Abstract
Circadian rhythms, endogenous cycles of about 24 h in physiology, are generated by a master clock located in the suprachiasmatic nucleus of the hypothalamus and other clocks located in the brain and peripheral tissues. Circadian disruption is known to increase the incidence of various illnesses, such as mental disorders, metabolic syndrome, and cancer. At the molecular level, periodicity is established by a set of clock genes via autoregulatory translation–transcription feedback loops. This clock mechanism is regulated by post-translational modifications such as phosphorylation and ubiquitination, which set the pace of the clock. Ubiquitination in particular has been found to regulate the stability of core clock components but also other clock protein functions. Mutation of genes encoding ubiquitin ligases can cause either elongation or shortening of the endogenous circadian period. Recent research has also started to uncover roles for deubiquitination in the molecular clockwork. Here, we review the role of the ubiquitin pathway in regulating the circadian clock and we propose that ubiquitination is a key element in a clock protein modification code that orchestrates clock mechanisms and circadian behavior over the daily cycle.
INTRODUCTION: THE MOLECULAR CIRCADIAN CLOCK
Circadian rhythms are endogenous ∼24 h cycles in physiology and behavior generated by a master clock in the suprachiasmatic nucleus of the hypothalamus, and clocks located in most other tissues. Circadian clocks enable organisms to anticipate predictable daily occurrences, such as changes in light, temperature, or food availability (). The importance of circadian clocks is illustrated by the impacts of circadian disruption in humans. For example, shift work increases the risk of developing various illnesses, such as mental disorders, metabolic syndrome, and cancer ().
At the molecular level, the circadian clock relies on self-sustained transcription-translation feedback loops involving “clock genes” (Figure 1; ). In mammals, CLOCK and BMAL1 dimerize and activate Period (Per) 1 and 2 and Cryptochrome (Cry) 1 and 2 genes. The PER1/2 and CRY1/2 proteins then enter the nucleus and inhibit the activity of CLOCK/BMAL1, thereby repressing their own transcription. However, the mechanism is more complex, with additional interlocking feedback loops, including one that involves the induction of the Rev-erb and Ror genes, whose protein products regulate Bmal1 gene transcription. One consequence of these feedback loops is that the mRNAs and proteins of many clock genes present circadian rhythms in their abundance. Moreover, hundreds of clock-controlled genes, which do not participate in the clock mechanism, but whose transcription is under the control of the clock molecular machinery, also present rhythms at the RNA and protein levels (; ), thus linking the circadian clock with cellular physiology.
FIGURE 1
The timing of these feedback loops is dictated by post-translational modifications (PTMs; ; ). Indeed, clock proteins are subject to phosphorylation, ubiquitination, acetylation, SUMOylation, and other PTMs (Figure 1). Ubiquitination is of particular interest due to the diversity of signals that it can generate. In particular, its direct role in determining protein half-life is crucial for proteins with a daily rhythm in abundance. In this article, we review the current state of knowledge on ubiquitination of clock proteins and their ubiquitin-modifying enzymes in animal models, with a special focus on the mammalian clock (Table 1).
Table 1
| Clock protein | Mammalian enzyme | Phenotype of mice, tissues, or cells upon loss-of-function mutation or knock-down of ubiquitin-modifying enzyme | Drosophila homolog | References |
|---|---|---|---|---|
| Ubiquitin ligases | ||||
| CRY1/2 | FBXL3 | Long free-running period of locomotor activity rhythms, cultured fibroblasts and SCN; stabilized CRYs; dampened and delayed rhythms of Per and Cry mRNAs; phenotype partly rescued by Fbxl21 loss-of-function. | , , , | |
| FBXL21 | Normal or short free-running period of locomotor activity rhythms; short period of cultured fibroblasts, SCN and pituitary; destabilized CRYs (when subcellular fractions were studied, the mutation stabilized CRY1 in cytoplasm and destabilized it in the nucleus); mutation partly rescues Fbxl3 loss-of-function. | , , | ||
| PER1/2 | β-TRCP1 (FBW1A), β-TRCP2 (FBW1B) | Dampened or long-period rhythms in fibroblasts; stabilized PERs; β-Trcp1 KO mice have no circadian-related phenotype. | SLIMB | , , , |
| REV-ERBα | HUWE1 (ARF-BP1) | Stabilized REV-ERBα, decreased Bmal1/Cry1 expression (knock-down of both HUWE1 and PAM together). | CG8184 | |
| PAM (MYCBP2) | Stabilized REV-ERBα, decreased Bmal1/Cry1 expression (knock-down of both HUWE1 and PAM together). | Highwire | ||
| FBXL3? | Long-period phenotype of Fbxl3 mutants rescued by Rev-erbα KO; dampened but more sustained REV-ERBα levels in Fbxl3 mutants, and prolonged REV-ERBα transcriptional activity. | |||
| BMAL1 | UBE3A | Dampening and longer period of circadian rhythms in cultured fibroblasts. | dUBE3A | |
| Deubiquitinating enzymes | ||||
| CRY1 | USP2 | Decreased CRY1 protein levels in liver (with Usp2 knock-down). | ||
| PER1 | USP2 | Slightly elongated free-running period of locomotor activity rhythms; altered response to light; altered clock gene expression and increased levels of ubiquitinated PER1 in fibroblasts; no change in PER1 stability; alteration in the timing of PER1 intracellular localization. | , | |
| BMAL1 | USP2 | Normal free-running period and slightly altered light response; reduced BMAL1 levels in the SCN. | , | |
Ubiquitin-modifying enzymes involved in the regulation of mammalian clock proteins.
UBIQUITINATION IN THE CIRCADIAN CLOCK
UBIQUITINATION OF CRYPTOCHROMES BY FBXL UBIQUITIN LIGASES
N-Ethyl-N-nitrosourea screens led to the discovery of mice exhibiting free-running periods of locomotor activity rhythms ∼2–3 h longer than normal (; ). These mice had loss-of-function mutations in the gene encoding the F-box protein FBXL3. Loss of FBXL3 activity leads to CRY protein stabilization due to decreased ubiquitination (). Further work on FBXL3 revealed that ubiquitination of CRY1/2 by the FBXL3-containing SCF E3 ubiquitin ligase complex was necessary for the timely degradation of the CRY proteins and the reactivation of BMAL1/CLOCK (). A prolonged inhibition of BMAL1/CLOCK-mediated transcription in the mutant mice leads to reduced peak levels and delayed rhythms of the Per and Cry mRNAs in mutant mouse SCN, cerebellum, and liver (; ).
Interestingly, FBXL3 cannot undergo SCF complex formation in the absence of its CRY substrates (). X-ray crystallography revealed that FBXL3 binds to the FAD-binding pocket of mammalian CRY, which may also be bound by FAD or PER proteins (; ), which suggests a mechanism for the protection of CRYs from degradation in the presence of PER ().
An FBXL3 paralog, FBXL21, was identified in sheep, where it was also found to bind to CRY1, thereby affecting transcriptional activation by CLOCK/BMAL1 (). Despite the high similarity between FBXL3 and FBXL21, they appear to have non-redundant roles within the clock. Indeed, while Fbxl3 gene mutant or knock-out (KO) mice display a long free-running period of locomotor activity rhythms, Fbxl21-mutant or KO mice present either a short () or a normal () period. Moreover, when the mutant lines are crossed, the Fbxl21 mutation attenuates the long-period phenotype of Fbxl3-mutant mice.
The distinct roles of the FBXL proteins may be based on the timing of their expression and that of their substrates. While Fbxl3 is expressed at constant levels over the day, Fbxl21 expression has a pronounced circadian rhythm in the mouse SCN, with a peak by the end of the subjective day (), thus restricting its action to only part of the cycle. Interestingly, while FBXL3 protein levels do not vary over time, its action on CRYs is conditional on their phosphorylation by AMPK, whose expression and nuclear abundance vary over the day (). Ligase intracellular localization also plays a role: while FBXL3 protein is restricted to the nucleus, FBXL21 is located both in the nucleus and cytoplasm (; ). The work of both laboratories supports a two-step mode of action of FBXL21. First, FBXL21 allows CRYs to accumulate in the cytoplasm. This occurs when CRY levels rise around the end of the day or beginning of the night. Shortly thereafter, after CRYs have entered the nucleus, FBXL21 might counteract FBXL3: FBXL21 binds CRYs more stably and with a higher affinity than FBXL3, suggesting that FBXL21 may in part stabilize CRYs by preventing FBXL3 binding (). Then, when FBXL21 levels have decreased, FBXL3 can finally act on CRYs and target them to degradation. It is interesting to note that if this model is further confirmed, the roles of FBXL21 in the clock will turn out to be partly non-degradative (regulation of nuclear entry, protection from the action of another F-box protein, FBXL3), in contrast to other ubiquitin ligases involved in the clock, which target clock proteins to proteasomal degradation. Finally, CRY ubiquitination mechanisms might be even more complex, as it was suggested that another ubiquitin ligase might be involved in regulating CRY accumulation (; ).
UBIQUITINATION OF PERIOD PROTEINS BY β-TRCP UBIQUITIN LIGASES
In Drosophila, the F-box component of an SCF ligase, SLIMB, was shown to be critical for ubiquitination and degradation of PER protein over the course of the circadian cycle (; ). SLIMB binds to PER after its phosphorylation by Doubletime (DBT), in particular on serine 47 within the SLIMB recognition site ().
SLIMB has two homologs in mammals, β-TRCP1/FBW1A and β-TRCP2/FBW1B. Similarly to the action of DBT and SLIMB on PER in the fly, β-TRCP1/2 are recruited to PER2 following phosphorylation of this protein by the kinases CK1δ and CK1ε (mammalian homologs of DBT), which leads to polyubiquitination and subsequent degradation of PER2 (). Indeed, expression of a dominant negative form of β-TRCP leads to the inhibition of PER2 ubiquitination and degradation. β-TRCP1/2 also interact with PER1 in a CK1ε-dependent manner and a knockdown of both β-TRCPs was found to stabilize PER1, and reduce levels of transcriptional activation by CLOCK/BMAL1 (). Accordingly, preventing the action of β-TRCP1/2 on PER proteins leads to long-period or dampened circadian rhythms in cultured fibroblasts (; ). Surprisingly though, mice lacking β-TRCP1 neither show alteration in circadian locomotor behavior nor differences in SCN PER2 levels when compared to WT controls, suggesting either that the SCN clock behaves differently from clocks in fibroblasts or that there is redundancy at the level of the ubiquitin ligases (). Finally, similarly to PER proteins protecting CRYs (see Ubiquitination of Cryptochromes by FBXL Ubiquitin Ligases), PER proteins are protected from ubiquitination and degradation upon association with CRYs ().
UBIQUITINATION OF REV-ERBα
The stability of REV-ERBα is also regulated by a sequence of phosphorylation, ubiquitination, and proteasomal degradation. Indeed, REV-ERBα is stabilized following phosphorylation by GSK3β (). Treating cells with lithium, a GSK3β inhibitor, leads to the quick degradation of REV-ERBα and therefore, to increased expression of Bmal1 (Figure 1). Subsequent work identified HUWE1/ARF-BP1 and PAM/MYCBP2 as E3 ligases involved in this lithium-induced REV-ERBα degradation (). Their depletion in cells stabilized REV-ERBα, decreased Bmal1 gene expression, and disrupted oscillations of other clock genes. HUWE1 and PAM may not be the only ubiquitin ligases acting on REV-ERBα. In Fbxl3-mutant mice, REV-ERBα levels are higher and consequently its repression of Bmal1 and Cry1 genes is enhanced. Creation of double-mutant Fbxl3/Rev-erbα-/- mice rescues the Fbxl3-mutant phenotype () indicating that FBXL3, in addition to its role on CLOCK/BMAL1-mediated transcription via destabilization of CRYs, also has an effect on REV-ERBα-mediated repression of target genes. Although this effect may be indirect, it does indicate a role for this F-box protein as a coordinator of different clock transcription factors.
UBIQUITINATION OF BMAL1
Many studies have indicated a tight regulation of BMAL1 stability. Indeed, BMAL1 undergoes different phosphorylation events that either target it for ubiquitination and degradation (e.g., GSK3β, ) or on the contrary for deubiquitination and stabilization (e.g., PKCγ, ). Importantly, BMAL1 ubiquitination and proteasome-mediated proteolysis appear to coincide with the time of highest transcriptional activity (; ; ), whereas in conditions where CLOCK/BMAL1 activity is repressed (e.g., presence of CRYs), BMAL1 is stabilized (; ). However, no BMAL1-specific ubiquitin ligase had been uncovered until a recent report, which described UBE3A as an E3 ligase that binds and destabilizes BMAL1 (). Knockdown of this ligase in mammalian cells and in Drosophila clock neurons leads to a strong dampening of circadian oscillations or even arrhythmicity.
SUMOylation IN THE CIRCADIAN CLOCK
The small ubiquitin-related modifier (SUMO) proteins also play a role in the clock. Like other PTMs, SUMOylation is reversible and the conjugation/deconjugation mechanisms are reminiscent of the ubiquitin pathway (). In contrast to ubiquitination though, SUMOylation does not directly target proteins for degradation but rather regulates other functions such as nuclear localization, protein–protein interactions, transcriptional activity and, interestingly, ubiquitination itself (; ).
SUMOylation was first implicated in the clock following the discovery of a SUMOylation consensus motif in BMAL1 (). Co-expression of BMAL1 and SUMO showed that BMAL1 could indeed be SUMOylated. In the liver, this occurs in a rhythmic manner, with peak SUMOylation in the second half of the light phase. This timing coincides with peak BMAL1 phosphorylation and activity, suggesting an interplay between these PTMs. In further support of this, a functional CLOCK protein is required for both BMAL1 SUMOylation and phosphorylation (; ; ). SUMOylated BMAL1 is most abundant when the CLOCK/BMAL1 targets Dbp and Rev-erbα show their highest mRNA levels, again supporting that SUMOylation of BMAL1 is involved in its transcriptional activity (). Indeed, BMAL1 binding to the Dbp promoter was reduced when the lysine required for SUMOylation was mutated (). Interestingly, SUMOylation of BMAL1 is a prerequisite for its subsequent ubiquitination, again highlighting the interplay of different PTMs in the circadian clock.
DEUBIQUITINATION IN THE CIRCADIAN CLOCK
Given the importance of ubiquitination within the clock, it appears reasonable to assume that deubiquitination plays a role as well. Interestingly, the mRNA levels of a deubiquitinating enzyme (DUB), ubiquitin-specific protease 2 (USP2), show rhythmicity in most tissues examined (; ; ). This is notable, because among the hundreds of clock-controlled transcripts, only a small minority cycles in multiple locations. The circadian rhythm of Usp2 is blunted in Clock mutant and Bmal1 KO mice (; ), and the Usp2 promoter is activated by CLOCK/BMAL1 (), indicating that Usp2 is a direct target of these transcription factors. In addition to its circadian regulation, Usp2 expression is also induced by starvation and it was therefore proposed that USP2 integrates nutritional and circadian timing cues (). In turn, liver USP2 appears to be involved in the generation of a diurnal rhythm in glucose metabolism ().
However, the circadian role of USP2 is not limited to mediating the rhythmic control of cellular processes by the molecular clock. Since the short list of genes rhythmic in multiple tissues is enriched for clock components, USP2 was hypothesized to exert a role within the clock mechanism. To address this, Usp2 KO mice were generated by two laboratories. In one case, they revealed no alteration of the free-running period of locomotor rhythms (). In contrast, our Usp2 KOs display a period longer than WT littermates (), implying a role within the clockwork. In line with this, the absence of USP2 affects the mRNA levels of several clock genes (; , ), and USP2 interacts with clock proteins. In our hands, whereas it forms a complex with several clock proteins, USP2 directly binds only to PER1 (). Accordingly, PER1 is deubiquitinated in the presence of USP2, but notably, this does not lead to PER1 stabilization. Instead, USP2 appears to regulate PER1 intracellular localization (). Interestingly, the only other DUB that to our knowledge has been implicated in clock mechanisms, Drosophila USP8, also seems to act in a non-degradative manner: it deubiquitinates CLOCK, thereby inhibiting transcriptional activity of CLOCK/CYCLE (CYCLE is the Drosophila homolog of BMAL1; ).
In contrast, the work of other groups showed a stabilization of other clock proteins due to deubiquitination by USP2. BMAL1 levels are lower in the SCN of Usp2 KO mice (), whereas in cultured cells, USP2 stabilized BMAL1 () and reduced its ubiquitination (). Interestingly, a report suggested the involvement of PKCγ-triggered deubiquitination of BMAL1 in the resetting of peripheral clocks by feeding schedules, but the DUB involved in this pathway remains unknown (). In addition to PER1 and BMAL1, USP2 deubiquitinates CRY1 in cultured cells in response to a serum shock, and in the mouse liver, Usp2 knockdown increases CRY ubiquitination and decreases CRY1 protein levels ().
Data also support a role for USP2 in the response of the clock to external cues. We found that Usp2 KO mice exhibit larger phase delays than WT mice after light treatment in the first part of the night, and reduced phase advances, upon light treatment later in the night (). Thus, USP2 appears to be involved in the response of the SCN clock to light, which is also supported by data of , which show increased phase-shifting in response to low irradiance light in the early night. USP2 may also mediate the response of the clock to inflammation, as the expression of the gene is increased in response to TNFα treatment, and CRY1 protein induction in response to this cytokine is abrogated when Usp2 expression is knocked down ().
Together, these studies ascribe a pivotal role to USP2, and deubiquitination in general, not only in the circadian clock mechanism, but also as an integrator of environmental and physiological signals, and in output pathways linking the molecular clockwork to cellular and physiological functions.
A CLOCK PROTEIN MODIFICATION CODE?
Overall, the work described above underscores the importance of PTMs within the circadian timing mechanism. Given that different modifications often converge on the same clock protein, we propose the existence of a clock protein modification code whereby the fate/function of a given protein is determined by the precise combination and/or the consecutive occurrence of different PTMs. This clock protein modification code is proposed to exist at different levels:
- 1.
Interplay of different PTMs: PTMs often occur sequentially. In particular, there are numerous examples of phosphorylation at specific sites being a pre-requisite for subsequent ubiquitination of the target protein (), as occurs in many clock proteins. Another example of sequential modification is the SUMOylation of BMAL1 as a pre-requisite for its ubiquitination (). Moreover, different combinations of PTMs on a protein can lead to distinct outcomes. For example, dual SUMOylation and ubiquitination of BMAL1 result in the BMAL1 localization to the nuclear bodies and active transcription (), whereas later in the circadian cycle, additional events, possibly including further ubiquitination, lead to degradation of the protein. Combinations of different phosphorylation events can also regulate protein fate differentially: for example, in Drosophila, PER phosphorylation by DBT is modulated by prior action of another kinase, NEMO, and consequently, these kinases have opposing effects on PER stability ().
- 2.
Ubiquitin code: There is a large diversity in the ubiquitination of proteins (): they can be mono- or polyubiquitinated; in the latter case, ubiquitin chains can be linear or branched, and the linkages between ubiquitin monomers can be via different lysines. These different ubiquitination states can be generated by various ligases/conjugating enzymes and DUBs. This ubiquitin code can be read by proteins containing ubiquitin-binding domains (UBDs). The effects of ubiquitination can therefore be diverse depending on the type of modifications and the presence of particular UBD-containing proteins: not only targeting to the proteasome, but also regulation of intracellular localization, activity, protein–protein interaction, etc. (). As non-degradative functions of clock protein ubiquitination have started to be identified (see previous sections), it is now important to characterize precisely the ubiquitin code (location and type of ubiquitination) on clock proteins and identify the specific UBD-containing proteins that recognize the code and translate it into specific effects on clock proteins.
- 3.
PTMs around the clock: PTMs of clock proteins are orchestrated across the 24 h cycle. For example, BMAL1 undergoes a series of PTMs associated with a variation in activity and partner binding. Peak phosphorylation and SUMOylation of BMAL1 occurs in the late subjective day in mouse peripheral tissues (; ), and SUMOylation is a pre-requisite for ubiquitination (). The occurrence of these PTMs coincides with peak transcriptional activity of the CLOCK/BMAL1 dimer (; ). Further, this appears to be regulated by another PTM, O-GlcNAcylation, which opposes the ubiquitination of BMAL1 (). This maximal activity of CLOCK/BMAL1 results in expression of CRY proteins that then repress CLOCK/BMAL1, at a time that is synchronous with the BMAL1 dephosphorylation and stabilization (; ). BMAL1 acetylation by CLOCK also occurs at this time and leads to increased recruitment of CRY (). CRYs themselves are good examples of substrates for sequential PTMs over the 24 h day and across the progression of the clock feedback loop (see Ubiquitination of Cryptochromes by FBXL Ubiquitin Ligases). Therefore, each clock protein undergoes a daily wave of PTMs, in a sequential and often conditional manner, which determines the expression, localization, and activity of the protein and its partners.
CONCLUSION
In conclusion, ubiquitination and deubiquitination are involved in the regulation of key core clock components. On one hand, ubiquitin ligases are selectively acting on one or a few clock proteins. A given clock protein can even be the target of two or three different E3 ligases, depending on the time of day and cellular compartment. On the other hand, DUBs seem less specific, and only one was identified as a mammalian clock component so far: USP2. This DUB regulates the stability and function of PER1, CRY1, BMAL1 and perhaps other clock proteins, as well as components of the input and output pathways of the clock. Moreover, there is a complex interplay of ubiquitination with other PTMs. It will be crucial in future years to precisely define ubiquitin chain configurations and conjugation sites on clock proteins, to unravel the precise regulation of their addition and removal and identify all the actors involved. Furthermore, ubiquitination becomes an attractive drug target. Indeed, recent chemical screens of compounds binding CRY proteins have identified molecules modulating their ubiquitin-induced degradation (), suggesting the possibility of therapeutic resetting of the circadian clock by drug-mediated ubiquitin modulation of clock components.
Statements
Acknowledgments
The authors thank Dr. Kai-Florian Storch for critical review of the manuscript and all members of Nicolas Cermakian’s laboratory for discussions. This work was supported by grants from the Natural Sciences and Engineering Research Council (RGPIN 249731-12) to Nicolas Cermakian and the Canadian Institutes of Health Research (MOP 115106) to Simon S. Wing. Katarina Stojkovic was supported by a fellowship from McGill Faculty of Medicine and Nicolas Cermakian by a salary award from the Fonds de Recherche du Québec—Santé.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
REFERENCES
1
BuschmannT.FuchsS. Y.LeeC. G.PanZ. Q.RonaiZ. (2000). SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53.Cell101753–762. 10.1016/S0092-8674(00)80887-9
2
BusinoL.BassermannF.MaiolicaA.LeeC.NolanP. M.GodinhoS. I.et al (2007). SCFFbxl3 controls the oscillation of the circadian clock by directing the degradation of cryptochrome proteins.Science316900–904. 10.1126/science.1141194
3
CardoneL.HirayamaJ.GiordanoF.TamaruT.PalvimoJ. J.Sassone-CorsiP. (2005). Circadian clock control by SUMOylation of BMAL1.Science3091390–1394. 10.1126/science.1110689
4
CardozoT.PaganoM. (2004). The SCF ubiquitin ligase: insights into a molecular machine.Nat. Rev. Mol. Cell Biol.5739–751. 10.1038/nrm1471
5
ChiuJ. C.KoH. W.EderyI. (2011). NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed.Cell145357–370. 10.1016/j.cell.2011.04.002
6
ChiuJ. C.VanselowJ. T.KramerA.EderyI. (2008). The phospho-occupancy of an atypical SLIMB-binding site on PERIOD that is phosphorylated by DOUBLETIME controls the pace of the clock.Genes Dev.221758–1772. 10.1101/gad.1682708
7
CzarnaA.BerndtA.SinghH. R.GrudzieckiA.LadurnerA. G.TiminszkyG.et al (2013). Structures of Drosophila cryptochrome and mouse cryptochrome1 provide insight into circadian function.Cell1531394–1405. 10.1016/j.cell.2013.05.011
8
DardenteH.FortierE. E.MartineauV.CermakianN. (2007). Cryptochromes impair phosphorylation of transcriptional activators in the clock: a general mechanism for circadian repression.Biochem. J.402525–536. 10.1042/BJ20060827
9
DardenteH.MendozaJ.FustinJ. M.ChalletE.HazleriggD. G. (2008). Implication of the F-Box Protein FBXL21 in circadian pacemaker function in mammals.PLoS ONE3:e3530. 10.1371/journal.pone.0003530
10
DesterroJ. M.RodriguezM. S.HayR. T. (1998). SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation.Mol. Cell2233–239. 10.1016/S1097-2765(00)80133-1
11
DibnerC.SchiblerU.AlbrechtU. (2010). The mammalian circadian timing system: organization and coordination of central and peripheral clocks.Annu. Rev. Physiol.72517–549. 10.1146/annurev-physiol-021909-135821
12
DuguayD.CermakianN. (2009). The crosstalk between physiology and circadian clock proteins.Chronobiol. Int.261479–1513. 10.3109/07420520903497575
13
EideE. J.WoolfM. F.KangH.WoolfP.HurstW.CamachoF.et al (2005). Control of mammalian circadian rhythm by CKIepsilon-regulated proteasome-mediated PER2 degradation.Mol. Cell. Biol.252795–2807. 10.1128/MCB.25.7.2795-2807.2005
14
EvansJ. A.DavidsonA. J. (2013). Health consequences of circadian disruption in humans and animal models.Prog. Mol. Biol. Transl. Sci.119283–323. 10.1016/B978-0-12-396971-2.00010-5
15
GallegoM.VirshupD. M. (2007). Post-translational modifications regulate the ticking of the circadian clock.Nat. Rev. Mol. Cell Biol.8139–148. 10.1038/nrm2106
16
GodinhoS. I.MaywoodE. S.ShawL.TucciV.BarnardA. R.BusinoL.et al (2007). The after-hours mutant reveals a role for Fbxl3 in determining mammalian circadian period.Science316897–900. 10.1126/science.1141138
17
GossanN. C.ZhangF.GuoB.JinD.YoshitaneH.YaoA.et al (2014). The E3 ubiquitin ligase UBE3A is an integral component of the molecular circadian clock through regulating the BMAL1 transcription factor.Nucleic Acids Res.425765–5775. 10.1093/nar/gku225
18
GrimaB.LamourouxA.ChelotE.PapinC.Limbourg-BouchonB.RouyerF. (2002). The F-box protein slimb controls the levels of clock proteins period and timeless.Nature420178–182. 10.1038/nature01122
19
HerideC.UrbeS.ClagueM. J. (2014). Ubiquitin code assembly and disassembly.Curr. Biol.24R215–R220. 10.1016/j.cub.2014.02.002
20
HiranoA.YumimotoK.TsunematsuR.MatsumotoM.OyamaM.Kozuka-HataH.et al (2013). FBXL21 regulates oscillation of the circadian clock through ubiquitination and stabilization of cryptochromes.Cell1521106–1118. 10.1016/j.cell.2013.01.054
21
HirayamaJ.SaharS.GrimaldiB.TamaruT.TakamatsuK.NakahataY.et al (2007). CLOCK-mediated acetylation of BMAL1 controls circadian function.Nature4501086–1090. 10.1038/nature06394
22
HirotaT.LeeJ. W.JohnP. C.St.SawaM.IwaisakoK.NoguchiT.et al (2012). Identification of small molecule activators of cryptochrome.Science3371094–1097. 10.1126/science.1223710
23
KitaY.ShiozawaM.JinW.MajewskiR. R.BesharseJ. C.GreeneA. S.et al (2002). Implications of circadian gene expression in kidney, liver and the effects of fasting on pharmacogenomic studies.Pharmacogenetics1255–65. 10.1097/00008571-200201000-00008
24
KoH. W.JiangJ.EderyI. (2002). Role for slimb in the degradation of Drosophila period protein phosphorylated by doubletime.Nature420673–678. 10.1038/nature01272
25
KomanderD.ClagueM. J.UrbeS. (2009). Breaking the chains: structure and function of the deubiquitinases.Nat. Rev. Mol. Cell Biol.10550–563. 10.1038/nrm2731
26
KondratovR. V.ChernovM. V.KondratovaA. A.GorbachevaV. Y.GudkovA. V.AntochM. P. (2003). BMAL1-dependent circadian oscillation of nuclear CLOCK: posttranslational events induced by dimerization of transcriptional activators of the mammalian clock system.Genes Dev.171921–1932. 10.1101/gad.1099503
27
KondratovR. V.KondratovaA. A.LeeC.GorbachevaV. Y.ChernovM. V.AntochM. P. (2006). Post-translational regulation of circadian transcriptional CLOCK(NPAS2)/BMAL1 complex by CRYPTOCHROMES.Cell Cycle5890–895. 10.4161/cc.5.8.2684
28
KurabayashiN.HirotaT.SakaiM.SanadaK.FukadaY. (2010). DYRK1A and glycogen synthase kinase 3beta, a dual-kinase mechanism directing proteasomal degradation of CRY2 for circadian timekeeping.Mol. Cell. Biol.301757–1768. 10.1128/MCB.01047-09
29
KwonI.LeeJ.ChangS. H.JungN. C.LeeB. J.SonG. H.et al (2006). BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer.Mol. Cell. Biol.267318–7330. 10.1128/MCB.00337-06
30
LamiaK. A.SachdevaU. M.DitacchioL.WilliamsE. C.AlvarezJ. G.EganD. F.et al (2009). AMPK regulates the circadian clock by cryptochrome phosphorylation and degradation.Science326437–440. 10.1126/science.1172156
31
LeeJ.LeeY.LeeM. J.ParkE.KangS. H.ChungC. H.et al (2008). Dual modification of BMAL1 by SUMO2/3 and ubiquitin promotes circadian activation of the CLOCK/BMAL1 complex.Mol. Cell. Biol.286056–6065. 10.1128/MCB.00583-08
32
LiM. D.RuanH. B.HughesM. E.LeeJ. S.SinghJ. P.JonesS. P.et al (2013). O-GlcNAc signaling entrains the circadian clock by inhibiting BMAL1/CLOCK ubiquitination.Cell Metab.17303–310. 10.1016/j.cmet.2012.12.015
33
LuoW.LiY.TangC. H.AbruzziK. C.RodriguezJ.PescatoreS.et al (2012). CLOCK deubiquitylation by USP8 inhibits CLK/CYC transcription in Drosophila.Genes Dev.262536–2549. 10.1101/gad.200584.112
34
MoluskyM. M.LiS.MaD.YuL.LinJ. D. (2012a). Ubiquitin-specific protease 2 regulates hepatic gluconeogenesis and diurnal glucose metabolism through 11beta-hydroxysteroid dehydrogenase 1.Diabetes611025–1035. 10.2337/db11-0970
35
MoluskyM. M.MaD.BuelowK.YinL.LinJ. D. (2012b). Peroxisomal localization and circadian regulation of ubiquitin-specific protease 2.PLoS ONE7:e47970. 10.1371/journal.pone.0047970
36
MullerS.HoegeC.PyrowolakisG.JentschS. (2001). SUMO, ubiquitin’s mysterious cousin.Nat. Rev. Mol. Cell Biol.2202–210. 10.1038/35056591
37
OhsakiK.OishiK.KozonoY.NakayamaK.NakayamaK. I.IshidaN. (2008). The role of {beta}-TrCP1 and {beta}-TrCP2 in circadian rhythm generation by mediating degradation of clock protein PER2.J. Biochem.144609–618. 10.1093/jb/mvn112
38
OishiK.MiyazakiK.KadotaK.KikunoR.NagaseT.AtsumiG.et al (2003). Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes.J. Biol. Chem.27841519–41527. 10.1074/jbc.M304564200
39
ReischlS.VanselowK.WestermarkP. O.ThierfelderN.MaierB.HerzelH.et al (2007). Beta-TrCP1-mediated degradation of PERIOD2 is essential for circadian dynamics.J. Biol. Rhythms22375–386. 10.1177/0748730407303926
40
RippergerJ. A.SchiblerU. (2006). Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions.Nat. Genet.38369–374. 10.1038/ng1738
41
SaharS.ZocchiL.KinoshitaC.BorrelliE.Sassone-CorsiP. (2010). Regulation of BMAL1 protein stability and circadian function by GSK3beta-mediated phosphorylation.PLoS ONE5:e8561. 10.1371/journal.pone.0008561
42
ScomaH. D.HumbyM.YadavG.ZhangQ.FogertyJ.BesharseJ. C. (2011). The de-ubiquitinylating enzyme, USP2, is associated with the circadian clockwork and regulates its sensitivity to light.PLoS ONE6:e25382. 10.1371/journal.pone.0025382
43
ShiG.XingL.LiuZ.QuZ.WuX.DongZ.et al (2013). Dual roles of FBXL3 in the mammalian circadian feedback loops are important for period determination and robustness of the clock.Proc. Natl. Acad. Sci. U.S.A.1104750–4755. 10.1073/pnas.1302560110
44
ShiroganeT.JinJ.AngX. L.HarperJ. W. (2005). SCFbeta-TRCP controls clock-dependent transcription via casein kinase 1-dependent degradation of the mammalian period-1 (Per1) protein.J. Biol. Chem.28026863–26872. 10.1074/jbc.M502862200
45
SiepkaS. M.YooS. H.ParkJ.SongW.KumarV.HuY.et al (2007). Circadian mutant overtime reveals F-box protein FBXL3 regulation of cryptochrome and period gene expression.Cell1291011–1023. 10.1016/j.cell.2007.04.030
46
StorchK. F.LipanO.LeykinI.ViswanathanN.DavisF. C.WongW. H.et al (2002). Extensive and divergent circadian gene expression in liver and heart.Nature41778–83. 10.1038/nature744
47
StratmannM.SuterD. M.MolinaN.NaefF.SchiblerU. (2012). Circadian Dbp transcription relies on highly dynamic BMAL1-CLOCK interaction with E boxes and requires the proteasome.Mol. Cell48277–287. 10.1016/j.molcel.2012.08.012
48
TongX.BuelowK.GuhaA.RauschR.YinL. (2012). USP2a protein deubiquitinates and stabilizes the circadian protein CRY1 in response to inflammatory signals.J. Biol. Chem.28725280–25291. 10.1074/jbc.M112.340786
49
XingW.BusinoL.HindsT. R.MarionniS. T.SaifeeN. H.BushM. F.et al (2013). SCF(FBXL3) ubiquitin ligase targets cryptochromes at their cofactor pocket.Nature49664–68. 10.1038/nature11964
50
YagitaK.TamaniniF.YasudaM.HoeijmakersJ. H.Van Der HorstG. T.OkamuraH. (2002). Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein.EMBO J.211301–1314. 10.1093/emboj/21.6.1301
51
YanJ.WangH.LiuY.ShaoC. (2008). Analysis of gene regulatory networks in the mammalian circadian rhythm.PLoS Comput. Biol.4:e1000193. 10.1371/journal.pcbi.1000193
52
YangY.DuguayD.BédardN.RachalskiA.BaquiranG.NaC. H.et al (2012). Regulation of behavioral circadian rhythms and clock protein PER1 by the deubiquitinating enzyme USP2.Biol. Open1789–801. 10.1242/bio.20121990
53
YangY.DuguayD.FahrenkrugJ.CermakianN.WingS. S. (2014). USP2 regulates the intracellular localization of PER1 and circadian gene expression.J. Biol. Rhythms(in press).
54
YinL.JoshiS.WuN.TongX.LazarM. A. (2010). E3 ligases Arf-bp1 and Pam mediate lithium-stimulated degradation of the circadian heme receptor Rev-erb alpha.Proc. Natl. Acad. Sci. U.S.A.10711614–11619. 10.1073/pnas.1000438107
55
YinL.WangJ.KleinP. S.LazarM. A. (2006). Nuclear receptor Rev-erbalpha is a critical lithium-sensitive component of the circadian clock.Science3111002–1005. 10.1126/science.1121613
56
YooS. H.MohawkJ. A.SiepkaS. M.ShanY.HuhS. K.HongH. K.et al (2013). Competing E3 ubiquitin ligases govern circadian periodicity by degradation of CRY in nucleus and cytoplasm.Cell1521091–1105. 10.1016/j.cell.2013.01.055
57
YumimotoK.MuneokaT.TsuboiT.NakayamaK. I. (2013). Substrate binding promotes formation of the Skp1-Cul1-Fbxl3 (SCF(Fbxl3)) protein complex.J. Biol. Chem.28832766–32776. 10.1074/jbc.M113.511303
58
ZhangL.AbrahamD.LinS. T.OsterH.EicheleG.FuY. H.et al (2012). PKCgamma participates in food entrainment by regulating BMAL1.Proc. Natl. Acad. Sci. U.S.A.10920679–20684. 10.1073/pnas.1218699110
Summary
Keywords
circadian clock, clock gene, ubiquitin, ubiquitin ligase, deubiquitinase, stability
Citation
Stojkovic K, Wing SS and Cermakian N (2014) A central role for ubiquitination within a circadian clock protein modification code. Front. Mol. Neurosci. 7:69. doi: 10.3389/fnmol.2014.00069
Received
14 May 2014
Accepted
08 July 2014
Published
07 August 2014
Volume
7 - 2014
Edited by
Ashok Hegde, Wake Forest School of Medicine, USA
Reviewed by
Jason DeBruyne, Morehouse School of Medicine, USA; Kazuhiro Yagita, Kyoto Prefectural University of Medicine, Japan
Copyright
© 2014 Stojkovic, Wing and Cermakian.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Nicolas Cermakian, Douglas Mental Health University Institute, McGill University, 6875 LaSalle Boulevard, Montréal, QC H4H 1R3, Canada e-mail: nicolas.cermakian@mcgill.ca
This article was submitted to the journal Frontiers in Molecular Neuroscience.
Disclaimer
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.