Regulation of PP2AC Carboxylmethylation and Cellular Localisation by Inhibitory Class G-Protein Coupled Receptors in Cardiomyocytes

Abstract

The enzymatic activity of the type 2A protein phosphatase (PP2A) holoenzyme, a major serine/threonine phosphatase in the heart, is conferred by its catalytic subunit (PP2AC). PP2AC activity and subcellular localisation can be regulated by reversible carboxylmethylation of its C-terminal leucine309 (leu309) residue. Previous studies have shown that the stimulation of adenosine type 1 receptors (A1.Rs) induces PP2AC carboxylmethylation and altered subcellular distribution in adult rat ventricular myocytes (ARVM). In the current study, we show that the enzymatic components that regulate the carboxylmethylation status of PP2AC, leucine carboxylmethyltransferase-1 (LCMT-1) and phosphatase methylesterase-1 (PME-1) are abundantly expressed in, and almost entirely localised in the cytoplasm of ARVM. The stimulation of Gi-coupled A1.Rs with N6-cyclopentyladenosine (CPA), and of other Gi-coupled receptors such as muscarinic M2 receptors (stimulated with carbachol) and angiotensin II AT2 receptors (stimulated with CGP42112) in ARVM, induced PP2AC carboxylmethylation at leu309 in a concentration-dependent manner. Exposure of ARVM to 10 µM CPA increased the cellular association between PP2AC and its methyltransferase LCMT-1, but not its esterase PME-1. Stimulation of A1.Rs with 10 µM CPA increased the phosphorylation of protein kinase B at ser473, which was abolished by the PI3K inhibitor LY294002 (20 µM), thereby confirming that PI3K activity is upregulated in response to A1.R stimulation by CPA in ARVM. A1.R-induced PP2AC translocation to the particulate fraction was abrogated by adenoviral expression of the alpha subunit (Gαt1) coupled to the transducin G-protein coupled receptor. A similar inhibitory effect on A1.R-induced PP2AC translocation was also seen with LY294002 (20 µM). These data suggest that in ARVM, A1.R-induced PP2AC translocation to the particulate fraction occurs through a GiPCR-Gβγ-PI3K mediated intracellular signalling pathway, which may involve elevated PP2AC carboxylmethylation at leu309.

Methylation of protein phosphatase 2A—Influence of regulators and environmental stress factors

“…Methylation of PP2A-C can lead to more frequent localization to certain membranes or membrane rafts. Longman, Ranieri, Avkiran, and Snabaitis (2014) reported that stimulation of A1 receptors (a type of G-coupled receptors in mammals) increased the association of LCMT1 with PP2A-C and thereby augmented the carboxymethylation of the C subunits and facilitated association of PP2A-C with membrane-rich particulate compartments. Sontag, Nunbhakdi-Craig, and Sontag (2013) reported coenrichment of LCMT1, methylated PP2A-C, and Bα in cholesterol-rich membrane rafts of mammalian N2a cells.…”

Regulation of Ca2+ transient by PP2A in normal and failing heart

“…The constitutively active Pak1, the downstream effectors for Cdc42 and Rac1 induces activation of PP2A and dephosphorylation of myofilament regulatory proteins (Ke et al, 2004 ). PI3K is another possible link between Gi and PP2A activities that enhances carboxylmethylation at leu309 (Longman et al, 2014 ) (Figure 2 ).…”

Global loss of leucine carboxyl methyltransferase-1 causes severe defects in fetal liver hematopoiesis

“…Regarding signaling, LCMT-1 methylation of the PP2A C subunit is stimulated by multiple GiPCR agonists in adult rat ventricular myocytes (Ref. 27 and references therein); LCMT-1 is a negative regulator of Akt and p70/p85 S6 kinase (28); and LCMT-1 helps prevent anchorage-independent growth of cells (28).…”