Zhang Yi-Qiang, Herman Brian
Department of Cellular and Structural Biology, University of Texas HSC at San Antonio, San Antonio, Texas 78249, USA.
J Cell Biochem. 2006 Oct 1;99(2):575-88. doi: 10.1002/jcb.20946.
Apoptosis repressor with a CARD domain (ARC) has been demonstrated to protect heart cells against ischemia/reperfusion (I/R) injury. In this study, we investigated the mechanism by which ARC protects heart cells against oxidative stress. We monitored the extent of apoptosis and activity of multiple components of the intrinsic apoptotic pathway in rat cardiac myoblast cell line H9c2 with either reduced or increased expression of ARC during oxidative stress. Overexpression of ARC-inhibited oxidative stress-induced caspase-2/3 activation, cytochrome c release, and translocation of Bax to mitochondria. Furthermore, phosphorylation of ARC at threonine 149 was found to be critical to its function. ARC containing a T149A mutation failed to translocate to mitochondria, did not inhibit caspase-2 activation, and had a dominant negative effect against the protective effect of endogenous ARC during oxidative stress. In addition, wild-type ARC but not the T149A mutant inhibited cell death induced by overexpression of caspase-2. Using a yeast two-hybrid (YTH) screening approach and co-immunoprecipitation (Co-IP), we found that protein phosphatase 2C (PP2C) interacted with ARC and that PP2C mediated-dephosphorylation of ARC inhibited its anti-apoptotic activity. Eliminating either the N-terminal CARD domain or the C-terminal P/E domain also abolished the anti-apoptotic function of ARC, suggesting that full-length ARC is required for its apoptotic inhibition. These results indicate that ARC plays an important role in protection of H9c2 cells against oxidative stress-induced apoptosis by phosphorylation-dependent suppression of the mitochondria-mediated intrinsic pathway, partially initiated through the activation of caspase-2.
含CARD结构域的凋亡抑制因子（ARC）已被证明可保护心脏细胞免受缺血/再灌注（I/R）损伤。在本研究中，我们探究了ARC保护心脏细胞免受氧化应激的机制。我们监测了在氧化应激期间ARC表达降低或增加的大鼠心肌成肌细胞系H9c2中凋亡的程度以及内源性凋亡途径多个组分的活性。ARC的过表达抑制了氧化应激诱导的半胱天冬酶-2/3激活、细胞色素c释放以及Bax向线粒体的转位。此外，发现ARC第149位苏氨酸的磷酸化对其功能至关重要。含有T149A突变的ARC无法转位至线粒体，不能抑制半胱天冬酶-2的激活，并且在氧化应激期间对内源性ARC的保护作用具有显性负效应。此外，野生型ARC而非T149A突变体抑制了由半胱天冬酶-2过表达诱导的细胞死亡。使用酵母双杂交（YTH）筛选方法和免疫共沉淀（Co-IP），我们发现蛋白磷酸酶2C（PP2C）与ARC相互作用，并且PP2C介导的ARC去磷酸化抑制了其抗凋亡活性。去除N端CARD结构域或C端P/E结构域也消除了ARC的抗凋亡功能，这表明全长ARC是其抑制凋亡所必需的。这些结果表明，ARC通过磷酸化依赖性抑制线粒体介导的内源性途径（部分通过半胱天冬酶-2的激活启动），在保护H9c2细胞免受氧化应激诱导的凋亡中发挥重要作用。
