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

Michael R Longman,Metin Avkiran,Andrew K Snabaitis,Antonella Ranieri

doi:10.1371/journal.pone.0086234

Michael R Longman, Metin Avkiran + Show 2 more

Open Access

https://doi.org/10.1371/journal.pone.0086234

Copy DOI

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.

Highlights

The type 2A protein phosphatase (PP2A) is a serine/threonine protein phosphatase that is ubiquitously expressed in all eukaryotic cells and may account for up to 1% of total cellular protein [1]
The importance of PP2AC leu309 carboxylmethylation by leucine carboxylmethyltransferase-1 (LCMT-1) for recruitment of regulatory B subunits to the PP2AA/C heterodimer can be considered a sliding scale, whereby it is an absolute prerequisite for PPP2R2/B subunit recruitment by the PP2AA/C heterodimer and progressively less important for the recruitment of PPP2R5/B9, PPP2R3/B0 and the striatin/B90 subunits to PP2AA/C [8]
We demonstrate that (i) the LCMT-1/phosphatase methylesterase-1 (PME-1)/PP2AC/B55a intracellular signalling axis is mainly localised in the cytoplasm, (ii) PP2AC carboxylmethylation can be induced by multiple GiPCR agonists, (iii) A1.R stimulation increases the cellular association between PP2AC and LCMT-1 and (iv) A1.R-induced PP2AC translocation to a membrane-rich particulate fraction occurs through a Gbc phosphoinositide 3-kinase (PI3K) pathway

Summary

Introduction

The type 2A protein phosphatase (PP2A) is a serine/threonine protein phosphatase that is ubiquitously expressed in all eukaryotic cells and may account for up to 1% of total cellular protein [1]. The PP2A catalytic subunit undergoes reversible post-translational phosphorylation and carboxylmethylation, both of which can alter catalytic activity and cellular distribution of PP2A. The carboxylmethylation of leu309 increases the binding affinity of the PP2AA/C heterodimer for some, but not all, regulatory B subunits, which have been classified into four separate sub families and are encoded by 15 human genes: PPP2R2/B (A–D), PPP2R5/ B9 (A–E), PPP2R3/B0 (A–C) and the striatins/B90 (1, 3 and 4). The importance of PP2AC leu309 carboxylmethylation by LCMT-1 for recruitment of regulatory B subunits to the PP2AA/C heterodimer can be considered a sliding scale, whereby it is an absolute prerequisite for PPP2R2/B subunit recruitment by the PP2AA/C heterodimer and progressively less important for the recruitment of PPP2R5/B9, PPP2R3/B0 and the striatin/B90 subunits to PP2AA/C [8]

Methods

Results

Conclusion