The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKI (casein kinase I) has been postulated to prime PER2 for degradation. Supporting this idea, CKI inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKI inhibition also slows the degradation of PER2 in cells. CKI-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein ␤-TrCP to a specific site, and dominant negative ␤-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.Diverse organisms from prokaryotes to mammals coordinate behavioral and physiological rhythms with the daily dark-light cycle by means of a circadian clock. In mammals, the master circadian clock is located in the suprachiasmatic nucleus of the brain, and it entrains peripheral cell-autonomous clocks throughout the body. In mice, a positively acting heterodimeric transcription factor composed of the PAS-bHLH proteins CLOCK (CLK) and BMAL1 drives transcription of tissuespecific circadian output genes, as well as its own negative regulators, the Period (denoted mPer1, mPer2, and mPer3), and Cryptochrome (mCry1 and mCry2) genes. The mammalian PER and CRY proteins form multimeric complexes that enter the nucleus and repress the transcriptional activity of CLK/ BMAL1, modulating circadian output (reviewed in references 29 and 41). Additional stabilizing feedback loops, including inhibition of Bmal1 transcription by REV-ERB␣ (37), further contribute to the timing and robustness of the cycle. The daily rhythmic degradation of PERIOD proteins leading to derepression of CLK/BMAL1 is postulated to be critical to the proper functioning of the clock. Therefore, the mechanism and control of this process are of great interest.Genetic studies have identified CKIε (casein kinase Iε) as a key regulator of metazoan circadian rhythm and both genetic and biochemical studies suggest that the PER proteins are important substrates (reviewed in reference 10). CKIε was first implicated as a circadian regulator in Drosop...

Synchronization between the environmental lighting cycle and the biological clock in the suprachiasmatic nucleus (SCN) is correlated with phosphorylation of the Ca 2ϩ /cAMP response element binding protein (CREB) at the transcriptional activating site Ser 133 . Mechanisms mediating the formation of phospho-CREB (P-CREB) and their relation to clock resetting are unknown. To address these issues, we probed the signaling pathway between light and P-CREB. Nocturnal light rapidly and transiently induced P-CREB-like immunoreactivity (P-CREB-lir) in the rat SCN. Glutamate (Glu) or nitric oxide (NO) donor administration in vitro also induced P-CREB-lir in SCN neurons only during subjective night. Clock-controlled sensitivity to phase resetting by light, Glu, and NO is similarly restricted to subjective night. The effects of NMDA and nitric oxide synthase (NOS) antagonists on Glu-mediated induction of P-CREB-lir paralleled their inhibition of phase shifting. Significantly, among neurons in which P-CREB-lir was induced by light were NADPH-diaphorase-positive neurons of the SCN's retinorecipient area. Glu treatment increased the intensity of a 43 kDa band recognized by anti-P-CREB antibodies in subjective night but not day, whereas anti-␣CREB-lir of this band remained constant between night and day. Inhibition of NOS during Glu stimulation diminished the anti-P-CREB-lir of this 43 kDa band. Together, these data couple nocturnal light, Glu, NMDA receptor activation and NO signaling to CREB phosphorylation in the transduction of brief environmental light stimulation of the retina into molecular changes in the SCN resulting in phase resetting of the biological clock.

Circadian rhythms in Drosophila melanogaster depend on a molecular feedback loop generated by oscillating products of the period (per) and timeless (tim) genes. In mammals, three per homologs are cyclically expressed in the suprachiasmatic nucleus (SCN), site of the circadian clock, and two of these, mPer1 and mPer2, are induced in response to light. Although this light response distinguishes the mammalian clock from its Drosophila counterpart, overall regulation, including homologous transcriptional activators, appears to be similar. Thus, the basic mechanisms used to generate circadian timing have been conserved. However, contrary to expectations, the recently isolated mammalian tim homolog was reported not to cycle. In this study, we examined mRNA levels of the same tim homolog using a different probe. We observed a significant (approximately threefold) diurnal variation in mTim expression within mouse SCN using two independent methods. Peak levels were evident at the day-to-night transition in light-entrained animals, and the oscillation persisted on the second day in constant conditions. Furthermore, light pulses known to induce phase delays caused significant elevation in mTim mRNA. In contrast, phase-advancing light pulses did not affect mTim levels. The mTim expression profile and the response to nocturnal light are similar to mPer2 and are delayed compared with mPer1. We conclude that temporal ordering of mTim and mPer2 parallels that of their fly homologs. We predict that mTIM may be the preferred functional partner for mPER2 and that expression of mTim and mPer2 may, in fact, be driven by mPER1.

Behavioral and electrophysiological evidence indicates that the biological clock in the hypothalamic suprachiasmatic nuclei (SCN) can be reset at night through release of glutamate from the retinohypothalamic tract and subsequent activation of nitric oxide synthase (NOS). However, previous studies using NADPH‐diaphorase staining or immunocytochemistry to localize NOS found either no or only a few positive cells in the SCN. By monitoring conversion of l‐[3H]arginine to l‐[3H]citrulline, this study demonstrates that extracts of SCN tissue exhibit NOS specific activity comparable to that of rat cerebellum. The enzymatic reaction requires the presence of NADPH and is Ca2+/calmodulin‐dependent. To distinguish the neuronal isoform (nNOS; type I) from the endothelial isoform (type III), the enzyme activity was assayed over a range of pH values. The optimal pH for the reaction was 6.7, a characteristic value for nNOS. No difference in nNOS levels was seen between SCN collected in day versus night, either by western blot or by enzyme activity measurement. Confocal microscopy revealed for the first time a dense plexus of cell processes stained for nNOS. These data demonstrate that neuronal fibers within the rat SCN express abundant nNOS and that the level of the enzyme does not vary temporally. The distribution and quantity of nNOS support a prominent regulatory role for this nitrergic component in the SCN.

No abstract