Regulation of Bnip3 death pathways by calcium, phosphorylation, and hypoxia-reoxygenation.
Bnip3 is a proapoptotic member of the Bcl-2 family of death-regulating proteins that promote the intrinsic pathway of programmed cell death. The Bnip3 death program requires membrane insertion through an N-terminal transmembrane domain that directs the protein to mitochondrial and endoplasmic reticular (ER) membranes. We have reported that simulated ischemia induces transcription of the Bnip3 gene, and Bnip3 protein is stabilized by acidosis. Bnip3 programmed death is atypical, with features of both apoptosis and necrosis. Here we demonstrate that hypoxia-reoxygenation and agents that activate protein kinase C, including calcium ionophore, phorbol 12-myristate 13-acetate, and okadaic acid, also induce Bnip3. The molecular size of Bnip3 predicted from the amino acid sequence is 21.5 kDa, but the protein typically migrates in SDS-PAGE as a 31-kDa monomer and 60-kDa dimer. Treatment of cell extracts containing Bnip3 with phosphatase yielded a series of rapidly migrating species, the smallest of which corresponded with the theoretic molecular size of Bnip3. Conversely, treatment of cells with okadaic acid eliminated the rapidly migrating species, suggesting that Bnip3 phosphorylation is a dynamic process. Elevated levels of the phosphoprotein correlated with initiation of Bnip3-dependent death, whereas the dephosphorylated species correlated with extreme acidosis.
Graham
Regina M
RM
Department of Molecular and Cellular Pharmacology and the Vascular Biology Institute, University of Miami School of Medicine, Miami, Florida 33136, USA.
Thompson
John W
JW
Wei
Jianqin
J
Bishopric
Nanette H
NH
Webster
Keith A
KA
D000138:Acidosis; D000469:Alkaline Phosphatase / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology*; D002118:Calcium / Q000502:physiology*; D018929:Cell Culture Techniques; D016923:Cell Death; D015687:Cell Hypoxia / Q000502:physiology*; D003326:Coronary Circulation; D003331:Coronary Vessels / Q000502:physiology; D006321:Heart / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D009206:Myocardium / Q000166:cytology; D010101:Oxygen Consumption; D010766:Phosphorylation; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics
C090121:BNIP3 protein, human; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D000469:Alkaline Phosphatase; D002118:Calcium