Physical association of CIB1 with ASK1. (A) A schematic representation of ASK1 indicating the thioredoxin binding region (gray box), the TRAF2 binding region (dotted box), and the kinase domain (hatched box) is shown in Upper. The indicated ³⁵S-labled ASK1 variants were reacted with GST-CIB1 immobilized on glutathione-agarose beads. The bead-bound ³⁵S-labled proteins were eluted and detected by SDS/PAGE and autoradiography. The gel was also stained with Coomassie brilliant blue. A portion (25%) of the ³⁵S-labled protein input to the binding reaction was also subjected to SDS/PAGE and autoradiography. (B) The 293T cells were left untreated or treated with 1 mM H₂O₂ or 20 ng/mL TNF-α for 20 min, lysed, and subjected to immunoprecipitation (IP) with antibodies to ASK1 or with rabbit preimmune IgG (control). The resulting precipitates and cell lysates were subjected to immunoblot (IB) analysis with antibodies to CIB1 or to ASK1.

CIB1 inhibits the recruitment of TRAF2 to ASK1, the phosphorylation of ASK1 on Thr⁸³⁸, and the interaction between ASK1 and MKK3. (A) The 293T cells were transfected for 48 h with vectors for Flag-CIB1, HA-ASK1, and Flag-TRAF2, as indicated. The cell lysates were subjected to immunoprecipitation with anti-HA antibody, and the resulting precipitates were immunoblotted with anti-Flag antibody. (B and D) HeLa cells expressing a control siRNA or CIB1 siRNA were untreated or treated with 1 mM H₂O₂ for 20 min, lysed, and subjected to immunoprecipitation with anti-ASK1 antibody. The resulting precipitates were subjected to immunoblot analysis with antibodies to TRAF2 (B) or to the phospho-Thr⁸³⁸ form of ASK1 (pASK1) (D). (C) The 293T cells were transfected for 48 h with plasmid vectors for HA-ASK1 and Flag-CIB1 as indicated. The cells were left untreated or treated with 1 mM H₂O₂ for 20 min, lysed, and subjected to immunoprecipitation with anti-HA antibody. The precipitates were subjected to immunoblot analysis with antibody to the phospho-Thr⁸³⁸ form of ASK1 (pASK1). (E) The 293T cells were transfected for 48 h with vectors for Flag-CIB1, HA-MKK3, and Myc-ASK1 as indicated, incubated in the absence or presence of 1 mM H₂O₂ for 20 min, lysed, and subjected to immunoprecipitation with anti-Myc antibody. The resulting precipitates were immunobloted with anti-HA antibody. (F) HeLa cells expressing a control siRNA or CIB1 siRNA were untreated or treated with 1 mM H₂O₂ for 20 min, lysed, and subjected to immunoprecipitation with anti-MKK3 antibody. The resulting precipitates were examined by immunoblotting with anti-ASK1 antibody.

KCl-elicited Ca²⁺ influx prevents the inhibitory action of CIB1 on 6-OHDA-induced ASK1 activation and apoptosis. (A) SH-SY5Y cells were incubated first with 6-OHDA (100 μM) for 50 min and then without or with KCl (40 mM) either alone or together with BAPTA-AM (2.5 μM) for 10 min, as indicated. Cell lysates were subjected to immunoprecipitation with anti-ASK1 antibody or rabbit preimmune IgG (P.I.), and the resulting precipitates were examined by immunoblotting with antibody to CIB1 (Left) or to TRAF2 (Right). (B) SH-SY5Y cells expressing a control siRNA or CIB1 siRNA were treated with 6-OHDA, KCl, BAPTA-AM as in A. Cell lysates were assayed for ASK1 activity by immune complex kinase assay. (C) SH-SY5Y cells expressing a control siRNA or CIB1 siRNA were incubated consecutively with 6-OHDA (100 μM) for 50 min, with KCl (40 mM) either alone or together with BAPTA-AM (2.5 μM) for 30 min, and in DMEM containing 2% FBS for 12 h, as indicated. Cells were analyzed for apoptosis by flow cytometry. The percentages of apoptotic cells in a representative experiment are indicated.

CIB1 inhibits ASK1 activity. (A) The 293T cells were transfected for 48 h with a plasmid vector for Flag-ASK1 and were treated with 1 mM H₂O₂ for 20 min where indicated. Cell lysates were subjected to immunoprecipitation with anti-Flag antibody. The resulting precipitates were incubated for 30 min at 20 °C with the indicated amounts of GST-CIB1 or GST in 50 μL of Hepes buffer (pH 7.4), and were then assayed for ASK1 activity. The relative activity of ASK1 is shown below each lane. (B) The 293T cells were transfected for 48 h with a vector encoding HA-ASK1 alone or together with a vector for Flag-CIB1, and were then incubated in the absence or presence of H₂O₂ (1 mM) or TNF-α (20 ng/mL) for 20 min. Cell lysates were assayed for ASK1 activity by immune complex kinase assay. (C) HeLa cells expressing a control siRNA or CIB1 siRNA were left untreated or treated with H₂O₂ (1 mM) or TNF-α (20 ng/mL) for 20 min. Where indicated, HeLa cells expressing CIB1 siRNA were transiently transfected for 48 h with a vector encoding Flag₃-CIB1* before the chemical treatment. Flag₃-CIB1* containing three silent third-codon point mutations (GAC → GAT, CTT → CTC, AGC → AGT) within the region targeted by CIB1 siRNA was generated by site-directed mutagenesis of human CIB1 gene with the use of a Quickchange kit (Stratagene). Cell lysates were subjected to immunoprecipitation with anti-ASK1 antibody, and the resulting precipitates were assayed for ASK1 activity. The precipitates were also subjected to immunoblotting with anti-ASK1 antibody. Cell lysates were also examined directly by immunoblot analysis with antibodies to CIB1 or to ASK1. eCIB1, endogenous CIB1. (D) HeLa cells expressing a control siRNA or CIB1 siRNA were untreated or treated with H₂O₂ (1 mM) for 20 min. Cell lysates were subjected to immunoprecipitation with antibodies to ASK1, and the resulting precipitates were subjected to immunoblot analysis with antibodies to CIB1 or to thioredoxin-1 (Trx1).

Depletion of CIB1 by RNAi potentiates ASK1 activation and apoptosis induced by 6-OHDA or by TNF-α. (A) SH-SY5Y cells expressing a control siRNA or CIB1 siRNA were incubated in the absence or presence of 6-OHDA (100 μM) for 1 h, lysed, and examined for ASK1 activity by immune complex kinase assay (Left). Alternatively, the cells were incubated first in the absence or presence of 6-OHDA (100 μM) for 2 h and then without 6-OHDA for an additional 12 h. The cells were stained with annexin V and PI, and the percentage of apoptotic cells was determined by flow cytometry (Right). (B) MCF7 cells expressing a control siRNA or CIB1 siRNA were incubated in the absence or presence of TNFα (20 ng/mL) for 20 min, lysed, and assayed for ASK1 activity (Left) by immune complex kinase assay. Alternatively, the cells were incubated in the absence or presence of TNF-α (20 ng/mL) plus cycloheximide (3 ng/mL) for 20 h and then examined for apoptosis (Right) as in A. Data from apoptosis assays in A and B are means ± SD of values from three independent experiments.

KCl-elicited Ca²⁺ influx reverses the inhibitory action of CIB1 on 6-OHDA-induced ASK1 activation and cell death in rat mesencephalic dopaminergic neurons. Primary mesencephalic neurons were transfected for 48 h with control or rat CIB1 siRNA oligonucleotides. The neurons were untreated or treated with 50 μM 6-OHDA for 50 min and then incubated for 10 min in the absence or presence of KCl (40 mM) alone or together with BAPTA-AM (2.5 μM), as indicated. The cells were lysed and then assayed for ASK1 activity (A) or incubated for an additional 24 h in Neurobasal medium, and subjected to immunocytochemistry with anti-TH antibody (B). (B) Shown are representative images of the stained cells (Lower) (scale bar, 100 μm), and the viability of TH-positive neurons (Upper) as a percentage of that for untreated control siRNA-transfected cells. Data are means ± SD of values from three independent experiments. *, P < 0.01 represents the result of Student's t test for control siRNA-transfected cells with no treatment versus those with 6-OHDA treatment. **, P < 0.01 for differences between control siRNA-transfected cells treated with 6-OHDA and those treated with 6-OHDA + KCl. ***, P < 0.01 for differences between control siRNA-transfected cells treated with 6-OHDA + KCl and those treated with 6-OHDA + KCl + BAPTA-AM. *, P < 0.01 for differences between CIB1 siRNA-transfected cells with no treatment and those with 6-OHDA treatment. ●, P < 0.01 for differences between control siRNA-transfected cells treated with 6-OHDA and CIB1 siRNA-transfected cells treated with 6-OHDA. NS, no significance between the indicated paired data.