Cell Culture and Transfection
. Human embryonic kidney (HEK) 293 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Scientific, Waltham, MA). All of media were supplemented with 10% fetal bovine serum (FBS, Thermo Scientific) and penicillin/streptomycin in the presence of 5% CO
. For transfection, 4 × 10
cells were seeded before the day of transfection and transfected with DNA using OmicsFect
(Omics Biotechnology, Taiwan).
Antibodies and Plasmid Constructions.
Anti-JNK was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-SAPK/JNK (specific for phospho-Thr183 and phosho-Tyr185) was from Cell Signaling Technology (Danvers, MA). Monoclonal Anti-FLAG was from Sigma-Aldrich. His-tagged DUSP14-WT was constructed in pET28a plasmid (Novagen, Madison, WI) for protein expression in
and FLAG-tagged DUSP14-WT and DUSP14 D80A mutant were constructed in pcDNA3.1 (Invitrogen, Carlsbad, California).
Purification of the six-His-tagged Proteins.
PTP expression plasmids were constructed in pET-28a (+) and transformed into BL21 (DE3)-RIL
. Recombinant proteins were induced with 1 mM isopropyl-β-D-thiogalactopyranoside at 20 °C for 16 h. Cells were harvested and then lysed by sonication in 50 mM Tris–HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole, 1% NP-40, 1 mM phenylmethylsulphonyl fluoride (PMSF). The lysates were clarified at 13,000 rpm for 30 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), 300 mM NaCl, and 20 mM imidazole and then eluted with 20 mM TrisHCl (pH 8.0), 300 mM NaCl, and 250 mM imidazole. The eluted proteins were dialyzed overnight against 20 mM Tris–HCl, 150 mM NaCl, 20% glycerol, and 0.5 mM PMSF before storage at −80 °C.
In vitro Phosphatase Assays and Kinetic Analysis.
Phosphatase activities were measured using the substrate 3-
-methylfluorescein phosphate (OMFP; Sigma, St. Louis, MO) at concentrations that varied according to the
(Michaelis constant) of each enzyme in a 96-well microtiter plate assay based on methods described previously.
PTP inhibitor V and OMFP were solubilized in DMSO. All reac-tions were performed at a final concentration of 1% DMSO. The final incubation mixture (150 μL) was optimized for enzyme activity and was composed of 30 mM Tris–HCl (pH 7.0), 75 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1 mM dithiothreitol (DTT), 0.33% bovine serum albumin (BSA) and 100 nM of each PTP. Reactions were initiated by addition of OMFP and incubated for 30 min at 37 °C. Fluorescence emission from the product was measur-ed with a multiwall plate reader (Synergy H1; excitation filter, 485 nm; emission filter, 535 nm). The reaction was linear over the experimental time period and was directly proportional to both enzyme and substrate concentration. The half-maximal inhibition constant (IC
) was defined as the concentration of inhibitor that caused a 50% decrease in the PTP activity. Half-maximal inhibition constants and best curve fit for Lineweaver-Burk plots were determined by using the curve fitting program PRISM 3.0 (GraphPad Soft-ware). All experiments were performed in triplicate and were repeated at least three times.
. The inhibition constant (
) to DUSP14 phosphatase for the inhibitor was determined by measuring the initial rates at several OMFP concentrations for each fixed concentration of the inhibitor. The data were fitted to the following equation to obtain the inhibition constant of reversible competitive inhibitors. The slopes obtained were replotted against the inhibitor concentrations. The
value was obtained from the slopes of these replots.
(1 + [I]/
[S] + 1/V
Effect of PTP Inhibitor V on DUSP14 Expressed in Mammalian Cells.
HEK 293 cells were transfected with FLAG-DUSP14 WT or DUSP14 D80A expression plasmid. After 48 h of transfection, cells were washed twice with phosphate buffered saline (PBS) buffer and lysed in PTP lysis buffer (0.5% NP-40, 0.5% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1% glycerol, 1 mM PMSF, and 1 μg/mL aprotinin) for 30 min at 4 °C. Cleared cell lysates from centrifugation were mixed with washed FLAG M2-agarose (Sigma-Aldrich, St. Louis, MO) and incubated for 16 h at 4 °C using rotation device. After incubation, FLAG M2-agarose was washed three times with PTP lysis buffer and measured their phosphatase activities.
Dephosphorylation Assays with Active Phosphorylated JNK.
The six-His-tagged DUSP14 (1 μg) was combined with active phosphorylated JNK (10 ng) in PTP assay buffer (30 mM Tris–HCl (pH 7.0), 75 mM NaCl, 1 mM EDTA, 0.1 mM DTT, 0.33% BSA) and incubated for 30 min at 37 °C in a 30 μL reaction volume. To determine whether PTP inhibitor V inhibits the DUSP14 effect on JNK
, 100 nM of DUSP14 was mixed with 10 ng of active phosphorylated JNK and various concentrations of PTP inhibitor V (0, 10, 50, or 100 μM) in a 30 μL reaction volume and incubated for 30 min at 37 °C. The products of dephosphorylation reac-tions were subjected to SDS-PAGE and then immunoblotted with an anti-phospho-JNK antibody.
Transfected cells with or without FLAG-DUSP14 WT or DUSP14 mutant expression plasmids for 48 h were washed twice with phosphate buffer-ed saline (PBS) buffer and lysed in 1x SDS sample buffer (12 mM Tris-HCl pH 6.8, 0.4% SDS, 5% glycerol, 1% β-mercaptoethanol, 0.02% bromophenol blue) and boiled at 100 °C for 5 min. Sampled were run in SDS-10% polyacryl-amide gels and transferred to nitrocellulose membrane. Then the membranes were blocked with 5% skim milk for 1 h and incubated with an appropriate antibody, followed by incubation with a HRP-conjugated secondary antibody. The protein bands were visualized by the ECL detection system (Pierce, Rockford, IL).
This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science, ICT & Future Planning (NRF-2011-0030029 and NRF- NRF-2012R1A1B3001937).
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