Global Analysis of Redox Modification on Protein Kinase Function

 描述(由申请人提供)：在许多与健康和疾病相关的信号通路中，活性氧物种(ROS)正在成为关键的第二信使。虽然在理解细胞内调节ROS水平的机制方面已经取得了很大进展，但对ROS产生下游发生的分子信号事件知之甚少。蛋白磷酸酶可能是最具特征的ROS效应因子，这表明ROS依赖和磷酸化依赖的途径之间很容易发生串扰。最近的研究表明，几种蛋白激酶也受到ROS修饰的直接调控，这一观点得到了支持。事实上，氧化还原敏感激酶中特定半胱氨酸残基的可逆氧化已被证明影响其活性(无论是积极的还是消极的)、亚细胞定位和蛋白质-蛋白质相互作用。在少数病例中，受影响的激酶中修饰的半胱氨酸在同一激酶家族的其他成员中是保守的。这增加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敏感性 在其他有代表性的家族成员中已知被氧化的位置含有保守的半胱氨酸残基的激酶。在目标2中，我们将使用由19,000个独特的人类蛋白质(约占人类蛋白质组的90%)组成的功能性“HuProt”蛋白质芯片来探索氧化对氧化还原敏感蛋白激酶底物选择的影响。我们的假设是，氧化将导致蛋白质在微阵列上的不同磷酸化，甚至可能改变给定蛋白的底物偏好，从而使不同的底物集合被氧化和还原形式的蛋白所靶向。这些研究不仅将为氧化诱导底物专一性的变化提供独特的见解，而且由于HuProt微阵列上有大量的蛋白质，它们还有望极大地扩大参考状态(即在还原条件下)每种酶的已知底物数量。我们将基于我们在AIMS 1和2中的发现，通过对培养细胞中与生理相关的氧化还原信号通路激活后的氧化还原敏感激酶及其底物进行有针对性的分析。

 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.