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.
