Proline rich.pdf (1.3 MB)

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

Download (1.3 MB)
journal contribution
posted on 16.01.2018 by P. Belvitch, D. Adyshev, V.R. Elangovan, M.E. Brown, C. Naureckas, A.N. Rizzo, J.H. Siegler, J.G. Garcia, S.M. Dudek
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.

Funding

Grant Support P01 HL 58064/HL/NHLBI NIH HHS/United States R01 HL 88144/HL/NHLBI NIH HHS/United States R01 HL088144/HL/NHLBI NIH HHS/United States T32 HL 082547/HL/NHLBI NIH HHS/United States T32 HL082547/HL/NHLBI NIH HHS/United States

History

Publisher Statement

This is the author’s version of a work that was accepted for publication in Microvascular Research. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Microvascular Research, 2014. 95(1): 94-102. DOI: 10.1016/j.mvr.2014.07.007.

Publisher

Elsevier

issn

0026-2862

Issue date

01/09/2014

Exports

Categories

Exports