Computational Prediction and Experimental Verification of New MAP Kinase Docking Sites and Substrates Including Gli Transcription Factors
Figure 5
Identification of a D-site in the PPM1J phosphatase.
(A) Wild-type (WT) and N-terminal truncated versions (NT w/o D) of PPM1J were fused to GST and tested for binding to JNK 1–3. The sequence of the D-site is shown. (B) As shown in A, 35S-radiolabeled JNK1, 2 or 3 (∼1 pmole) were tested for binding to 40 µg of GST (lane 1) or 30 µg of GST-PPMIJ K17–506 (containing the D-site, lanes 3 and 4) or GST-PPMIJ K80–506 (lacking the D-site, lanes 5 and 6). Lane 1 shows a 5% of the total JNK input. The lower panel shows Coomassie Blue (CB) staining of the sedimented GST-fusion proteins. Other details as in Fig. 4C. (C) Wild-type (WT) and D-site-mutant (DSM) versions of PPM1J were tested as substrates for in vitro phosphorylation by active JNK1-3. (D) As shown in C, 1 µM of each GST-PPM1J protein was incubated with 0, 0.1 or 1 mU JNK1-3 and [γ-32]ATP for 20 min. Incorporation of radioactive phosphate into the substrate, as assessed by autoradiography, is shown in 3 panels, and a representative Coomassie blue (CB) stained gel, to demonstrate equal loading of the substrates proteins, is shown. (E) Wild-type and D-site mutant versions of PPM1J were C-terminally-tagged with the V5 epitope, expressed in Cos-1 cells, immunoprecipitated, and used as substrates for JNK1-mediated phosphorylation. (F). As shown in E, Cos-1 cells were transfected with either empty vecor (EV), PPM1J-V5 WT, or PPM1J-V5 DSM. 16 h post-transfection, the cells were harvested, lysed, and immunoprecipitated with anti-V5 antibodies. MAPK buffer, [γ-32]ATP, and active JNK1 were added to the immunoprecipitated pellets, and phosphorylation of the immunoprecipitated proteins was visualized by PhosphorImager. In addition, portions (20 µg) of each lysate were separated by SDS-PAGE and immunoblotted (WB, for Western Blot) with either anti-V5 (1∶5000) or anti-total JNK (1∶500) antibodies.