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

Yuko Harada, Mihoko Sakai, Nobuhiro Kurabayashi, Tsuyoshi Hirota, Yoshitaka Fukada
2005 Journal of Biological Chemistry  
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
more » ... f 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 critical role in generating the circadian rhythm in mammals. In addition to the transcriptional regulation of the clockrelated genes, protein phosphorylation plays important roles for regulation of the phase and period length of the molecular clock (13-21). The clock proteins including CLOCK, BMAL1, mPER1, and mPER2 undergo temporal change in phosphorylation in the mouse liver (22), a well characterized peripheral tissue that contains the circadian clock. It was unclear as to whether mCRYs are phosphorylated in vivo. Mammalian PERs are phosphorylated by casein kinase I⑀ (CKI⑀), and the phosphorylation reduces the stability of PER (23). A defect in hamster CKI⑀ corresponds to the short period tau mutation (24). Similarly, PER in Drosophila is phosphorylated and destabilized by Doubletime (DBT), a Drosophila homolog of CKI⑀, and the mutant alleles of dbt result in alteration of the period length of the circadian rhythm (14, 17) . Mutant alleles of Shaggy (SGG)/glycogen synthase kinase-3 (GSK-3) also affect the period length of molecular oscillation of the clock in Drosophila (25), and SGG phosphorylates TIM (25), an essential clock component serving as a binding partner of PER (13). Interestingly, mammalian GSK-3 has been related to circadian clockwork in mice (26), but its target protein(s) yet remains to be elucidated. In every case no information is available about the in vivo phosphorylation site(s) in these clock proteins. We have recently found that mCRY2 is phosphorylated at Ser-265 and Ser-557 by mitogen-activated protein kinase (MAPK) in vitro (27). Likewise, Ser-247 of mCRY1, which corresponds to Ser-265 of mCRY2, is phosphorylated by MAPK in vitro (27) , whereas mCRY1 does not have the Ser or Thr resi-
doi:10.1074/jbc.m506225200 pmid:15980066 fatcat:r4bxwnuas5bdnfqynx4ytepuvm