title: Calcineurin regulates endothelial barrier function by interaction with and dephosphorylation of myosin phosphatase.
abstract: OBJECTIVE
  Calcineurin (CN) influences myosin phosphorylation and alters endothelial barrier function; however, the molecular mechanism is still obscure. Here we examine whether CN controls myosin phosphorylation via mediating the phosphorylation state of Thr696 in myosin phosphatase (MP) target subunit 1 (MYPT1), the phosphorylation site inhibitory to the catalytic activity of MP.
  
  
  RESULTS
  Exposure of bovine or human pulmonary artery endothelial cells (BPAECs or HPAECs) to the CN inhibitor cyclosporin A (CsA) induces a rise in intracellular Ca(2+) and increases the phosphorylation level of cofilin(Ser3) and MYPT1(Thr696) in a Ca(2+)-and Rho-kinase-dependent manner. An active catalytic fragment of CN overexpressed in tsA201 cells decreases endogenous MYPT-phospho-Thr696 (MYPT1(pThr696)) levels. Purified CN dephosphorylates (32)P-labelled MYPT1, suggesting direct action of CN on this substrate. Interaction of MYPT1 with CN is revealed by MYPT1 pull-down experiments and colocalization in both BPAECs and HPAECs as well as by surface plasmon resonance (SPR)-based binding studies. Stabilization of the MYPT1-CN complex occurs via the MYPT1(300PLIEST305) sequence similar to the CN substrate-docking PxIxIT-motif. Thrombin induces a transient increase of MYPT1(pThr696) in BPAECs, whereas its combination with CsA results in maintained phosphorylation levels of both MYPT1(pThr696) and myosin. These phosphorylation events might correlate with changes in endothelial permeability since CsA slows down the recovery from the thrombin-induced decrease of the transendothelial electrical resistance of the BPAEC monolayer.
  
  
  CONCLUSIONS
  CN may improve endothelial barrier function via inducing dephosphorylation of cofilin(pSer3) and by interaction with MYPT1 and activating MP through MYPT1(pThr696) dephosphorylation, thereby affecting actin polymerization and decreasing myosin phosphorylation.
authors.affiliation: Department of Medical Chemistry, Medical and Health Science Center,  University of Debrecen, Nagyerdei krt 98, Debrecen H-4032,  Hungary.
authors.forename: Bernadett
authors.initials: B
authors.lastname: Kolozsvári
authors.forename: Éva
authors.initials: É
authors.lastname: Bakó
authors.forename: Bálint
authors.initials: B
authors.lastname: Bécsi
authors.forename: Andrea
authors.initials: A
authors.lastname: Kiss
authors.forename: Ágnes
authors.initials: Á
authors.lastname: Czikora
authors.forename: Attila
authors.initials: A
authors.lastname: Tóth
authors.forename: György
authors.initials: G
authors.lastname: Vámosi
authors.forename: Pál
authors.initials: P
authors.lastname: Gergely
authors.forename: Ferenc
authors.initials: F
authors.lastname: Erdődi
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000818:Animals; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism*; D065095:Calcineurin Inhibitors; D002118:Calcium / Q000378:metabolism; D002199:Capillary Permeability / Q000187:drug effects; D002417:Cattle; D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D017097:Electric Impedance; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D006801:Humans; D018994:Myosin Light Chains / Q000378:metabolism*; D043263:Myosin-Light-Chain Phosphatase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D012694:Serine; D020349:Surface Plasmon Resonance; D013912:Threonine; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D054460:rho-Associated Kinases / Q000378:metabolism
chemical_list: D051339:Actin Depolymerizing Factors; D065095:Calcineurin Inhibitors; D018994:Myosin Light Chains; D013912:Threonine; D012694:Serine; D016572:Cyclosporine; D054460:rho-Associated Kinases; D019703:Calcineurin; D043263:Myosin-Light-Chain Phosphatase; C490372:PPP1R12A protein, human; D013917:Thrombin; D002118:Calcium