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Biochemical and Cellular Analysis of Redox Modification on Protein Kinase Substrate Selection

氧化还原修饰对蛋白激酶底物选择的生化和细胞分析

基本信息

批准号：
10224757
负责人：
Robert Howard Newman
金额：
$ 36万
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：
2018
资助国家：
美国
起止时间：
2018-09-10 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10224757
关键词：
Affect Antioxidants Biochemical Cardiovascular Diseases Cell Proliferation Cell Respiration Cells Complement Cyclic AMP-Dependent Protein Kinases Diabetes Mellitus Differentiated Gene Disease Event Family Family member Foundations Future Gene Expression Generations Goals Health Hydrogen Peroxide Hypoxia Image In Situ Kinetics Length MAPK1 gene Malignant Neoplasms Mediating Modeling Modification Molecular Mutate Mutation Mutation Analysis Oxidation-Reduction Oxidative Stress Oxides Pathologic Pathologic Processes Pathway interactions Peptides Phosphorylation Phosphotransferases Physiological Physiological Processes Platelet-Derived Growth Factor Play Process Protein Kinase Protein Microchips Protein phosphatase Proteins Proteomics Reactive Oxygen Species Regulation Research Role Second Messenger Systems Series Signal Pathway Signal Transduction Site Solid Specificity Substrate Interaction Surface Plasmon Resonance Testing Therapeutic cell growth regulation experimental study insight interest member migration mutant oxidation preference protein protein interaction tandem mass spectrometry

项目摘要

PROJECT SUMMARY 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. A growing body of evidence suggests that protein kinases are directly regulated by ROS modification. For instance, 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 many 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. To explore this possibility further, we recently used functional protein microarrays to examine the impact of oxidation on the global substrate selection of a series of AGC and CMCG kinase family members. These studies suggest that H2O2-dependent oxidation shifts the substrate preference of many kinases, leading to distinct substrate profiles in the oxidized and reduced states. Interestingly, in most cases, both increases and decreases in substrate phosphorylation were observed. As a consequence, reversible oxidation may play an important role in controlling the signaling specificity of redox-sensitive kinases in cells. To investigate these questions further, we propose 1) to examine the molecular mechanisms underlying the H2O2-induced shifts in kinase substrate selection (Aims 1 & 2) and 2) to begin to explore the functional consequences of redox modification on kinase-dependent signaling processes inside the cell (Aim 3). During these studies, we will focus on two representative AGC and CMGC family members, namely PKA and ERK2. Not only will this provide new insights into the redox regulation of these important kinases, but it will also lay a foundation for the analysis of other redox sensitive kinases identified in the microarray experiments. Together, these studies will offer unique insights into ROS-mediated regulation of kinase function and provide the foundation for future studies into crosstalk between ROS- and phosphorylation-dependent signaling pathways in physiological and pathological states.

项目摘要活性氧 (ROS) 正在成为许多信号通路中关键的第二信使与健康和疾病有关。虽然在理解这一机制方面已经取得了很大进展哪些 ROS 水平在细胞内受到调节，但对于发生的分子信号传导事件知之甚少 ROS 生成的下游。越来越多的证据表明，蛋白激酶直接作用于由 ROS 修饰调节。例如，氧化还原中特定半胱氨酸残基的可逆氧化敏感激酶已被证明可以影响其活性（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半胱氨酸是在同一激酶家族的其他成员中保守。这增加了可逆氧化的可能性可能是调节细胞内激酶功能的通用方法。为了进一步探索这种可能性，我们最近使用功能性蛋白质微阵列来检查氧化对整体底物的影响选择一系列 AGC 和 CMCG 激酶家族成员。这些研究表明 H2O2 依赖性氧化改变了许多激酶的底物偏好，导致氧化后的底物特征不同和简化的状态。有趣的是，在大多数情况下，底物磷酸化都会增加和减少被观察到。因此，可逆氧化可能在控制信号传导方面发挥重要作用细胞中氧化还原敏感激酶的特异性。为了进一步研究这些问题，我们建议 1) 检查 H2O2 诱导的激酶底物选择变化背后的分子机制（目标 1 和 2）以及 2) 开始探索氧化还原修饰对激酶依赖性信号传导的功能性影响细胞内的过程（目标 3）。在这些研究中，我们将重点关注两个具有代表性的 AGC 和 CMGC 家庭成员，即 PKA 和 ERK2。这不仅将为氧化还原调节提供新的见解这些重要的激酶，但它也将为其他氧化还原敏感激酶的分析奠定基础在微阵列实验中鉴定。总之，这些研究将为 ROS 介导提供独特的见解激酶功能的调节，并为未来研究 ROS-和 ROS 之间的串扰奠定基础生理和病理状态下的磷酸化依赖性信号通路。

项目成果

期刊论文数量（8）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Impact of Redox Modification on PKA Substrate Selection.

氧化还原修饰对 PKA 底物选择的影响。

DOI：
发表时间：
2022
期刊：
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
影响因子：
0
作者：
Gay,NaudiaM;Ekhator,EseS;Delva-Wiley,Jeannette;Newman,RobertH
通讯作者：
Newman,RobertH

Redox Modification of PKA-Cα Differentially Affects Its Substrate Selection.

DOI：
10.3390/life13091811
发表时间：
2023-08-26
期刊：
Life (Basel, Switzerland)
影响因子：
0
作者：
通讯作者：

Impact of Redox Modification on MAPK Global Substrate Selection.

氧化还原修饰对 MAPK 全局底物选择的影响。

DOI：
发表时间：
2022
期刊：
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
影响因子：
0
作者：
Adams,LaquaundraL;Postiglione,Anthony;Odelade,AnuE;Keyes,JeremiahD;Poole,LeslieB;Newman,RobertH
通讯作者：
Newman,RobertH

RF-MaloSite and DL-Malosite: Methods based on random forest and deep learning to identify malonylation sites