Cytoskeleton and Signal Transduction in Host Defense

描述（由申请人提供）：白细胞细胞骨架与迁移、粘附、吞噬作用和细胞分裂密切相关;因此，细胞骨架组装的调节在宿主防御中具有重要作用。虽然细胞骨架被广泛理解为参与这些功能所需的形状变化，但细胞骨架的一个不太受重视但同样重要的作用是，它可以充当信号级联组装的支架，特别是响应细胞粘附。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，关于细胞骨架的组装如何影响信号级联的知之甚少。几年前，我们假设白细胞特异性肌动蛋白交联蛋白L-纤维蛋白（LPL）在细胞骨架和信号转导之间的串扰中起重要作用;该资助支持了我们所有的研究来验证这一假设。LPL是皮质细胞骨架的普遍和受调节的组分，其可以响应于许多白细胞活化剂（包括细菌病原体相关分子模式（PAMP）、免疫复合物、趋化肽、细胞因子和趋化因子）而在其氨基末端附近的Ser 5上磷酸化。在上一个资助期，我们已经表明，细胞渗透肽，重建LPL磷酸化位点可以激活白细胞整合素，这种效果需要磷酸化的肽。对于理解LPL在调节白细胞功能中的遗传方法，我们通过同源重组产生了LPL缺陷小鼠。这些小鼠表明，LPL是嗜中性粒细胞和巨噬细胞中整合素信号传导的重要方面所需的，因此，LPL的缺乏导致宿主对金黄色葡萄球菌的防御缺陷。在本申请中，我们建议继续我们的遗传和生物化学方法，以分子水平了解LPL在白细胞中整合素依赖性信号传导中的作用。具体而言，我们建议确定：1）在PMN和巨噬细胞中整合素连接后的信号传导中需要LPL的分子基础（“由外向内信号传导”）; 2）与整合素活化信号传导相关的LPL磷酸化的机制和生物学作用（“由内向外信号传导”）。从这些研究中，我们将获得更多的了解参与炎症，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.