Fig 1.
Involvement of DUSP22 in MAPK phosphorylation.
(A) HCT 116 cells were transfected with FLAG-DUSP22 WT or FLAG-DUSP22 C88S expression plasmid. After 48 h, cells were lysed in PTP lysis buffer. Cell lysates were immunoprecipitated with the anti-FLAG M2 affinity gel for 1 h at 4°C. Then, phosphatase activity was measured as described in the Materials and methods and presented as fluorescence intensity (in arbitrary unit). Immunoblot analysis was performed with cell lysates using an anti-FLAG or an anti-tubulin antibody. IB, immunoblot. (B) Transfected HCT 116 cells were further incubated in the presence or absence of H2O2 (1 mM) for 1 h and lysed in PTP lysis buffer. Cell lysates were subjected to immunoblotting with antibodies as indicated. Twenty micrograms of cell lysates were loaded in each lane for detection. Those levels were quantified by analysis with LabWorks software (UVP Inc., Upland, CA) and normalized to corresponding total protein levels. Right panel: Relative fold induction of phosphorylated MAPK level after normalization. Tubulin served as a loading control. All data are representative of three independent experiments. Statistical significance was determined by one-way ANOVA. *P< 0.01 compared with control. (C) HEK 293 cells were co-transfected using HA-JNK1 expression plasmid together with FLAG-DUSP22 WT or FLAG-DUSP22 C88S expression plasmid. After 48 h, cells were additionally incubated in the presence or absence of H2O2 (1 mM) for 1 h and lysed in lysis buffer. HA-JNK1 was immunoprecipitated with an anti-HA antibody. Immunoprecipitates were subjected to an in vitro kinase assay using 1 μg of GST-c-Jun as a substrate. Kinase activity was normalized to the expression level of c-Jun and presented as fold increase. CB, coomassie blue staining.
Fig 2.
Interaction of DUSP22 with ASK1, JNK1/2, and MKK7.
(A) HEK 293 cells were co-transfected using FLAG-DUSP22 expression plasmid with or without HA-ASK1 expression plasmid. After 48 h, lysates were prepared and incubated with the anti-FLAG M2 affinity gel. Pulled-down DUSP22 complexes were subjected to SDS-PAGE. Immunoblot analysis was performed using an anti-HA antibody. IP, immunoprecipitation. (B) HEK 293 cells were co-transfected with HA-DUSP22 expression plasmid with or without FLAG-ASK1 expression plasmid. After immunoprecipitation, pulled-down ASK1 complex was detected as described above. HEK 293 cells were transfected with FLAG-DUSP22 expression plasmid together with (C) HA-ERK1, HA-JNK1, or HA-p38γ (D) HA-MKK4 or HA-MKK7. Cells were lysed in PTP lysis buffer and immunoprecipitated with the anti-FLAG M2 affinity gel. The immunoprecipitates were subjected to SDS-PAGE and then immunoblotting with anti-HA and anti-FLAG antibodies.
Fig 3.
Interaction of DUSP22 mutant with ASK1, MKK7, and JNK1 and dimerization of DUSP22.
FLAG-DUSP22 WT or FlAG-DUSP22 C88S expression plasmid was co-transfected together with (A) HA-ASK1, (B) HA-MKK7, (C) HA-JNK1, or (D) HA-JNK2 expression plasmid. After 48 h, cells were lysed in lysis buffer and immunoprecipitated with the anti-FLAG M2 affinity gel. Pulled-down DUSP22 WT and mutant complexes were detected by immunoblotting with anti-HA and anti-FLAG antibodies. After 48 h, cells were lysed and immunoprecipitated with the anti-FLAG M2 affinity gel. The immunoprecipitates were subjected to SDS-PAGE and then immunoblotting with an anti-HA antibody.
Fig 4.
(A) Tethering mechanism of DUSP22 as a scaffold protein. (B) HEK 293 cells were co-transfected using 3 μg each of FLAG-ASK1 and GST-MKK7 expression plasmids in 100 mm tissue culture dishes and then split into 60 mm tissue-culture dishes. After 24 h, HA-DUSP22 (0, 0.75, 1.5, 3 μg) expression plasmid was transfected in a dose-dependent manner into HEK 293 cells. After 48 h of transfection, cells were lysed and immunoprecipitated with the anti-FLAG M2 affinity gel. Then, the immunoprecipitates were subjected to SDS-PAGE and then immunoblotting with an anti-HA, anti-GST, or anti-FLAG antibody. (C) FLAG-DUSP22 WT (0, 0.5, 0.75, 1.25, 2, 3 μg) or (D) FLAG-DUSP22 C88S (0, 0.4, 0.6, 1, 1.6, 2.4 μg) expression plasmid was transiently transfected in a dose-dependent manner into HEK 293 cells. After 48 h, cell lysates were subjected to immunoblotting with anti-JNK and anti-p-JNK antibodies. Relative JNK phosphorylation levels were quantified by analysis with LabWorks software and normalized to corresponding total JNK protein levels.
Fig 5.
(A) Cell viability was determined as described in Materials and Methods. HEK 293 cells were transfected with FLAG-DUSP22 (WT or C88S mutant, 1 μg) expression plasmid. Data represent means ± SEM of six observations from three independent experiments. The expression levels of DUSP22 were determined by immunoblotting with an anti-FLAG antibody. IB, immunoblot. *p < 0.01 versus the control sample by Student’s t-test. (B) HCT 116 cells were transfected with 2 μg of FLAG-DUSP22 WT or FLAG-DUSP22 C88S expression plasmid. After 48 h of transfection, cell lysates were subjected to immunoblotting with indicated antibodies. Fifty microgram of cell lysate was loaded in each lane and the blot was probed with antibodies specific to p-JNK, cleaved PARP, or cleaved caspase-3. Twenty microgram of cell lysate was used for detection of JNK and FLAG. HCT 116 cells were transfected with (C) and (E) FLAG-DUSP22 WT (0, 0.5, 1, 2, 3, 4 μg) or (D) and (F) FLAG-DUSP22 C88S (0, 0.5, 1, 2, 3, 4 μg) for 48 h and then incubated for 1 h in the absence or presence of H2O2. Total cell lysates were subjected to immunoblot analyses with appropriate antibodies. Right panel: Relative fold of cleaved PARP levels after normalization to corresponding tubulin levels. All data are representative of three independent experiments.