Characterization of the role of AKT1 in glucose metabolism

AKT1 在葡萄糖代谢中的作用表征

• 批准号: 580214-2022
• 负责人: Heinemann, IlkaIU
• 金额: $ 1.82万
• 依托单位: University of Western Ontario
• 依托单位国家: 加拿大
• 项目类别: Alliance Grants
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744039
• 关键词: Characterization; role; AKT1; glucose; metabolism

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.

蛋白激酶B（AKT 1）是一种丝氨酸/苏氨酸激酶，在调节细胞存活途径中发挥核心作用。AKT 1在其非磷酸化形式下是无活性的，需要诱导磷酸化来研究AKT 1生物学。研究细胞中AKT 1生物学的典型方法依赖于通过磷酸化激活AKT 1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在于缺乏特异性，其中许多其他激酶和途径同时被激活。因此，我们开发了一种新的系统，将活性AKT 1传递到人类细胞，避免使用非特异性刺激。我们以前建立了一种方法，从大肠杆菌中产生AKT 1磷酸化变体与程序磷酸化。这些AKT 1变体与细胞穿透肽标签融合，允许使用细胞自身的蛋白质输入系统将预活化的AKT 1直接摄取到细胞中。这种新的方法使我们能够调查AKT 1生物学，而不使用非特异性的细胞stimulation.In初步的数据，我们表明，预活化的TAT-磷酸AKT 1诱导已知的AKT 1底物糖原合成酶激酶3a的选择性磷酸化，这表明AKT在葡萄糖代谢中的高度特异性作用。我们现在将研究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。研究响应于AKT信号传导的代谢组将使我们能够定义AKT激活细胞的化学足迹。我们与世界代谢组学专家的合作将揭示AKT如何调节细胞代谢。虽然人们对代谢组学越来越感兴趣，但加拿大很少有研究人员能够应用这种技术，而且所需的质谱设备也很有限。将代谢组学作为AKT信号研究的另一个维度，对于加拿大领导人来说是一个独特的机会，可以扩大我们对AKT 1生物学的了解。