NOVEL SIGNALLING PATHWAY IN NEUTROPHILS

描述(改编自申请人的摘要)：宽泛的、长期的 这项提议的目的是阐明 参与吞噬细胞刺激的分子事件 白细胞。从这些研究中获得的知识将是高度相关的 对宿主防御机制的理解。此外，研究 这里描述的可能最终会提出新的战略，以增强 传染病过程中的抗微生物机制。受刺激 中性粒细胞经历了各种变化，包括趋化作用， 溶酶体酶的释放和超氧化物的产生。超氧化物 是依赖氧气的抗菌剂武库的关键成分 这些细胞。这些细胞反应经常涉及到磷酸化。 富含丙氨酸的肌动蛋白C底物(Marcks-Protein) 和NADPH-氧化酶系统的47 kDa亚基(P47-Phox) 多个站点。这些蛋白质参与细胞的重排。 肌动蛋白、细胞骨架和超氧化物的生成。这个 研究人员最近观察到，中性粒细胞含有四种新的 和分子质量为69、63、49的未鉴定的蛋白激酶 和40 kDa，在这些细胞的刺激下迅速激活 一种趋化性多肽。这些新的激酶可以催化 含有磷酸化位点的多肽的磷酸化 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蛋白的激活 磷脂酰肌醇3-激酶(PI 3-K)及其可能的产生 C-3-磷酸化磷脂酰肌醇(PPI)。因此，他可能已经 发现D-3-PPI在中性粒细胞中的第二个信使角色 下游目标派3-K！ 具体地说，这个项目集中在信号中的四个未勘探区域 中性粒细胞的转导途径。它们是：(1)净化和 63 kDa蛋白激酶作为这个家族的范例的特征 (2)建立结构-功能关系 这种酶，(3)揭示了调节 该激酶(例如，照亮上游事件)，以及(4) 确定该酶在中性粒细胞刺激中的确切作用 (例如，揭开生理底物)。技术问题 生物化学(酶学)、分子生物学(克隆和测序) 并将使用细胞生物学(例如，免疫定位研究)。 经典的和更现代的程序(亲和色谱柱、高效液相系统) 将被用来提纯这种酶。定性研究将 包括对该蛋白质进行测序并确定其结构基序 在确定底物专一性的同时，辅因子要求 以及由该激酶识别的最小共识序列。特例 将注意揭示pI3-K在体内的确切作用。 激活63 kDa的蛋白激酶。翻译后修饰 (例如，磷酸化)将被寻求。最终的目标是锻造 分离的63 kDa蛋白的调控特性之间存在牢固的联系 蛋白激酶与相关刺激反应现象的调控 完好无损的细胞。

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.