Chemical biology investigation of cell death pathways activated by endoplasmic reticulum stress reveals cytoprotective modulators of ASK1.
The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is caused by many disease-relevant conditions, inducing conserved signaling events collectively known as the unfolded protein response. When ER stress is excessive or prolonged, cell death (usually occurring by apoptosis) is triggered. We undertook a chemical biology approach for investigating mechanisms of ER stress-induced cell death. Using a cell-based high throughput screening assay to identify compounds that rescued a neuronal cell line from thapsigargin-induced cell death, we identified benzodiazepinones that selectively inhibit cell death caused by inducers of ER stress (thapsigargin and tunicamycin) but not by inducers of extrinsic (tumor necrosis factor) or intrinsic (mitochondrial) cell death pathways. The compounds displayed activity in several cell lines and primary cultured neurons. Mechanism of action studies revealed that these compounds inhibit ER stress-induced activation of p38 MAPK and kinases responsible for c-Jun phosphorylation. Active benzodiazepinones suppressed cell death at the level of apoptotic signal kinase-1 (ASK1) within the IRE1 pathway but without directly inhibiting the kinase activity of ASK1 or >400 other kinases tested. Rather, active compounds enhanced phosphorylation of serine 967 of ASK1, promoting ASK1 binding to 14-3-3, an event associated with suppression of ASK1 function. Reducing ASK1 protein expression using small interfering RNA also protected cells from ER stress-induced apoptosis, confirming the importance of this protein kinase. Taken together, these findings demonstrate an essential role for ASK1 in cell death induced by ER stress. The compounds identified may prove useful for revealing endogenous mechanisms that regulate inhibitory phosphorylation of ASK1.
Kim
InKi
I
Burnham Center for Chemical Genomics, Burnham Institute for Medical Research, La Jolla, California 92037, USA.
Shu
Chih-Wen
CW
Xu
Wenjie
W
Shiau
Chung-Wai
CW
Grant
Daniel
D
Vasile
Stefan
S
Cosford
Nicholas D P
ND
Reed
John C
JC
D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D001570:Benzodiazepinones / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D048848:MAP Kinase Kinase Kinase 5 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051379:Mice; D009474:Neurons / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000502:physiology; D017510:Protein Folding; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D013312:Stress, Physiological / Q000187:drug effects / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D048948:14-3-3 Proteins; D000900:Anti-Bacterial Agents; D001570:Benzodiazepinones; D004791:Enzyme Inhibitors; D016755:Proto-Oncogene Proteins c-jun; D014409:Tumor Necrosis Factor-alpha; D014415:Tunicamycin; D019284:Thapsigargin; D048051:p38 Mitogen-Activated Protein Kinases; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; C482859:Map3k5 protein, mouse