Cytoskeleton and Signal Transduction in Host Defense

描述(申请人提供)：白细胞细胞骨架与迁移、黏附、吞噬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，但细胞骨架的一个较少被认识但同样重要的作用是，它可以作为组装信号级联的支架，特别是在响应细胞黏附时。近年来，关于信号级联如何影响肌动蛋白聚合的研究已经有了很多。然而，关于细胞骨架的组装如何影响信号级联，我们知道的要少得多。几年前，我们假设白细胞特异的肌动蛋白交联蛋白L-血浆蛋白(LPL)在细胞骨架和信号转导之间的串扰中发挥重要作用；这一资助支持了我们所有验证这一假说的研究。LPL是皮质细胞骨架中普遍存在且受调节的成分，可在其氨基末端附近的Ser5上被磷酸化，以响应许多白细胞激活剂，包括细菌病原体相关分子模式(PAMPs)、免疫复合体、趋化肽、细胞因子和趋化因子。在上一次资助期间，我们已经证明了重建LPL磷酸化位点的细胞预示性多肽可以激活白细胞整合素，并且这种作用需要多肽的磷酸化。为了从遗传学角度理解LPL在调节白细胞功能中的作用，我们通过同源重组创造了一只LPL缺陷小鼠。这些小鼠表明，中性粒细胞和巨噬细胞中整合素信号的重要方面需要LPL，因此，LPL的缺失会导致宿主对金黄色葡萄球菌的防御出现缺陷。在目前的应用中，我们建议继续我们的遗传和生化方法，以分子理解LPL在白细胞整合素依赖信号转导中的作用。具体地说，我们建议确定：1)在PMN和巨噬细胞中整合素连接后LPL信号传递所需的分子基础(“自外向内信号”)；2)与整合素激活信号相关的LPL磷酸化的机制和生物学作用(“内向外信号”)。通过这些研究，我们将对炎症、免疫和宿主防御的分子机制有更多的了解，从而增加控制传染病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.