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...

Circadian molecular oscillation is generated by a transcription/translation-based feedback loop in which CRY proteins play critical roles as potent inhibitors for E-box-dependent clock gene expression. Although CRY2 undergoes rhythmic phosphorylation in its C-terminal tail, structurally distinct from the CRY1 tail, little is understood about how protein kinase(s) controls the CRY2-specific phosphorylation and contributes to the molecular clockwork. Here we found that Ser557 in the C-terminal tail of CRY2 is phosphorylated by DYRK1A as a priming kinase for subsequent GSK-3␤ (glycogen synthase kinase 3␤)-mediated phosphorylation of Ser553, which leads to proteasomal degradation of CRY2. In the mouse liver, DYRK1A kinase activity toward Ser557 of CRY2 showed circadian variation, with its peak in the accumulating phase of CRY2 protein.Knockdown of Dyrk1a caused abnormal accumulation of cytosolic CRY2, advancing the timing of a nuclear increase of CRY2, and shortened the period length of the cellular circadian rhythm. Expression of an S557A/S553A mutant of CRY2 phenocopied the effect of Dyrk1a knockdown in terms of the circadian period length of the cellular clock. DYRK1A is a novel clock component cooperating with GSK-3␤ and governs the Ser557 phosphorylation-triggered degradation of CRY2.

The circadian clock is phase-delayed or -advanced by light when given at early or late subjective night, respectively. Despite the importance of the time-of-day-dependent phase responses to light, the underlying molecular mechanism is poorly understood. Here, we performed a comprehensive analysis of light-inducible genes in the chicken pineal gland, which consists of light-sensitive clock cells representing a prototype of the clock system. Light stimulated expression of 62 genes and 40 ESTs by >2.5-fold, among which genes responsive to the heat shock and endoplasmic reticulum stress as well as their regulatory transcription factors heat shock factor (HSF)1, HSF2, and X-box-binding protein 1 (XBP1) were strongly activated when a light pulse was given at late subjective night. In contrast, the light pulse at early subjective night caused prominent induction of E4bp4, a key regulator in the phase-delaying mechanism of the pineal clock, along with activation of a large group of cholesterol biosynthetic genes that are targets of sterol regulatory element-binding protein (SREBP) transcription factor. We found that the light pulse stimulated proteolytic formation of active SREBP-1 that, in turn, transactivated E4bp4 expression, linking SREBP with the light-input pathway of the pineal clock. As an output of light activation of cholesterol biosynthetic genes, we found light-stimulated pineal production of a neurosteroid, 7α-hydroxypregnenolone, demonstrating a unique endocrine function of the pineal gland. Intracerebroventricular injection of 7α-hydroxypregnenolone activated locomotor activities of chicks. Our study on the genome-wide gene expression analysis revealed time-of-daydependent light activation of signaling pathways and provided molecular connection between gene expression and behavior through neurosteroid release from the pineal gland.