Figure 5.

Role of oxidation in H₂O₂-induced Ask1 signaling pathways. (A) Schematic representation of the various cysteine substitution mutants of the N (N-terminal), K (kinase), and C (C-terminal) domain of Ask1 used in this figure. The numbers indicated within each domain correspond to the cysteine residues that have been replaced by alanine residues. All constructs have an HA-tag at the C-terminal end. (B–E) 293T cells were transfected with the various Ask1 constructs as indicated at the top of each panel and defined in A, or transfected with the empty vector pcDNA3 (EV). Twenty-four hours after transfection, the cells were left untreated (−) or treated (+) with 0.1 mM (B and E) or 1 mM H₂O₂ (C and D) for 3 min, or with 10 μM thapsigargin for 10 min (E). Extracts were migrated on nonreducing gels and immunoblotted with anti-HA or on reducing gels and probed with phospho-specific Ask1 (P-Ask1), JNK (P-JNK), MKK4 (P-MKK4), or MKK7 (P-MKK7) antibodies. The expected position of the monomeric Ask1 constructs is indicated by closed arrowheads. Higher molecular weight Ask1 complexes are identified by open arrowheads. Lane numbers are referred to in the text. (F) HeLa cells were transfected with the pcDNA3 empty vector (EV), pcDNA3-Ask1-HA (Ask1), pcDNA3-Ask1K709M (KM), or pcDNA3-Ask1ΔCys-HA (ΔCys). Forty-eight hours later, the cells were left untreated (Control) or exposed to 500 μM H₂O₂ or to 10 μM thapsigargin for 3 h. The percentage of HA-positive cells showing condensed or fragmented nuclei was determined by immunofluorescence microscopy. In the EV group, all cells were considered in the counts. The data are means + SEM from four (Control and H₂O₂) or three (Thapsigargin) distinct experiments. **p < 0.01; *p < 0.05 (one-way analysis of variance), comparing apoptosis in cells transfected with Ask1-HA or ΔCys-HA with EV transfected cells within the same treatment (H₂O₂ or thapsigargin).