Relevance of Akt sumoylation in mouse embryonic stem cells

CAMILA VAZQUEZ ECHEGARAY; VICTORIA PETRONE; ANABELLA SREBROW; SOLEDAD COSENTINO; CLAUDIA SOLARI; LINO BARAÑAO; MARCOS FRANCIA; AYELEN TORO; ARIEL WAISMAN; ALEJANDRA GUBERMAN

Buenos Aires

Congreso; LXII Reunión Anual de la Sociedad Argentina de Investigación Clínica; 2017

Embryonic stem cells (ESC) have the ability to self-renew indefinitely and to give rise to cells of all three germ layers. The PI3K/Akt pathway is involved in survival and proliferation and is key for maintaining the levels and activity of ESC?s essential tran-scription factors (TF) Oct4, Sox2 and Nanog. Recently, it has been reported that SUMO conjugation to Akt1 regulates the activity of thiskinase, with consequences in the splicing pattern, proliferation andcell cycle in different cultured cell lines. However, the relevance of this post-translational modification (PTM) of Akt1 hasn?t been studied in ESC.Our hypothesis is that SUMOylation of Akt1 is relevant for survival, proliferation and maintenance of ESC fundamental properties. This work was aimed to study the effect of this PMT of Akt1 on the regulation of Nanog expression, and to set up the conditions to induce apoptosis and detect this process in mouse ESC.First, ESC were transfected with expression vectors for Akt1 variants with different capacity of being SUMOylated along with a Luciferase reporter vector driven by Nanog promoter. We found a differential activity of this promoter depending on Akt SUMOylation levels, suggesting that this Akt PTM could modulate Nanog expression in ESC (Analyzed using randomized block design ANOVA and Tukey test). To further determine the possible involvement of Akt1 SUMOylation in the regulation of ESC?s survival/apoptosis, we first set up the conditions for inducing apoptosis in our cell culture system. Culturing ESC in the absence of the cytokine LIF and the 2i inhibitor for up to 4 days induced apoptosis, as detected by DNA ladder assay, as well as by PARP-1 and Caspase 3 cleavage by Western Blot. Our preliminary results suggest that the SUMOylation of Akt1 couldplay a role on the expression of Nanog. We consider that studying the regulation of the fundamental properties of ESC ESC is essential for their future application in regenerative medicine.