GSK-3 Beta Does Not Stabilize Cryptochrome in the Circadian Clock of Drosophila
GSK-3 Beta Does Not Stabilize Cryptochrome in the Circadian Clock of Drosophila
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DOI：
10.1371/journal.pone.0146571
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发表时间：
2016-01-07
期刊：
影响因子：
3.7
通讯作者：
Peschel, Nicolai
中科院分区：
文献类型：
--
作者：
Fischer, Robin;Helfrich-Foerster, Charlotte;Peschel, Nicolai
隐花色素(CRY)是果蝇生物钟的主要感光器。它通过促进蛋白酶体中时钟蛋白Timless(TIM)的光诱导降解来重置生物钟。在持续的灯光下，由于蒂姆不在，时钟停止，苍蝇变得心律不齐。除TIM降解外，光还能诱导CREY降解。这取决于CRY与几种蛋白质的相互作用，如E3泛素连接酶JetLag(JET)和Ramshackle(BRWD3)。然而，Cay似乎也可以通过与GSK-3βFly同源蛋白--激酶Shaggy(SGG)的相互作用而稳定下来。因此，据报道，在某些背时钟神经元上有SGG过度表达的果蝇在持续的光照下保持节律。我们对CRY、Ramshackle和SGG之间的相互作用很感兴趣，并开始在S2细胞中进行蛋白质相互作用的研究。令我们惊讶的是，我们无法复制这一结果，SGG的过度表达确实稳定了CREY，无论是在S2细胞中，还是在相关的时钟神经元中。新加坡政府的做法恰恰相反。此外，在背部时钟神经元过度表达SGG的果蝇和野生型果蝇一样，变得心律失常。然而，我们可以重现已发表的SGG与TIM的相互作用，因为SGG在外侧时钟神经元的过度表达缩短了它们的自由奔跑周期。我们得出结论：SGG不直接与CRY相互作用，而是与TIM相互作用。此外，我们可以证明，一种非特异性抗体解释了观察到的对哭泣的稳定作用。
Cryptochrome (CRY) is the primary photoreceptor of Drosophila's circadian clock. It resets the circadian clock by promoting light-induced degradation of the clock protein Timeless (TIM) in the proteasome. Under constant light, the clock stops because TIM is absent, and the flies become arrhythmic. In addition to TIM degradation, light also induces CRY degradation. This depends on the interaction of CRY with several proteins such as the E3 ubiquitin ligases Jetlag (JET) and Ramshackle (BRWD3). However, CRY can seemingly also be stabilized by interaction with the kinase Shaggy (SGG), the GSK-3 beta fly orthologue. Consequently, flies with SGG overexpression in certain dorsal clock neurons are reported to remain rhythmic under constant light. We were interested in the interaction between CRY, Ramshackle and SGG and started to perform protein interaction studies in S2 cells. To our surprise, we were not able to replicate the results, that SGG overexpression does stabilize CRY, neither in S2 cells nor in the relevant clock neurons. SGG rather does the contrary. Furthermore, flies with SGG overexpression in the dorsal clock neurons became arrhythmic as did wild-type flies. Nevertheless, we could reproduce the published interaction of SGG with TIM, since flies with SGG overexpression in the lateral clock neurons shortened their free-running period. We conclude that SGG does not directly interact with CRY but rather with TIM. Furthermore we could demonstrate, that an unspecific antibody explains the observed stabilization effects on CRY.