Wu Cheng-Wei, Bell Ryan A, Storey Kenneth B
Institute of Biochemistry, Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada.
Institute of Biochemistry, Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON K1S 5B6, Canada.
Biochim Biophys Acta. 2015 Nov;1850(11):2196-202. doi: 10.1016/j.bbagen.2015.07.004. Epub 2015 Jul 17.
The insulin signaling pathway functions as a major regulator of many metabolic and cellular functions, and has been shown to be reversibly suppressed in many species during hibernation. This study characterized the regulation of PTEN phosphatase, a negative regulator of the insulin receptor network, over the torpor-arousal cycle of hibernation in the skeletal muscle of Ictidomys tridecemlineatus.
Western blotting and RT-PCR were used to analyze post-translational and transcriptional regulations of PTEN respectively. Enzymatic activities were determined by the malachite green assay, while protein stability was assessed the using pulse-proteolysis method.
During torpor, the ratio of non-phosphorylated PTEN (S380/T382/T383) was significantly elevated by 1.4-fold during late torpor compared with euthermic controls; this was coupled with an increase in substrate affinity for PIP3 (by 56%) in late torpor. Two proteolytic cleavage PEST motifs were identified in the C-terminus that overlapped with the phosphorylation sites of PTEN; pulse-proteolysis analysis of PTEN protein showed a decrease in protein stability during late torpor (Cm of urea decreased by 21%). Furthermore, the increase in PTEN activity observed was correlated with a decrease in PDK-1 phosphorylation by 32%, suggesting a downstream effect of PTEN activation during torpor. Transcriptional analysis showed that mRNA expression of pten and pdk-1 remain unchanged during hibernation, suggesting post-translation modification as the primary regulatory mechanism of PTEN function.
Phosphorylation plays an important role in the regulation of PTEN enzymatic activity and protein stability.
Activation of PTEN during torpor can regulate insulin signaling during periods of low energy state.
胰岛素信号通路是许多代谢和细胞功能的主要调节因子，并且已表明在许多物种冬眠期间该通路会受到可逆性抑制。本研究对十三条纹地松鼠骨骼肌在冬眠的蛰伏-觉醒周期中，胰岛素受体网络的负调节因子PTEN磷酸酶的调节作用进行了表征。
分别使用蛋白质免疫印迹法和逆转录-聚合酶链反应分析PTEN的翻译后调节和转录调节。通过孔雀绿测定法测定酶活性，同时使用脉冲蛋白水解法评估蛋白质稳定性。
在蛰伏期间，与正常体温对照组相比，非磷酸化PTEN（S380/T382/T383）的比例在蛰伏后期显著升高1.4倍；这与蛰伏后期对PIP3的底物亲和力增加（56%）相关。在C末端鉴定出两个与PTEN磷酸化位点重叠的蛋白水解切割PEST基序；PTEN蛋白的脉冲蛋白水解分析显示，蛰伏后期蛋白质稳定性降低（尿素Cm降低21%）。此外，观察到的PTEN活性增加与PDK-1磷酸化降低32%相关，表明蛰伏期间PTEN激活的下游效应。转录分析表明，冬眠期间pten和pdk-1的mRNA表达保持不变，表明翻译后修饰是PTEN功能的主要调节机制。
磷酸化在PTEN酶活性和蛋白质稳定性的调节中起重要作用。
蛰伏期间PTEN的激活可在低能量状态期间调节胰岛素信号传导。
