Global Analysis of Redox Modification on Protein Kinase Function

 描述（由申请人提供）：活性氧（ROS）是与健康和疾病相关的许多信号通路中的关键第二信使。虽然在理解细胞内ROS水平调节的机制方面取得了很大进展，但对ROS产生下游发生的分子信号传导事件知之甚少。蛋白磷酸酶可能是最好的特征活性氧效应，表明串扰容易发生之间的活性氧和磷酸化依赖的途径。最近的研究表明，几种蛋白激酶也直接受ROS修饰的调节，这一观点得到了支持。事实上，氧化还原敏感激酶中特定Cys残基的可逆氧化已被证明会影响其活性（正面或负面）、亚细胞定位和蛋白质-蛋白质相互作用。在许多情况下，受影响的激酶中的修饰的Cys在同一激酶家族的其他成员中是保守的。这提出了可逆氧化可能是调节细胞内激酶功能的一般手段的可能性。因此，在目标1中，我们将检查氧化还原灵敏度 在其他代表性家族成员中已知被氧化的位置含有保守Cys残基的激酶。在目标2中，我们将使用由19，000种独特的人类蛋白质（占人类蛋白质组的约90%）组成的功能性“HuProt”蛋白质微阵列，探索氧化对氧化还原敏感性激酶底物选择的影响。我们的假设是，氧化将导致微阵列上蛋白质的差异磷酸化，甚至可能改变给定激酶的底物偏好，使得不同的底物组被氧化和还原形式的激酶靶向。这些研究不仅将提供对氧化诱导的底物特异性变化的独特见解，而且由于HuProt微阵列上的大量蛋白质，它们还有望显著扩大参比状态下每种激酶的已知底物的数量（即，在还原条件下）。我们将在目标1和目标2的基础上，通过对培养细胞中生理相关氧化还原信号通路激活后的氧化还原敏感性激酶及其底物进行靶向分析。

 DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) are emerging as critical second messengers in many signaling pathways related to health and disease. While much progress has been made in understanding the mechanisms by which ROS levels are regulated inside cells, less is known about the molecular signaling events that occur downstream of ROS generation. Protein phosphatases are perhaps the best-characterized ROS effectors, suggesting that crosstalk readily occurs between ROS- and phosphorylation-dependent pathways. This notion is supported by recent studies demonstrating that several protein kinases are also directly regulated by ROS modification. Indeed, the reversible oxidation of specific Cys residues in redox-sensitive kinases has been shown to influence their activity (either positively or negatively), subcellular localization, and protein-protein interactions. In mny cases, the modified Cys in the affected kinase is conserved among other members in the same kinase family. This raises the possibility that reversible oxidation may be a general means of regulating kinase function inside cells. Therefore, in Aim 1, we will examine the redox sensitivity of kinases containing conserved Cys residues at positions known to be oxidized in other representative family members. In Aim 2, we will explore the impact of oxidation on substrate selection by redox-sensitive kinases using functional "HuProt" protein microarrays composed of 19,000 unique human proteins (representing ~90% of the human proteome). Our hypothesis is that oxidation will lead to differential phosphorylation of proteins on the microarrays and may even shift the substrate preference of a given kinase such that distinct sets of substrates are targeted by the oxidized and reduced forms of the kinase. Not only will these studies offer unique insights into oxidation-induced changes in substrate specificity but, due to the large number of proteins on the HuProt microarrays, they also promise to dramatically expand the number of known substrates for each kinase in the reference state (i.e., under reducing conditions). We will build upon our findings in Aims 1 and 2 through targeted analysis of redox-sensitive kinases and their substrates following activation of physiologically-relevant redox signaling pathways in cultured cells.