Chemical biology investigation of cell death pathways activated by endoplasmic reticulum stress reveals cytoprotective modulators of ASK1 - PubMed (original) (raw)
Benzodiazepinone compounds increase Ser-967 phosphorylation of ASK1 and association with 14-3-3. A, phosphorylation of ASK1 at various sites was monitored by phospho-specific antibody immunoblotting. 293T cells were transfected with pcDNA-HA-ASK1, and 1 day later the cells were incubated with DMSO or 100 μ
m
compounds in DMSO for 2 h. Compounds included five active benzodiazepinone hits (CID-2891837, CID-2891533, CID-2891714, CID-2882794, and CID-2879344), an active hit compound representing another chemical class with a different mechanism (EI14), and an inactive benzodiazepinone analogue (CID-2882293). Cell extracts were prepared and subjected to immunoblotting using anti-phospho-ASK1 antibodies specific for Ser-83, Thr-845, or Ser-967 or with anti-HA antibody. B, relative densities of phosphorylated ASK1 bands (gray = Ser-83; black = Thr-845; white = Ser-967) were determined by densitometry mode of ImageJ software and normalized relative to ASK1 protein, calculating intensity relative to cells cultured with DMSO without TG. C, compounds from_A_ were compared with respect to cytoprotective activity against TG-induced killing of undifferentiated CSM14.1 cells. Active benzodiazepinone compounds are represented by black bars, and controls are depicted with gray bars. Relative cell viability was based on measurements of ATP content (mean ± S.D., n = 3). D, 293T cells were transfected with pcDNA-HA-ASK1 and pEBG-GST-14-3-3, and then 1 day later cells were incubated with DMSO, CID-2891837 (active compound) or CID-2882293 (inactive compound) in DMSO for 2 h, before TG (20 μ
m
) treatment for various times as indicated. Cell extracts were prepared, and GST-14-3-3 protein was recovered using glutathione-Sepharose, followed by immunoblotting (IB) analysis using anti-HA antibody to detect HA-ASK1 protein and anti-GST antibody to confirm pulldown of comparable amounts of GST-14-3-3 protein. Aliquots of the same cell lysates (50 μg) were analyzed by immunoblotting using anti-phospho-ASK1(Ser-967) and HA antibodies. E, benzodiazepinone compounds modulate interaction of endogenous ASK1 and 14-3-3 in OVCAR5 and SNB19 cells. Cells (5 × 106) were cultured in 100-mm dishes and incubated overnight, and then compound CID-2891837 (active), CID-2882293 (inactive), or DMSO was added, followed 2 h later by TG (20 μ
m
) treatment for 30 min. Cell extracts were prepared and normalized for total protein content, and endogenous ASK1 protein was immunoprecipitated (IP). Immune complexes were analyzed by immunoblotting using antibodies specific for ASK1, ASK1 (phospho-Ser-967), or 14-3-3. Aliquots of the cell lysates (50 μg) were analyzed directly by immunoblotting using antibodies specific for 14-3-3 to confirm comparable levels of this protein in all samples. F, HeLa cells were transfected with control (Ctrl.) RNAi (scrambled) or two different RNAi reagents targeting ASK1. After 24 h, cells were treated with 15 μ
m
TG for 24 h for analysis of relative cell viability by ATP content (mean ± S.D., n = 3) (bottom). Because of low endogenous levels of ASK1 protein, ASK1 siRNA-transfected cells were transfected with pcDNA-HA-ASK1, and after 24 h, cells were lysed for analysis of HA-ASK1 and β-actin protein levels by immunoblotting (top), to confirm RNAi efficiency.