Global Analysis of Redox Modification on Protein Kinase Function

氧化还原修饰对蛋白激酶功能的整体分析

基本信息

批准号：
9272407
负责人：
Robert Howard Newman
金额：
$ 11.52万
依托单位：
NORTH CAROLINA AGRI & TECH ST UNIV
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-05-01 至 2018-09-09
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9272407
关键词：
Affect Antioxidants Biological Assay Cardiovascular Diseases Cell Proliferation Cell Respiration Cells Complement Coupled Cultured Cells Cyclic AMP-Dependent Protein Kinases Diabetes Mellitus Differentiated Gene Disease Docking Enzymes Event Family Family member Foundations Gene Expression Generations Goals Health Human Hydrogen Peroxide MAPK1 gene Malignant Neoplasms Mediating Modeling Modification Molecular Oxidases Oxidation-Reduction Oxidative Stress Oxides Pathologic Pathologic Processes Pathway interactions Peptides Phosphorylation Phosphotransferases Physiological Platelet-Derived Growth Factor Positioning Attribute Principal Investigator Process Protein Kinase Protein Microchips Protein phosphatase Proteins Proteome Reactive Oxygen Species Regulation Research Role Second Messenger Systems Signal Pathway Signal Transduction Site Site-Directed Mutagenesis Substrate Specificity Testing Therapeutic Time Western Blotting antioxidant enzyme catalase cell growth regulation insight member migration novel oxidation preference programs protein protein interaction public health relevance spatiotemporal

项目摘要

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.

描述（由适用提供）：在与健康和疾病有关的许多信号通路中，活性氧（ROS）成为关键的第二使者。尽管在理解细胞内部ROS水平受调节的机制方面取得了很多进展，但对ROS生成下游的分子信号事件的了解却较少。蛋白质磷酸酶可能是特征最佳的ROS作用，这表明串扰很容易发生在ROS-和磷酸化依赖性途径之间。最近的研究支持了这一概念，该研究表明，几种蛋白激酶也受ROS修饰直接调节。实际上，已经证明特定Cys的可逆氧化保留在氧化还原敏感的激酶中会影响其活性（积极或负面的），亚细胞定位和蛋白质 - 蛋白质相互作用。在MNY病例中，在同一激酶家族的其他成员中构成了受影响的激酶中的修饰CYS。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AIM 1中，我们将检查氧化还原灵敏度含有组成Cys的激酶保留在已知在其他代表性家庭成员中被氧化的位置。在AIM 2中，我们将使用功能性“ Huprot”蛋白微阵列探索氧化对氧化剂对底物选择的影响。我们的假设是，氧化将导致微阵列上蛋白质的差异化磷酸化，甚至可能改变给定激酶的底物偏好，从而使不同的底物集合被激酶的氧化和还原形式靶向。这些研究不仅会为氧化引起的底物特异性变化提供独特的见解，而且由于Huprot微阵列上的大量蛋白质，它们还有望大大扩大参考状态下每个激酶的已知底物数量（即在还原条件下）。在AIMS 1和2中，我们将通过对氧化还原敏感激酶及其底物进行针对性分析在培养细胞中物理相关的氧化还原信号通路后的底物来构建我们的发现。