Quantitative Phosphoproteomics of the Angiotensin AT <sub>2</sub> -Receptor Signaling Network Identifies HDAC1 (Histone-Deacetylase-1) and p53 as Mediators of Antiproliferation and Apoptosis
A. Augusto Peluso, Stefan J. Kempf, Thiago Verano‐Braga, Lucas Rodrigues‐Ribeiro, Lene E. Johansen, Mie R. Hansen, Gitte Kitlen, Andreas Houe Haugaard, Colin Sumners, Henrik J. Ditzel, Robson A.S. Santos, Michael Bäder, Martin R. Larsen, U. Muscha Steckelings
Abstract
Background: Angiotensin AT 2 -receptor signaling is atypical for a G-protein coupled receptor and incompletely understood. To obtain novel insights into AT 2 -receptor signaling, we mapped changes in the phosphorylation status of the entire proteome of human aortic endothelial cells in response to AT 2 -receptor stimulation. Methods: Phosphorylation status of human aortic endothelial cells after stimulation with C21 (1 µM; 0, 1, 3, 5, 20 minutes) was determined utilizing time-resolved quantitative phosphoproteomics. Specific changes in protein phosphorylation and acetylation were confirmed by Western Blotting. Functional tests included resazurin assay for cell proliferation, and caspase 3/7 luminescence assay or FACS analysis of annexin V expression for apoptosis. Results: AT 2 -receptor stimulation significantly altered the phosphorylation status of 172 proteins (46% phosphorylations, 54% dephosphorylations). Bioinformatic analysis revealed a cluster of phospho-modified proteins involved in antiproliferation and apoptosis. Among these proteins, HDAC1 (histone-deacetylase-1) was dephosphorylated at serine 421/423 involving serine/threonine phosphatases. Resulting HDAC1 inhibition led to p53 acetylation and activation. AT 2 -receptor stimulation induced antiproliferation and apoptosis, which were absent when cells were co-incubated with the p53 inhibitor pifithrin-α, thus indicating p53-dependence of these AT 2 -receptor mediated functions. Conclusions: Contrary to the prevailing view that AT 2 -receptor signaling largely involves phosphatases, our study revealed significant involvement of kinases. HDAC1 inhibition and resulting p53 activation were identified as novel, AT 2 -receptor coupled signaling mechanisms. Furthermore, the study created an openly available dataset of AT 2 -receptor induced phospho-modified proteins, which has the potential to be the basis for further discoveries of currently unknown, AT 2 -receptor coupled signaling mechanisms.