Project #1 studies have demonstrated that the gene encoding the multi-functional cytoskeletal protein, myosinlight chain kinase (MLCK), contains coding polymorphisms which are highly associated with susceptibilityto acute lung injury (ALI). The non-muscle isoform, nmMLCK, is a critical cytoskeletal effector which regulatesthe participation of the EC actin cytoskeleton in vascular barrier disruption, in barrier restoration, in lunginflammatory 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 paracelular gaps. In contrast, EC barrier-protective agonistsinduce the rapid translocation of MLCK to lamellipodial membrane protrusions (to close paracellular gapsand restore barrier integrity) and to cortical actin networks (to enhance linkage to junctional complexes andincrease barrier properties). The mechanism by which the full length nmMLCKI (and its five alternativelyspliced variants) is targeted to specific cellular sites is entirely unknown. Furthermore, the influence of ALIassociatednmMLCK coding SNPs (Pro21His, Pro147Ser, Val261Ala) on MLCK structure/function are similarlyunknown. 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 studiesto characterize nmMLCK (nmMLCKI, nmMLCK splice variants, MLCK-coding SNPs) utilizing kinaseand actin polymerization assays, GFP/YFP-MLCK fusion proteins and cytoskeletal binding assays. SA #2will examine the influence of kinase-mediated PTMs (Src, Abl, ERK and PKA) on site-specific MLCKresponses (nmMLCKI, nmMLCK-variants, nmMLCK-SNPs) utilizing mass spectroscopy, phosphopeptidemapping, GFP-MLCK fusion proteins, and binding partner assays. SA #3 will examine MLCK regulation ofactin polymeriza-tion 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 utilizeavailable and novel genetically-engineered mice to further define the in vivo role of nmMLCK splice variants(+/- SNPs) in lung inflammatory injury. These translational studies integrate across our entire PPG and leadto mechanistic insights into EC barrier regulation and the development of novel edema-reducing therapies.
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