTPN2 expression plasmid for 48 h. After incubation, cells were washed several times with 1 x phosphate buffer saline (PBS, pH 7.4) to remove the inhibitor remained in the media. The cells were lysed with the lysis buffer containing 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 0.5% IGEPAL CA-630, 0.5% Triton X-100, 1% Glycerol, 1 mM EDTA, 1 mM PMSF, and 3 μM DTT for 15 min at 4 °C. Cell lysates were centrifuged at 13,000 rpm for 30 min at 4 °C and the soluble fractions were immunopre-cipitated with anti-FLAG M2-agarose for 3 h at 4 °C. After binding, the beads were washed with lysis buffer and the bound proteins were incubated with PTP inhibitor V and OMFP. The reaction buffer was comprised of 30 mM Tris-HCl (pH 7.0), 75 mM NaCl, 1 mM EDTA, 0.4 mM DTT, and 0.132% BSA. The reaction mixtures were incubated at 37 °C for 30 min and fluorescence was measured using a microplate reader (excitation filter, 485 nm; emission filter, 535 nm).

Inhibition Study. The inhibition constant (Ki) to PTPN2 for the inhibitor was determine by measuring the initial rates at several OMFP concentration for each fixed concentration of the inhibitor. The data were fitted to the following equa-tion to obtain the inhibition constant of reversible competi-tive inhibitors. The slops obtained were replotted against the inhibitor concentrations. The Ki value was obtained from the slopes of these replots.18

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