The Role of β-TrCP1 and β-TrCP2 in Circadian Rhythm Generation by Mediating Degradation of Clock Protein PER2
The Role of β-TrCP1 and β-TrCP2 in Circadian Rhythm Generation by Mediating Degradation of Clock Protein PER2
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DOI：
10.1093/jb/mvn112
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发表时间：
2008-11-01
影响因子：
2.7
通讯作者：
Ishida, Norio
中科院分区：
文献类型：
--
作者：
Ohsaki, Kanae;Oishi, Katsutaka;Ishida, Norio
哺乳动物的生物钟蛋白经历了一个每天的积累周期，随后是磷酸化和降解。时钟蛋白进行降解的机制尚未完全了解。生物钟蛋白PERIOD2 （PER2）被证明是F-box蛋白β - trcp1（泛素E3连接酶的一个组成部分）的潜在靶标。在这里，我们证明了β - trcp2和β - trcp1在体外靶向PER2蛋白。我们还发现PER2的β - trcp结合位点（m2）同时被β - trcp1和β - trcp2识别。LuciferasePER2融合系统显示m2位点对PER2的稳定性负责。通过实时报告者实验分析β - trcp1和β - trcp2在昼夜节律产生中的作用，发现sirna介导的β - trcp1和/或β - trcp2的抑制可减弱NIH3T3细胞的昼夜节律振荡。然而，β - trcp1缺失的小鼠表现出正常的周期长度、光诱导的行为相移反应和正常的PER2表达，这表明β - trcp1对于视交叉上核的中央时钟是必不可少的。我们的研究表明，β - trcp1和β - trcp2参与了培养细胞中细胞自主昼夜节律的产生，尽管β - trcp2在视交叉上核中央时钟中的作用仍有待阐明。
The mammalian circadian clock proteins undergo a daily cycle of accumulation followed by phosphorylation and degradation. The mechanism by which clock proteins undergo degradation has not been fully understood. Circadian clock protein PERIOD2 (PER2) is shown to be the potential target of F-box protein beta-TrCP1, a component of ubiquitin E3 ligase. Here, we show that beta-TrCP2 as well as beta-TrCP1 target PER2 protein in vitro. We also identified beta-TrCP binding site (m2) of PER2 being recognized by both beta-TrCP1 and beta-TrCP2. LuciferasePER2 fusion system revealed that m2 site was responsible for the stability of PER2. The role of beta-TrCP1 and beta-TrCP2 in circadian rhythm generation was analysed by real-time reporter assay revealing that siRNA-mediated suppressions of beta-TrCP1 and/or beta-TrCP2 attenuate circadian oscillations in NIH3T3 cell. beta-TrCP1-deficient mice, however, showed normal period length, light-induced phase-shift response in behaviour and normal expression of PER2, suggesting that beta-TrCP1 is dispensable for the central clock in the suprachiasmatic nucleus. Our study indicates that beta-TrCP1 and beta-TrCP2 were involved in the cell autonomous circadian rhythm generation in culture cells, although the role of beta-TrCP2 in the central clock in the suprachiasmatic nucleus remains to be elucidated.