Control of Cell Respiration and Apoptosis by Phosphorylation of Cytochrome c

总结 细胞色素c（Cytc）在线粒体呼吸和2型 凋亡我们已经开发了新的协议来纯化线粒体蛋白质， 保持其生理调节特性和翻译后修饰， 我们发现Cytc在心脏中的不同酪氨酸残基上被磷酸化， 肝组织最近，我们在纯化的Cytc上定位了另外两个磷酸化位点， 来自牛和大鼠的肾脏，Thr 28和Ser 47，这是该赠款的主要重点 应用程序.在强有力的初步数据的支持下，我们的总体假设是， 磷酸化调节Cytc的两个主要功能，线粒体呼吸和 凋亡我们的长期目标是了解细胞信号转导对Cytc的调控 在正常和病理条件下的路径。作为实现我们目标的第一步， 将测试四个特定的假设：1）测试肾脏Cytc可以被 Thr 28和Ser 47上的磷酸化，这些修饰影响了细胞的基本功能。 2）验证Cytc Thr 28和Ser 47 磷酸化影响线粒体呼吸和细胞凋亡; 3）鉴定激酶 和控制Cytc磷酸化的磷酸酶;和4）为了证明 生理效应。将从奶牛和大鼠中分离磷酸化Cytc 肾脏和磷酸化模拟突变体Cytc将被过表达和纯化， 细菌，然后使用质谱法，光谱 方法和蛋白质晶体学（目的1）。磷酸化和拟磷酸化 将对突变的Cytc进行全面的功能分析，包括 体外呼吸和凋亡测量，并通过体内研究， 在Cytc敲除细胞中稳定表达磷酸化模拟Cytc（Aim 2）。接下来我们将 探索信号通路和识别激酶的基础工作， 使用亲和纯化/质谱技术作用于Cytc的磷酸酶 (Aim 3）。最后，我们将重新引入拟磷酸化和非磷酸化突变体， Cytc基因敲除小鼠体内，研究Cytc基因修饰和调控对细胞周期的影响。 动物水平（目标4）。我们希望这项研究将揭示细胞色素c，一种蛋白质， 做出生与死的决定，受细胞信号的调节， 理解线粒体呼吸和细胞凋亡的机会， 在呼吸和凋亡失调的病理条件下控制它。

SUMMARY Cytochrome c (Cytc) plays a central role in mitochondrial respiration and type 2 apoptosis. We have developed new protocols to purify mitochondrial proteins while maintaining their physiological regulatory properties and posttranslational modifications, and we discovered that Cytc is phosphorylated on distinct tyrosine residues in heart and liver tissue. Recently, we have mapped two more phosphorylation sites on Cytc purified from cow and rat kidneys, Thr28 and Ser47, which are the primary focus of this grant application. Supported by strong preliminary data it is our overall hypothesis that Cytc phosphorylation regulates the two main functions of Cytc, mitochondrial respiration and apoptosis. Our long term goal is to understand the regulation of Cytc by cell signaling pathways under normal and pathological conditions. As a first step towards our goal we will test four specific hypotheses: 1) to test the hypothesis that kidney Cytc can be phosphorylated on Thr28 and Ser47, and that these modifications affect the basic properties of the molecule; 2) to test the hypothesis that Cytc Thr28 and Ser47 phosphorylation affects mitochondrial respiration and apoptosis; 3) to identify kinases and phosphatases that control Cytc phosphorylation; and 4) to demonstrate the physiological effect in mice. Phosphorylated Cytc will be isolated from cow and rat kidneys and phosphomimetic mutant Cytc will be overexpressed and purified from bacteria, followed by structural characterization using mass spectrometry, spectroscopic methods, and protein crystallography (Aim 1). Phosphorylated and phosphomimetic mutant Cytc will be subjected to a comprehensive set of functional analyses including in vitro respiration and apoptosis measurements, and by in vivo studies with phosphomimetic Cytc stably expressed in Cytc knockout cells (Aim 2). We will next lay the ground work to explore the signaling pathways and identify kinases and phosphatases that act on Cytc using affinity purification/mass spectrometry techniques (Aim 3). Finally, we will reintroduce phosphomimetic and non-phosphorylatable mutant Cytc into Cytc knockout mice to study the effect of Cytc modification and regulation at the animal level (Aim 4). We expect that this research will reveal that Cytc, a protein that makes life and death decisions, is subject to regulation by cell signaling, opening new opportunities for the understanding of mitochondrial respiration and apoptosis, and to control it in pathological conditions in which respiration and apoptosis are dysregulated.