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

• 批准号: 7916331
• 负责人: CHARLES S RUBIN
• 金额: $ 32.87万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7916331
• 关键词: 1,2-diacylglycerol; Animals; Area; Bacterial Infections; Binding; Biochemical; Biological Assay; Biological Models; Caenorhabditis elegans; Cardiovascular Diseases; Cell membrane; Cells; Chemotaxis; Diglycerides; Epithelium; Family; Fluorescence Microscopy; Functional disorder; Genes; Genetic; Genetic Programming; Goals; 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; 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; Route; 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; public health relevance; research study; second messenger; therapeutic target

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（由申请方提供）：蛋白激酶D（PKD）亚型是神经控制、DAG调节的信号级联中的PKC效应物。关于PKD在正常分化细胞中的调节、底物和功能知之甚少。将通过诱变、生物化学和体内分析来研究命名为DKF-2A和DKF-2B的秀丽隐杆线虫PKD，以确定4个结构域的性质如何控制PKD的质膜募集、活化和细胞内路由。实验将严格评估C1 a和C1 b结构域对DAG介导的体内DKF-2A/2B的易位和活化的贡献相等的想法，并确定活化环（A环）中的两个P-丝氨酸是否差异性地调节催化活性。DKF-2A和2B由一个基因编码，但这2种激酶可能受到差异调节，并在体内支配不同的功能。DKF-2缺陷型C. elegans以及在无效和野生型（WT）背景中表达DKF-2A或2B转基因的动物将被表征以发现D激酶的生理功能。使用荧光显微镜和结合A环中关键磷酸化位点的IgG对WT、突变体和转基因（TG）动物进行的研究将阐明体内单个细胞中DKF-2A/2B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质的mRNA组的表达。表达DKF-2同种型的细胞将通过使用驱动GFP标记的DKF-2蛋白的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病原菌侵害的蛋白质的表达;神经元DKF-2B介导趋化性。这些知识将被利用来开发基于DKF-2调节的mRNA和蛋白质、趋化性和对细菌感染的抗性的测量的测定，其量化（并允许可视化）体内DKF-2A或2B活性。该检测试剂盒可进行3条线的深入研究。(1)C1 a，C1 b，PH和A-环结构域的机制和调节特性，在体外生物化学分析测定，将定量分析在体内的情况下，通过表达相关的DKF-2突变体蛋白质的“报告菌株”的秀丽隐杆线虫。(2)体内活化试验将与遗传学相结合，以确定哪些异源三聚体G蛋白、PLC和PKC构成控制肠细胞和神经元中DKF-2A和2B活性的上游信号传导途径。(3)DKF-2亚型磷酸化和控制转录调节因子HDA-4（一种组蛋白脱乙酰酶）和p38 MAP激酶级联的成员NSY-1的活性的可能性将通过体内分析进行严格评估。计划的实验将揭示信号分子，机制和途径，耦合外部刺激PKD控制的生理过程中正常分化的细胞。对C. elegans模型将揭示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调节促进心脏肥大（收缩功能障碍和心力衰竭的前兆）的遗传程序，其将PKD和PKD底物鉴定为心血管疾病的高优先级治疗靶标。此外，我们对模型系统的初步研究表明，PKD将激素信号与先天免疫反应的控制联系起来，这些先天免疫反应保护肠道和其他上皮细胞免受入侵的细菌病原体的侵害。