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Properties, Regulation and Functions of Diacylglycerol-Activated Protein Kinase D

二酰甘油激活蛋白激酶 D 的性质、调控和功能

基本信息

批准号：
8118804
负责人：
CHARLES S RUBIN
金额：
$ 32.54万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-08-01 至 2013-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8118804
关键词：
1,2-diacylglycerol Animals Area Bacterial Infections Binding Biochemical Biological Assay Biological Models Caenorhabditis elegans Cardiovascular Diseases Cell membrane Cells Chemotaxis Complementary DNA Diglycerides Epithelium Family Fluorescence Microscopy Functional disorder Genes Genetic Genetic Programming Goals Health Heart Hypertrophy Heart failure Heterotrimeric GTP-Binding Proteins Histone Deacetylase Hormonal Imagery Immune Immune response In Vitro Individual Inflammatory Intestines Invaded Investigation Knowledge Learning Link MAP Kinase Modules Measurement Mediating Messenger RNA Microarray Analysis Modeling Mutagenesis Names Natural Immunity Neurons Nomenclature Organism Output Pathway interactions Phosphorylation Site Phosphotransferases Physiological Physiological Processes Property Protein Isoforms Proteins Regulation Reporter Role Second Messenger Systems Serine Signal Pathway Signal Transduction Signaling Molecule Signaling Protein Stimulus Stress System Tissues Transgenes Transgenic Animals bacterial resistance base in vivo member mitogen-activated protein kinase p38 mutant pathogen pathogenic bacteria promoter protein expression protein kinase D research study second messenger therapeutic target

项目摘要

DESCRIPTION (provided by applicant): Protein kinase D (PKD) isoforms are PKC effectors in hormonally-controlled, DAG-regulated signaling cascades. Little is known about PKD regulation, substrates and functions in normal differentiated cells. C.elegans PKDs named DKF-2A and DKF-2B will be studied by mutagenesis, biochemical and in vivo analysis to determine how properties of 4 structural domains control plasma membrane recruitment, activation and intracellular routing of PKDs. Experiments will rigorously evaluate the idea that both C1a and C1b domains contribute equally to DAG-mediated translocation and activation of DKF-2A/2B in vivo and determine if two P- serines in the activation loop (A-loop) differentially regulate catalytic activity. DKF-2A and 2B are encoded by one gene, but the 2 kinases may be differentially regulated and govern distinct functions in vivo. DKF-2 deficient (null) C. elegans, as well as animals expressing DKF-2A or 2B transgenes in null and wild type (WT) backgrounds will be characterized to discover physiological functions of D kinases. Studies on WT, mutant and transgenic (TG) animals, using fluorescence microscopy and IgGs that bind crucial phosphorylation sites in the A-loop, will elucidate relationships among DKF-2A/2B activation, translocation and stability in individual cells in vivo. Microarray analysis will determine if DKF-2A and 2B regulate expression of groups of mRNAs encoding functionally related proteins. Cells expressing DKF-2 isoforms will be identified by using gene promoters that drive targeted expression of GFP-tagged DKF-2 proteins. Preliminary results indicate that DKF-2 isoforms link DAG signals to two critical physiological processes: DKF-2A controls expression of proteins that protect intestinal cells against pathogenic bacteria; neuronal DKF-2B mediates chemotaxis. This knowledge will be exploited to develop assays, based on measurements of DKF-2 regulated mRNAs and proteins, chemotaxis, and resistance to bacterial infection, that quantify (and allow visualization) of DKF-2A or 2B activity in vivo. The assays enable 3 lines of incisive investigation. (1) Mechanistic and regulatory properties of C1a, C1b, PH and A-Loop domains, determined heretofore by in vitro biochemical analysis, will be quantitatively analyzed in an in vivo context by expressing relevant DKF-2 mutant proteins in the "reporter strains" of C.elegans. (2) In vivo activation assays will be combined with genetics to determine which heterotrimeric G proteins, PLCs and PKCs constitute upstream signaling pathways that control DKF-2A and 2B activity in intestinal cells and neurons. (3) The possibility that DKF-2 isoforms phosphorylate and control activities of a transcriptional regulator, HDA-4 (a histone deacetylase) and a member of a p38 MAP kinase cascade, NSY-1, will be rigorously assessed by in vivo analysis. Planned experiments will reveal signaling molecules, mechanisms and pathways that couple external stimuli to PKD-controlled physiological processes in normal differentiated cells. Studies on the C. elegans model will reveal how PKDs link DAG second messenger to regulation of chemotaxis and innate immunity. The results and will guide examination of these currently unexplored areas in mammalian systems. PUBLIC HEALTH RELEVANCE: Acquisition of new knowledge regarding protein kinase D (PKD, DKF) regulation and physiological functions will advance understanding of how tissues counter environmental immune and inflammatory stresses. PKDs regulate a genetic program that promotes cardiac hypertrophy (a precursor of contractile dysfunction and heart failure), which identifies PKDs and PKD substrates as high priority therapeutic targets for cardiovascular diseases. In addition, our preliminary studies on a model system reveal that PKDs link hormonal signals to control of innate immune responses that protect intestine and other epithelia against invading bacterial pathogens.

描述（由申请人提供）：蛋白激酶D （PKD）异构体是PKC在激素控制、dag调节的信号级联中的效应物。对正常分化细胞中PKD的调控、底物和功能知之甚少。我们将通过诱变、生化和体内分析对秀丽隐杆线虫pkd DKF-2A和DKF-2B进行研究，以确定4个结构域的特性如何控制pkd的质膜募集、激活和细胞内通路。实验将严格评估C1a和C1b结构域对dag介导的DKF-2A/2B的易位和激活有同等作用的观点，并确定激活环（a环）中的两个P-丝氨酸是否对催化活性有差异调节。DKF-2A和2B由一个基因编码，但这两种激酶在体内可能受到不同的调控，并支配不同的功能。DKF-2缺陷（null）秀丽隐杆线虫，以及表达DKF-2A或2B转基因的动物在null和野生型（WT）背景下将被表征，以发现D激酶的生理功能。利用荧光显微镜和结合a环关键磷酸化位点的igg对WT、突变体和转基因（TG）动物进行研究，将阐明DKF-2A/2B在体内单个细胞中的活化、易位和稳定性之间的关系。微阵列分析将确定DKF-2A和2B是否调节编码功能相关蛋白的mrna组的表达。表达DKF-2亚型的细胞将通过使用驱动gfp标记的DKF-2蛋白靶向表达的基因启动子来鉴定。初步结果表明，DKF-2亚型将DAG信号与两个关键的生理过程联系起来：DKF-2A控制保护肠细胞免受致病菌侵害的蛋白质表达；神经元DKF-2B介导趋化作用。这些知识将被用于开发基于DKF-2调节的mrna和蛋白质、趋化性和对细菌感染的抗性测量的分析，这些分析可以量化（并允许可视化）DKF-2A或2B在体内的活性。这些分析可以进行3条线的深入调查。(1)通过体外生化分析确定的C1a、C1b、PH和A-Loop结构域的机制和调控特性，将通过在秀丽隐杆线虫“报告菌株”中表达相关的DKF-2突变蛋白，在体内环境下定量分析。(2)体内激活试验将与遗传学相结合，以确定哪些异三聚体G蛋白、plc和PKCs构成了控制肠细胞和神经元中DKF-2A和2B活性的上游信号通路。(3) DKF-2亚型磷酸化并控制转录调节因子HDA-4（一种组蛋白去乙酰化酶）和p38 MAP激酶级联成员NSY-1活性的可能性将通过体内分析进行严格评估。计划中的实验将揭示正常分化细胞中外部刺激与pkd控制的生理过程耦合的信号分子、机制和途径。对秀丽隐杆线虫模型的研究将揭示pkd如何将DAG第二信使与趋化性和先天免疫调节联系起来。研究结果将指导对哺乳动物系统中这些目前尚未探索的领域的研究。公共卫生相关性：获得关于蛋白激酶D （PKD， DKF）调节和生理功能的新知识将促进对组织如何对抗环境免疫和炎症应激的理解。PKD调节促进心脏肥厚（收缩功能障碍和心力衰竭的前兆）的遗传程序，这将PKD和PKD底物确定为心血管疾病的优先治疗靶点。此外，我们对模型系统的初步研究表明，pkd将激素信号与控制先天免疫反应联系起来，从而保护肠道和其他上皮细胞免受入侵细菌病原体的侵害。