Functional characterization of a mitochondrial Ser/Thr protein phosphatase in cell death regulation.
Functional characterization of a mitochondrial Ser/Thr protein phosphatase in cell death regulation.
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DOI：
10.1016/s0076-6879(09)05014-9
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发表时间：
2009
影响因子：
--
通讯作者：
Wang, Yibin
中科院分区：
文献类型：
--
作者：
Lu, Gang;Sun, Haipeng;Korge, Paavo;Koehler, Carla M.;Weiss, James N.;Wang, Yibin
蛋白质磷酸化是一种重要的翻译后修饰形式，对细胞信号传导至关重要，也发生在线粒体蛋白质组中。然而，仅进行了非常有限的研究来表征神经靶向蛋白激酶或磷酸酶。最近，我们发现了一个新的PP2C家族成员（PP2Cm），它是一种线粒体蛋白磷酸酶，在正常发育和细胞存活中起着重要作用。在这一章中，我们将描述的方法应用于识别PP2Cm作为一个居民线粒体蛋白磷酸酶的基础上序列分析和生化特性。我们还将提供用于建立PP2Cm的细胞内定位的实验方案，以实现PP2Cm在培养细胞和完整组织中的损失和获得功能，并评估PP2Cm缺乏对细胞死亡，线粒体氧化磷酸化和渗透性转换孔开放的影响。
Protein phosphorylation is a major form of posttranslational modification critical to cell signaling that also occurs in mitochondrial proteome. Yet, only very limited studies have been performed to characterize mitochondrial-targeted protein kinases or phosphatases. Recently, we identified a novel member of PP2C family (PP2Cm) that is a resident mitochondrial protein phosphatase which plays an important role in normal development and cell survival. In this chapter, we will describe the methods applied in the identification of PP2Cm as a resident mitochondrial protein phosphatase based on sequence analysis and biochemical characterization. We will also provide experimental protocols used to establish the intracellular localization of PP2Cm, to achieve loss and gain function of PP2Cm in cultured cells and intact tissue, and to assess the impact of PP2Cm deficiency on cell death, mitochondria oxidative phosphorylation and permeability transition pore opening.