LPS REGULATION OF MACROPHAGE FUNCTION

蛋白激酶C（PKC）诱导的巨噬细胞磷酸化是必需的 对细菌脂多糖（LPS）的全功能反应。 的 本项目的目的是了解LPS调节 巨噬细胞中PKC依赖的信号通路。 我们的重点是 PKC底物的分子表征， 豆蔻酰化和磷酸化受LPS调节， 因此，作为LPS诱导的细胞凋亡的效应分子的主要候选者， 应答 我们纯化、克隆并测序了一个68 KPKC底物， 其豆蔻酰化和膜结合由LPS诱导。 我们将 确定是否有68 K周期往返于膜，由肉豆蔻酸 酸，由豆蔻酰化-68K结合蛋白靶向，并由 蛋白质的磷酸化状态。 68 K的精确点 将定义肉豆蔻酰化的调节。 位点特异性诱变 将用于确定肉豆蔻酸是否将68 K引导到 膜，并需要其随后的磷酸化的PKC。 我们 将确定参与取代豆蔻酰化的磷酸化位点 68 K的去磷酸化，并评估58 K的去磷酸化是否促进了细胞膜的 重新附着到膜上。 在膜上，68 K存在于点状 与局灶性粘连相对应的结构。 我们将确定 这些结构的分子组成部分，并定义它们如何与 肌动蛋白细胞骨架 68 K和PKC在调节细胞凋亡中的作用 粘着斑和肌动蛋白细胞骨架之间的相互作用将是 在各种条件下探索，包括吞噬作用和 趋化性 将建立透化细胞系统，其中 进一步研究68 K磷酸化对肌动蛋白细胞骨架的影响， organization. 用纯化的68 K和肌动蛋白进行的体外实验将确定 68 K与肌动蛋白结合的位点，并将阐明68 K与肌动蛋白结合的功能。 58 K磷酸化对肌动蛋白结构的影响。 两个PKC 由LPS诱导豆蔻酰化的底物也代表良好的 作为LPS依赖性应答的效应分子的候选物。 我们将 纯化40 K和42 K蛋白，克隆编码40 K和42 K蛋白的cDNA， 并研究LPS诱导的肉豆蔻酰化和 磷酸化在其亚细胞位置上。 LPS启动PKC诱导的花生四烯酸代谢的机制 将得到进一步表征。 我们将研究LPS是否会增加 环氧化酶的转录、翻译和活性， 脂氧合酶以及LPS是否促进这些酶的结合 酶与细胞膜。

Protein kinase C (PKC)-induced phosphorylation in macrophages is necessary for a full functional response to bacterial lipopolysaccharides (LPS). The aim of this project is to understand the mechanism by which LPS regulates PKC-dependent signaling pathways in macrophages. Our focus is the molecular characterization of PKC substrates whose synthesis, myristoylation and phosphorylation are regulated by LPS, and which are therefore primary candidates as effector molecules of LPS-induced responses. We have purified, cloned and sequenced a 68K PKC substrate whose myristoylation and membrane association is induced by LPS. We will determine whether 68K cycles to and from the membrane, directed by myristic acid, targeted by myristoylated-68K binding proteins, and regulated by the phosphorylation state of the protein. The precise point at which 68K myristoylation is regulated will be defined. Site-specific mutagenesis will be utilized to determine whether myristic acid directs 68K to the membrane and is required for its subsequent phosphorylation by PKC. We will define the phosphorylation sites involved in displacing myristoylated 68K from the membrane, and assess whether dephosphorylation of 58K promote reattachment to the membrane. At the membrane 68K resides in punctate structures corresponding to focal adhesions. We will identify the molecular components of these structures and define how they associate with the actin cytoskeleton. The role of 68K and PKC in regulating the interaction between focal adhesion and the actin cytoskeleton will be explored under a variety of conditions, including phagocytosis and chemotaxis. A permeabilized cell system will be established in which to further investigate the effect of 68K phosphorylation on actin-cytoskeleton organization. In vitro experiments with purified 68K and actin will define the site of binding of 68K with actin, and will clarify the functional consequences of 58K phosphorylation on actin structure. Two other PKC substrates whose myristoylation is induced by LPS also represent good candidates as effector molecules for LPS-dependent responses. We will purify these 40K and 42K proteins, clone and sequence the cDNA's encoding them, and study the effects of LPS-induced myristoylation and phosphorylation on their subcellular location. The mechanism by which LPS primes PKC-induced arachidonic acid metabolism will be further characterized. We will examine whether LPS increases the transcription, translation and activities of the cyclooxygenase and lipoxygenase enzymes and whether LPS promotes the association of these enzymes with the plasma membrane.