Department of Chemistry and Molecular Biology, University of Gothenburg, Sweden.
FEBS J. 2014 Apr;281(7):1901-17. doi: 10.1111/febs.12753. Epub 2014 Mar 4.
The AMP-activated protein kinase, AMPK, controls energy homeostasis in eukaryotic cells but little is known about the mechanisms governing the dynamics of its activation/deactivation. The yeast AMPK, SNF1, is activated in response to glucose depletion and mediates glucose de-repression by inactivating the transcriptional repressor Mig1. Here we show that overexpression of the Snf1-activating kinase Sak1 results, in the presence of glucose, in constitutive Snf1 activation without alleviating glucose repression. Co-overexpression of the regulatory subunit Reg1 of the Glc-Reg1 phosphatase complex partly restores glucose regulation of Snf1. We generated a set of 24 kinetic mathematical models based on dynamic data of Snf1 pathway activation and deactivation. The models that reproduced our experimental observations best featured (a) glucose regulation of both Snf1 phosphorylation and dephosphorylation, (b) determination of the Mig1 phosphorylation status in the absence of glucose by Snf1 activity only and (c) a regulatory step directing active Snf1 to Mig1 under glucose limitation. Hence it appears that glucose de-repression via Snf1-Mig1 is regulated by glucose via at least two independent steps: the control of activation of the Snf1 kinase and directing active Snf1 to inactivating its target Mig1.
AMP 激活的蛋白激酶（AMPK）控制真核细胞的能量稳态，但对于其激活/失活动力学的调控机制知之甚少。酵母 AMPK，即 SNF1，会对葡萄糖耗尽做出响应而被激活，并通过使转录阻遏物 Mig1 失活来介导葡萄糖去阻遏。在这里，我们发现过量表达 Snf1 激活激酶 Sak1 会导致在有葡萄糖的情况下，Snf1 持续激活，而不会减轻葡萄糖的抑制作用。Glc-Reg1 磷酸酶复合物的调节亚基 Reg1 的共过表达部分恢复了 Snf1 的葡萄糖调控。我们生成了一组基于 Snf1 途径激活和失活的动态数据的 24 个动力学数学模型。最能再现我们实验观察结果的模型具有以下特点：(a) 葡萄糖对 Snf1 磷酸化和去磷酸化的调控；(b) 在没有葡萄糖的情况下，Snf1 活性决定 Mig1 的磷酸化状态；(c) 在葡萄糖限制下，将活性 Snf1 导向 Mig1 的调节步骤。因此，似乎通过 Snf1-Mig1 的葡萄糖去阻遏是通过至少两个独立的步骤来调节的：Snf1 激酶的激活控制和将活性 Snf1 导向其靶标 Mig1 失活。
