NOVEL SIGNALLING PATHWAY IN NEUTROPHILS

中性粒细胞中的新信号通路

• 批准号: 2905760
• 负责人: JOHN A BADWEY
• 金额: $ 20.54万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2905760
• 关键词: NAD(P)H dehydrogenase; affinity chromatography; biological signal transduction; chemical kinetics; chemoattractants; confocal scanning microscopy; enzyme activity; enzyme mechanism; enzyme structure; enzyme substrate; high performance liquid chromatography; immunocytochemistry; leukocyte activation /transformation; molecular cloning; myristates; neutrophil; nucleic acid sequence; phosphatidylinositol 3 kinase; phosphorylation; posttranslational modifications; protein kinase; protein purification; protein sequence

DESCRIPTION (Adapted from applicant's abstract): The broad, long term objective of this proposal is to elucidate the exact sequence of molecular events that are involved in the stimulation of phagocytic leukocytes. Knowledge gained from these studies will be highly relevant to an understanding of host-defense mechanisms. Moreover, studies described here may ultimately suggest novel strategies for enhancing antimicrobial mechanisms during infectious disease. Stimulated neutrophils undergo a variety of changes that include chemotaxis, lysosomal enzyme release and the production of superoxide. Superoxide is a key component of the oxygen-dependent, antimicrobial arsenal of these cells. These cellular responses frequently involve phosphorylation of the myristolyated alanine-rich C kinase substrate (MARCKS-protein) and the 47 kDa subunit of the NADPH-oxidase system (p47-phox) on multiple sites. These proteins are involved in rearrangements of the actin cytoskeleton and superoxide generation, respectively. The investigator has recently observed that neutrophils contain four novel and uncharacterized protein kinases with molecular masses of 69, 63, 49 and 40 kDa that are rapidly activated upon stimulation of these cells with a chemotactic peptide. These novel kinases can catalyze the phosphorylation of peptides which contain the phosphorylation sites of p47-phox and MARCKS. Most importantly, the stimulation of these novel protein kinases appears to be dependent upon the activation of phosphatidylinositol 3- kinase (PI 3-K) and perhaps the production of C-3-phosphorylated phosphoinositides (PPIs). Thus, he may have uncovered a second messenger role for D-3-PPIs in neutrophils and the downstream targets of PI 3-K! Specifically, this project focuses on four unexplored areas in the signal transduction pathways of neutrophils. These are: (1) purifying and characterizing the 63 kDa protein kinase as a paradigm for this family of novel kinases, (2) establishing the structure-function relationships of this enzyme, (3) uncovering the regulatory mechanisms that modulate this kinase (e.g., illuminating the upstream events), and (4) establishing the exact role of this enzyme in neutrophil stimulation (e.g., uncovering the physiological substrates). Techniques of biochemistry (enzymology), molecular biology (cloning and sequencing) and cell biology (e.g., immunolocalization studies) will be employed. Classical and more modern procedures (affinity columns, HPLC systems) will be used to purify the enzyme. Characterization studies will include sequencing this protein and determining its structural motifs along with establishing the substrate specificity, cofactor requirements and the minimal consensus sequence recognized by the kinase. Particular attention will be paid to uncovering the exact role of PI 3-K in the activation of the 63 kDa kinase. Post-translational modifications (e.g., phosphorylation) will be sought. The ultimate goal is to forge a solid link between the regulatory properties of the isolated 63 kDa kinase and the control of the relevant stimulus-response phenomena in intact cells.

描述（改编自申请人的摘要）：广泛的、长期的 该提案的目的是阐明具体的顺序 参与刺激吞噬细胞的分子事件 白细胞。从这些研究中获得的知识将具有高度相关性 了解宿主防御机制。此外，研究 这里描述的最终可能会提出新的策略来增强 传染病期间的抗菌机制。受刺激 中性粒细胞经历多种变化，包括趋化性、 溶酶体酶的释放和超氧化物的产生。超氧化物 是氧依赖性抗菌库的关键组成部分 这些细胞。这些细胞反应经常涉及磷酸化 肉豆蔻酰化富含丙氨酸的 C 激酶底物（MARCKS 蛋白） 以及 NADPH 氧化酶系统 (p47-phox) 的 47 kDa 亚基 多个站点。这些蛋白质参与重排 分别是肌动蛋白细胞骨架和超氧化物生成。 这 研究人员最近观察到中性粒细胞含有四种新的 以及分子量为 69、63、49 的未表征蛋白激酶 和 40 kDa 在刺激这些细胞后迅速激活 与趋化肽。 这些新型激酶可以催化 含有磷酸化位点的肽的磷酸化 p47-phox 和 MARCKS。最重要的是这些小说的刺激 蛋白激酶似乎依赖于 磷脂酰肌醇 3-激酶 (PI 3-K) 以及可能的产生 C-3-磷酸化磷酸肌醇（PPI）。 因此，他可能有 发现了 D-3-PPI 在中性粒细胞中的第二信使作用 PI 3-K 的下游靶点！ 具体来说，该项目重点关注信号中四个未探索的领域 中性粒细胞的转导途径。它们是：（1）净化和 将 63 kDa 蛋白激酶描述为该家族的范例 新型激酶的研究，(2) 建立结构-功能关系 这种酶，（3）揭示调节机制 该激酶（例如，阐明上游事件），以及 (4) 确定该酶在中性粒细胞刺激中的确切作用 （例如，揭示生理基础）。技术 生物化学（酶学）、分子生物学（克隆和测序） 将采用细胞生物学（例如免疫定位研究）。 经典和更现代的程序（亲和柱、HPLC 系统） 将用于纯化酶。 表征研究将 包括对该蛋白质进行测序并确定其结构基序 随着建立底物特异性、辅因子要求 以及激酶识别的最小共有序列。 特别的 我们将重点关注揭示 PI 3-K 在 63 kDa 激酶的激活。 翻译后修饰 （例如，磷酸化）将被寻求。 最终目标是打造 分离的 63 kDa 的调控特性之间存在牢固的联系 激酶和相关刺激反应现象的控制 完整的细胞。