Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.

TitleProline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics.
Publication TypeJournal Article
Year of Publication2014
AuthorsBelvitch P, Adyshev D, Elangovan VR, Brown ME, Naureckas C, Rizzo AN, Siegler JH, Garcia JGN, Dudek SM
JournalMicrovasc Res
Volume95
Pagination94-102
Date Published2014 Sep
ISSN Number1095-9319
KeywordsAntigens, CD, Binding Sites, Cadherins, Capillary Permeability, Cell Membrane, Cells, Cultured, Cortactin, Cytoskeleton, Endothelial Cells, Humans, Immunoprecipitation, Kymography, Lung, Lysophospholipids, Microscopy, Fluorescence, Mutagenesis, Site-Directed, Myosin-Light-Chain Kinase, Proline-Rich Protein Domains, Protein Interaction Domains and Motifs, Sphingosine, Stress Fibers, Thrombin, Time Factors, Transfection
Abstract

<p>Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed an increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated an increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. the wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function.</p>

DOI10.1016/j.mvr.2014.07.007
Alternate JournalMicrovasc. Res.
PubMed ID25072537
PubMed Central IDPMC4188692
Grant ListR01 HL 88144 / HL / NHLBI NIH HHS / United States
T32 HL082547 / HL / NHLBI NIH HHS / United States
T32 HL 082547 / HL / NHLBI NIH HHS / United States
P01 HL 58064 / HL / NHLBI NIH HHS / United States
R01 HL088144 / HL / NHLBI NIH HHS / United States
P01 HL058064 / HL / NHLBI NIH HHS / United States