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

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 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 increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. 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.

α-Enolase Causes Proinflammatory Activation of Pulmonary Microvascular Endothelial Cells and Primes Neutrophils Through Plasmin Activation of Protease-Activated Receptor 2

“…Recently, identical proteomic analyses of the plasma from rats that underwent hemorrhagic shock demonstrated that α-enolase significantly increased; moreover, these identical proteins are also found in stored, but not fresh, PRBCs, which may be used to resuscitate these injured patients (7;31). The function of α-enolase in conjunction with plasminogen in PMN-mediated lung injury and organ dysfunction is largely unexplored; while thrombin has been implicated in the activation of endothelial cells resulting in decreased barrier function and fibrin deposition, a hall mark of ALI/ARDS (33–35). …”

Loss of cortactin causes endothelial barrier dysfunction via disturbed adrenomedullin secretion and actomyosin contractility

“…Cortactin is ubiquitously expressed except for most hematopoietic cells and contains the following key domains: an N-terminal acidic domain that can bind to the Arp2/3 complex, an F-actin binding domain, a proline-rich region containing serine and tyrosine phosphorylation sites and a SH3 domain that mediates interaction with several proteins involved in actin polymerization (N-WASP), cell–cell adhesion (ZO-1) and membrane dynamics (dynamin) 16 17 18 . Cortactin can interact with myosin-light chain kinase (MLCK) in endothelial cells 19 20 , but a direct contribution of cortactin to MLC phosphorylation and actomyosin contractility has not been investigated. Instead, it has been shown that cortactin translocates to endothelial cell contacts in response to shear-stress where it may contribute to actin remodeling 21 .…”

Myosin light chain kinase (MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions

“…We utilized an established method for live cell imaging and kymograph analysis. 8 , 26 Briefly, EC grown on coverslips were loaded into a recording chamber (ALA Scientific Instruments, Westbury, NY, USA) in culture media at 37℃. Images were acquired by a Zeiss 710 laser scanning confocal microscope (every 6 s) and cells were observed under basal conditions and 10 min post administration of 1 μM S1P.…”

“…Images were analyzed using ImageJ and kymographic analysis was performed using the Multiple Kymograph Plugin to assess membrane dynamics as we have described previously. 8 , 26 …”

“…The essential role of the non-muscle myosin light chain kinase isoform or nmMLCK 1 in the complex regulation of endothelial cell cytoskeleton rearrangement, especially during disruption and restoration of vascular integrity, has been well defined. 2 – 8 Acute and chronic cardiovascular diseases, including acute lung injury (ALI) and its more severe form acute respiratory distress syndrome (ARDS), 9 have obvious uncontrolled pulmonary vascular hyper-permeability which has been determined to be the major cause of pulmonary alveolar edema. 10 The major translational product of the MYLK gene in endothelium is the non-muscle isoform of MLCK (nmMLCK, 210 kd).…”