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 severe asthma. 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 paracelular 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 alternatively spliced variant nmMLCK2) is targeted to specific cellular sites is entirely unknown. Furthermore, the influence of the asthma-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 case control studies in African Americans with asthma who reside in Harlem, NY to validate our earlier observations in asthma cohorts from Chicago and Barbados.
Specific Aim #2 studies will characterize nmMLCK (nmMLCK1, nmMLCK2, MLCK-coding SNPs) utilizing kinase and actin polymerization assays, GFP/YFP-MLCK fusion proteins and cytoskeletal binding assays. SA #3 will examine the influence of kinase-mediated PTMs (Src, Abl, and PKA) on site-specific MLCK responses (nmMLCK1, nmMLCK2, nmMLCK-SNPs) utilizing mass spectroscopy, phosphopeptide mapping, GFP-MLCK fusion proteins, and binding partner assays. SA #4 will utilize available and novel genetically-engineered mice to further define the site specific in vivo involvement of nmMLCK ( SNPs) in lung inflammatory injury. We believe these integrated translational studies will lead to mechanistic insights into asthma pathobiology and the development of novel edema-reducing therapies.
Asthma is a disorder affecting over 20 million Americans with unacceptable morbidity and mortality, particularly in under-represented minority populations such as African Americans. Our proposal will offer clues for the involvement and importance of the cytoskeleton in the development and severity of asthma.
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