Graham Regina M, Thompson John W, Wei Jianqin, Bishopric Nanette H, Webster Keith A
Department of Molecular and Cellular Pharmacology and the Vascular Biology Institute, University of Miami School of Medicine, Miami, Florida 33136, USA.
Antioxid Redox Signal. 2007 Sep;9(9):1309-15. doi: 10.1089/ars.2007.1726.
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.
Bnip3是Bcl-2家族中一种促凋亡的死亡调节蛋白成员，可促进程序性细胞死亡的内在途径。Bnip3死亡程序需要通过N端跨膜结构域插入膜中，该结构域将蛋白质导向线粒体和内质网（ER）膜。我们曾报道，模拟缺血可诱导Bnip3基因转录，且酸中毒可使Bnip3蛋白稳定。Bnip3程序性死亡是非典型的，具有凋亡和坏死的特征。在此我们证明，缺氧复氧以及激活蛋白激酶C的试剂，包括钙离子载体、佛波醇12-肉豆蔻酸酯13-乙酸酯和冈田酸，也可诱导Bnip3。根据氨基酸序列预测的Bnip3分子大小为21.5 kDa，但该蛋白在SDS-PAGE中通常以31 kDa的单体和60 kDa的二聚体形式迁移。用磷酸酶处理含有Bnip3的细胞提取物会产生一系列快速迁移的条带，其中最小的条带与Bnip3的理论分子大小相对应。相反，用冈田酸处理细胞可消除快速迁移的条带，这表明Bnip3磷酸化是一个动态过程。磷蛋白水平升高与Bnip3依赖性死亡的启动相关，而脱磷酸化的条带与极端酸中毒相关。
