Abstract 20512: Cortactin Binds to a Novel Focal Adhesion Protein, EVL, During Endothelial Cytoskeleton Rearrangement
The endothelial cell (EC) cytoskeleton is a complex array of proteins intimately involved in the diverse cell shape changes critical to dynamic regulation of lung vascular barrier integrity, particularly in the phases of onset and recovery during ARDS and VILI. Cortactin is structurally designed to be a central actin filament regulatory protein with an SH3 domain that directly binds non-muscle myosin light chain kinase (nmMLCK) and other effectors, and an acidic N-terminus that directly binds to Arp2/3, the protein complex regulating actin branching. In this study, we demonstrate cortactin binding to a focal adhesion protein, EVL, an actin polymerization-enhancing effector involved in linear actin filament formation and cortactin-mediated EC cytoskeleton regulation. To confirm the binding of cortactin to other cytoskeleton effector proteins, including nmMLCK, we generated a unique GFP-tagged proline-deficient nmMLCK mutant predicted to alter cortactin SH3 binding to MLCK. EC transfected with GFP-tagged proline-deficient nmMLCK mutant construct exhibit significantly higher stress fiber density and reduced localization in the cell periphery, highlighting the regulatory influence of cortactin on nmmLCK spatial localization. Cortactin-nmMLCK interaction is increased by tyrosine phosphorylation of either nmMLCK or cortactin by c-Abl, during S1P mediated cytoskeleton rearrangement. Importantly, c-Abl-mediated nmMLCK phosphorylation reverses nmMLCK’s inhibitory effects on cortactin/Arp2/3-induced pyrene-labeled actin polymerization. Cortactin binds to EVL, at basal level, with binding promoted during cytoskeleton rearrangement stimulated by S1P. The binding with EVL is critical for the cortactin translocation and interaction with nmMLCK in lamellipodia, as the silencing of EVL by siRNA significantly attenuated these essential cellular events (lamellipodia formation, cortactin translocation to lamellipodia, and binding of cortactin with nmMLCK) during cytoskeleton rearrangement. These findings reveal novel cellular mechanisms of endothelial cytoskeleton rearrangement and barrier function in ARDS and VILI.
Author Disclosures: J.B. Mascarenhas: None. M.E. Brown: None. T. Wang: None. J.G. Garcia: None. D.M. Steven: None.
- © 2014 by American Heart Association, Inc.