Regulation of PP2AC carboxylmethylation and cellular localisation by inhibitory class G-protein coupled receptors in cardiomyocytes.
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 N(6)-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.
School of Pharmacy and Chemistry, Faculty of Science, Engineering and Computing, Kingston University, Kingston-upon-Thames, Surrey, United Kingdom.
Michael R
MR
Longman
King's College London British Heart Foundation Centre, Cardiovascular Division, The Rayne Institute, St Thomas' Hospital, London, United Kingdom.
Antonella
A
Ranieri
King's College London British Heart Foundation Centre, Cardiovascular Division, The Rayne Institute, St Thomas' Hospital, London, United Kingdom.
Metin
M
Avkiran
School of Pharmacy and Chemistry, Faculty of Science, Engineering and Computing, Kingston University, Kingston-upon-Thames, Surrey, United Kingdom ; King's College London British Heart Foundation Centre, Cardiovascular Division, The Rayne Institute, St Thomas' Hospital, London, United Kingdom.
Andrew K
AK
Snabaitis
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D002867:Chromones; D003593:Cytoplasm / Q000378:metabolism*; D047468:Immunoprecipitation; D008745:Methylation / Q000187:drug effects; D009025:Morpholines; D032383:Myocytes, Cardiac / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011497:Protein O-Methyltransferase / Q000378:metabolism*; D054648:Protein Phosphatase 2 / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D011945:Receptors, Angiotensin / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*
D002867:Chromones; D009025:Morpholines; D011945:Receptors, Angiotensin; D043562:Receptors, G-Protein-Coupled; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C048599:N(6)-cyclopentyladenosine; D011497:Protein O-Methyltransferase; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; C514014:Ppp2ca protein, rat; D054648:Protein Phosphatase 2; D000241:Adenosine