NOVEL SIGNALLING PATHWAY IN NEUTROPHILS

描述（改编自申请人的摘要）：广泛，长期 本提案的目的是阐明 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提出了新的策略， 感染性疾病期间的抗菌机制。刺激 嗜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 是氧依赖性抗菌剂库的关键组成部分， 这些细胞。这些细胞反应经常涉及磷酸化 富含丙氨酸的C激酶底物（MARCKS蛋白） 和NADPH氧化酶系统的47 kDa亚基（p47-phox）， 多个站点。这些蛋白质参与了蛋白质的重排。 肌动蛋白细胞骨架和超氧化物的产生。 的 研究者最近观察到中性粒细胞含有四种新 和分子量为69、63、49的未表征蛋白激酶 和40 kDa的蛋白质在这些细胞受到刺激后迅速活化 一种趋化肽 这些新的激酶可以催化 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）和可能的生产 C-3-磷酸化磷酸肌醇（PPI）。 因此，他可能 揭示了D-3-PPI在中性粒细胞中的第二信使作用， PI 3-K的下游目标 具体来说，这个项目集中在信号中四个未探索的领域 中性粒细胞的转导途径。这些是：（1）净化和 将63 kDa蛋白激酶表征为该家族的范例 （2）建立结构-功能关系 （3）揭示调节这种酶的调节机制， 这种激酶（例如，照亮上游事件），以及（4） 确定这种酶在中性粒细胞刺激中的确切作用 (e.g.,揭开生理基质）。技术 生物化学（酶学）、分子生物学（克隆和测序） 和细胞生物学（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 包括对这种蛋白质进行测序并确定其结构基序 沿着建立底物特异性、辅因子要求 和激酶识别的最小共有序列。 特别 将注意揭示PI 3-K的确切作用， 激活63 kDa激酶。 翻译后修饰 (e.g.,磷酸化）。 最终目标是打造 分离的63 kDa的调节特性之间的坚实联系 激酶和相关刺激反应现象的控制， 完整的细胞

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.