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: 7660361
• 负责人: CHARLES S RUBIN
• 金额: $ 33.2万
• 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/7660361
• 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（由申请人提供）：蛋白激酶D（PKD）同工型是荷尔蒙控制，DAG调节的信号级联的PKC效应子。关于正常分化细胞中PKD调控，底物和功能知之甚少。 C.将通过诱变，生化和体内分析来研究名为DKF-2A和DKF-2B的元素PKD，以确定4个结构结构域的特性如何控制质膜募集，激活和PKD的细胞内路由。实验将严格评估C1A和C1B结构域在体内同样有助于DKF-2A/2B的DKF-2A/2B激活，并确定激活环（A-loop）中的两个P-丝氨酸是否差异地调节催化活性。 DKF-2A和2B由一个基因编码，但是2个激酶可以受到差异调节并控制体内不同的功能。 DKF-2缺陷（NULL）秀丽隐杆线虫以及表达DKF-2A或2B转基因的动物在零和野生型（WT）背景中将被表征以发现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荧光显微镜和IgG的WT，突变和转基因动物（TG）动物的研究将阐明体内单个细胞中DKF-2A/2B激活，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。通过使用驱动靶向GFP标记的DKF-2蛋白的靶向表达的基因启动子来鉴定表达DKF-2同工型的细胞。初步结果表明，DKF-2同工型将DAG信号与两个关键的生理过程联系起来：DKF-2A控制着保护肠细胞免受致病细菌的蛋白质的表达；神经元DKF-2B介导趋化性。基于DKF-2调节的mRNA和蛋白质，趋化性以及对细菌感染的抗性的测量，将利用这些知识来开发测定法，从而量化了（并允许可视化）DKF-2A或2B活性的体内活性。这些测定能够进行3行临时调查。 （1）通过体外生化分析确定的C1a，C1b，pH和A环域的机械和调节性能将通过在体内环境中进行定量分析，通过表达相关的DKF-2突变蛋白在“ c.elegans”的“报告者”中的相关DKF-2突变蛋白。 （2）体内激活测定法将与遗传学结合，以确定哪种异三聚体G蛋白，PLC和PKC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同工型磷酸化和转录调节剂HDA-4（组蛋白脱乙酰基酶）和p38 MAP激酶Cascade，NSY-1的成员的可能性将通过VIVO分析进行严格评估。计划的实验将揭示将外部刺激与正常分化细胞中PKD控制的生理过程相结合的信号分子，机制和途径。关于秀丽隐杆线虫模型的研究将揭示PKDS如何将DAG Second Messenger连接到趋化性和先天免疫力的调节。结果并将指导检查哺乳动物系统中这些目前未开发的区域。公共卫生相关性：获取有关蛋白激酶D（PKD，DKF）调节和生理功能的新知识将提高对组织如何应对环境免疫和炎症应激的理解。 PKD调节了促进心脏肥大（收缩功能障碍和心力衰竭的先驱）的遗传程序，该程序将PKD和PKD底物确定为心血管疾病的高优先治疗靶标。此外，我们对模型系统的初步研究表明，PKD将激素信号连接到控制先天免疫反应，以保护肠和其他上皮症免受入侵细菌病原体的影响。