CLOCK phosphorylation

Cycling protein phosphorylation plays a role in the mammalian circadian clock as well. PERI, PER2 and BMAL all show temporal changes in electrophoretic mobility that are eliminated by phosphatase treatment (Lee et al 2001). Although these PER phosphorylations are likely to reflect CKl activity they may not be the only clock-related substrates of this enzyme family. CRYl and CRY2, for instance, can be phosphorylated by CKls in vitro when present in a CRY/PER/CKle complex (Eide et al 2002). Two isoforms of mammalian CLOCK (orthologue of Drosophila CLK) also appear to be phosphorylated, resulting in mobility shifts by Western analysis (Lee et al 2001). The kinase(s) responsible for CLOCK phosphorylation is (are) unknown. 

The results obtained with the model for the mammalian circadian clock provide cues for circadian-rhythm-related sleep disorders in humans [117]. Thus permanent phase shifts in LD conditions could account for (a) the familial advanced sleep phase syndrome (FASPS) associated with PER hypopho-sphorylation [118, 119] and (b) the delayed sleep phase syndrome, which is also related to PER [120]. People affected by FASPS fall asleep around 7 30 p.m. and awake around 4 30 a.m. The duration of sleep is thus normal, but the phase is advanced by several hours. Moreover, the autonomous period measured for circadian rhythms in constant conditions is shorter [121]. The model shows that a decrease in the activity of the kinase responsible for PER phosphorylation is indeed accompanied by a reduction of the circadian period in continuous darkness and by a phase advance upon entrainment of the rhythm by the LD cycle [114]. 

Recent experimental studies have uncovered a direct link between the cell cycle and circadian rhythms. Thus, the circadian clock protein BMALl induces the expression of the gene Weel, which codes for the protein kinase that inactivates through phosphorylation the kinase cdkl that controls the G2/M transition [149]. This link allows the coupling of cell division to the circadian clock and explains how the latter may entrain the cell cycle clock in a variety of cell types. 

Hardin It is phosphorylated, but it is hke Clock in that we see it phosphorylated the entire time. We don t see its phosphorylation state changing with the cycle. As soon as you see it, it is phosphorylated and this doesn t change a lot. 

There is growing evidence that clock proteins are regulated dynamically in both temporal (production and degradation) and spatial (nuclear and cytoplasmic) dimensions. The phosphorylation of mPERl and mPER2 by casein kinase le (CKIe) is known as an important step for the accumulation of negatively active clock proteins (Lowrey et al 2000) as in Drosophila (Kloss et al 1998). 

Garceau NY, Liu Y, Loros JJ, Dunlap JC 1997 Alternative initiation of translation and time-specific phosphorylation yield multiple forms of the essential clock protein FREQUENCY. Cell 89 469-476... 

Liu Y, Loros J, Dunlap JC 2000 Phosphorylation of the Neurospora clock protein FREQUENCY determines its degradation rate and strongly influences the period length of the circadian clock. Proc Natl Acad Sci USA 97 234—239 Loros JJ, Dunlap JC 2001 Genetic and molecular analysis of circadian rhythms in Neurospora. Annu Rev Physiol 63 757—794... 

Yang Y, Cheng P, Zhi G, Liu Y 2001 Identification of a calcium/calmodulin-dependent protein kinase that phosphorylates the Neurospora clock protein FREQUENCY. J Biol Chem 276 41064-41072... 

Reddy AB, Field MD, Maywood ES Hastings MH 2002 Differential resynchronisation of circadian clock gene expression within the suprachiasmatic nuclei of mice subjected to experimental jet-lag . J Neurosci 22 7326—7330 Schurov IL, McNulty S, Best JD, Sloper PJ, Hastings MH 1999 Glutamatergic induction of CREB phosphorylation and Fos expression in primary cultures of the suprachiasmatic hypothalamus in vitro is mediated by co-ordinate activity of NMDA and non-NMDA receptors. J Neuroendocrinol 11 43-51... 

Kloss B, Price JL, Saez L et al 1998 The Drosophila clock gene double-time encodes a protein closely related to human casein kinase Is. Cell 94 97—107 Kloss B, Rothenfluh A, Young MW, Saez L 2001 Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless In the Drosophila clock. Neuron 30 699-706... 

Lee C, Bae K, Edery I 1998 The Drosophila CLOCK protein undergoes daily rhythms in abundance, phosphorylation and interactions with the PER-TIM complex. Neuron 4 857-867... 

To compare the molecular mechanisms of circadian clocks in divergent species, we cloned and analysed circadian mRNA expression of human clock genes in serum shocked fibroblasts. We also developed novel antisera and examined the temporal expression of three PER proteins (hPERl, hPER2, and hPER3). The results showed robust circadian profiles of hPER protein abundance, phosphorylation and degradation. 

Fig. 5). The phosphorylation and proteasome degradation of circadian clock proteins may play an important role in maintaining the circadian clock even in humans. 

FIG. 5. Role of phosphorylation and degradation of clock protein PER in human normal fibroblasts. After the phosphorylation of hPERl by casein kinase, the ubiquitin-proteosome pathway may be involved in its degradation in human cells. 

Camacho F, Cilio M, Guo Y et al 2001 Human casein kinase Idelta phosphorylation of human circadian clock proteins period 1 and 2. FEES Lett 489 159-165 Delaunay F, Thisse C, Marchand O, Laudet V, Thisse B 2000 An inherited functional circadian clock in zebrafish embryos. Science 289 297-300 Dunlap J 1998 Circadian rhythms. An end in the beginning. Science 280 1548-1549 Ebisawa T, Uchiyama M, Kajimura N et al 2001 Association of structural polymorphisms in the human period3 gene with delayed sleep phase syndrome. EMBO Rep 2 342-346 Edery I, Zwiebel LJ, Dembinska ME, Rosbash M 1994 Temporal phosphorylation of the Drosophila period protein. Proc Natl Acad Sci USA 91 2260-2264 Ishida N, Kaneko M, AUada R 1999 Biological clocks. Proc Natl Acad Sci USA 96 8819-8820... 

A genetic screen for novel clock mutations in Drosophila sA to the discovery of the first kinase that plays a role in circadian rhythmicity (Price et al 1998). Doubletime dht) is a member of the casein kinase 1 (CKl) family, and is 86% identical to human casein kinase Is (CKls) within the kinase domain (Kloss et al 1998). Originally, two mutations were isolated that produce short (18 h dht ) and long (27 h dbt ) behavioural rhythms. Since then, many period-altering mutations and loss-of-function alleles have been described. The majority of these mutations reside in the kinase domain of dbt, altering its ability to bind and/or phosphorylate its substrate (Fig. 1). All of the DBT point mutations analysed alter the phosphorylation state and accumulation of PER, indicating that DBT affects... 

Although a substantial body of evidence indicates that the functions of DBT and SGG depend on PER and TIM phosphorylation respectively, these may not be the only substrates in the Drosophila clock. For example, in mammals it was recently found that casein kinase 1 phosphorylates CRY and BMALl (the orthologue of CYC) in addition to PER (see below) (Eide et al 2002). 

In addition to CKle, a second casein kinase 1 orthologue, CK1(5, has been implicated in the mammalian circadian clock. CK15 and CKle both bind and phosphorylate mammalian PER proteins in vitro (Keesler et al 2000, Vielhaber et al 2000, Camacho et al 2001), and are physically associated with PER and CRY in vivo (Lee et al 2001). In tau mutants, PER proteins continue to be phosphorylated in spite of the lowered function measured for CKle in vitro (Lee et al 2001). It has been suggested that the residual phosphorylation might be supplied by PER-associated CK15 in the mutants, and that CKle and CK1(5 have overlapping functions in the mammahan circadian system (Lee et al 2001). 

In the Drosophila circadian clock, three proteins are rhythmically phosphorylated throughout the circadian cycle PER, TIM and dCLK. The electrophoretic mobility of these three proteins all undergo changes during the circadian day by Western analysis. In all cases, phosphatase treatment reduced or eliminated the slower migrating bands, suggesting that the mobility shifts are due to phosphorylation (Edery et al 1994, Zeng et al 1996, Lee et al 1998). While there is evidence that DBT phosphorylates PER and SGG phosphorylates TIM, it is possible that other kinases phosphorylate these proteins as well. TIM, for instance, is phosphorylated by a tyrosine kinase before it is ubiquitinated and... 

Unhke other clock proteins, neither DBT/CKl nor SGG/GSK3 is rhythmically expressed. Their substrates, however, are rhythmically phosphorylated throughout the circadian day. How, then, are the activities of these kinases regulated ... 

Kume K, Zylka MJ, Sriram S et al 1999 mCRYl and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98 193-205 Lee C, Bae K, Edery I 1998 The Drosophila CLOCK protein undergoes daily rhythms in abundance, phosphorylation, and interactions with the PER-TIM complex. Neuron 21 857-867... 

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