Translate text to:
In JoVE (1)
Other Publications (23)
- Nature Neuroscience
- Molecular and Cellular Biology
- Methods in Enzymology
- Journal of Biochemistry
- Journal of Biological Rhythms
- Proceedings of the National Academy of Sciences of the United States of America
- Journal of Biological Rhythms
- Molecular and Cellular Biology
- Genes & Development
- BMC Genomics
- Current Biology : CB
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Science (New York, N.Y.)
- The Journal of Neuroscience : the Official Journal of the Society for Neuroscience
- Molecular Cell
- Genes & Development
- Genes & Development
Articles by Isaac Edery in JoVE
Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila
Joanna C. Chiu1,2, Kwang Huei Low1,3, Douglas H. Pike1, Evrim Yildirim1,3, Isaac Edery1,3
1Center for Advanced Biotechnology and Medicine, Rutgers University, 2Current Address: Department of Entomology, College of Agricultural and Environmental Sciences, University of California, Davis, 3Department of Molecular Biology and Biochemistry, Rutgers University
Other articles by Isaac Edery on PubMed
Nature. Dec, 2002 | Pubmed ID: 12442174
Protein phosphorylation has a key role in modulating the stabilities of circadian clock proteins in a manner specific to the time of day. A conserved feature of animal clocks is that Period (Per) proteins undergo daily rhythms in phosphorylation and levels, events that are crucial for normal clock progression. Casein kinase Iepsilon (CKIepsilon) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals. This was first shown in Drosophila with the characterization of Doubletime (Dbt), a homologue of vertebrate casein kinase Iepsilon. However, it is not clear how Dbt regulates the levels of Per. Here we show, using a cell culture system, that Dbt promotes the progressive phosphorylation of Per, leading to the rapid degradation of hyperphosphorylated isoforms by the ubiquitin-proteasome pathway. Slimb, an F-box/WD40-repeat protein functioning in the ubiquitin-proteasome pathway interacts preferentially with phosphorylated Per and stimulates its degradation. Overexpression of slimb or expression in clock cells of a dominant-negative version of slimb disrupts normal rhythmic activity in flies. Our findings suggest that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb.
Neuron. Mar, 2002 | Pubmed ID: 11931742
In the Drosophila circadian clock, daily cycles in the RNA levels of dclock (dClk) are antiphase to those of period (per). We altered the timing/levels of dClk expression by generating transgenic flies whereby per circadian regulatory sequences were used to drive rhythmic transcription of dClk. The results indicate that posttranscriptional mechanisms make substantial contributions to the temporal changes in the abundance of the dCLK protein. Circadian regulation is largely unaffected in the transgenic per-dClk flies despite higher mean levels of dCLK. However, in per-dClk flies the duration of morning activity is lengthened in light-dark cycles and light pulses evoke longer lasting bouts of activity. Our findings suggest that, in addition to a role in generating circadian rhythms, dCLK modulates the direct effects of light on locomotion.
Nature Neuroscience. Mar, 2003 | Pubmed ID: 12563262
The posttranslational modification of clock proteins is critical for the function of circadian oscillators. By genetic analysis of a Drosophila melanogaster circadian clock mutant known as Andante, which has abnormally long circadian periods, we show that casein kinase 2 (CK2) has a role in determining period length. Andante is a mutation of the gene encoding the beta subunit of CK2 and is predicted to perturb CK2beta subunit dimerization. It is associated with reduced beta subunit levels, indicative of a defect in alpha:beta association and production of the tetrameric alpha2:beta2 holoenzyme. Consistent with a direct action on the clock mechanism, we show that CK2beta is localized within clock neurons and that the clock proteins Period (Per) and Timeless (Tim) accumulate to abnormally high levels in the Andante mutant. Furthermore, the nuclear translocation of Per and Tim is delayed in Andante, and this defect accounts for the long-period phenotype of the mutant. These results suggest a function for CK2-dependent phosphorylation in the molecular oscillator.
Splicing of the Period Gene 3'-terminal Intron is Regulated by Light, Circadian Clock Factors, and Phospholipase C
Molecular and Cellular Biology. Apr, 2004 | Pubmed ID: 15060157
The daily timing of circadian ( congruent with 24-h) controlled activity in many animals exhibits seasonal adjustments, responding to changes in photoperiod (day length) and temperature. In Drosophila melanogaster, splicing of an intron in the 3' untranslated region of the period (per) mRNA is enhanced at cold temperatures, leading to more rapid daily increases in per transcript levels and earlier "evening" activity. Here we show that daily fluctuations in the splicing of this intron (herein referred to as dmpi8) are regulated by the clock in a manner that depends on the photoperiod (day length) and temperature. Shortening the photoperiod enhances dmpi8 splicing and advances its cycle, whereas the amplitude of the clock-regulated daytime decline in splicing increases as temperatures rise. This suggests that at elevated temperatures the clock has a more pronounced role in maintaining low splicing during the day, a mechanism that likely minimizes the deleterious effects of daytime heat on the flies by favoring nocturnal activity during warm days. Light also has acute inhibitory effects, rapidly decreasing the proportion of dmpi8-spliced per transcript, a response that does not require a functional clock. Our results identify a novel nonphotic role for phospholipase C (no-receptor-potential-A [norpA]) in the temperature regulation of dmpi8 splicing.
Analyzing the Degradation of PERIOD Protein by the Ubiquitin-proteasome Pathway in Cultured Drosophila Cells
Methods in Enzymology. 2005 | Pubmed ID: 15817301
Time-of-day specific changes in the levels of key clock proteins are critical for the normal progression of circadian pacemakers. Evidence indicates a major role for the ubiquitin-proteasome pathway (UPP) in the temporal control of clock protein stability. A conserved feature of animal clocks is that PERIOD (PER) proteins undergo daily rhythms in abundance. The stability of PER proteins is regulated by differential phosphorylation, whereby hyperphosphorylated isoforms are selectively degraded by the UPP. The use of transformed stable cell lines has been instrumental in advancing our understanding of the mechanisms underlying the intersection of the UPP and clock protein metabolism. This article describes several standard methodologies used to analyze the UPP-mediated degradation of Drosophila PER (dPER) expressed in cultured Drosophila cells (Ko et al., 2002). Although this article focuses on dPER as a case study, general issues are discussed that should have broad application to other cell culture-based systems and clock proteins. For example, we discuss (i) advantages?disadvantages of cultured cells, (ii) types of expression vectors and "peptide tags" for recombinant protein production and surveillance, and (iii) standard approaches to determine whether a protein of interest is modified by ubiquitin and degraded by the proteasome. Prior to the discussion on methodologies, the article provides a brief overview of diverse strategies by which clock proteins in a variety of systems are regulated by the UPP.
Regulating a Circadian Clock's Period, Phase and Amplitude by Phosphorylation: Insights from Drosophila
Journal of Biochemistry. Nov, 2006 | Pubmed ID: 17012288
Much progress has been made in understanding the molecular underpinnings governing circadian ( approximately 24 h) rhythms. Despite the increased complexity in metazoans whereby inter-cellular networks form the basis for driving overt rhythms, such as wake-sleep cycles in animals, single isolated cells can exhibit all the formal properties of a circadian pacemaker. How do these cell-autonomous rhythm generators operate? Breakthrough studies in Drosophila melanogaster led to the realization that the molecular logic underlying circadian clocks are highly shared. Most notably, interconnected transcriptional-translational feedback loops produce coordinated rhythms in "clock" RNAs and proteins that are required for the daily progression of clocks, synchronization to local time and transducing temporal signals to downstream effector pathways. More recent findings indicate prominent roles for reversible phosphorylation of clock proteins in the core oscillatory mechanism. In this review we focus on findings in Drosophila to explore the multiple levels that reversible phosphorylation plays in clock function. Specific clock proteins in this system are subjected to different phosphorylation programs, which affect three key properties of a circadian oscillator, its period, amplitude and phase. The role of phosphorylation in clocks is of clear relevance to human health because mutations that affect the PERIOD (PER) phosphorylation program are associated with familial sleep disorders. In addition, the central role of phosphorylation in the assembly of a circadian oscillator was dramatically shown recently by the ability to reconstitute a circadian phosphorylation/dephosphorylation cycle in vitro, suggesting that the dynamics of clock protein phosphorylation are at the "heart" of circadian time-keeping.
Clock-gated Photic Stimulation of Timeless Expression at Cold Temperatures and Seasonal Adaptation in Drosophila
Journal of Biological Rhythms. Aug, 2006 | Pubmed ID: 16864646
Numerous lines of evidence indicate that the initial photoresponse of the circadian clock in Drosophila melanogaster is the light-induced degradation of TIMELESS (TIM). This posttranslational mechanism is in sharp contrast to the well-characterized pacemakers in mammals and Neurospora, where light evokes rapid changes in the transcriptional profiles of 1 or more clock genes. The authors show that light has novel effects on D. melanogaster circadian pacemakers, acutely stimulating the expression of tim at cold but not warm temperatures. This photoinduction occurs in flies defective for the classic visual phototransduction pathway or the circadian-relevant photoreceptor CRYPTOCHROME (CRY). Cold-specific stimulation of tim RNA abundance is regulated at the transcriptional level, and although numerous lines of evidence indicate that period (per) and tim expression are activated by the same mechanism, light has no measurable acute effect on per mRNA abundance. Moreover, light-induced increases in the levels of tim RNA are abolished or greatly reduced in the absence of functional CLOCK (CLK) or CYCLE (CYC) but not PER or TIM. These findings add to a growing number of examples where molecular and behavioral photoresponses in Drosophila are differentially influenced by "positive" (e.g., CLK and CYC) and "negative" (e.g., PER and TIM) core clock elements. The acute effects of light on tim expression are temporally gated, essentially restricted to the daily rising phase in tim mRNA levels. Because the start of the daily upswing in tim expression begins several hours after dawn in long photoperiods (day length), this gating mechanism likely ensures that sunrise does not prematurely stimulate tim expression during unseasonally cold spring/summer days. The results suggest that the photic stimulation of tim expression at low temperatures is part of a seasonal adaptive response that helps advance the phase of the clock on cold days, enabling flies to exhibit preferential daytime activity despite the (usually) earlier onset of dusk. Taken together with prior findings, the ability of temperature and photoperiod to adjust trajectories in the rising phases of 1 or more clock RNAs constitutes a major mechanism contributing to seasonal adaptation of clock function.
Balance Between DBT/CKIepsilon Kinase and Protein Phosphatase Activities Regulate Phosphorylation and Stability of Drosophila CLOCK Protein
Proceedings of the National Academy of Sciences of the United States of America. Apr, 2006 | Pubmed ID: 16603629
The first circadian-relevant kinase to be identified was DOUBLE-TIME (DBT) in Drosophila, a homolog of vertebrate CKIepsilon, which regulates the progressive phosphorylation and stability of PERIOD (PER) proteins in animals. A negative feedback loop wherein PER directly inhibits the transcriptional activity of the CLOCK-CYCLE (CLK-CYC) heterodimer is central to the generation of molecular rhythms and normal progression of the clock in Drosophila. We show that DBT activity is required for the phase-specific hyperphosphorylation of CLK in vivo, an event that correlates with times of maximal repression in per RNA levels. The ability of DBT to hyperphosphorylate CLK, enhance its degradation, and evoke modest inhibition of CLK-dependent transactivation from circadian promoter elements was directly shown in cultured Drosophila cells. Intriguingly, DBT seems to function in close partnership with the PER-relevant protein phosphatase 2A, resulting in dynamic equilibrium between hypo- and hyperphosphorylated isoforms of CLK. This balancing mechanism might act to stabilize the limiting levels of CLK against stochastic fluctuations minimizing the propagation of "molecular noise" in the feedback circuitry. Also, the subcellular localization of CLK was altered from predominately nuclear to strong cytoplasmic staining in the presence of PER. These results suggest that, in contrast to mammalian clocks, circadian transcriptional inhibition in Drosophila involves displacement of the positive factors from chromatin. These results also demonstrate that DBT can target both negative and positive factors in circadian feedback loops and support a conserved role for dynamic regulation of reversible phosphorylation in directly modulating the activities of circadian transcription factors.
Cis-combination of the Classic Per(S) and Per(L) Mutations Results in Arrhythmic Drosophila with Ectopic Accumulation of Hyperphosphorylated PERIOD Protein
Journal of Biological Rhythms. Dec, 2007 | Pubmed ID: 18057324
The 1st circadian "clock" gene identified was the X-linked period (per) gene in Drosophila melanogaster. In the pioneering initial report, Konopka and Benzer (1971) characterized 3 alleles of per that shortened (per (S); approximately 19 h), lengthened (per (L); approximately 29 h), or abolished (per (0)) circadian behavioral rhythms. They also showed that transheterozygotes carrying the per (S) and per (L) mutations exhibit robust behavioral rhythms with nearly normal periods of approximately 23 h, highlighting the semidominant nature of many clock mutants. In this study, per (0) flies bearing a doubly mutated per transgene that carries both the per (S) and per (L) alleles (per (0); per (S/L)) were analyzed for behavioral and molecular rhythms. Unlike singly mutated versions, the per (0);per ( S/L) transgenic flies are arrhythmic in constant dark conditions and exhibit little, if any, entrainment to daily light-dark cycles. In a wildtype per (+) background, expression of per ( S/L) abolishes behavioral rhythms, indicating that it functions in a transdominant negative fashion. Biochemical analysis of head extracts revealed that only hyperphosphorylated isoforms of the PERS/L protein are detected throughout a daily cycle, and the levels remain constant. Intriguingly, little if any PERS/L is observed in key pacemaker neurons that control daily activity rhythms, consistent with the notion that hyperphosphorylated isoforms of PER are unstable. Nonetheless, PERS/L is detected in ectopic cells in the brain, in which it exhibits an unusual localization, mainly staining the periphery of the nucleus. These results suggest that posttranslational mechanisms play a key role in limiting the accumulation of PER to specific cells. On a broader scope, our results indicate that the semidominant effects of period-altering alleles observed in trans are not necessarily preserved in the cis-configuration and that novel phenotypes can emerge.
A DOUBLETIME Kinase Binding Domain on the Drosophila PERIOD Protein is Essential for Its Hyperphosphorylation, Transcriptional Repression, and Circadian Clock Function
Molecular and Cellular Biology. Jul, 2007 | Pubmed ID: 17452449
A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins from hypo- to hyperphosphorylated species, events that are highly dependent on casein kinase 1 epsilon (termed DOUBLETIME [DBT] in Drosophila melanogaster) and necessary for normal clock progression. Drosophila PER (dPER) functions in the negative limb of the clockworks by presumably binding to the transcription factor CLOCK (CLK) and inhibiting its transactivation activity. Here, we identify a small region on dPER that is conserved with mammalian PERs and contains the major in vivo DBT binding domain, termed dPDBD (for dPER DBT binding domain). This domain is required for the manifestation of molecular and behavioral rhythms in vivo. In the absence of the dPDBD, the dPER protein is present at constant high levels throughout a daily cycle, undergoes little phosphorylation, and is severely impaired in its ability to function as a transcriptional repressor. Our findings indicate that the binding of dPER to CLK is not sufficient for transcriptional inhibition, implicating a more indirect mode of action whereby dPER acts as a molecular bridge to "deliver" DBT and/or other factors that directly repress CLK-dependent gene expression.
Cell. Apr, 2007 | Pubmed ID: 17418778
The daily activity of the fruit fly Drosophila is controlled by both a "morning" and an "evening" circadian clock. In this issue Stoleru et al. (2007) demonstrate that day length determines which clock dominates the neural circuitry governing circadian behavior. Thus, these findings suggest a mechanism by which the system for circadian timing adapts to changes in the seasons to impose appropriate rhythms of daily activity.
Natural Variation in the Splice Site Strength of a Clock Gene and Species-specific Thermal Adaptation
Neuron. Dec, 2008 | Pubmed ID: 19109911
We show that multiple suboptimal splice sites underlie the thermal-sensitive splicing of the period (per) 3'-terminal intron (dmpi8) from D. melanogaster, enabling this species to prolong its midday "siesta," a mechanism that likely diminishes the deleterious effects of heat during the longer summer days in temperate climates. In D. yakuba and D. santomea, which have a more ancestral distribution indigenous to Afro-equatorial regions wherein day length and temperature exhibit little fluctuation throughout the year, the splicing efficiencies of their per 3'-terminal introns do not exhibit thermal calibration, consistent with the little effect of temperature on the daily distribution of activity in these species. We propose that the weak splice sites on dmpi8 underlie a mechanism that facilitated the acclimation of the widely colonized D. melanogaster (and possibly D. simulans) to temperate climates and that natural selection operating at the level of splicing signals plays an important role in the thermal adaptation of life forms.
The Phospho-occupancy of an Atypical SLIMB-binding Site on PERIOD That is Phosphorylated by DOUBLETIME Controls the Pace of the Clock
Genes & Development. Jul, 2008 | Pubmed ID: 18593878
A common feature of animal circadian clocks is the progressive phosphorylation of PERIOD (PER) proteins, which is highly dependent on casein kinase Idelta/epsilon (CKIdelta/epsilon; termed DOUBLETIME [DBT] in Drosophila) and ultimately leads to the rapid degradation of hyperphosphorylated isoforms via a mechanism involving the F-box protein, beta-TrCP (SLIMB in Drosophila). Here we use the Drosophila melanogaster model system, and show that a key step in controlling the speed of the clock is phosphorylation of an N-terminal Ser (S47) by DBT, which collaborates with other nearby phosphorylated residues to generate a high-affinity atypical SLIMB-binding site on PER. DBT-dependent increases in the phospho-occupancy of S47 are temporally gated, dependent on the centrally located DBT docking site on PER and partially counterbalanced by protein phosphatase activity. We propose that the gradual DBT-mediated phosphorylation of a nonconsensus SLIMB-binding site establishes a temporal threshold for when in a daily cycle the majority of PER proteins are tagged for rapid degradation. Surprisingly, most of the hyperphosphorylation is unrelated to direct effects on PER stability. We also use mass spectrometry to map phosphorylation sites on PER, leading to the identification of a number of "phospho-clusters" that explain several of the classic per mutants.
BMC Genomics. 2008 | Pubmed ID: 18284684
MicroRNAs (miRNAs) are short non-coding RNA molecules that target mRNAs to control gene expression by attenuating the translational efficiency and stability of transcripts. They are found in a wide variety of organisms, from plants to insects and humans. Here, we use Drosophila to investigate the possibility that circadian clocks regulate the expression of miRNAs.
Current Biology : CB. Feb, 2008 | Pubmed ID: 18261909
We sought to determine if the innate immune response is under circadian regulation and whether this impacts overall health status. To this end, we used infection of Drosophila with the human opportunistic pathogenic bacteria Pseudomonas aeruginosa as our model system . We show that the survival rates of wild-type flies vary as a function of when, during the day, they are infected, peaking in the middle of the night. Although this rhythm is abolished in clock mutant flies, those with an inactive period gene are highly susceptible to infection, whereas mutants with impairment in other core clock genes exhibit enhanced survival. After an initial phase of strong suppression, the kinetics of bacterial growth correlate highly with time of day and clock mutant effects on survival. Expression profiling revealed that nighttime infection leads to a clock-regulated transient burst in the expression of a limited number of innate immunity genes. Circadian modulation of survival also was observed with another pathogen, Staphylococcus aureus. Our findings suggest that medical intervention strategies incorporating chronobiological considerations could enhance the innate immune response, boosting the efficacy of combating pathogenic infections.
Neuron. Oct, 2009 | Pubmed ID: 19874783
In this issue of Neuron, Sehadova et al. show that synchronization of circadian clocks in the brains of Drosophila by daily temperature changes requires chordotonal organs, mechanosensory structures that function as stretch receptors in insects. This is strikingly different from the more direct path by which brain clocks perceive light.
Two Distinct Modes of PERIOD Recruitment Onto DCLOCK Reveal a Novel Role for TIMELESS in Circadian Transcription
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2010 | Pubmed ID: 20980603
Negative transcriptional feedback loops are a core feature of eukaryotic circadian clocks and are based on rhythmic interactions between clock-specific repressors and transcription factors. In Drosophila, the repression of dCLOCK (dCLK)-CYCLE (CYC) transcriptional activity by dPERIOD (dPER) is critical for driving circadian gene expression. Although growing lines of evidence indicate that circadian repressors such as dPER function, at least partly, as molecular bridges that facilitate timely interactions between other regulatory factors and core clock transcription factors, how dPER interacts with dCLK-CYC to promote repression is not known. Here, we identified a small conserved region on dPER required for binding to dCLK, termed CBD (for dCLK binding domain). In the absence of the CBD, dPER is unable to stably associate with dCLK and inhibit the transcriptional activity of dCLK-CYC in a simplified cell culture system. CBD is situated in close proximity to a region that interacts with other regulatory factors such as the DOUBLETIME kinase, suggesting that complex architectural constraints need to be met to assemble repressor complexes. Surprisingly, when dPER missing the CBD (dPER(Î”CBD)) was evaluated in flies the clock mechanism was operational, albeit with longer periods. Intriguingly, the interaction between dPER(Î”CBD) and dCLK is TIM-dependent and modulated by light, revealing a novel and unanticipated in vivo role for TIM in circadian transcription. Finally, dPER(Î”CBD) does not provoke the daily hyperphosphorylation of dCLK, indicating that direct interactions between dPER and dCLK are necessary for the dCLK phosphorylation program but are not required for other aspects of dCLK regulation.
Science (New York, N.Y.). Oct, 2010 | Pubmed ID: 20947752
A Hierarchical Phosphorylation Cascade That Regulates the Timing of PERIOD Nuclear Entry Reveals Novel Roles for Proline-directed Kinases and GSK-3beta/SGG in Circadian Clocks
The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Sep, 2010 | Pubmed ID: 20861372
The daily timing of when PERIOD (PER) proteins translocate from the cytoplasm to the nucleus is a critical step in clock mechanisms underpinning circadian rhythms in animals. Numerous lines of evidence indicate that phosphorylation plays a prominent role in regulating various aspects of PER function and metabolism, including changes in its daily stability and subcellular distribution. In this report, we show that phosphorylation of serine 661 (Ser661) by a proline-directed kinase(s) is a key phospho-signal on the Drosophila PER protein (dPER) that regulates the timing of its nuclear accumulation. Mutations that block phosphorylation at Ser661 do not affect dPER stability but delay its nuclear entry in key pacemaker neurons, yielding longer behavioral rhythms. Intriguingly, abolishing phosphorylation at Ser661 also attenuates the extent of dPER hyperphosphorylation in vivo, suggesting the phosphorylated state of Ser661 regulates phosphorylation at other sites on dPER. Indeed, we identify Ser657 as a site that is phosphorylated by the glycogen synthase kinase GSK-3Î² (SHAGGY; SGG) in a manner dependent on priming at Ser661. Although not as dramatic as mutating Ser661, mutations that abolish phosphorylation at Ser657 also lead to longer behavioral periods, suggesting that a multi-kinase hierarchical phosphorylation module regulates the timing of dPER nuclear entry. Together with evidence in mammalian systems, our findings implicate proline-directed kinases in clock mechanisms and suggest that PER proteins are key downstream targets of lithium therapy, a potent inhibitor of GSK-3Î² used to treat manic depression, a disorder associated with clock malfunction in humans.
A Morning-induced, Phosphorylation-gated Repressor Times Evening Gene Expression: a New Way for Circadian Clocks to Use an Old Trick
Molecular Cell. Dec, 2011 | Pubmed ID: 22152469
In this issue of Molecular Cell, Sancar et al. (2011) show that a morning-induced transcriptional repressor with a phosphorylation-gated half-life is a key cog in driving evening gene expression, adding new insights into how circadian clocks achieve phase-specific gene expression.
Genes & Development. Nov, 2011 | Pubmed ID: 22085960
In this issue of Genes & Development, Abruzzi et al. (pp. 2374-2386) use chromatin immunoprecipitation (ChIP) tiling array assays (ChIP-chip) to show that physical interactions between circadian (â‰…24-h) clock machineries and genomes are more widespread than previously thought and provide novel insights into how clocks drive daily rhythms in global gene expression.
NEMO/NLK Phosphorylates PERIOD to Initiate a Time-delay Phosphorylation Circuit That Sets Circadian Clock Speed
Cell. Apr, 2011 | Pubmed ID: 21514639
The speed of circadian clocks in animals is tightly linked to complex phosphorylation programs that drive daily cycles in the levels of PERIOD (PER) proteins. Using Drosophila, we identify a time-delay circuit based on hierarchical phosphorylation that controls the daily downswing in PER abundance. Phosphorylation by the NEMO/NLK kinase at the "per-short" domain on PER stimulates phosphorylation by DOUBLETIME (DBT/CK1Î´/É›) at several nearby sites. This multisite phosphorylation operates in a spatially oriented and graded manner to delay progressive phosphorylation by DBT at other more distal sites on PER, including those required for recognition by the F box protein SLIMB/Î²-TrCP and proteasomal degradation. Highly phosphorylated PER has a more open structure, suggesting that progressive increases in global phosphorylation contribute to the timing mechanism by slowly increasing PER susceptibility to degradation. Our findings identify NEMO as a clock kinase and demonstrate that long-range interactions between functionally distinct phospho-clusters collaborate to set clock speed.
Genes & Development. Feb, 2012 | Pubmed ID: 22327476
Post-translational modifications of one or more central "clock" proteins, most notably time-of-day-dependent changes in phosphorylation, are critical for setting the pace of circadian (â‰…24 h) clocks. In animals, PERIOD (PER) proteins are the key state variable regulating circadian clock speed and undergo daily changes in abundance and cytoplasmic-nuclear distribution that are partly driven by a complex phosphorylation program. Here, we identify O-GlcNAcylation (O-GlcNAc) as a critical post-translational modification in circadian regulation that also contributes to setting clock speed. Knockdown or overexpression of Drosophila O-GlcNAc transferase (ogt) in clock cells either shortens or lengthens circadian behavioral rhythms, respectively. The Drosophila PERIOD protein (dPER) is a direct target of OGT and undergoes daily changes in O-GlcNAcylation, a modification that is mainly observed during the first half of the night, when dPER is predominantly located in the cytoplasm. Intriguingly, the timing of when dPER translocates from the cytoplasm to the nucleus is advanced or delayed in flies, wherein ogt expression is reduced or increased, respectively. Our results suggest that O-GlcNAcylation of dPER contributes to setting the correct pace of the clock by delaying the timing of dPER nuclear entry. In addition, OGT stabilizes dPER, suggesting that O-GlcNAcylation has multiple roles in circadian timing systems.