Characterization of the role of AKT1 in glucose metabolism

蛋白激酶B(AKT1)是一种丝氨酸/苏氨酸激酶，在调节细胞生存途径中起着核心作用。AKT1在其非磷酸化形式下是不活跃的，需要诱导磷酸化来研究AKT1生物学。在细胞中研究AKT1生物学的典型方法依赖于通过磷酸化激活AKT1的非特异性生长因子或胰岛素刺激。这种刺激的一个显著缺点是缺乏特异性，许多其他的激酶和通路同时被激活。因此，我们开发了一种新的系统，将活性AKT1运送到人类细胞，绕过了非特异性刺激的使用。我们以前建立了一种方法，通过程序磷酸化从大肠杆菌中产生AKT1磷酸化变异体。这些AKT1变异体与细胞穿透性多肽标签融合，允许预先激活的AKT1通过细胞自己的蛋白质输入系统直接被细胞摄取。这一新的方法使我们能够在不使用非特异性细胞刺激的情况下研究AKT1生物学。在初步数据中，我们发现预激活的TAT-磷酸AKT1诱导已知的AKT1底物糖原合成酶激酶3a的选择性磷酸化，表明AKT在葡萄糖代谢中具有高度特异性的作用。我们现在将调查这些变化是否反映在代谢组水平上。代谢物是小的化学物质，如糖、葡萄糖，可转化为能量。研究AKT信号反应的代谢组将使我们能够确定AKT激活细胞的化学足迹。我们与世界代谢学专家的合作将揭示AKT如何调节细胞代谢。虽然代谢组学越来越引起人们的兴趣，但加拿大很少有研究人员能够应用这项技术，而且获得所需的质谱学设备的机会也很有限。将代谢组学作为AKT信号研究的另一个维度，是加拿大在努力扩大我们对AKT1生物学知识的努力中的领先地位的一个独特机会。

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.