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Calcineurin regulates endothelial barrier function by interaction with and dephosphorylation of myosin phosphatase

Bernadett Kolozsvári, Éva Bakó, Bálint Bécsi, Andrea Kiss, Ágnes Czikora, Attila Tóth, György Vámosi, Pál Gergely, Ferenc Erdődi
DOI: http://dx.doi.org/10.1093/cvr/cvs255 494-503 First published online: 6 August 2012

Abstract

Aims 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.

Methods and 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 Ca2+ and increases the phosphorylation level of cofilinSer3 and MYPT1Thr696 in a Ca2+-and Rho-kinase-dependent manner. An active catalytic fragment of CN overexpressed in tsA201 cells decreases endogenous MYPT-phospho-Thr696 (MYPT1pThr696) levels. Purified CN dephosphorylates 32P-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 MYPT1300PLIEST305 sequence similar to the CN substrate-docking PxIxIT-motif. Thrombin induces a transient increase of MYPT1pThr696 in BPAECs, whereas its combination with CsA results in maintained phosphorylation levels of both MYPT1pThr696 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.

Conclusion CN may improve endothelial barrier function via inducing dephosphorylation of cofilinpSer3 and by interaction with MYPT1 and activating MP through MYPT1pThr696 dephosphorylation, thereby affecting actin polymerization and decreasing myosin phosphorylation.

  • Myosin phosphatase
  • Calcineurin (protein phosphatase-2B)
  • Myosin phosphatase target subunit 1 (MYPT1)
  • Endothelial barrier function
  • Transendothelial electrical resistance
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