Quantitative Phosphoproteomics of the Angiotensin AT2-Receptor Signaling Network Identifies HDAC1 (Histone-Deacetylase-1) and p53 as Mediators of Antiproliferation and Apoptosis

A Augusto B Peluso, Stefan J Kempf, Thiago Verano-Braga, Lucas Rodrigues-Ribeiro, Lene Egedal Johansen, Mie Rytz Hansen, Gitte Kitlen, Andreas Houe Haugaard, Colin Sumners, Henrik J Ditzel, Robson A Santos, Michael Bader, Martin R Larsen, U Muscha Steckelings*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

BACKGROUND: Angiotensin AT2-receptor signaling is atypical for a G-protein coupled receptor and incompletely understood. To obtain novel insights into AT2-receptor signaling, we mapped changes in the phosphorylation status of the entire proteome of human aortic endothelial cells in response to AT2-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: AT2-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 serine421/423 involving serine/threonine phosphatases. Resulting HDAC1 inhibition led to p53 acetylation and activation. AT2-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 AT2-receptor mediated functions.

CONCLUSIONS: Contrary to the prevailing view that AT2-receptor signaling largely involves phosphatases, our study revealed significant involvement of kinases. HDAC1 inhibition and resulting p53 activation were identified as novel, AT2-receptor coupled signaling mechanisms. Furthermore, the study created an openly available dataset of AT2-receptor induced phospho-modified proteins, which has the potential to be the basis for further discoveries of currently unknown, AT2-receptor coupled signaling mechanisms.

Original languageEnglish
JournalHypertension (Dallas, Tex. : 1979)
Volume79
Issue number11
Pages (from-to)2530-2541
ISSN0194-911X
DOIs
Publication statusPublished - Nov 2022

Keywords

  • angiotensin receptors
  • apoptosis
  • endothelial cells
  • histone deacetylase 1
  • phosphoproteomics

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