Institute for Advanced Biosciences, Keio University, Tsuruoka, Japan.
Systems Biology Program, Graduate School of Media and Governance, Keio University, Fujisawa, Japan.
Genes Cells. 2019 Dec;24(12):768-780. doi: 10.1111/gtc.12726. Epub 2019 Nov 6.
The anhydrobiotic tardigrade, Hypsibius exemplaris, was previously considered to require de novo gene expression and protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activity for successful anhydrobiosis. These indicate that H. exemplaris has signal transduction systems responding to desiccation stress, with the involvement of phosphorylation events. To this end, we carried out time-series phosphoproteomics of H. exemplaris exposed to mild desiccation stress and detected 48 phosphoproteins with significant differential regulations. Among them, immediate and successive reduction of phosphorylation levels of AMP-activated protein kinase (AMPK) was observed. The subsequent chemical genetic approach showed that AMPK was activated during the preconditioning stage for anhydrobiosis, and inhibition of its activity impaired successful anhydrobiosis. As PP2A is known to dephosphorylate AMPK in other organisms, we suggested that decreased phosphorylation levels of AMPK upon mild desiccation stress were caused by dephosphorylation by PP2A. Accordingly, phosphoproteomics of animals pre-treated with the PP1/PP2A inhibitor cantharidic acid (CA) lacked the decrease in phosphorylation levels of AMPK. These observations suggest that AMPK activity is required for successful anhydrobiosis in H. exemplaris, and its phosphorylation state is possibly regulated by PP2A.
先前人们认为，耐旱缓步动物水熊虫（Hypsibius exemplaris）需要从头开始表达基因，并需要蛋白磷酸酶 1（PP1）和蛋白磷酸酶 2A（PP2A）活性才能成功进入耐旱状态。这表明 H. exemplaris 具有响应干燥胁迫的信号转导系统，涉及磷酸化事件。为此，我们对暴露于轻度干燥胁迫下的 H. exemplaris 进行了时间序列磷酸蛋白质组学研究，检测到 48 种具有显著差异调控的磷酸化蛋白。其中，观察到 AMP 激活蛋白激酶（AMPK）的磷酸化水平立即和连续降低。随后的化学遗传学方法表明，AMPK 在进入耐旱状态的预适应阶段被激活，其活性的抑制会损害成功的耐旱状态。由于已知 PP2A 在其他生物体中使 AMPK 去磷酸化，我们推测在轻度干燥胁迫下 AMPK 的磷酸化水平降低是由 PP2A 去磷酸化引起的。因此，用蛋白磷酸酶 1/2A 抑制剂斑蝥素（CA）预处理动物的磷酸蛋白质组学研究中缺乏 AMPK 磷酸化水平的降低。这些观察结果表明，AMPK 活性是 H. exemplaris 成功耐旱的必需条件，其磷酸化状态可能受 PP2A 调控。
