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.

项目摘要在许多信号通路中，活性氧（ROS）作为关键的第二使者出现与健康和疾病有关。尽管在理解机制方面取得了很多进展哪些ROS水平是常规的细胞内部，对OCUR的分子信号事件知之甚少 ROS生成的下游。越来越多的证据表明蛋白激酶是直接的由ROS修饰调节。例如，氧化还原特定Cys残基的可逆氧化敏感激酶已被证明会影响其活性（积极或负面），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CYS是在同一激酶家族的其他成员中构成。这提高了可逆氧化的潜力可能是调节细胞内激酶功能的一般含义。为了进一步探索这个立场，我们最近使用的功能性蛋白质微阵列检查氧化对全局底物的影响选择一系列AGC和CMCG激酶家庭成员。这些研究表明H2O2依赖性氧化移动了许多激酶的底物偏好，从而导致氧化中不同的底物谱。和减少状态。有趣的是，在大多数情况下，底物磷酸化的增加和减少观察到。结果，可逆氧化可能在控制信号中起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转移（AIMS 1＆2）和 2）开始探索氧化还原修饰对激酶依赖性信号的功能后果细胞内部的过程（AIM 3）。在这些研究中，我们将重点关注两个代表性的AGC和CMGC 家庭成员，即PKA和ERK2。该提供商不仅将对氧化还原监管的新见解这些重要的激酶，但它也将为分析其他氧化还原敏感激酶的分析奠定基础在微阵列实验中确定。这些研究将共同提供对ROS介导的独特见解激酶功能的调节，并为将来研究ROS-和ROS之间的串扰奠定了基础物理和病理状态中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