Abstract
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.
Highlights
The circadian clock is an ancient mechanism in almost every living being on this planet, including bacteria, plants and animals [1]
Co-expression of SGG in S2 cells was reported to result in a strong stabilization of CRY–even under constant light (LL) conditions [21], whereas JET makes CRY less stable, when the two proteins are co-expressed under the same conditions in LL [13]
The Glycogen Synthase-kinase 3 beta plays an important role in regulating the circadian clock of different animals, like mice, humans or Drosophila
Summary
The circadian clock is an ancient mechanism in almost every living being on this planet, including bacteria, plants and animals [1]. This sophisticated evolved machinery allows all organisms, to adapt to the daily 24 hours environmental changes on this planet–caused by the earth’s rotation round its own axis [2]. On the molecular level the clock consists of a transcriptionally regulated negative feedback loop. In Drosophila, the two clock proteins Period (PER) and Timeless (TIM) inhibit the transcription factors Clock (CLK) and Cycle (CYC). CYC and CLK act as transcriptional activators of per and tim. The negative feedback loop runs in about 150 neurons inside Drosophila’s brain [3].
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