Angiotensin II promotes the subcellular distribution of mTOR pathway components activation in H295R adrenal human cells.

LUCÍA HERRERA; KATIA E. HELFENBERGER; CECILIA PODEROSO; GIULIANA ARGENTINO; PAOLA FINOCCHIETTO

Congreso; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2019

Target of rapamycin (TOR) forms two kinase complexes named mTOR complex 1 and 2 (mTORC1/2). mTORC1 regulates cellular growth by phosphorylation of a different set of components as p70 S6 kinase and S6 ribosomal protein (S6), among others, which promotes protein synthesis by cap-dependent translation through the eukaryotic initiation factor 4E. Canonical PI3K/Akt pathway activates mTORC1 and Akt is able to activate mTORC2. Glycogen-synthase kinase-3 (GSK-3) pathway negatively regulates mTORC1 by phosphorylation and activation of an mTOR inhibitory protein. We have previously shown that mTORC1/2 is activated in adrenal cells by angiotensin II (Ang II) with the involvement of a key steroidogenic enzyme, acyl-CoA synthetase 4. Subcellular localization may be a general principle used by hormones and growth factors to endorse precise spatial and temporal control of specialized cellular functions. mTORC1/2 localized in distinct subcellular compartments suggested to be an important mechanism to achieve signaling specificity. Thus, the aim of this work was to analyze the subcellular distribution of some components of mTORC1/2 pathway under Ang II stimulation in human H295R adrenal cells. We performed subcellular fractionation and observed by immunoblot that Ang II promotes mitochondrial Akt phosphorylation in a time-dependent manner (a peak at 2h and decrease thereafter, 2h vs. control; ***p0.001). Ang II elicits a significant increase of phospho-S6 (pS6) in lysate, mitochondrial and post-mitochondrial fractions, as fast as 30 min (***p0.001). We showed that mitochondrial S6 remains phosphorylated until 6h, in contrast with a marked post-mitochondrial S6 inactivation (mito 6h vs post 6h; ***p0.001). We could also detect mitochondrial phospho-GSK-3 in basal and Ang II-stimulated cells. These results indicate that Ang II-mediated response promotes subcellular distribution of mTORC pathway components in H295R adrenal human cells.