Ser-557-phosphorylated mCRY2 Is Degraded upon Synergistic Phosphorylation by Glycogen Synthase Kinase-3β

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 ϳ4 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-3␤ (GSK-3␤), resulting in efficient degradation of mCRY2 by a proteasome pathway. As assessed by phosphorylation of GSK-3␤ at Ser-9, which negatively regulates the kinase activity, GSK-3␤ 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.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). These rhythms are synchronized with (entrained to) environmental time cues such as light, and the rhythms are sustained with a period of ϳ24 h even in the absence of the time cues. In mammals, the suprachiasmatic nucleus in the anterior hypothalamus serves as the central clock of the circadian timing system (3-5). Peripheral tissues throughout the body also have circadian clocks, and both the central and peripheral clocks generate the 24-h rhythm with molecular machinery very similar to each other (6 -8).The molecular mechanism of the circadian oscillator has been investigated extensively by genetic and molecular studies on Drosophila and mice. In the mouse molecular clock, a heterodimer of the two transcription factors, CLOCK and BMAL1, activates E-box-dependent transcription of two cryptochrome genes, mCry1 1 and mCry2, and three period genes, mPer1, mPer2, and mPer3 (9, 10). Translated mCRY and mPER proteins translocate to the nucleus where mCRY proteins act as predominant negative regulators by interacting directly with CLOCK/BMAL1 heterodimer to inhibit the transactivation from the E-box (10, 11). 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. Importantly, mice lacking both mCry1 and mCry2 exhibit arrhythmic behavior immediately after being placed in constant darkness (12), indicating their c...

Role of Phosphorylation in the Mammalian Circadian Clock

“…Whereas Shaggy phosphorylates dTIM in Drosophila, substrates of mammalian GSK3β are CRY2 (Harada et al 2005), Rev-erbα (Yin et al 2006) and PER2 (Iitaka et al 2005). Here, GSK3β-mediated phosphorylation regulates the stability of CRY2 and Rev-erbα as well as the subcellular localization of PER2.…”

A Common Origin: Signaling Similarities in the Regulation of the Circadian Clock and DNA Damage Responses

“…51) GSK-3b induces this phosphorylation only if Ser-557 is also phosphorylated. 51) fects on circadian clock elements, CLOCK targets key components of the cell cycle machinery. It is well established that post-translational modifications are vital for the regulation of the cell cycle and DDR.…”

“…107 Phosphorylation (Ser557) MAPK/ERK (MAPK/ERK phosphorylates Function unknown but phospho-Ser-557-CRY2 is localized the Ser-557 of CRY2 in vitro, but it is specifically in the nucleus and displays robust circadian unlikely to contribute to this variation. 51,122) phosphorylation in vivo. )…”

“…For example, in mammals, glycogen synthase kinase-3b (GSK-3b), the key kinase regulating the Wnt signaling pathway, phosphorylates CRY2 and PER2 and thereby controls CRY2 protein stability and PER2 subcellular localization. 48,51) Another example is the stimulation of Dec1 gene expression that is induced by the TGF-b/activin signaling pathway during alkaline shockinduced entrainment of mammalian peripheral clocks. 49) The signaling pathways involving these mediators play essential roles in development, proliferation, DDR, and cell death processes, suggesting that these processes and the circadian clock are separate nodes of a common regulatory network.…”

Phosphorylation of the Cryptochrome 1 C-terminal Tail Regulates Circadian Period Length

“…Although the mechanism by which CRYs repress CLOCK/BMAL1 activity is not well understood, the PHR domain appears to be sufficient for this function as long as it includes a coiled coil domain near the junction between the PHR and the tail (5-7). The role of the C-terminal tails is more mysterious, although the CRY1 C terminus may have a role in nuclear localization and may facilitate BMAL1 binding (7,8), and phosphorylation of the CRY2 C terminus destabilizes the protein (9,10).…”