cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity
cAMP-dependent tyrosine phosphorylation of subunit I inhibits cytochrome c oxidase activity
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DOI：
10.1074/jbc.m411335200
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发表时间：
2005-02-18
影响因子：
4.8
通讯作者：
Hüttemann, M
中科院分区：
文献类型：
--
作者：
Lee, I;Salomon, AR;Hüttemann, M
针对线粒体的信号通路知之甚少。在这里，我们研究磷酸化的cAMP依赖性途径的细胞色素c氧化酶（考克斯），电子传递链的末端酶的亚基。使用抗磷酸化抗体，我们表明，牛肝考克斯亚基I是酪氨酸磷酸化的茶碱，磷酸二酯酶抑制剂，产生高cAMP水平的存在下，但不是在其缺席。通过质谱鉴定的磷酸化位点是考克斯催化亚基I的酪氨酸304。亚基I磷酸化导致细胞色素c的V-max降低和K-m增加，并使反应动力学从双曲线转变为S曲线，使得考克斯在高达10 μ M的细胞色素c底物浓度下被完全或强烈抑制，甚至在存在变构激活剂ADP的情况下。为了评估我们在生理学背景下使用分离的酶的发现，我们在人HepG 2细胞和牛肝组织上测试了饥饿信号胰高血糖素。胰高血糖素导致考克斯失活，在与腺苷酸环化酶激活剂毛喉素孵育后也观察到这种作用。因此，胰高血糖素受体/G蛋白/cAMP途径调节考克斯活性。在用于缓解哮喘的治疗浓度下，茶碱引起肺考克斯抑制并降低细胞ATP水平，提示其临床作用机制。
Signaling pathways targeting mitochondria are poorly understood. We here examine phosphorylation by the cAMP-dependent pathway of subunits of cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain. Using anti-phospho antibodies, we show that cow liver COX subunit I is tyrosine-phosphorylated in the presence of theophylline, a phosphodiesterase inhibitor that creates high cAMP levels, but not in its absence. The site of phosphorylation, identified by mass spectrometry, is tyrosine 304 of COX catalytic subunit I. Subunit I phosphorylation leads to a decrease of V-max and an increase of K-m for cytochrome c and shifts the reaction kinetics from hyperbolic to sigmoidal such that COX is fully or strongly inhibited up to 10 muM cytochrome c substrate concentrations, even in the presence of allosteric activator ADP. To assess our findings with the isolated enzyme in a physiological context, we tested the starvation signal glucagon on human HepG2 cells and cow liver tissue. Glucagon leads to COX inactivation, an effect also observed after incubation with adenylyl cyclase activator forskolin. Thus, the glucagon receptor/G-protein/cAMP pathway regulates COX activity. At therapeutic concentrations used for asthma relief, theophylline causes lung COX inhibition and decreases cellular ATP levels, suggesting a mechanism for its clinical action.