DESCRIPTION (provided by applicant): Our studies have demonstrated that the gene encoding the multi-functional cytoskeletal protein, myosin light chain kinase (MLCK), contains coding polymorphisms which are highly associated with susceptibility to acute lung injury (ALI). The non-muscle isoform, nmMLCK, is a critical cytoskeletal effector which regulates the participation of the EC actin cytoskeleton in vascular barrier disruption, in barrier restoration, in lung inflammatory cell trafficking and in vascular responses to mechanical stretch. Following edemagenic agents, MLCK phosphorylates MLCs on Ser19 and Thr18, producing barrier-disrupting cytoplasmic stress fibers, spatially-localized actomyosin contraction and paracellular gaps. In contrast, EC barrier-protective agonists induce the rapid translocation of MLCK to lamellipodial membrane protrusions (to close paracellular gaps and restore barrier integrity) and to cortical actin networks (to enhance linkage to junctional complexes and increase barrier properties). The mechanism by which the full length nmMLCK1 (and its five alternatively spliced variants) is targeted to specific cellular sites is entirely unknown. Furthermore, the influence of ALI-associated nmMLCK coding SNPs (Pro21His, Pro147Ser, Val261Ala) on MLCK structure/function are similarly unknown. We hypothesize that site-specific nmMLCK regulation involves post-translational modifications (PTMs) and results in variant- and SNP-specific MLCK activities. Specific Aim (SA) #1 will conduct studies to characterize nmMLCK (nmMLCK1, nmMLCK splice variants, MLCK-coding SNPs) utilizing kinase and actin polymerization assays, GFP/YFP-MLCK fusion proteins and cytoskeletal binding assays. SA #2 will examine the influence of kinase-mediated PTMs (Src, Abl, ERK and PKA) on site-specific MLCK responses (nmMLCK1, nmMLCK-variants, nmMLCK-SNPs) utilizing mass spectroscopy, phosphopeptide mapping, GFP-MLCK fusion proteins, and binding partner assays. SA #3 will examine MLCK regulation of actin polymerization and focal adhesion remodeling in EC lamellipodia (critical to paracellular gap closure) using GFP-MLCK- and paxillin fusion proteins coupled to atomic force microscopy. SA #4 will utilize available and novel genetically-engineered mice to further define the in vivo role of nmMLCK splice variants ( SNPs) in lung inflammatory injury. We predict that translational studies will lead to mechanistic insights into EC barrier regulation and the development of novel edema-reducing therapies. PROJECT NARRATIVE: Acute inflammatory lung injury is a disorder affecting over 100,000 patients per year with an unacceptable mortality of 30-35 %. The essential features of diverse lung vascular pathobiology remains unclear; however, the EC cytoskeleton and nmMLCK appear to be critical participants. As this cytoskeletal effector is essentially involved in both barrier dysfunction and barrier recovery enhancement, there is a critical need to fully understand MLCK involvement in barrier regulation and the effects of MLCK PTMs/SNPs in these processes.
|Effective start/end date||9/1/08 → 8/31/12|
- National Institutes of Health: $164,217.00