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Cardiovascular Research 2007 74(3):487-496; doi:10.1016/j.cardiores.2007.02.013
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Copyright © 2007, European Society of Cardiology

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

F.V. Härtela, C.W. Rodewalda, M. Aslama, D. Gündüza, L. Haferb, J. Neumannb, H.M. Pipera and T. Nolla,*

aPhysiologisches Institut, Justus-Liebig-Universität Aulweg 129, D-35392 Giessen, Germany
bInstitut für Pharmakologie und Toxikologie, Martin-Luther-Universität Halle-Wittenberg, Germany

* Corresponding author. Tel.: +49 641 99 47343; fax: +49 641 99 47219. Email address: thomas.noll{at}physiologie.med.uni-giessen.de

Objectives: 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 PP1{delta} 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 PP1{delta} and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.

KEYWORDS ATP; Myosin light chain phosphatase; Myosin protein targeting subunit; Protein phosphatase inhibitor-2

* This article was handled by Mark Post of Maastricht University (Maastricht, Netherlands), who served as Guest Editor.

Time for primary review 28 days


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