title: 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 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.
authors: Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA.
authors: Patrick
authors: P
authors: Belvitch
authors: Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA.
authors: Djanybek
authors: D
authors: Adyshev
authors: Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA.
authors: Venkateswaran R
authors: VR
authors: Elangovan
authors: Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA.
authors: Mary E
authors: ME
authors: Brown
authors: Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA.
authors: Caitlin
authors: C
authors: Naureckas
authors: Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA.
authors: Alicia N
authors: AN
authors: Rizzo
authors: Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA.
authors: Jessica H
authors: JH
authors: Siegler
authors: University of Arizona Health Sciences Center, Tucson, AZ, USA.
authors: Joe G N
authors: JG
authors: Garcia
authors: Division of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois Hospital and Health Science System, Chicago, IL, USA. Electronic address: sdudek@uic.edu.
authors: Steven M
authors: SM
authors: Dudek
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D001665:Binding Sites; D015820:Cadherins / Q000378:metabolism; D002199:Capillary Permeability; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D051356:Cortactin / Q000378:metabolism; D003599:Cytoskeleton / Q000201:enzymology*; D042783:Endothelial Cells / Q000201:enzymology*; D006801:Humans; D047468:Immunoprecipitation; D007734:Kymography; D008168:Lung / Q000098:blood supply*; D008246:Lysophospholipids / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055232:Proline-Rich Protein Domains; D054730:Protein Interaction Domains and Motifs; D013110:Sphingosine / Q000031:analogs & derivatives / Q000378:metabolism; D022502:Stress Fibers / Q000201:enzymology; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D000099152:Cadherin 5
chemical_list: D015703:Antigens, CD; D015820:Cadherins; D051356:Cortactin; D008246:Lysophospholipids; D009219:Myosin-Light-Chain Kinase; D013110:Sphingosine; D013917:Thrombin; D000099152:Cadherin 5; C495868:CTTN protein, human; C060506:sphingosine 1-phosphate
keywords: ARDS; Barrier function; Cortactin; Cytoskeleton; Endothelium; Membrane dynamics; Non-muscle myosin light chain kinase; Stress fibers