Project #1 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 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 nmMLCKI (and its five alternatively spliced variants) is targeted to specific cellular sites is entirely unknown. Furthermore, the influence of ALIassociated 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 (nmMLCKI, 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 (nmMLCKI, 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 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 utilize available 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 lead to mechanistic insights into EC barrier regulation and the development of novel edema-reducing therapies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL058064-17
Application #
8374683
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
2014-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
17
Fiscal Year
2012
Total Cost
$300,649
Indirect Cost
$109,155
Name
University of Illinois at Chicago
Department
Type
DUNS #
098987217
City
Chicago
State
IL
Country
United States
Zip Code
60612
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Camp, Sara M; Chiang, Eddie T; Sun, Chaode et al. (2015) Pulmonary endothelial cell barrier enhancement by novel FTY720 analogs: methoxy-FTY720, fluoro-FTY720, and ?-glucuronide-FTY720. Chem Phys Lipids 191:16-24
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