A16523 Mapping of the angiotensin AT2 receptor-coupled signalling network by time-resolved quantitative phosphoproteomics in human aortic endothelial cells identified HDAC-1 and p53 to be involved in AT2 receptor-mediated anti-proliferation

Abstract

Objectives: The angiotensin AT2-receptor (AT2R) is a main component of the protective arm of the renin-angiotensin system. In order to get new and deeper insights into AT2R mediated signalling, this study mapped the AT2R-coupled phosphoproteome in human aortic endothelial cells (HAEC) by quantitative phosphoproteomics. Methods: HAEC were stimulated with the AT2R agonist C21 (1 μM) for 1, 3, 5 or 20 minutes. Following protein digestion and TMT peptide labelling, samples were subjected to TiO2−SIMAC phospho-peptide enrichment and LC−MS/MS. Data were analysed using Proteome Discoverer and the human sequence libraries SwissProt and UniProt. For confirmation of phosphoproteomics results, expression of p53 and phosphorylation of HDAC-1 were determined by Western Blot. Cellular translocation of HDAC-1 and p53 was assessed by immunofluorescence and activity of HDAC-1 by deacetylation assay. HAEC proliferation was determined by resazurin assay and apoptosis by caspase 3/7 assay. Results: AT2R stimulation of HAEC changed the phosphorylation status of 265 proteins at 471 sites (281 phosphorylations and 190 dephosphorylations). Gene Ontology and STRING analysis revealed the predominant phospho-modification of proteins involved in anti-proliferation, cell differentiation and apoptosis including HDAC-1, p53, BCL2 or HSP90. AT2R-mediated inhibition of HDAC-1 and activation of p53 were confirmed by additional experiments demonstrating changes in cellular localisation of HDAC-1 and p53, in total expression of p53, and in enzymatic activity and phosphorylation of HDAC1. Conclusion: This study mapped the AT2R coupled phospho-signalling network. It identified dephosphorylation and deactivation of HDAC-1 as well as nuclear importation and increased expression of p53 as novel, potential mediators of AT2R-coupled anti-proliferation.