Abstract
Cryptochrome 1 and 2 act as essential components of the central and peripheral circadian clocks for generation of circadian rhythms in mammals. Here we show that mouse cryptochrome 2 (mCRY2) is phosphorylated at Ser-557 in the liver, a well characterized peripheral clock tissue. The Ser-557-phosphorylated form accumulates in the liver during the night in parallel with mCRY2 protein, and the phosphorylated form reaches its maximal level at late night, preceding the peak-time of the protein abundance by approximately 4 h in both light-dark cycle and constant dark conditions. The Ser-557-phosphorylated form of mCRY2 is localized in the nucleus, whereas mCRY2 protein is located in both the cytoplasm and nucleus. Importantly, phosphorylation of mCRY2 at Ser-557 allows subsequent phosphorylation at Ser-553 by glycogen synthase kinase-3beta (GSK-3beta), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3beta at Ser-9, which negatively regulates the kinase activity, GSK-3beta exhibits a circadian rhythm in its activity with a peak from late night to early morning when Ser-557 of mCRY2 is highly phosphorylated. Altogether, the present study demonstrates an important role of sequential phosphorylation at Ser-557/Ser-553 for destabilization of mCRY2 and illustrates a model that the circadian regulation of mCRY2 phosphorylation contributes to rhythmic degradation of mCRY2 protein.
Highlights
The physiology and behavior of living organisms from bacteria to humans show daily fluctuations, and those controlled by autonomous clocks are termed circadian rhythms [1, 2]
MCry1 knock-out and mCry2 knockout mice display accelerated and delayed free-running periodicity, respectively [12]. This observation suggests that mCRY1 and mouse cryptochrome 2 (mCRY2) participate in the clockwork in different ways, and it is intriguing to elucidate the mechanism how each mCRY protein is regulated and how they contribute to the molecular oscillation
It has been suggested that mCRY1 and mCRY2 proteins may be differently regulated by and/or contribute to the clockwork in manners that are distinguishable from each other
Summary
□S The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1S. The negative regulation in turn results in decrease of the protein levels of mCRYs and mPERs and allows the molecular cycle to start again with the activation of the E-box-dependent transcription. Mice lacking both mCry and mCry exhibit arrhythmic behavior immediately after being placed in constant darkness [12], indicating their critical role in generating the circadian rhythm in mammals. Ser-557 phosphorylation of mCRY2 allows subsequent phosphorylation by GSK-3, leading to proteasome-dependent degradation of mCRY2 These results demonstrate an important role of Ser557 phosphorylation as the primary step of clearance of mCRY2 and provide the first evidence for the molecular link between GSK-3 and the clock protein of vertebrates
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