Characterization of the role of AKT1 in glucose metabolism

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Protein kinase B (AKT1) is a serine/threonine kinase that plays a central role in regulating cell survival pathways. AKT1 is inactive in its unphosphorylated form, requiring induced phosphorylation to investigate AKT1 biology. Typical approaches to study AKT1 biology in cells rely on unspecific growth factor or insulin stimulation that activates AKT1 via phosphorylation. A significant drawback of this stimulation lies in the lack of specificity, where many other kinases and pathways are activated at the same time. We therefore developed a novel system to deliver active AKT1 to human cells, circumventing the use of unspecific stimulation. We previously established a method to produce AKT1 phospho-variants from Escherichia coli with programmed phosphorylation. These AKT1 variants were fused with a cell penetrating peptide tag, allowing a direct uptake of pre-activated AKT1 to the cells using the cells very own protein import systems. This new method allows us to investigate AKT1 biology without using unspecific cell stimulation.In preliminary data, we showed that pre-activated TAT-phosphoAKT1 induced selective phosphorylation of the known AKT1 substrate glycogen synthase kinase 3a indicating a highly specific role for AKT in glucose metabolism. We will now investigate whether these changes are reflected on the metabolome level. Metabolites are small chemicals, such as the sugar glucose, that are converted into e.g. energy. Investigating the metabolome in response to AKT signalling will allow us to define the chemical footprints of AKT activated cells. Our collaboration with a world expert in metabolomics will uncover how cellular metabolism is regulated by AKT. While metabolomics is of increasing interest, very few researchers in Canada are able to apply this technique, and access to the required mass spectrometry equipment is limited. Adding metabolomics as another dimension to AKT signaling research is a unique opportunity for Canadian leadership in the endeavour broaden our knowledge on AKT1 biology.