Quantitative phosphoproteomics of the angiotensin AT(2)-receptor signaling network identifies HDAC1 (Histone-Deacetylase-1) and p53 as mediators of antiproliferation and apoptosis

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Item Type:	Article
Title:	Quantitative phosphoproteomics of the angiotensin AT(2)-receptor signaling network identifies HDAC1 (Histone-Deacetylase-1) and p53 as mediators of antiproliferation and apoptosis
Creators Name:	Peluso, A.A.B., Kempf, S.J., Verano-Braga, T., Rodrigues-Ribeiro, L., Johansen, L.E., Hansen, M.R., Kitlen, G., Haugaard, A.H., Sumners, C., Ditzel, H.J., Santos, R.A., Bader, M., Larsen, M.R. and Steckelings, U.M.
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.
Keywords:	Acetylation, Apoptosis, Endothelial Cells, Histone Deacetylase 1, Phosphoproteomics
Source:	Hypertension
ISSN:	0194-911X
Publisher:	American Heart Association
Volume:	79
Number:	11
Page Range:	2530-2541
Date:	November 2022
Official Publication:	https://doi.org/10.1161/hypertensionaha.121.18620
PubMed:	View item in PubMed

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