Control of mammalian circadian rhythm by CKIε-regulated proteasome-mediated PER2 degradation
Control of mammalian circadian rhythm by CKIε-regulated proteasome-mediated PER2 degradation
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DOI：
10.1128/mcb.25.7.2795-2807.2005
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发表时间：
2005-04-01
影响因子：
5.3
通讯作者：
Virshup, DM
中科院分区：
文献类型：
--
作者：
Eide, EJ;Woolf, MF;Virshup, DM
哺乳动物的昼夜节律调节蛋白PER1和PER2经历每日的积累循环，随后是磷酸化和降解。尽管据推测这些抑制剂的磷酸化调节的蛋白水解对于生物钟的功能至关重要，但尚未表明抑制这一过程会改变哺乳动物的昼夜节律。我们已经开发了一种基于细胞的PER2降解模型。细胞可渗透的蛋白磷酸酶抑制剂花萼海绵诱癌素A诱导的小鼠PER2（mPER2）过度磷酸化之后，迅速发生泛素化，并由26S蛋白酶体降解。蛋白酶体介导的降解在昼夜节律生物钟中至关重要，因为蛋白酶体抑制剂会导致Rat - 1细胞的昼夜节律周期显著延长。酪蛋白激酶Iε（CKIε）被假定为使PER2易于降解。支持这一观点的是，在同步化的Rat - 1细胞中，抑制CKIε也会导致昼夜节律周期显著延长。抑制CKIε还会减缓细胞中PER2的降解。CKIε介导的PER2磷酸化将泛素连接酶衔接蛋白β - TrCP招募到一个特定位点，并且显性负性β - TrCP阻断mPER2的磷酸化依赖性降解。这些结果为所观察到的哺乳动物PER2的磷酸化 - 降解循环提供了一种生化机制和功能相关性。基于细胞培养的生化分析与基于细胞的节律测量相结合，补充了遗传学研究，以阐明控制哺乳动物生物钟的基本机制。
The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKI epsilon (casein kinase I epsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKI epsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKI epsilon inhibition also slows the degradation of PER2 in cells. CKI epsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.