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

描述（由申请人提供）：蛋白激酶 D (PKD) 同工型是激素控制、DAG 调节的信号级联中的 PKC 效应子。对于正常分化细胞中的 PKD 调节、底物和功能知之甚少。将通过诱变、生化和体内分析来研究名为 DKF-2A 和 DKF-2B 的线虫 PKD，以确定 4 个结构域的特性如何控制 PKD 的质膜募集、激活和细胞内路由。实验将严格评估 C1a 和 C1b 结构域对 DAG 介导的易位和体内 DKF-2A/2B 激活的同等贡献，并确定激活环（A 环）中的两个 P-丝氨酸是否差异调节催化活性。 DKF-2A 和 2B 由一种基因编码，但这 2 种激酶可能受到差异性调节并在体内控制不同的功能。 DKF-2 缺陷（无效）线虫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。表达 DKF-2 同工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进行鉴定。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 控制保护肠道细胞免受病原菌侵害的蛋白质的表达；神经元 DKF-2B 介导趋化作用。这些知识将用于开发基于 DKF-2 调节的 mRNA 和蛋白质、趋化性和细菌感染抵抗力的测量的测定方法，从而量化（并允许可视化）体内 DKF-2A 或 2B 活性。这些分析能够进行 3 条深入的研究。 (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 突变蛋白在体内进行定量分析。 (2) 体内激活测定将与遗传学相结合，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 活性的上游信号通路。 (3) 将通过体内分析严格评估 DKF-2 同种型磷酸化并控制转录调节因子 HDA-4（一种组蛋白脱乙酰酶）和 p38 MAP 激酶级联成员 NSY-1 的活性的可能性。计划中的实验将揭示将外部刺激与正常分化细胞中 PKD 控制的生理过程耦合起来的信号分子、机制和途径。对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的调节联系起来。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检查。公共健康相关性：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 调节促进心脏肥大（收缩功能障碍和心力衰竭的前兆）的遗传程序，该程序将 PKD 和 PKD 底物确定为心血管疾病的高度优先治疗靶点。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细菌病原体的入侵。

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.