Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration.
Mammalian liver cytochrome c is tyrosine-48 phosphorylated in vivo, inhibiting mitochondrial respiration.
复制标题
哺乳动物肝脏细胞色素 c 在体内被酪氨酸 48 磷酸化，抑制线粒体呼吸。
DOI：
10.1016/j.bbabio.2008.04.023
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Hüttemann,Maik
中科院分区：
文献类型：
--
作者：
Yu,Hong;Lee,Icksoo;Salomon,ArthurR;Yu,Kebing;Hüttemann,Maik
细胞色素c（Cytochrome c，Cyt c）是线粒体电子传递链（electron transfer chain，ETC）的一部分，从bc 1复合物接受电子并将电子传递给细胞色素c氧化酶（Cytochrome c oxidase，CcO）。ETC产生线粒体膜电位，其被ATP合酶用于产生ATP。此外，从线粒体释放Cyt c通常使细胞经历凋亡。考虑到它在生命（呼吸）和死亡（细胞凋亡）决策中的核心作用，人们预计Cyt c功能会受到严格调节。可逆磷酸化是一种主要的细胞调节机制，但靶向线粒体氧化磷酸化系统的细胞信号传导的作用还没有很好地理解，并且迄今为止仅鉴定了少量可以磷酸化的蛋白质。我们最近发现，从牛心脏组织中分离的Cyt C在体内酪氨酸97上磷酸化，这导致与CcO反应中的呼吸抑制。在这项研究中，我们从不同的器官，牛肝，在保持生理磷酸化状态的条件下分离的细胞色素c。磷酸酪氨酸特异性抗体的Western分析表明，肝细胞色素c磷酸化。令人惊讶的是，通过固定化金属亲和色谱/纳米液相色谱/电喷雾离子化质谱（IMAC/nano-LC/ESI-MS），磷酸化位点明确指定为Tyr-48，而不是先前在牛心脏中鉴定的磷酸化Tyr-97。与Tyr-97一样，Tyr-48在真核生物中是保守的。作为Tyr-48磷酸化的一个可能的后果，我们分析了离体牛肝CcO的体外反应动力学，揭示了显着的差异。Tyr-48磷酸化Cyt c的最大周转率为3.7 s-1，而去磷酸化导致活性增加2.2倍，达到8.2 s-1。Tyr-48磷酸化的Cyt c晶体结构的基础上的影响进行了讨论。
Cytochrome c (Cyt c) is part of the mitochondrial electron transport chain (ETC), accepting electrons from bc1complex and transferring them to cytochrome c oxidase (CcO). The ETC generates the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. In addition, the release of Cyt c from the mitochondria often commits a cell to undergo apoptosis. Considering its central role in life (respiration) and death (apoptosis) decisions one would expect tight regulation of Cyt c function. Reversible phosphorylation is a main cellular regulatory mechanism, but the effect of cell signaling targeting the mitochondrial oxidative phosphorylation system is not well understood, and only a small number of proteins that can be phosphorylated have been identified to date. We have recently shown that Cyt c isolated from cow heart tissue is phosphorylated on tyrosine 97 in vivo, which leads to inhibition of respiration in the reaction with CcO. In this study we isolated Cyt c from a different organ, cow liver, under conditions preserving the physiological phosphorylation state. Western analysis with a phosphotyrosine specific antibody suggested that liver Cyt c is phosphorylated. Surprisingly, the phosphorylation site was unambiguously assigned to Tyr-48 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS), and not to the previously identified phospho-Tyr-97 in cow heart. As is true of Tyr-97, Tyr-48 is conserved in eukaryotes. As one possible consequence of Tyr-48 phosphorylation we analyzed the in vitro reaction kinetics with isolated cow liver CcO revealing striking differences. Maximal turnover of Tyr-48 phosphorylated Cyt c was 3.7 s−1whereas dephosphorylation resulted in a 2.2 fold increase in activity to 8.2 s−1. Effects of Tyr-48 phosphorylation based on the Cyt c crystal structure are discussed.