Interplay of myosin phosphatase and protein phosphatase-2A in the regulation of endothelial nitric-oxide synthase phosphorylation and nitric oxide production

Abstract

The inhibitory phosphorylation of endothelial nitric oxide (NO) synthase (eNOS) at Thr497 (eNOSpThr497) by protein kinase C or RhoA-activated kinase is a major regulatory determinant of eNOS activity. The signalling mechanisms involved in the dephosphorylation of eNOSpThr497 have not yet been clarified. This study identifies myosin phosphatase (MP) holoenzyme consisting of protein phosphatase-1 catalytic subunit (PP1c) and MP target subunit-1 (MYPT1) as an eNOSpThr497 phosphatase. In support of this finding are: (i) eNOS and MYPT1 interacts in various endothelial cells (ECs) and in in vitro binding assays (ii) MYPT1 targets and stimulates PP1c toward eNOSpThr497 substrate (iii) phosphorylation of MYPT1 at Thr696 (MYPT1pThr696) controls the activity of MP on eNOSpThr497. Phosphatase inhibition suppresses both NO production and transendothelial resistance (TER) of ECs. In contrast, epigallocatechin-3-gallate (EGCG) signals ECs via the 67 kDa laminin-receptor (67LR) resulting in protein kinase A dependent activation of protein phosphatase-2A (PP2A). PP2A dephosphorylates MYPT1pThr696 and thereby stimulates MP activity inducing dephosphorylation of eNOSpThr497 and the 20 kDa myosin II light chains. Thus an interplay of MP and PP2A is involved in the physiological regulation of EC functions implying that an EGCG dependent activation of these phosphatases leads to enhanced NO production and EC barrier improvement.

Inhibition of protein phosphatase-1 and -2A by ellagitannins: structure-inhibitory potency relationships and influences on cellular systems

“…Although, partial selectivity of several toxins (such as okadaic acid and tautomycin) in the inhibition of PP1 and PP2A are proven in vitro , there are still concerns with respect to their specificity in cellular systems 6 due to their differences in membrane permeability and intracellular concentrations. Nevertheless, calyculin A and tautomycin have been shown to specifically inhibit PP2A and PP1 in Balb/c 3T3 cells 7 , respectively, and this distinction between PP1 and PP2A by these toxins was also confirmed in THP1 leukemic 8 and endothelial 9 cells by phosphatase activity assays. Even though, the use of toxins to differentiate between the cellular actions of phosphatase types is limited due to cytotoxicity of these compounds.…”

“…There is also an apparent controversy concerning the cellular effects of EGCG: it inhibits phosphatase activity of various cell lines at quite high (100–500 µM) concentration 13 while it activates PP2A at 5–20 µM concentrations in a laminin-receptor mediated manner 14 . As a consequence, PP1-type myosin phosphatase is also stimulated by PP2A driven dephosphorylation of its myosin phosphatase target subunit-1 (MYPT1) at phosphorylation sites inhibitory on PP1c 9 suggesting a specific interplay of PP1 and PP2A in cellular phosphatase activation.…”

Myosin Phosphatase Is Implicated in the Control of THP-1 Monocyte to Macrophage Differentiation

“…Alternatively, MP can be activated by enhancing the activity of the phosphatase(s) responsible for the dephosphorylation of inhibitory phosphosites of MYPT1. In this regard, the green tea polyphenol epigallocatechin-3-gallate (EGCG) was shown to induce the dephosphorylation of MYPT1 pT696 by stimulating PP2A via the 67 kDa laminin receptor (67LR) [ 26 , 27 ]. We have demonstrated recently that the EGCG/67LR/PP2A pathway also functions in THP-1 cells [ 28 ].…”

“…It is a relatively new finding that MP can be activated by potentiating MYPT1 dephosphorylation through the initiation of the 67LR/PKA/PP2A pathway by EGCG [ 26 , 27 ]. Indeed, EGCG has a variety of pharmacological actions including cardiovascular protective effects [ 42 ].…”

SALM4 regulates angiogenic functions in endothelial cells through VEGFR2 phosphorylation at Tyr1175

“…Acute brain I/R damage can increase VEGF-A expression in neurons, astrocytes, macrophages, and ECs. Increased VEGF-A influences vascular permeability through PI3K-AKT phosphorylation, which activates eNOS (52)(53)(54). Phosphorylation of VEGFR2, a major receptor for VEGF-A, leads to degradation of endothelial junction molecules after stroke (55).…”