title: Extracellular ATP induces assembly and activation of the myosin light chain phosphatase complex in endothelial cells.
abstract: OBJECTIVE
  Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood.
  
  
  METHODS
  Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2).
  
  
  RESULTS
  Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1delta catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells.
  
  
  CONCLUSIONS
  The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1delta and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.
authors.affiliation: Physiologisches Institut, Justus-Liebig-Universität Aulweg 129, D-35392 Giessen, Germany.
authors.forename: F V
authors.initials: FV
authors.lastname: Härtel
authors.forename: C W
authors.initials: CW
authors.lastname: Rodewald
authors.forename: M
authors.initials: M
authors.lastname: Aslam
authors.forename: D
authors.initials: D
authors.lastname: Gündüz
authors.forename: L
authors.initials: L
authors.lastname: Hafer
authors.forename: J
authors.initials: J
authors.lastname: Neumann
authors.forename: H M
authors.initials: HM
authors.lastname: Piper
authors.forename: T
authors.initials: T
authors.lastname: Noll
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000577:Amides / Q000494:pharmacology; D015153:Blotting, Western; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors; D008387:Marine Toxins; D043263:Myosin-Light-Chain Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009708:Nucleotidases / Q000037:antagonists & inhibitors; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D058915:Purinergic P1 Receptor Antagonists; D011725:Pyridines / Q000494:pharmacology; D013806:Theophylline / Q000031:analogs & derivatives / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection / Q000379:methods; D054460:rho-Associated Kinases; D000097847:Protein Phosphatase Inhibitory Proteins
chemical_list: D000255:Adenosine Triphosphate; D000577:Amides; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008387:Marine Toxins; D043263:Myosin-Light-Chain Phosphatase; D009708:Nucleotidases; D010080:Oxazoles; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases; D011506:Proteins; D058915:Purinergic P1 Receptor Antagonists; D011725:Pyridines; D013806:Theophylline; D013917:Thrombin; D054460:rho-Associated Kinases; C092431:6-N,N-diethyl-beta,gamma-dibromomethylene-D-ATP; C024833:protein phosphatase inhibitor-2; D000097847:Protein Phosphatase Inhibitory Proteins; C108830:Y 27632; C059041:calyculin A; C028322:8-phenyltheophylline