Redox status regulates Akt1 activity and cell cycle through mitochondrial phosphorylation of Thr 308.

ANTICO ARCIUCH, VG; FRANCO, MC; CARRERAS, MC; PODEROSO, JJ.

Washington DC, EEUU

Congreso; 14th Annual Meeting of the Society for Free Radical Biology and Medicine.; 2007

Society for Free Radical Biology and Medicine.

Akt is an important regulator of cellular processes such as proliferation and apoptosis, angiogenesis and glucose metabolism. Akt becomes activated by different stimuli that include growth factors, cytokines, steroid hormones and cellular stress. For its complete activation, Akt1 requires to be sequentially phosphorylated in Ser473 and Thr308. The aim of this work was to study Akt1 activation and translocation to mitochondria by H2O2 in connection with cell cycle progression of NIH/3T3 cell line. Proliferation and apoptosis were evaluated by [H3]thymidine incorporation, flow cytometry with propidium iodide and annexin V staining. Subcellular distribution of P-Akt (nuclei, mitochondria and cytosol) was followed by transient transfection with pcDNA3-Akt1 wt and negative dominant and analyzed by western blot anti HA-tag. Akt1 activity was detected by an in vitro kinase assay using GSK-3 fusion protein as a substrate. PDK1-Akt1 interaction was assesed by an in vitro binding assay using previously oxydized recombinant Akt1 protein. Differentially, 50 µM H2O2 promoted proliferation, and > 250 mM H2O2 apoptosis with cytochrome c release to cytosol and a decrease in Bcl-xL content. At 50 µM H2O2, Akt1 activation and translocation to nucleus were rapid and persistent while translocation to mitochondria was transitory. On the contrary, 250 µM H2O2 induced transient activation and transitory translocation to nucleus while it was retained in mitochondria where Thr308 phosphorylation was prevented in the apoptotic condition. In this context, Akt1 activity decreased by 43% in mitochondria after 15 minutes exposure to 250 µM H2O2. The high H2O2 concentration effect was caused by a decrease of Akt1 binding to the upstream kinase, PDK1. The results therefore surmise that mitochondria regulates Akt1 phosphorylation in Thr 308 and that, high H2O2 yield impedes Akt1-PDK1 interaction in the organelles, where the partially inactive kinase is retained leading to the apoptotic condition.