Cytoskeleton and Signal Transduction in Host Defense

描述（由申请人提供）：白细胞骨架与迁移、粘附、吞噬和细胞分裂密切相关；由于这些原因，细胞骨架组装的调节在宿主防御中起着至关重要的作用。虽然人们普遍认为细胞骨架参与了这些功能所需的形状变化，但细胞骨架的一个不太为人所知但同样重要的作用是，它可以作为信号级联组装的支架，特别是在响应细胞粘附时。近年来，人们对信号级联如何影响肌动蛋白聚合有了很多了解。然而，对于细胞骨架的组装如何影响信号级联，我们所知甚少。几年前，我们假设白细胞特异性肌动蛋白交联蛋白l -活蛋白（LPL）在细胞骨架和信号转导之间的串扰中起重要作用；这笔拨款支持了我们所有验证这一假设的研究。LPL是皮质细胞骨架的一种普遍和受调控的成分，在对许多白细胞激活因子（包括细菌病原体相关分子模式（PAMPs）、免疫复合物、趋化肽、细胞因子和趋化因子）的反应中，LPL可以在其氨基末端附近的Ser5上磷酸化。在上一个授权期内，我们已经证明，重建LPL磷酸化位点的细胞渗透肽可以激活白细胞整合素，并且这种作用需要肽的磷酸化。为了从遗传学的角度理解LPL在白细胞功能调控中的作用，我们通过同源重组建立了LPL缺陷小鼠。这些小鼠表明LPL是中性粒细胞和巨噬细胞整合素信号传导的重要方面所必需的，因此，LPL的缺失会导致宿主对金黄色葡萄球菌的防御缺陷。在目前的应用中，我们建议继续我们的遗传和生化方法来理解LPL在白细胞中整合素依赖信号传导中的作用。具体而言，我们建议确定：1)PMN和巨噬细胞中整合素连接后LPL信号传导需求的分子基础（“外向内信号传导”）；2) LPL磷酸化与整合素激活信号相关的机制和生物学作用（“内向外信号传导”）。从这些研究中，我们将进一步了解炎症、免疫和宿主防御的分子机制，从而增加控制感染性和炎症性疾病的机会。

DESCRIPTION (provided by applicant): The leukocyte cytoskeleton is intimately involved in migration, adhesion, phagocytosis, and cell division; for these reasons, regulation of cytoskeletal assembly has an essential role in host defense. While cytoskeleton is broadly understood to be involved in the shape changes required for these functions, a less appreciated but equally fundamental role for the cytoskeleton is that it can act as a scaffold for the assembly of signaling cascades, particularly in response to cell adhesion. In recent years quite a lot has been learned about how signaling cascades affect actin polymerization. However, much less is known about how assembly of cytoskeleton affects signa ng cascades. Some years ago, we hypothesized that the leukocyte-specific actin crosslinking protein L-plastin (LPL) has an important role in crosstalk between cytoskeleton and signal transduction; this grant has supported all our studies to test this hypothesis. LPL is a prevalent and regulated component of the cortica cytoskeleton that can become phosphorylated on Ser5 near its aminoterminus in response to many leukocyte activators, including bacterial pathogen-associated molecular patters (PAMPs), immune complexes, chemotactic peptides, cytokines, and chemokines. In the last grant period, we have shown that cell-permeant peptides that recreate the LPL phosphorylation site can activate leukocyte integrins and that this effect requires phosphorylation of the peptide. For genetic approaches to understanding LPL in regulation of leukocyte function, we have created a mouse deficient in LPL by homologous recombination. These mice show that LPL is required for important aspects of integrin signaling in neutrophils and macrophages and that, as a consequence, absence of LPL causes a defect in host defense against Staphylococcus aureus. In the current application, we propose to continue our genetic and biochemical approaches to a molecular understanding of the role of LPL in integrin-dependent signaling in leukocytes. Specifically, we propose to determine: 1) the molecular basis of the requirement for LPL in signaling following integrin ligation in PMN and macrophages ("outside-in signaling"); 2) the mechanisms and I biological roles of LPL phosphorylation relevant to signaling for integrin activation ("inside-out signaling"). From these studies we will obtain an increased understanding of molecular mechanisms involved in inflammation, immunity, and host defense, leading to increased opportunities for control of infectious and inflammatory diseases.