Regulation of Respiration and Apoptosis by Cytochrome c Phosphorylation.

描述（由申请人提供）：我们最近发现细胞色素c （Cytc）通过哺乳动物肝脏和心脏两个不同部位的酪氨酸磷酸化直接受到细胞信号的调节，这一发现是通过我们开发的分离技术实现的，该技术可以保持蛋白质的生理磷酸化状态。我们的总体假设是，在细胞凋亡过程中，Cytc磷酸化调节了Cytc的两个主要功能，即线粒体呼吸和Cytc释放。我们的长期目标是了解Cytc磷酸化对呼吸和凋亡的影响，并确定指向Cytc的细胞信号通路。在初步数据的支持下，我们将研究三个特定的目的：1)验证Cytc在体内以一种独特的组织特异性方式磷酸化的假设，以及这种磷酸化导致Cytc结构变化的假设；2)验证Cytc磷酸化导致呼吸控制并阻止其参与细胞凋亡的假设；3)系统地鉴定磷酸化Cytc的线粒体酪氨酸激酶。磷酸化的Cytc将从牛肝脏和心脏组织中分离出来，并使用质谱和蛋白质晶体学进行结构表征（目的1）。磷酸化的Cytc将进行功能分析，包括体外呼吸和细胞凋亡测量，同时在Cytc敲除细胞系中进行Cytc构建的体内诱变研究（目的2）。鉴定作用于Cytc的激酶的实验将包括蛋白质组学方法、激酶定位和酵母双杂交研究（目标3）。本研究有望揭示Cytc，长期以来被认为是呼吸和凋亡的中心分子，受细胞信号传导的调节，为理解和控制这两个关键的生物过程开辟新的机会。

DESCRIPTION (provided by applicant): We recently discovered that cytochrome c (Cytc) is directly regulated by cell signaling via tyrosine phosphorylation on two distinct sites in mammalian liver and heart, a finding made possible by isolation techniques developed by us that preserve the physiological phosphorylation status of the protein. It is our overall hypothesis that Cytc phosphorylation regulates the two main functions of Cytc, mitochondrial respiration and Cytc release during apoptosis. Our long term goal is to understand the effect of Cytc phosphorylation on respiration and apoptosis and to identify the cell signaling pathways directed to Cytc. Supported by preliminary data, three specific aims will be investigated: 1) to test the hypotheses that Cytc is phosphorylated in vivo in a distinct tissue-specific manner and that this phosphorylation results in structural changes of Cytc; 2) to test the hypotheses that phosphorylation of Cytc leads to controlled respiration and prevents its participation in apoptosis; and 3) to systematically identify mitochondrial tyrosine kinases that phosphorylate Cytc. Phosphorylated Cytc will be isolated from cow liver and heart tissue and structurally characterized using mass spectrometry and protein crystallography (Aim 1). Phosphorylated Cytc will be subjected to functional assays including in vitro respiration and apoptosis measurements, accompanied by mutagenesis studies in vivo with Cytc constructs in a Cytc knockout cell line (Aim 2). Experiments to identify kinases that act on Cytc will include a proteomic approach, kinase localization, and yeast two-hybrid studies (Aim 3). This research is expected to reveal that Cytc, long recognized as a central molecule in respiration and apoptosis, is subject to regulation by cell signaling, opening new opportunities for the understanding and control of those two key biological processes. PUBLIC HEALTH RELEVANCE: Our laboratory discovered that Cytochrome c (Cytc), which plays a key role in programmed cell death (apoptosis) and cellular respiration, is regulated by cell signaling networks that reversibly phosphorylate Cytc. In this study, we will analyze the structural changes brought about by phosphorylation, determine whether and to what extent phosphorylation affects the functions of Cytc in apoptosis and respiration, and identify the kinases that phosphorylate Cytc. Since apoptosis and cellular respiration are involved in many diseases such as cancer and neurodegeneration, this study is expected to lead to better understanding and control of such diseases.