Extracellular ATP induces assembly and activation of the myosin light chain phosphatase complex in endothelial cells

• Published in: Cardiovascular research Vol. 74; no. 3; pp. 487 - 496
• Main Authors: HÄRTEL, F. V, RODEWALD, C. W, ASLAM, M, GÜNDÜZ, D, HAFER, L, NEUMANN, J, PIPER, H. M, NOLL, T
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.06.2007
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - pharmacology; Amides - pharmacology; Biological and medical sciences; Blotting, Western; Cardiology. Vascular system; Cells, Cultured; Endothelial Cells - drug effects; Endothelial Cells - enzymology; Enzyme Activation; Enzyme Inhibitors - pharmacology; Humans; Immunoprecipitation; Intracellular Signaling Peptides and Proteins - antagonists & inhibitors; Medical sciences; Myosin-Light-Chain Phosphatase - antagonists & inhibitors; Myosin-Light-Chain Phosphatase - metabolism; Nucleotidases - antagonists & inhibitors; Oxazoles - pharmacology; Phosphoprotein Phosphatases - antagonists & inhibitors; Phosphoprotein Phosphatases - metabolism; Phosphorylation; Protein Phosphatase 1; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Proteins - genetics; Proteins - metabolism; Purinergic P1 Receptor Antagonists; Pyridines - pharmacology; rho-Associated Kinases; Theophylline - analogs & derivatives; Theophylline - pharmacology; Thrombin - pharmacology; Transfection - methods; Protein inhibitor; Endothelial cell; Myosin light chain phosphatase; Targeting; Enzyme; Phosphoprotein phosphatase; Phosphoric monoester hydrolases; Protein phosphatase inhibitor-2; Esterases; Activation; Light peptide chain; Subunit; Complexes; Extracellular; Phlebology; Protein; Myosin; Myosin protein targeting subunit; Hydrolases; Circulatory system; Cardiology; ATP; [Myosin-light-chain]-phosphatase
• ISSN: 0008-6363; 1755-3245
• DOI: 10.1016/j.cardiores.2007.02.013

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. 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). 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. 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.