Quantitative Phosphoproteomics of the Angiotensin AT(2)-Receptor Signaling Network Identifies HDAC1 (Histone-Deacetylase-1) and p53 as Mediators of Antiproliferation and Apoptosis

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 mu M; 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-alpha, 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.