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

描述(申请人提供)：蛋白激酶D(PKD)亚型是荷尔蒙控制的、DAG调节的信号级联中的PKC效应物。对正常分化细胞中PKD的调节、底物和功能知之甚少。本文将通过诱变、生化和体内分析来研究线虫PKD(命名为DKF-2A和DKF-2B)，以确定4个结构域的性质如何控制PKD的质膜募集、激活和细胞内路径。实验将严格评估C1a和C1b结构域在体内DAG介导的DKF-2A/2B转位和激活中同等作用的观点，并确定激活环(A-环)中的两个P-丝氨酸是否对催化活性起不同的调节作用。DKF-2A和DKF-2B由一个基因编码，但这两个激酶在体内可能受到不同的调控，并控制着不同的功能。DKF-2缺陷型(空)线虫以及在缺失和野生型(WT)背景下表达DKF-2A或2B转基因基因的动物将被用来研究D激酶的生理功能。对WT、突变和转基因(TG)动物的研究，利用荧光显微镜和A环中关键的磷酸化位点结合的IGG，将阐明DKF-2A/2B在体内单个细胞中的激活、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质的mRNAs组的表达。表达DKF-2亚型的细胞将通过使用基因启动子来鉴定，这些启动子驱动GFP标记的DKF-2蛋白的靶向表达。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控制保护肠道细胞免受病原菌侵袭的蛋白质的表达；神经元DKF-2B介导趋化作用。这些知识将被利用来开发基于DKF-2调节的mRNAs和蛋白质、趋化性和对细菌感染的抵抗力的测量方法，以量化(并允许可视化)DKF-2A或2B在体内的活性。这些化验方法可以进行三条线的深入调查。(1)迄今为止通过体外生化分析确定的C1a、C1b、PH和A-Loop结构域的机械和调节特性，将通过在线虫“报告菌株”中表达相关的DKF-2突变蛋白，在体内进行定量分析。(2)体内激活试验将与遗传学相结合，以确定哪些异源三聚体G蛋白、PLC和PKCs构成控制肠道细胞和神经元中DKF-2A和2B活性的上游信号通路。(3)体内分析将严格评估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激酶级联成员NSY-1活性的可能性。计划中的实验将揭示将外部刺激与正常分化细胞中PKD控制的生理过程相耦合的信号分子、机制和路径。对线虫模型的研究将揭示PKD如何将DAG第二信使与趋化和先天免疫的调节联系起来。这一结果将指导对哺乳动物系统中这些目前尚未探索的区域的检查。与公共健康相关：获得有关蛋白激酶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调节促进心肌肥大(收缩功能障碍和心力衰竭的先兆)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点。此外，我们对模型系统的初步研究显示，PKD将激素信号与控制先天性免疫反应联系起来，以保护肠道和其他上皮细胞免受入侵的细菌病原体的侵袭。

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.