BREAKTHROUGHS AND VIEWS Circadian Rhythm Biochemistry: From Protein Degradation to Sleep and Mating Norio Ishida,* , † , ‡ ,1 Koyomi Miyazaki,* and Takaomi Sakai* ,2 *Clock Cell Biology, National Institute of Advanced Industrial Science and Technology (AIST), IMCB 6-5, 1-1-1 Higashi, Tsukuba 305-8566, Japan; †Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan; and ‡Center for Interdisciplinary Research, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan Received June 15, 2001 The behavior and physiology of most organisms show circadian, 24-h rhythmicity. Circadian oscillators are thought to be controlled by negative-feedback loops in clock genes expressed in organisms as simple as bac- teria to mammals. Oscillating molecules that control their own expression in a circadian fashion seem to be very important for generating circadian rhythms in most organisms (1). In this minireview, the importance of protein degradation of circadian clock gene products as a molecular mechanism is summarized. We then discuss cell and tissue levels of clocks and the relation- ship between the molecular clock and sleep, and be- tween the molecular clock and mating behavior. PROTEIN DEGRADATION AND CIRCADIAN CLOCK The circadian expression in Drosophila of clock gene products, such as PER and TIM, is thought to be im- portant for driving overt rhythms. The constitutive expression of per by the heat shock (2) or rhodopsin (3) promoters restores rhythmicity of the null allele of per, suggesting that per mRNA cycling may not be required for protein cycling or for locomotor rhythms. Further- more, the constitutive expression of tim mRNA also supports protein cycling and behavioral rhythms in tim mutant flies (4). Sehgal et al. also showed that elimi- nating the oscillations of PER and TIM proteins by overexpression abrogated circadian rhythmicity. These data indicate that the circadian rhythmic expression of PER and TIM proteins is much more important than their rhythmic mRNA expression. Recent positional cloning has revealed that the tau locus (which shorten circadian rhythm) in hamsters is encoded by casein kinase I (5), a homology of the Drosophila clock gene double-time. The double-time gene product phosphorylates PER and causes protein degradation in Drosophila (6). Also in mammals, ca- sein kinase 1 (CK1) phosphorylates PER1, PER2 and PER3, then renders them unstable (7–9). Recent find- ings indicate that the human PER2 site phosphory- lated by CK1 is mutated in familial advanced sleep phase syndrome (10). This syndrome affects individu- als who are “morning larks” in whom a 4-h advance of sleep, temperature, and melatonin rhythms suggests that sleep is under the control of the molecular circa- dian clock. Drosophila TIM is degraded by a photic entrain- ment cue. In cultures, tyrosine phosphorylation- dependent TIM degradation is blocked by inhibitors of proteasome activity (11). These data suggest that the TIM degradation mechanism is involved through the ubiquitin–proteosome pathway. A new clock gene, shaggy/glycogen synthase kinase-3 (GSK-3) will be described soon, indicating a role for TIM phosphorylation (12). The data suggest that shaggy- dependent TIM phosphorylation increases PER/TIM heterodimerization or promotes the nuclear translo- cation of PER/TIM complexes in wild flies. TIM phos- phorylation by shaggy may be different from protein degradation. These data imply that the phosphorylation and protein degradation of clock gene products underlie the mechanism of circadian rhythm generation. The emergence of molecular components of the circadian clock sets the stage for elucidating the biochemical mechanisms in many diverse species from flies to humans. 1 To whom correspondence should be addressed at Clock Cell Bi- ology, National Institute of Advanced Industrial Science and Tech- nology (AIST), IMCB 6-5, 1-1-1 Higashi, Tsukuba 305-8566, Japan. Fax: +81-298-61-6095. E-mail: n.ishida@aist.go.jp. 2 Present address: Institute for Behavioral Sciences, Gunma Uni- versity School of Medicine, 3-39-22, Maebashi, 371-8511, Japan. E-mail: sakait@med.gunma-u.ac.jp. Biochemical and Biophysical Research Communications 286, 1–5 (2001) doi:10.1006/bbrc.2001.5207, available online at http://www.idealibrary.com on 1 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.