Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics |
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| Affiliation: | 1. Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA;2. University of Arizona Health Sciences Center, Tucson, AZ, USA;1. Laboratoire des Venins et Biomolécules Thérapeutiques, Institut Pasteur de Tunis, Tunisia;2. Aix Marseille Université, Institut National de la Santé et de la Recherche Médicale, UMR_S 911, Marseille, France;3. APHM, Hôpital Timone, Service Pharmacie, Marseille, France;4. Equipe CROPS, Institut de Chimie Radicalaire — UMR 7273, Université d''Aix-Marseille, Site de Saint Jérôme, Av. Escadrille Normandie Niemen, 13397 Marseille, France;5. Faculté de Médecine de Tunis, Tunisia;1. Department of Physiology and Pharmacology, Western University, London, ON, Canada;2. Department of Pharmacology, University of Alberta, Edmonton, AB, Canada;1. Department of Anesthesia, Dalhousie University, Halifax, NS, Canada;2. Department of Anesthesiology and Intensive Care Medicine, Ernst-Moritz-Arndt-Universität, Greifswald, Germany;3. Institute for Pathophysiology “Ljubodrag Buba Mihailović”, Faculty of Medicine, University of Belgrade, Belgrade, Serbia;1. Department of Physiology and Biophysics, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA;2. Naval Medical Research Center, Silver Spring, MD 20910, USA;1. Department of General Surgery, Jinling Hospital, Medical School of Nanjing University, China;2. Binjiang College, Zhejiang Chinese Medical University, China;1. Department of Physiology and Biophysics, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antônio Carlos 6627, Campus Pampulha, Cx Post 468, CEP 31270‐901 Belo Horizonte, MG, Brazil;2. Department of General Pathology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antônio Carlos 6627, Campus Pampulha, Cx Post 468, CEP 31270‐901 Belo Horizonte, MG, Brazil |
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| Abstract: | 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. |
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