GRP78 and Raf-1 cooperatively confer resistance to endoplasmic reticulum stress-induced apoptosis.
The chaperone glucose-regulated protein, 78/immunoglobulin binding protein (GRP78/Bip), protects cells from cytotoxicity induced by DNA damage or endoplasmic reticulum (ER) stress. In this study, we showed that GRP78 is a major inducible protein in human non-small cell lung cancer H460 cells treated with ER stress inducers, including A23187 and thapsigargin. AEBSF, an inhibitor of serine protease, diminished GRP78 induction, enhanced mitochondrial permeability, and augmented apoptosis in H460 cells during ER stress. Simultaneously, AEBSF promoted Raf-1 degradation and suppressed phosphorylation of Raf-1 at Ser338 and/or Tyr340 during ER stress. Coimmunoprecipitation assays and subcellular fractionations showed that GRP78 associated and colocalized with Raf-1 on the outer membrane of mitochondria, respectively. While treatment of cells with ER stress inducers inactivated BAD by phosphorylation at Ser75, a Raf-1 phosphorylation site; AEBSF attenuated phosphorylation of BAD, leading to cytochrome c release from mitochondria. Additionally, overexpression of GRP78 and/or Raf-1 protected cells from ER stress-induced apoptosis. Taken together, our results indicate that GRP78 may stabilize Raf-1 to maintain mitochondrial permeability and thus protect cells from ER stress-induced apoptosis.
Institute of Biotechnology, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
Chih-Wen
CW
Shu
Fang-Chun
FC
Sun
Jun-Hung
JH
Cho
Chih-Chien
CC
Lin
Pei-Feng
PF
Liu
Ping-Yen
PY
Chen
Margaret Dah-Tsyr
MD
Chang
Hua-Wen
HW
Fu
Yiu-Kay
YK
Lai
D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D045304:Cytochromes c / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000473:pathology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000187:drug effects / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism*; D013450:Sulfones / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D051025:bcl-Associated Death Protein / Q000378:metabolism
D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D013450:Sulfones; D051025:bcl-Associated Death Protein; D010768:Phosphoserine; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D019284:Thapsigargin; D045304:Cytochromes c; D019908:Proto-Oncogene Proteins c-raf