Protein Interactions

The PAS domain functions as a protein dimerization motif that mediates interaction with Timeless (that has shown no homology with PER, and no PAS domain). PER dimerization is regulated by phosphorylation, occurring as the protein enters the nucleus. Hyperphosphorylation may signal proteolysis, accounting for PER protein's rapid disappearance at the end of the dark portion of the diurnal cycle (Reppert, 1995 and Edery, 1994). cAMP-dependent protein kinase A (PKA) plays a role in circadian rhythm (Leine, 1994).

TIM and PER accumulate in the cytoplasm when independently expressed in cultured (S2) Drosophila cells. If coexpressed, however, the proteins move to the nuclei of these cells. Domains of PER and TIM have been identified that block nuclear localization of the monomeric proteins. These regions of PER and TIM interaction consist of the PAS domain of PER and an adjacent domain also required for cytoplasmic localization (CLD). The sequence of TIM involved in interaction with PER resides between amino acids 505 to 578. TIM and PER both contain domains required for cytoplasmic localization. The site in PER required for nuclear localization is a sequence between amino acids 453 and 511. The sequence of TIM required for cytoplasmic localization (the TIM CLD) is C-terminal. It is thought that the CLD interacts with a cytoplasmic factor that inhibits nuclear localization. The results indicate a mechanism for controlled nuclear localization in which suppression of cytoplasmic localization is accomplished by direct interaction of PER and TIM. No other clock functions are required for nuclear localization. The findings suggest that a checkpoint in the circadian cycle is established by requiring cytoplasmic assembly of a PER/TIM complex as a condition for nuclear transport of either protein (Saez, 1996).

To investigate the mechanism of phase shifing of circadian clocks by light stimulation, the effects of light pulses on the protein and messenger RNA products of the Drosophila clock gene period (per) were measured. Photic stimuli perturb the timing of the PER protein and messenger RNA cycles in a manner consistent with the direction and magnitude of the phase shift. The recently identified clock protein Timeless (TIM) interacts with PER in vivo, and this association is rapidly decreased by light. This disruption of the PER-TIM complex in the cytoplasm is accompanied by a delay in PER phosphorylation and nuclear entry and disruption in the nucleus by an advance in PER phosphorylation and disappearance. These results suggest a mechanism for how a unidirectional environmental signal elicits a bidirectional clock response (Lee, 1996).

Sim is a developmental basic helix-loop-helix (bHLH) transcription factor containing a Per-Arnt-Sim (PAS) region of homology. Sim, in analogy to the structurally related bHLH/PAS dioxin receptor, can stably associated with the molecular chaperone hsp90. In the case of the dioxin receptor, release of hsp90 and derepression of receptor function appear to be regulated by ligand binding and dimerization with Arnt, a non-hsp90-associated bHLH/PAS factor. Dimerization with Arnt very efficiently disrupts Sim-hsp90 interaction, a process that required both the bHLH and PAS dimerization motifs of Arnt. Moreover, hsp90 is also released upon dimerization of Sim with the Drosophila PAS factor Per, whereas the hsp90-associated dioxin receptor fails to interact with Sim. These results indicate that hsp90 may play a role in conditional regulation of Sim function, and that Per and possibly bHLH/PAS partner factors may activate Sim by inducing release of hsp90 during the dimerization process (McGuire, 1995).

Differential effects of light and heat on the Drosophila circadian clock proteins PER and TIM

Temperature also regulates the Drosophila biological clock. Circadian (approximately 24-h) rhythms are governed by endogenous biochemical oscillators (clocks) that in a wide variety of organisms can be phase shifted (i.e., delayed or advanced) by brief exposure to light and changes in temperature. However, it is not known how changes in temperature reset circadian timekeeping mechanisms. One circadian rhythm that is sensitive to temperature shift is locomotion. For example, flies raised at 25 degreesC were placed at 37 degreesC for 30 minutes at 15 hours (T15) after the last dark to light transition (three hours after the beginning of the dark period). Their behavior was monitored for 7 to 10 days in constant darkness. Heat treatment delays peak locomotion by 2.3 hours from its normal peak (12.7 hours after the dark to light transition). To begin to address the biochemical basis of the behavioral change, the effects of short-duration heat pulses were measured on the protein and mRNA products from the Drosophila circadian clock genes period (per) and timeless (tim) (Sidote, 1998).

Heat treatment at T15 (15 hours after the last dark to light transition, that is 3 hours into the dark period) elicits the rapid disappearance of both Per and Tim proteins. Clear reductions are first observed between 3 to 5 min following the start of the 37 degree incubation, and essentially undetectable levels are reached after 10 to 15 min of heat treatment. Similar heat-induced decreases in the levels of both proteins were observed at all times in a daily cycle. No changes in the levels of either per or tim transcripts are detected during the first 20 min of the heat pulses, strongly suggesting that a posttranscriptional mechanism is solely responsible for mediating the heat-induced decreases in the levels of Per and Tim. The magnitude of the phase shift in the locomotor activity rhythm is also proportional to the temperature of the pulse, consistent with a causal relationship between the heat-induced degradation of Per, Tim, or both and phase resetting. It is thought that the majority of the heat-induced disappearance of Per and Tim is due to protein degradation. It is noteworthy that brief pulses at 37 degreesC elicit a full-blown heat shock response in D. melanogaster, raising the likelyhood that this pathway participates in mediating the enhanced degradation of Per and Tim. For example, thermally denatured proteins are prime targets for proteolysis by the ubiquitin-proteasome system (Sidote, 1998).

Per protein level is sensitive to heat but not light, indicating that individual clock components can markedly differ in sensitivity to environmental stimuli. A similar resetting mechanism involving delays in the per-tim transcriptional-translational feedback loop likely underlies the observation that when heat and light signals are administered in the early night, they both evoke phase delays in behavioral rhythms. Heat induces the degradation of Per and Tim independently, since the heat induced degradation of each protein takes place in flies mutant for the other protein. The results indicate that Per and Tim can be independently regulated by heat and that this degradation does not require a functional clock (Sidote, 1998).

The light-induced degradation of Tim in the late night is accompanied by stable phase advances in the temporal regulation of the Per and Tim biochemical rhythms (Per and Tim protein and phosphorylation levels). A 37 degree heat pulse at T15 evokes stable phase delays of several hours in the biochemical oscillations of Per and Tim, consistent with the magnitude and direction of the phase shift in locomotor activity rhythms produced by identical temperature treatments. The heat-induced delays in the temporal regulation of Per and Tim abundance and phosphorylation are stable for at least 2 days after the environmental perturbation (Sidote, 1998)

The initial heat-induced degradation of Per and Tim in the late night, unlike treatment in the early night, is followed by a transient and rapid increase in the speed of the Per-Tim temporal program. The net effect of these heat-induced changes results in an oscillatory mechanism with a steady-state phase similar to that of the unperturbed control situation; at the same time, there is little effect on locomotion. These findings can account for the lack of apparent steady-state shifts in Drosophila behavioral rhythms by heat pulses applied in the late night (Sidote, 1998).

An intriguing observation is that Per is sensitive to heat but not light, whereas Tim is sensitive to both stimuli. Thus, individual clock components can markedly differ in sensitivity to the two most important environmental entraining cues. Although not well studied, it is highly likely that under natural conditions a wide variety of organisms manifest circadian rhythms that are influenced by multiple temporal cues. In the case of Drosophila, it appears that the photic and heat signal transduction pathways converge at the level of regulating the stability of one or more key clock proteins. The observation that Per and Tim interact to form a functional complex that is involved in an autoregulatory circuit that is central to the timekeeping mechanism might ensure that the effects of light and temperature on individual clock proteins are combined into a coherent temporal cue resulting in daily rhythms that are optimally adapted to the precise local conditions. It will be of interest to determine whether other circadian timekeeping devices are assembled with components that differ in sensitivity to different environmental entraining cues (Sidote, 1998).

The Drosophila CLOCK protein undergoes daily rhythms in abundance, phosphorylation, and interactions with the PER-Tim complex

Drosophila Clock protein (dClock) is a transcription factor that is required for the expression of the circadian clock genes period (per) and timeless (tim). dClock undergoes circadian fluctuations in abundance, is phosphorylated throughout a daily cycle, and interacts with Per, Tim, and/or the Per-Tim complex during the night but not during most of the day. Both Per and Tim copurify with dClock in a time-of-day-specific manner: Per and Tim are first detected at ZT12 (beginning of the dark period), followed by increases in amounts that reach peak values at ZT23.9 (just before the lights go on). Between ZT16 (a third of the way through lights off) and ZT23.9, the amounts of all three proteins in immune complexes increase, even though the total levels of Tim and Per in head extracts peak at ZT16 and ZT20, respectively. This suggests that during the night dClock is present in limiting amounts compared to Per and Tim. Despite the higher levels of immunoprecipitated dClock between ZT4 and ZT8 compared to values obtained between ZT12 and ZT16, very little, if any, Per and Tim are detected. A likely explanation for this is that between ZT4 and ZT8 the total levels of Per and especially those of Tim are at, or close to, trough values. Thus, the interaction of Per and Tim with dClock is mainly restricted to nighttime hours (Lee, 1998).

Analysis of immune complexes derived from a period mutant clearly indicate that in the absence of Per, Tim can still interact with dClock. Because Tim is apparently located exclusively in the cytoplasm in the absence of Per, this result could suggest that the nuclear localization of dClock also requires Per or a functional oscillator. Alternatively, low levels of Tim might be able to enter the nucleus in the absence of Per. In contrast, several attempts to visualize a specific interaction between Per and dClock in the absence of Tim were unsuccessful. There are at least two nonmutually exclusive reasons that might account for thr inability to detect Per in dClock-containing immune complexes prepared from tim mutant flies: (1) the levels of Per are very low in tim mutant flies and as such the amounts of Per that copurify with dClock are below the detection limit, and (2) the interaction of Per with dClock requires Tim, possibly via formation of the Per-Tim complex and/or a dependence for nuclear localization (Lee, 1998 and references).

Attempts were made to measure the relative amounts of dClock that interact with Per and Tim as a function of time in an LD cycle. Head extracts were incubated with antibodies against either Per or Tim, and immune complexes probed for dClock, Per, and Tim. At ZT20 almost identical levels of dClock copurify with antibodies directed against either Per or Tim. Equivalent amounts of Per were also present in both immune pellets, but 1.6-fold more Tim is immunoprecipitated with antibodies to Tim, as compared to those directed against Per. These results are almost identical with a previous study showing that (1) in head extracts prepared from flies collected at ZT20, 80% of the total amount of Per is bound to Tim in a 1:1 stoichiometric relationship, and (2) there is 1.5-1.8 times more Tim, as compared to Per. Thus, the current results suggest that at ZT20 the majority of the Per and Tim proteins that interact with dClock are in the form of a heterodimeric Per-Tim complex. During the early day, only low levels of dClock are detected in immune complexes obtained using either antibodies to Per or Tim, in agreement with results using anti-dClock antibodies. Furthermore, it is mainly versions of Per and Tim that are essentially free of one another that interact with dClock during the early day (Lee, 1998).

How might a trimeric complex containing Per, Tim, and dClock be assembled? Presumably the HLH domain of dClock does not participate in mediating protein-protein interactions in this putative trimeric complex, because neither Per nor Tim seems to have a similar dimerization region. The only other regions that have been shown to mediate protein-protein interactions are the PAS domain found in Per and dClock and a not so well characterized region in Tim that spans 400 amino acids and interacts with the PAS domain of Per. It is tempting to speculate that one or both of these domains has the capacity to engage in at least trimeric formation. Although these studies do not address the nature of the trimeric interaction, they indicate that PAS-containing proteins are not limited to binary interactions (Lee, 1998).

These results suggest that Per and Tim participate in transcriptional autoinhibition by physically interacting with dClock or a dClock-containing complex. Nevertheless, in the absence of Per or Tim, the levels of dClock are constitutively low, indicating that Per and Tim also act as positive elements in the feedback loop by stimulating the production of dClock. Although Per and Tim inhibit dClock activity, Per and Tim are required for the high-level production of dClock protein and mRNA. Thus, Per and Tim appear to be the main "motor" of the Drosophila circadian oscillator, driving both positive and negative elements of the transcriptional-translational feedback loop. These observations suggest an explanation for the previously unexplained finding that the levels of Per mRNA in per mutant flies are approximately half as high as those obtained at peak times in wild-type flies. In contrast, mutations that abolish Neurospora FRQ activity result in high levels of frq RNA, suggesting that the frq-based circadian oscillator in Neurospora is based on a more simple negative transcriptional feedback loop. How Per and Tim stimulate dClock expression is not clear. They may interact with other transcription factors and act as coactivators. Alternatively, they may block the function of negative factors leading to the stimulation of gene expression. In addition to regulating the transcriptional activity of the dClock-CYC complex, Per and Tim might also interact with other transcription factors that are not involved in the circadian oscillator and as such molecularly couple the timekeeping mechanism to downstream effector pathways (Lee, 1998).

Timeless-dependent positive and negative autoregulation in the Drosophila circadian clock

Phosphorylation is an important feature of pacemaker organization in Drosophila. Genetic and biochemical evidence suggests involvement of the casein kinase I homolog doubletime (dbt) in the Drosophila circadian pacemaker. Two novel dbt mutants have been characterized. Both cause a lengthening of behavioral period and profoundly alter period (per) and timeless (tim) transcript and protein profiles. The Per profile shows a major difference from the wild-type program only during the morning hours, consistent with a prominent role for Dbt during the Per monomer degradation phase. The transcript profiles are delayed, but there is little effect on the protein accumulation profiles, resulting in the elimination of the characteristic lag between the mRNA and protein profiles. These results and others indicate that light and post-transcriptional regulation play major roles in defining the temporal properties of the protein curves and suggest that this lag is unnecessary for the feedback regulation of per and tim protein on per and tim transcription (Suri, 2000).

Both mutations, when presented in the context of the highly similar yeast casein kinase I HRR25, severely reduce kinase activity on peptide substrates. The long-period phenotypes are likely caused by insufficient Dbt activity, so it takes longer to reach some required level of Per phosphorylation. It is also assumed that both mutants are expressed at a level similar to that of wild-type Dbt (Suri, 2000).

Both dbth and Dbtg/+ have ~29 hr periods and are similar in all other respects, suggesting that the phenotypes are not idiosyncratic features of the mutations but reflect the role of Dbt in the pacemaker. Although the mutant flies entrain to imposed 24 hr photoperiods, the LD locomotor activity patterns indicate that there is no anticipation of the morning or evening light/dark transitions, and the evening activity peak is delayed by several hours into the night. The altered LD patterns are probably a consequence of the longer periods. Indeed, flies that carry pers as well as dbth have a period of ~22.5 hr and manifest robust anticipation of both morning and evening transitions as well as an advanced evening activity peak. Both dbt mutant LD profiles resemble that of the 29 hr period perl mutant strain, consistent with this altered period notion (Suri, 2000).

The molecular features of the perl circadian program are difficult to compare with those of wild-type flies, because the mutant rhythms are weak and of low amplitude as well as long period even under 12 hr LD entraining conditions. In contrast, Per and Tim cycling in the long-period dbt mutants is robust. Protein levels are comparable with those in wild-type flies during the night, and levels in the two mutant strains appear even higher than wild-type levels during the daytime. Previous work suggests a role for Dbt-catalyzed phosphorylation in targeting Per for degradation: this probably reflects slower protein turnover during the morning in the dbt mutants. The Tim phosphorylation pattern in the mutants did not show any noticeable difference from the wild-type pattern. These observations suggest that the modest mutant effects on the Tim profiles are indirect, perhaps through a primary effect of the dbt mutants on Per (Suri, 2000).

Per phosphorylation is still readily observable in both mutant lines. In fact, there is a hint that Per is even hyperphosporylated in these strains. Although this might reflect phosphorylation events that never take place in a wild-type background, less active Dbt mutants might be expected to depress the magnitude as well as the kinetics of the temporal phosphorylation program. This suggests that Per might not be a direct Dbt substrate in vivo but is only influenced indirectly, through intermediates that are direct Dbt targets. For example, Dbt may phosphorylate and activate a direct Per kinase or a specific protease. In this context, Per has not yet been shown to be a direct Dbt substrate. It is also possible that Dbt is a functionally relevant but minor Per kinase. In this case, the bulk of the Per mobility shift on SDS-PAGE is a consequence of other kinases. Because Per persists for several hours longer in the mutants than in wild-type flies, the other kinases would continue to function and give rise to even more highly phosphorylated species than are usually observed. These would be an indirect consequence of weak dbt activity and delayed degradation. A final possibility is that the enhanced and delayed Per phosphorylation simply reflects some misregulation of Dbt activity (Suri, 2000).

Careful analysis of the Per and Tim protein profiles in the long-period dbt mutants suggests that Dbt acts in the late night and morning phase of the molecular cycle: the mutants leave the early evening protein profile almost unaltered. This indicates that dbt probably targets nuclear, monomeric Per. It has also been suggested that Dbt acts in the early night to destabilize cytoplasmic Per, thus delaying nuclear entry and repression. The dbt mutants reported here do not significantly change this early night, presumptive cytoplasmic phase of accumulation. It is possible that Dbt prefers free Per over Per complexed to Tim. If free Per is a better substrate, then Dbt mutants should show a greater effect in the late night and early morning, after a large fraction of Tim has disappeared. Alternatively, Dbt might influence only marginally the Per accumulation phase for some other reason. But dbt mutant larvae accumulate high levels of hypophosphorylated Per, which suggests that Dbt is the major Per kinase and strongly influences Per accumulation as well as degradation. There is evidence, however, that much of this Per accumulation occurs in cells and tissues where Per is not normally detectable, making the connection with the normal Per-Tim cycle uncertain (Suri, 2000).

To assess the effect of the dbt mutants on transcription, per and tim mRNA cycling was assayed in wild-type and dbt mutant flies. Both mutant profiles are delayed by 4-5 hr. This is presumably because of the delayed disappearance of Per as well as Tim, which has been suggested to repress per and tim transcription. This relationship is very similar to that previously reported for the perS mutant strain; in this case, the clock proteins disappear more quickly, leading to an advance in the RNA profiles. The perS effect is more pronounced on Per than on Tim, consistent with the notion that monomeric Per might be the major transcriptional repressor. In any case, comparable results in the three mutants indicate a solid relationship between the timing of the decline in protein levels and the timing of the subsequent increase in per and tim transcription (Suri, 2000).

Based on these observations, a possible model for Dbt function in the Drosophila pacemaker is presented. In the cytoplasm, normal destabilization of Per delays substantial buildup of Per-Tim complexes and the consequent nuclear transport of the dimeric Per-Tim complex. In the nucleus, Per destabilization relieves repression. In Dbt mutants, Per degradation is much slower. This prolongs repression and delays the per and tim mRNA upswing in the next cycle (Suri, 2000).

There is an impressive relationship between the per and tim RNA profiles in comparison to the evening locomotor activity peak. In all cases, these RNA and locomotor activity begin to increase at approximately the same time, i.e., around ZT7 in the middle of the daytime. Mutants or physiological manipulations that affect the timing of the RNA profiles affect the timing of the evening activity peak in parallel. This fits with the emerging view, from mammalian as well as Drosophila work, that cycling transcription plays an important role in circadian output as well as within the central pacemaker oscillator. A further implication of these relationships is that the protein oscillations from one day affect behavior as well as the RNA profiles on the next one: the morning decline and eventual disappearance of Per and Tim terminate a protein cycle from the previous day, which then causes the subsequent increases in both RNA levels and locomotor activity (Suri, 2000).

In contrast, the delayed Per and Tim disappearance in the mutants has little if any effect on the subsequent protein accumulation phase (ZT13-ZT20) under these standard LD conditions; it is hardly affected, and both proteins peak at approximately the same time as they do in the wild-type flies (ZT19-ZT21). Because of the delayed RNA rise in the mutants, the per and tim RNA accumulation profiles almost coincide with those of the proteins, between ZT15 and ZT21. This indicates that the timing of the RNA rise is insufficient to time the protein rise. The increase in protein levels may reflect protein half-life regulation, which is uncoupled from the underlying mRNA levels, at least under some circumstances (Suri, 2000).

The coincidence of the protein and RNA curves also raises doubts about the importance of the 4-6 hr lag between these two accumulation profiles. The data presented in this study indicate that the lag is dispensable for robust behavioral and molecular oscillations. This is especially relevant for the RNA fluctuations. Despite evidence that at least per mRNA fluctuations may not be necessary for core oscillator function, they normally correlate with other molecular and behavioral circadian fluctuations. Moreover, there are substantial data indicating that Per and Tim feedback regulate these transcriptional oscillations. There is also considerable experimental evidence as well as theoretical models, to suggest that the normal 4-6 hr lag between the RNA and protein curves is essential for generating these robust, high-amplitude transcriptional oscillations. The general view is that the protein accumulation delay gives enough time for transcription to increase substantially, before protein levels have increased sufficiently to inhibit transcription. The presence of robust transcriptional oscillations without the delayed protein accumulation makes this scheme less likely. It redirects focus toward some post-transcriptional delay (e.g., the timing of nuclear entry of the Per-Tim dimer), which is predicted to be functional and important for transcriptional feedback regulation. It is important to note that these conclusions are based on biochemical experiments with whole-head extracts. It is still possible that the mRNA-protein lag may be important in the specific pacemaker neurons of Drosophila (Suri, 2000).

All of these experiments were performed under LD conditions. When the light comes on at ZT24, it causes a rapid decline in Tim levels. In DD conditions, therefore, Tim levels are much higher in the early subjective day, as expected. But a major, unanticipated difference was that the Per and Tim profiles in the dbt mutant flies are profoundly delayed in DD, as evidenced by the late appearance of faster-migrating species. This occurs without a comparable change in the RNA profiles, giving rise to a quasi-normal lag between RNA and protein. The light-mediated advance of the protein curves and the absence of a comparable light reset of the RNA profile reinforce the independent regulation of the accumulation phase of the clock RNAs and proteins: only the RNA profiles are influenced by the declining phase of the protein cycle of the previous day, whereas only the protein profiles appear to be reset by the light entrainment stimulus. The data are therefore consistent with a post-translational route of light entrainment, perhaps mediated by some aspect of the normal light effect on Tim. This presumably contributes to the daily advance of the dbt mutant clock under LD conditions, which counteracts the 5 hr period-lengthening effect that would take place under DD conditions (Suri, 2000).

Further understanding of the role of Dbt in the clock will require experiments that directly address Dbt function and regulation. For example, it is possible that temporal regulation of Dbt activity makes a major contribution to the temporal phosphorylation profile and more generally to the normal timing of the circadian program. Additionally, the extent to which Dbt modifies other pacemaker proteins is not clear. It is possible that these other putative Dbt substrates may also be intimately connected to the pacemaker mechanism. Addressing these issues would provide a much deeper understanding of the role of phosphorylation in the pacemaker (Suri, 2000).

Interlocked feedback loops within the Drosophila circadian oscillator

Drosophila Clock (Clk) is rhythmically expressed, with peaks in mRNA and protein (Clk) abundance early in the morning. Clk mRNA cycling is shown here to be regulated by Period-Timeless (Per-Tim)-mediated release of Clk- and Cycle (Cyc)-dependent repression. Lack of both Per-Tim derepression and Clk-Cyc repression results in high levels of Clk mRNA, which implies that a separate Clk activator is present. These results demonstrate that the Drosophila circadian feedback loop is composed of two interlocked negative feedback loops: a per-tim loop, which is activated by Clk-Cyc and repressed by Per-Tim, and a Clk loop, which is repressed by Clk-Cyc and derepressed by Per-Tim (Glossop, 1999).

Comparatively little is known about the regulation of Clk mRNA cycling. The levels of Clk mRNA are low in mutants lacking Per (per01) or Tim (tim01) function, which suggests that Per and Tim activate Clk transcription in addition to their roles as transcriptional repressors. The mechanism of Per-Tim-dependent activation is not known, but three models have been proposed to account for this activation. In the first two models, Per and Tim promote Clk transcription by shuttling transcriptional activators into the nucleus or by coactivating a transcriptional complex. In the third model, Per or Tim or both inhibit the activity of a transcriptional repressor complex (Glossop, 1999).

To distinguish among these alternative models, Clk mRNA levels were measured in different clock gene mutant combinations. Because Clk and Cyc are both required for per and tim activation, it was predicted that mutants lacking functional Clk (ClkJrk) or Cyc (Cyc0) would exhibit low levels of Clk mRNA because the concentrations of the Per and Tim activators (of Clk) would be low. It was surprising to find that the level of Clk mRNA is indistinguishable from the wild-type peak in both mutants. The levels of Clk mRNA do not vary significantly over the circadian cycle in these mutants, which is consistent with the lack of a functional circadian oscillator (Glossop, 1999).

The high level of Clk mRNA in the absence of Clk-dependent Per accumulation indicates that Per-dependent Clk activation does not occur by nuclear localization of an activator or by coactivation. However, the possibility remains that low levels of per and tim transcripts in ClkJrk or Cyc0 mutants lead to some active Per-Tim dimer formation and subsequent activation of Clk transcription. To eliminate this possibility, Clk mRNA levels were measured in per01;ClkJrk and per01;Cyc0 double mutants. In both cases, the levels of Clk mRNA observed under light-dark (LD) or constant dark (DD) conditions are close to the peak level in wild-type flies, indicating that Per-Tim activates Clk transcription through derepression (Glossop, 1999).

The Clk repressor that is removed as a result of Per-Tim accumulation appears to be either Clk-Cyc itself or a repressor that is activated by Clk-Cyc. When comparing the levels of Clk between per01 flies and per01;ClkJrk or per01;Cyc0 double mutants, the presence of active Clk and Cyc results in the repression of Clk transcript accumulation. In per01 mutants, Clk mRNA is at low but detectable levels. This suggests that in the absence of Per-Tim derepression, Clk transcription reaches a steady state in which activation and Clk-Cyc-dependent repression equilibrate to produce low levels of Clk mRNA transcripts and, hence, of Clk protein. In per01 and tim01 mutants, per and tim transcription is constitutive and per and tim transcripts are relatively low in abundance. This result can be explained by the partial activation of per and tim by low levels of Clk-Cyc dimers in the absence of Per-Tim repression (Glossop, 1999).

On the basis of these observations, it is proposed that interlocked negative feedback loops mediate circadian oscillator function in Drosophila. Late at night, Per-Tim dimers in the nucleus bind to and sequester Clk-Cyc dimers. This interaction effectively inhibits Clk-Cyc function, which leads to the repression of per and tim transcription and the derepression of Clk transcription. As Per-Tim levels fall early in the morning (ZT 0-3), Clk-Cyc dimers are released and repress Clk expression, thereby decreasing Clk mRNA levels so that they are low by the end of the day (ZT 12). Concomitant with the drop in Clk mRNA levels (through Clk-Cyc-dependent repression) is the accumulation of per and tim mRNA (through E-box-dependent Clk-Cyc activation). As Clk mRNA falls to low levels early in the evening (ZT 15), the levels of Clk-Cyc also fall, leading to a decrease in per and tim transcription and an increase in Clk mRNA accumulation. A new cycle then begins as high levels of Per and Tim enter the nucleus and Clk starts to accumulate late at night (Glossop, 1999).

These observations also fit well with the regulation of Drosophila cryptochrome (cry), whose mRNA cycles in phase with that of Clk. Like Clk, CRY mRNA transcripts are constitutively low in per01 mutants and constitutively high in ClkJrk or Cyc0 single mutants and in per01;ClkJrk or per01;Cyc0 double mutants. These striking similarities between Clk and CRY mRNA phases (in the wild type) and Clk and CRYmRNA levels in circadian mutants suggest that the cry locus may be regulated by the same Per-Tim release of Clk-Cyc repression mechanism as Clk (Glossop, 1999).

These results reveal the existence of a Clk feedback loop and its regulatory interactions with the well-characterized per-tim feedback loop. One clear prediction from these experiments is that there is a separate activator of Clk expression. Such an activator is indicated by the high levels of Clk mRNA in the absence of Per and of either Clk or Cyc. This observation is somewhat surprising because the presence of this activator is independent of factors that control the expression of other clock genes (that is, Per, Clk, and Cyc) (Glossop, 1999).

Data supporting the existence of interlocked per-tim and Clk feedback loops were obtained from whole heads, raising the possibility that Clk expression in small subsets of 'clock-specific' cells such as the locomotor activity pacemaker cells (that is, lateral neurons) could be masked by Clk expression in other tissues. However, the autonomy and synchrony of per expression in diverse tissues in the head and body suggest that the circadian feedback loop mechanism is the same in all tissues and argue against fundamental tissue-specific differences in the feedback loop mechanism (Glossop, 1999).

An important aspect of circadian biology is how the clock regulates clock-controlled genes (CCGs). In mammals, it has been shown in vitro that CLOCK and BMAL-1 (the mammalian ortholog of Cyc) activate vasopressin gene transcription and that all three mouse Pers and Tim repress this activation, resulting in peak vasopressin mRNA transcripts by midmorning (ZT 6). Although this mode of regulation may be more general for CCGs whose mRNA transcripts peak in phase with per (or mPer), it does not explain how CCGs that cycle in antiphase are regulated. The results presented here provide a possible mechanism by which the clock regulates CCGs whose mRNAs cycle in antiphase to those of per. The similarities between Clk and cry mRNA profiles in the wild type and in several single and double circadian mutants suggest that Per-Tim release of Clk-Cyc repression may serve a more general role in regulating CCG mRNAs that cycle in antiphase to per mRNA (Glossop, 1999).

double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation

Three alleles of a novel Drosophila clock gene, double-time (dbt), have been isolated. Short- (dbtS) and long-period (dbtL) mutants alter both behavioral rhythmicity and molecular oscillations generated by previously identified clock genes, period and timeless. A third allele, dbtP, causes pupal lethality and eliminates circadian cycling of per and tim gene products in larvae. In dbtP mutants, Per proteins constitutively accumulate, remain hypophosphorylated, and no longer depend on Tim proteins for their accumulation. It is proposed that the normal function of Doubletime protein is to reduce the stability and thus the level of accumulation of monomeric Per proteins. This would promote a delay between per/tim transcription and Per/Tim complex function, which is essential for molecular rhythmicity (Price, 1998).

The Drosophila clock gene double-time encodes a protein closely related to human casein kinase Iepsilon

This paper reports the cloning of double-time (dbt). Doubletime protein is most closely related to human casein kinase Igamma/epsilon. Short- and long-period mutations (dbtS and dbtL), which alter period length of Drosophila circadian rhythms, produce single amino acid changes in conserved regions of the predicted kinase. A mutant causing pupal lethality (dbtP) eliminates rhythms of per and tim expression and constitutively overproduces hypophosphorylated Per proteins, abolishing most dbt expression. DBT mRNA appears to be expressed in the same cell types as are per and tim and shows no evident oscillation in wild-type heads. Dbt is capable of binding to Per in vitro and in Drosophila cells, suggesting that a physical association of Per and Dbt regulates Per phosphorylation and accumulation in vivo (Kloss, 1998).

Because Dbt and Per appear to be expressed in the same cells in the Drosophila brain and eyes, and patterns of Per phosphorylation and accumulation are altered in dbt mutants, it was asked whether functional interactions between Per and Dbt might include a physical association of these proteins. Two independent methods were employed to test and confirm such a physical interaction: in vitro binding studies, and coimmunoprecipitation of Dbt and Per from cultured Drosophila cells (S2) programmed to express both proteins. In vitro translation of Dbt from dbt cDNA reveals a protein of ~46 kDa as predicted from sequence analysis. GST (glutathione-S-transferase) fusions, involving varying segments of Per, were tested for evidence of affinity for this Dbt protein. The GST-Per fusions were immobilized on glutathione agarose beads and subsequently incubated with in vitro translated, 35S-labeled DBT. After extensive washing to remove nonspecifically bound proteins, SDS-PAGE analysis of labeled Dbt proteins bound to the beads showed that Dbt binds to Per 1-640 and Per 1-365, but the protein does not bind to Per 530-640 or GST alone. These results show that Dbt and Per can physically associate in vitro and that Dbt interacts directly with an N-terminal region of Per (Kloss, 1998).

Phosphorylation of Period is influenced by cycling physical associations of Double-time, Period, and Timeless in the Drosophila clock

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the Period protein. Whereas the period, timeless, and Clock genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and Dbt protein are constitutively expressed. Robust circadian changes in Dbt subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include Per, Tim, and Dbt, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of Per is strongly enhanced when Tim is removed from Per/Tim/Ddt complexes. The varying associations of Per, Ddt and Tim appear to determine the onset and duration of nuclear Per function within the Drosophila clock (Kloss, 2001).

Dbt RNA levels are constant throughout the day. In this respect, the same is true for Dbt protein levels, since there was no detectable circadian oscillation of Dbt accumulation in timed head extracts. Furthermore, a variety of mutations disrupting the circadian clock and molecular oscillations have no effect on the level of Dbt protein. Thus, production of Dbt protein is not under the control of clock genes. In contrast, the subcellular localization of Dbt in the lateral neurons and photoreceptor cells changes over the course of a daily cycle. Dbt is consistently detected in the nucleus. However, at the end of the day and in the early part of the night, a substantial increase is found in cytoplasmic Dbt, coincident with the cytoplasmic accumulation of Per proteins and Per/Tim complexes. Furthermore, when Per/Tim complexes translocate to the nucleus at ~ZT18, and early during the day when Per remains in the nucleus in absence of Tim, a substantial nuclear accumulation of Dbt is observed. These changes in subcellular location of Dbt appear to be influenced exclusively by the locus of Per accumulation (in the presence or absence of Tim). Tim protein has little or no effect on the localization of Dbt because Dbt is always detected in the nucleus in per01 flies, which lack Per and have a substantial amount of Tim in the cytoplasm. Consequently, there is circadian regulation of Dbt proteins, in the form of a changing subcellular distribution. The fact that the movement of Per and Tim from the cytoplasm to the nucleus predicts the distribution of Dbt implies a close correspondence between maximum levels of Per/Tim complex and cytoplasmic levels of Dbt. Such a relationship could indicate that Tim associates with cytoplasmic Per once the latter protein has effected cytoplasmic localization of most cellular Dbt (Kloss, 2001).

Because Dbt preferentially accumulates in nuclei in the absence of Per, cytoplasmic Per proteins must affect this default localization at certain times of day in wild-type flies. Although the half-life of Dbt has not been determined, Dbt RNA and proteins are constantly synthesized. Therefore, the subcellular fate of newly translated Dbt may simply depend on whether cytoplasmic Per is available to associate with Dbt and retard its nuclear translocation. Alternatively, accumulation of Dbt may involve mechanisms promoting both nuclear import and export, with the predominant localization of Dbt governed by the presence or absence of cytoplasmic Per. Regardless of the specific mechanism, since Dbt has also been implicated in vital developmental and cellular functions that are not mediated through Per, an important product of any device generating cycling subcellular localization of this kinase could be temporal regulation of its access to alternative substrates (Kloss, 2001).

Dbt has been shown to be a component of the cytoplasmic activity that destabilizes Per. Evidence was also found that Dbt influences the stability of nuclear Per proteins. However, it has been unclear whether Dbt acts in both subcellular compartments, or whether nuclear stability of Per is affected by a Dbt-dependent phosphorylation in the cytoplasm, with delayed effects once Per translocates into the nucleus. This study shows that Dbt proteins are found both in the cytoplasm and in the nucleus. Coupled with the finding that Per proteins are always found associated with Dbt, this suggests that Dbt is required both in the nucleus and in the cytoplasm for Per phosphorylations (Kloss, 2001).

The simultaneous changes in subcellular localization of Per, Tim, and Dbt make it likely that direct physical associations among these proteins cause the cycling Dbt localizations. Per and Dbt proteins can associate in vitro and in cultured cells. Per/Dbt complexes can be recovered at all times during the day from head extracts, regardless of whether the majority of these proteins are localized in the cytoplasm or in the nucleus. Thus, Per proteins are associated with Dbt proteins in vivo when Per is in a Per/Tim complex and when Per proteins are free from Tim (Kloss, 2001).

Conversely, while Dbt binds to Per and Per/Tim complexes, no evidence has been found that Tim protein, free from Per, associates with Dbt in vivo. This finding is in line with the conclusion that Dbt's effects on the circadian clock are primarily mediated through Per (Kloss, 2001).

Extensive efforts have failed to obtain a functional assay for bacterially produced, recombinant Dbt in vitro. The putative kinase domains of Dbt and its mammalian ortholog CKIepsilon are very closely related (86% aa identity), so it was surprising to find that recombinant, mammalian CKIepsilon readily phosphorylates Drosophila Per and human Per in vitro. These observations suggest that Dbt function might be tightly regulated in the fly. It has been established that truncation of mammalian CKIepsilon substantially increases its activity in vitro, and truncated forms of the enzyme were used in the above mentioned Per and hPer assays. Although a corresponding truncation of Dbt failed to generate activity, such studies of mammalian CKIepsilon also indicate more complex regulation for this kinase in vivo (Kloss, 2001).

Without direct kinetic measurements of the activity of Dbt at different times of day, it cannot be determine whether Dbt function is under circadian control. However, it can be asked whether Per phosphorylation in vivo is (1) dependent upon the presence of Dbt and (2) influenced by Tim. In timUL flies entrained to LD 12:12, where Per remains complexed with TimUL for a prolonged interval in the nucleus, Per remains hypophosphorylated during the dark phase. Because wild-type flies begin to phosphorylate their Per proteins during the dark phase of such LD cycles, the results with timUL suggest that Tim influences the timing of light-independent Per phosphorylation (Kloss, 2001).

Light-triggered removal of TimUL protein is correlated with a rapid and progressive increase in the level of Per phosphorylation. Because a similar, cytoplasmic association of Per and Dbt in tim01 flies results in cytoplasmic Per degradation, and such Per degradation requires Dbt, the most parsimonious explanation of these results should be that nuclear association of Per with TimUL protects Per from phosphorylation and, secondarily, from turnover. It has been shown that light eliminates Tim, but will not promote Per phosphorylation in a hypomorphic mutant of Dbt (dbtP). Thus, Per phosphorylation appears to be influenced by the formation of Per/Tim complexes, and only when Per is free from Tim is it subject to phosphorylation by a Dbt-dependent mechanism. While this view is favored, it is also possible that light directly activates elements of a Dbt-dependent mechanism to promote some Per phosphorylations, or that additional factors associate with Per (or Dbt) after Tim is removed by light. Such factors would then be essential for Dbt-regulated phosphorylation of Per (Kloss, 2001).

The following is a model for the accumulation, phosphorylation, and degradation of Per: Dbt-dependent phosphorylation of Per in the cytoplasm is thought to delay the accumulation of Per proteins until lights off. Increasing Tim levels result in stable Per/Tim/Dbt complexes containing hypophosphorylated Per. These complexes are translocated to nuclei, where continued physical association of Tim with Per prolongs the cycle. Subsequently, the formation of Per free from Tim allows the clock to advance by Dbt-dependent phosphorylation of nuclear Per. This phosphorylation could be indirectly controlled by Dbt. The cycle restarts after degradation of phosphorylated nuclear Per proteins. According to this model, Dbt would have opposing effects on the cycle at different times of day and in different subcellular compartments. This regulation would determine the onset and duration of Per's activity in the nucleus, and should therefore be required to establish rhythmicity and set the period of Drosophila's circadian clock (Kloss, 2001).

Light-dependent interaction between Drosophila CRY and the clock protein PER mediated by the carboxy terminus of CRY

The biological clock synchronizes the organism with the environment, responding to changes in light and temperature. Drosophila Cryptochrome (Cry), a putative circadian photoreceptor, interacts with the clock protein Timeless (Tim) in a light-dependent manner. Although Tim dimerizes with Period (Per), no association between Cry and Per has previously been revealed, and aspects of the light dependence of the Tim/Cry interaction are still unclear. Behavioral analysis of double mutants of per and cry suggest a genetic interaction between the two loci. To investigate whether this is reflected in a physical interaction, a yeast-two-hybrid system was employed that revealed a dimerization between Per and Cry. This is further supported by a coimmunoprecipitation assay in tissue culture cells. The light-dependent nuclear interactions of Per and Tim with Cry require the C terminus of Cry and may involve a trans-acting repressor. Thus, as in mammals, Drosophila Cry interacts with Per, and, as in plants, the C terminus of Cry is involved in mediating light responses (Rosato, 2001).

At 25°C, perS;cryb flies display predominantly 24 hr cycles in a LD 12:12 regime, although ~40% of the flies also have a minor 19 hr perS component. These two periodic components are not found together in either single mutant (Rosato, 2001).

Locomotor activity was monitored for perS, cryb, and the perS;cryb double mutants at 18°C and 28°C. Single mutant perS or cryb flies entrain to the LD 12:12 regime at both temperatures, showing a 24 hr period and distribution of activity around the times of light/dark transitions. In DD, they free-run with a period of about 24 hr for cryb and a period of about 19 hr for perS, with a modest temperature dependence. In DD conditions, perS;cryb flies behave virtually identically to perS mutants at both temperatures. However, the behavior of the double mutant changes dramatically in LD, in a temperature-dependent manner. At 18°C, all perS;cryb flies show a periodic component of about 24 hr, but about 60% of them also display a minor 19 hr component. At 28°C, 79% of the rhythmic flies display the endogenous 19 hr period as the main rhythmic component. The breakdown of entrainment at 28°C in double mutant flies could reflect a genuine genetic interaction between the cryb and perS mutations. Alternatively, perhaps the limits of entrainment at high temperature are reduced in cryb mutants so that perS;cryb flies might indeed entrain to a T cycle of 20 hr at this temperature (which is closer to the 19 hr endogenous period of perS), whereas cryb individuals (whose endogenous period is ~24 hr) might not. To test this hypothesis, the locomotor activity rhythms of single and double mutant flies was monitored at 28°C under an LD 10:10 regime. Both perS and cryb flies entrain under this condition. However, the double mutants may show some evidence of entrainment during the first two cycles of the new light/dark regime, but any entrainment soon breaks down, and the perS;cryb flies free-run, with their daytime activity advancing by about 90 min on each successive day. Therefore, the entrainment defect at high temperature shown by perS;cryb flies is the product of a specific interaction between the two mutations rather than a defect in the entrainment of cryb alone. In Drosophila, the visual system is involved in the reception of circadian-relevant light information. This system is perfectly functional in the double mutant and is revealed by the startle response that is evident at the transition points from dark to light (and vice versa) at both temperatures. Therefore, perS;cryb flies are able to detect light but are deficient in the transmission of light information to the clock mechanism in a temperature-dependent manner (Rosato, 2001).

The genetic interaction between perS and cryb prompted an investigatation of the possibility of a physical interaction between Per and Cry using a yeast-two-hybrid system. A full-length Cry protein, directly fused to LexA (bait), was challenged with Per(233-685) as prey. This fragment includes the major protein/protein interaction domains described for Per. A fragment of Tim(377-915) that is known to bind to Per and contains the relevant regions for Per/Tim dimerization as prey was also tested. No interactions were observed between LexA-Cry and both Per(233-685) and Tim(377-915) fragments in the dark. Cry has been shown to interact with full-length Tim, but not Per, under constant light. In light, LexA-Cry binds strongly to Per(233-685), but not to Tim(377-915). LexA-Cry was also challenged with full-length Per and Tim, both in darkness and light. No interactions were observed in the dark. Under constant light, only full-length Tim showed evidence of dimerization with LexA-Cry. Three conclusions are drawn from these results: Per and Tim interactions with LexA-Cry are light dependent; the N and/or the C terminus of Tim are required for the association with LexA-Cry, and there is an inconsistency between the results obtained from full-length Per and the fragment Per(233-685). In regard to the latter, the well-established Per/Tim interaction was retested using LexA-Tim bait with Per and Per(233-685) preys in darkness and light. No interactions were observed using full-length Per. Subsequent Western blot analysis has revealed that, in this system, full-length Per is poorly expressed, thereby explaining the lack of interactions in yeast with this construct. Nevertheless, a strong interaction between LexA-Cry and Per(233-685) could be demonstrated. This discrepancy between the current results and contradictory published results must reside in the different yeast-two-hybrid systems employed. Evidence was also found for a Tim-independent Cry/Per complex using coimmunoprecipitation (Rosato, 2001).

Cryptochromes are believed to interact with a signaling factor after light exposure, and evidence has been found in plants for a role of the C-terminal domain in signaling. Since the coimmunoprecipitation result supports the view that the interaction between LexA-Cry and Per(233-685) in yeast reflects a meaningful association between Per and Cry, the power of yeast genetics was exploited to test the regulatory role of the C terminus of Drosophila Cry. Twenty residues were deleted from the Cry C terminus to create CryDelta and it was challenged with Per(233-685) and full-length Tim in darkness and light. An interaction was evident in both conditions, with no obvious difference between them. It has been suggested that LexA-Cryb is unable to interact with Tim in yeast cells because it may have lost its photoresponsiveness. Both LexA-Cryb and LexA-CrybDelta, which are strongly expressed in yeast, are nevertheless unable to interact with Per(233-685) or with Tim. Given the light independence of CryDelta, it is suggested that the D[410]N substitution in Cryb probably confers a gross structural defect to LexA-Cryb, rather than simply affecting its photoreceptor ability (Rosato, 2001).

To further map the interaction between Cry and Per, LexA-CryDelta was challenged with several overlapping Per fragments. It was confirmed that LexA-Tim (377-915) interacts with the PAS A domain (Per[233-390]) and Per(233-685). LexA-CryDelta does not associate with Per(233-390), nor with the PAS A + B region (Per[233-485]), but interacts with the downstream C domain (Per[524-685], which includes the perS site. From these results, it is speculated that Tim and Cry may interact with different regions of the Per protein and, since Cry associates with region(s) of Tim external to the (377-915) fragment, it is hypothesized that Per, Tim, and Cry can be found in the same complex (Rosato, 2001).

LexA-Cry requires light in order to interact with Per(233-685) and Tim. However, it cannot be ruled out a priori that it is the temperature increase, caused by the continuous light exposure, rather than light per se, that triggers Cry's interactions. LexA-Cry was therefore challenged with Per(233-685) and Tim at 37°C in the dark, but no interactions were observed. Furthermore, since LexA-CryDelta does not require light, this variant was used to investigate the effect of temperature on Cry interactions (Rosato, 2001).

Yeast patches were grown on X-gal plates at 30°C and 37°C in parallel. It was noted that at 37°C, the LexA-CryDelta interaction with Per(233-685) is considerably weakened, whereas the control LexA-Tim(377-915)/Per(233-685) dimerization does not show any substantial temperature differences. The same temperature dependence is also observed when LexA-CryDelta is challenged with Tim and Per (524-685) (Rosato, 2001).

To further identify those regions of Cry that could suppress the negative effect of darkness, random Taq-induced mutations were introduced into full-length cry by PCR, and LexA-Cry* mutants were created by in vivo gap repair. The putative LexA-Crys* were challenged with Per(233-685) in the dark. A total of 14 bona fide light-independent mutations were identified that generated a Cry/Per interaction in darkness. The sequencing of these variants shows that all of these light-independent Crys* carry either a translational stop or a frame-shift at their C termini. Some of the mutants have additional amino acid substitutions scattered across the entire sequence, but because of their sporadic nature, it is very unlikely that these missense mutations are contributing to the light-independent phenotype (Rosato, 2001).

The results reported above support the view that the C terminus of Cry is responsible for the light dependence of the interactions with Per and Tim. Perhaps the removal of the C terminus changes Cry conformation to a form that is active in darkness. Alternatively, there could be a carboxy-terminal-bound, light-inhibited nuclear repressor of Cry in yeast. In fact, trans-acting factor in yeast can be mutated to disinhibit nuclear Cry activity in darkness, and currently, attempts are being made to identify the gene(s) involved (Rosato, 2001).

Thus, it has been shown that Cry binds Per in yeast and in a Drosophila cell culture system. As in yeast, the light-dependent activities of Cry in S2 cells have been reported only in the nucleus, where Cry is suggested to undergo a conformational change after light absorption, allowing it to bind to Tim (and now Per). However, Cry coimmunoprecipitates with Tim and Per in the cytoplasm of S2 cells under darkness, suggesting that light is not required to change Cry into its active conformation. Consequently, both in yeast and S2 cells, it is predicted that a nuclear factor may interact with the Cry C terminus in darkness to prevent it from interacting with the two clock proteins. Cry itself is probably not its own repressor, because full-length Cry was tested in a yeast-two-hybrid assay and it does not significantly self-associate in light or dark. However, mutagenesis of the yeast genome has identified two variants that can derepress the Cry/Per interaction in darkness. Isolation of this gene(s), irrespective of its function in yeast, will provide candidates for this nuclear repressor(s), which might have a clock relevant homolog(s) in Drosophila. An analogous situation has been reported in which Saccharomyces cerevisiae casein kinase I, HRR25 (without known clock function in yeast) binds and phosphorylates Per with affinities similar to the Drosophila casein kinase Iepsilon, Doubletime (Dbt). The signaling mechanism of cryptochrome is also mediated through the C terminus in Arabidopsis. A fusion between ß-glucuronidase (GUS) and the C-terminal domain (CCT) of either Cry1 or Cry2 (to create CCT1 and CCT2) mediates a constitutive light response. This means that 'isolated' CCTs display properties in the dark that are strikingly similar to those of light-activated Crys. Within the Cry molecule, the C-terminal domain is repressed under darkness, and light activation might be achieved either by an intramolecular or an intermolecular redox reaction, but the details of the light-induced activation of CCT are not known. In this study, it has been shown that the intermolecular model is the more appropriate to explain observations with Drosophila Cry. Light-induced activation of Cry removes a regulatory molecule, enabling the binding of Per and Tim, although the possibility exists that the regulatory molecule itself, rather than Cry, could act as the primary photopigment. It will be of interest to see if this model also applies to Arapidopsis cryptochromes. The C-terminal domain of Cry thus becomes a focal point for further studies, and it is probably not a coincidence that it is this region of the otherwise evolutionary conserved Cry molecule that is the most variable (Rosato, 2001).

It cannot be unequivocally concluded that the physical interaction revealed between Per and Cry is responsible for the genetic interaction that occurs in perS;cryb mutants at high temperature, even though this was the experiment that led the authors to test a possible Per/Cry dimerization. However, the Per/Cry interaction is temperature-sensitive in yeast, and it is the Per C domain (which includes the site of perS) that dimerizes with Cry, providing further circumstantial evidence that the genetic interaction between Per and Cry may correlate with the physical interaction. Furthermore, it is tempting to speculate that differences in the PerS/Cry physical interaction may be at the heart of reports that the perS mutants are hypersensitive to light and that flies carrying a small deletion (amino acids 515-568) within the Per C domain display short, poorly temperature-compensated rhythms and an altered behavioral response to light pulses. Perhaps a reduction in the strength of the PerS/Cry association, further decreased at 28°C below a critical threshold, might account for the entrainment defect of perS;cryb double mutants at high temperature. Finally, genetic interactions between short-period mutations perS and perT and the arrhythmic mutation dbtar implicate the C domain in the dynamics of Per phosphorylation by DBT. Taken together, these results suggest that the Per C domain may provide a convergence point for both Cry and Dbt, and it is anticipated that future research may disclose a prominent role for Cry in the fly circadian clock (Rosato, 2001).

A novel C-terminal domain of Drosophila Period inhibits Clock:Cycle-mediated transcription

The essence of the Drosophila circadian clock involves an autoregulatory feedback loop in which Period (Per) and Timeless (Tim) inhibit their own transcription by association with the transcriptional activators Clock (Clk) and Cycle (Cyc). Because Per, Clk, and Cyc each contain a PAS domain, it has been assumed that these interaction domains are important for negative feedback. However, a critical role for PAS-PAS interactions in Drosophila clock function has not been shown. Nuclear transport of Per is also believed to be an essential regulatory step for negative feedback, but this has not been directly tested, and the relevant nuclear localization sequence (NLS) has not been functionally mapped. These critical aspects of Per-mediated transcriptional inhibition have been evaluated in Drosophila Schneider 2 (S2) cells. The dCLK:CYC inhibition domain (CCID) of Per has been mapped; it lies in the C terminus, downstream of the PAS domain. Using deletion mutants and site-directed mutagenesis, a novel NLS has been identified in the CCID of Per that is a potent regulator of Per's nuclear transport in S2 cells. Nuclear transport, primarily through this novel NLS, is essential for the inhibitory activity of Per. The data indicate that nuclear Per inhibits Clk:Cyc-mediated transcription through a novel domain that additionally contains a potent NLS (Chang, 2003).

Thus, a key step in the Drosophila circadian negative feedback loop, Per inhibition of Clk:Cyc transcription, is not mediated by the PAS domain of Per. Instead, the previously uncharted C terminus of Per contains a novel domain (CCID; aa 764-1034) responsible for transcriptional inhibitory activity. The functional importance of this C-terminal region of Per is corroborated by an earlier in vivo experiment, in which a per transgene extending only up to amino acid 876 failed to rescue behavioral rhythms in per null mutant (per01) flies (Zehring, 1984). This truncated Per would still possess binding sites for Tim, Double-time, and Cryptochrome, but, as the experiments reveal, its CCID would be disrupted. The monopartite NLSs previously predicted by sequence analysis are relatively weak in regulating Per localization. Instead, a novel, bipartite NLS in the CCID is the dominant NLS in S2 cells. However, there may be several NLSs that contribute to Per nuclear transport in vivo since transgenic fly experiments suggest that there is a competent NLS in the first 95 amino acids of Per, and N-terminal fragments of Per do show some nuclear localization in S2 cells. The nuclear transport of Per is essential for its inhibition of Clk:Cyc-mediated transcription. These results advance understanding of Per function and thus understanding of the Drosophila circadian clock mechanism (Chang, 2003).

A role for casein kinase 2alpha in the Drosophila circadian clock

Circadian clocks drive rhythmic behavior in animals and are regulated by transcriptional feedback loops. For example, the Drosophila proteins Clock (Clk) and Cycle (Cyc) activate transcription of period (per) and timeless (tim). Per and Tim then associate, translocate to the nucleus, and repress the activity of Clk and Cyc. However, post-translational modifications are also critical to proper timing. Per and Tim undergo rhythmic changes in phosphorylation, and evidence supports roles for two kinases in this process: Doubletime (Dbt) phosphorylates Per, whereas Shaggy (Sgg) phosphorylates Tim. Yet Sgg and Dbt often require a phosphoserine in their target site, and analysis of Per phosphorylation in dbt mutants suggests a role for other kinases. The catalytic subunit of Drosophila casein kinase 2 (CK2alpha) is shown to be expressed predominantly in the cytoplasm of key circadian pacemaker neurons. CK2alpha mutant flies show lengthened circadian period, decreased CK2 activity, and delayed nuclear entry of Per. These effects are probably direct, since CK2alpha specifically phosphorylates Per in vitro. It is proposed that CK2 is an evolutionary link between the divergent circadian systems of animals, plants and fungi (Lin, 2002).

To identify new components of circadian clocks, about 2,000 ethylmethane-sulphonate (EMS)-mutagenized pers (short-period allele of per) lines were screened for circadian behavioral defects. A dominant mutant, Timekeeper (Tik), was identified. Tik homozygotes do not live to adulthood. Tik heterozygotes exhibit behavioral rhythms approximately 1.5 h longer than pers. In a per+ or perl (long-period allele of per) background, Tik lengthens the period of the behavioral rhythm by 3 h, reflecting an allele-specific interaction between per and Tik. A spontaneous partial revertant, TikR, was identified and this revertant could not be separated from Tik by recombination. The change in period in Tik mutants (about 3 h) exceeds that of nearly all heterozygous circadian mutants in Drosophila, suggesting that it might identify a protein of central importance in the circadian clock mechanism (Lin, 2002).

Tik maps to a region around the chromosomal centromere. Sequencing of the CK2alpha coding region from the wild-type parental strain and the initial Tik mutant identified two sequence changes, both resulting in coding changes: Met161Lys and Glu165Asp. The change from the non-charged Met 161 to the charged Lys occurs near the catalytic loop and within a hydrophobic binding pocket for ATP. The TikR mutant that was proposed to be a new Tik revertant allele was also sequenced. In addition to the two original Tik coding changes, an additional in-frame, 27-base-pair (bp) deletion was identified coupled to an in-frame, 6-bp insertion, resulting in a deletion of 7 amino acids (234–240) and another amino acid change (Arg242Glu). These mutations occur in residues that are highly conserved with their human counterpart (Lin, 2002).

Immunohistochemistry was performed on adult whole-mount dissected brains with an antiserum directed against a CK2 peptide sequence common to both fly and mammalian CK2alpha (anti-CK2alpha). Anti-CK2alpha specifically labels the cytoplasm and axonal projections of neurons in the lateral protocerebrum. To determine whether these neurons are the well-described pacemaker lateral neurons, double labelling was performed with an antiserum against Pigment-dispersing factor (Pdf). This neuropeptide is specifically localized to small and large lateral neurons critical to behavioral rhythms. Consistent with its proposed circadian role, CK2 co-localizes with Pdf in these adult neurons. CK2alpha is also probably expressed in the eyes, as CK2 activity is modestly reduced (about 20%) in eyes absent mutants. Of note, CK2alpha and Pdf also co-localize to neuronal termini, indicating that CK2 may regulate directly Pdf processing or release. CK2alpha seems to be constitutively cytoplasmic as a function of the time of day. The tissue specificity of CK2alpha expression suggests that it has a specific function in circadian clocks (Lin, 2002).

To ascertain CK2 function on Per and Tim, their levels and phosphorylation were examined in heterozygotes. Flies were entrained in a light/dark cycle and were transferred to constant darkness (DD). During the early subjective day (circadian time, CT, 0–12) levels of Per in the CK2alphaTik mutants are increased and show delayed disappearance. Furthermore, there seems to be a modest increase in less-phosphorylated forms of Per. During the subjective night, Per phosphorylation and to a lesser extent accumulation are delayed relative to wild type. An increase in the level of Per and Tim in CK2alphaTik mutants is delayed by approximately 3 h relative to wild type, consistent with the period-lengthening effect of CK2alphaTik mutants. Disappearance of Tim is also more strongly affected than accumulation in CK2alphaTik mutants. The Per and Tim profiles of CK2alphaTikR heterozygotes are largely unchanged, consistent with its heterozygous behavioral and biochemical phenotypes (Lin, 2002).

To examine the phenotype of homozygous CK2alpha mutants, immunofluorescence for Per was performed in third-instar larval brains. Per staining in wild-type larvae is predominantly nuclear by Zeitgeber time (ZT) 21 (three hours before 'lights on'). In CK2alphaTik and CK2alphaTikR homozygotes, however, Per nuclear entry is significantly delayed, with Per predominantly cytoplasmic at ZT21. By ZT1, the Per staining pattern in the CK2alpha mutants remains distinct from the wild-type nuclear pattern, appearing to be present in both cytoplasmic and nuclear compartments. These data demonstrate a strong effect of CK2alpha on Per nuclear entry and are consistent with the cytoplasmic expression of CK2alpha. Furthermore, no significant differences between CK2alphaTik and CK2alphaTikR were observed, consistent with biochemical data on recombinant proteins and their recessive lethality. These data support the model that CK2alphaTikR is a strong loss-of-function allele, lacking the strong dominant behavioral and biochemical effects of CK2alphaTik(Lin, 2002).

Bacterially expressed and purified CK2alpha can phosphorylate Per in vitro. Notably, this effect is specific, since no significant phosphorylation of the circadian transcription factor Cyc is observed. Tim (amino acids 1–1159) is phosphorylated by CK2alpha to a lesser extent than Per. These data are consistent with the direct regulation of Per and Tim by CK2 (Lin, 2002).

Taken together, this analysis indicates a dedicated and direct role of CK2 in the Drosophila circadian clock mechanism. CK2alphaTik has one of the strongest phenotypes of any heterozygous circadian rhythm mutant. The modest nature of the biochemical defect further supports the hypothesis that the circadian clock is highly sensitive to CK2 activity. The CK2alpha expression pattern indicates that it has a specific role in circadian rhythms. Its localization to neuronal termini raises the possibility that CK2 links the clock to circadian outputs (Lin, 2002).

It is suggested that CK2 directly phosphorylates Per and Tim in vivo, promoting their transition to the nucleus. The evidence for this pattern is compelling for Per. Allele-specific interactions between per and CK2alpha alleles support the model of a direct interaction. Given the cytoplasmic expression of CK2alpha, this phosphorylation may serve as a signal for nuclear entry and subsequent degradation, explaining the delayed nuclear entry and disappearance of these proteins in CK2alphaTik mutants (Lin, 2002).

This study also establishes an evolutionary connection between animal, plant and fungal circadian systems -- genetic studies have revealed clock components in plants (Arabidopsis) and fungi (Neurospora). These studies suggest that clocks have arisen several times in evolution; however, studies in both Arabidopsis and Neurospora have linked CK2 to circadian timekeeping. CK2 is therefore a gene involved in circadian rhythms that is shared between all three phylogenetic kingdoms. Indeed, the fly enzyme (amino acids 7–322) shares 77% and 72% identity with the Arabidopsis and Neurospora enzymes, respectively. It is proposed that the conserved role for CK2 is driven by the need to avoid mutagenic ultraviolet light. CK2 has a pivotal role in the response to ultraviolet radiation from yeast to humans. Consistent with this model, cryptochromes, components of plant and animal circadian systems, are homologous to ultraviolet-dependent DNA repair enzymes (Lin, 2002).

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, it has been shown that Casein kinase 2 (CK2) has a role in determining period length. Andante is a mutation of the gene encoding the ß subunit of CK2 and is predicted to perturb CK2ß subunit dimerization. It is associated with reduced ß subunit levels, indicative of a defect in alpha:ß association and production of the tetrameric alpha2:ß2 holoenzyme. Consistent with a direct action on the clock mechanism, it has been shown that CK2ß 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 (Akten, 2003).

It is of interest that the Andante mutation affects the nuclear entry of clock proteins in the small LNv population, but seems to have no effect on the large LNv neurons. Such a differential effect suggests that CK2 is important for oscillator function in one population but not the other. Indeed, the small LNv cells have been shown to be critical for the clock regulation of activity rhythms; the small cells send projections to a region of the dorsal brain implicated in clock output, and there is a circadian rhythm in the release of the clock output factor pigment dispersing factor (PDF) in these dorsal projections. Furthermore, it has been reported that the small but not the large LNv cells show Tim rhythmicity in constant darkness (DD). Finally, there are differences between the large and small LNv cells, with regard to the timing of Per and Tim nuclear entry. As CK2 deficits seem only to affect nuclear entry in the small LNv neurons, it is possible that this kinase regulates differences between the two neuronal populations (Akten, 2003).

Although it is likely that CK2 acts within clock cells to help specify period length, these studies indicate neither the cellular compartment in which the kinase acts nor the molecular substrates of the enzyme that are relevant for clock function. A delay in the nuclear accumulation of clock proteins, as seen in Andante, suggests that the kinase functions in the cytoplasm to promote nuclear entry. This function might be mediated by direct phosphorylation of a clock protein (such as Per or Tim) or by activation of a second kinase such as GSK-3/Shaggy, which has been implicated in promoting the nuclear entry of Tim. The elevated levels of Per and Tim observed in Andante suggest a decreased turnover of the clock proteins, and this might arise because of a defect in the targeted degradation of one or both proteins within the nucleus. Similar to the Doubletime kinase, CK2 might act both in the cytoplasm and nucleus of clock cells to determine the timing of nuclear entry and/or stability of clock proteins (Akten, 2003).

These studies of CK2 in Drosophila suggest that this kinase might have an important role in the regulation of circadian period in other animal species. Previous studies in the plant Arabidopsis and the fungus Neurospora have also implicated CK2 activity in circadian oscillator function. The Arabidopsis study showed that overexpression of the CK2ß3 subunit is associated with a shortening of circadian period, a result similar to that obtained in this study of Drosophila CK2ß. The Neurospora study showed abnormal phosphorylation of the Frequency (Frq) protein in a Neurospora CK2alpha mutant, but circadian behavior (such as conidiation rhythms) could not be examined in that study because of reduced viability. Although neither of these previous reports characterize a mutant with decreased CK2 activity, the results are consistent with studies of Andante and indicate an evolutionarily conserved role for CK2 in circadian oscillator function (Akten, 2003).

Ribosomal s6 kinase cooperates with casein kinase 2 to modulate the Drosophila circadian molecular oscillator

There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases, and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the mitogen-activated protein kinase pathway, but there is no direct evidence that it functions as a component of the circadian system. This study shows that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, a physical interaction was demonstrated between S6KII and the casein kinase 2 regulatory subunit (CK2beta), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. It is proposed that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism (Akten, 2009).

In vivo circadian function of casein kinase 2 phosphorylation sites in Drosophila PERIOD

Phosphorylation plays a key role in the precise timing of circadian clocks. Daily rhythms of phosphorylation of the Drosophila circadian clock component Period (Per) were first described more than a decade ago, yet little is known about their phosphorylation sites and their function in circadian behavior. Serines 151 and 153 in Per are shown to be required for robust in vitro phosphorylation by the casein kinase 2 (CK2) holoenzyme, a cytoplasmic kinase shown to be involved in circadian rhythms. Mutation of these sites in transgenic flies results in significant period lengthening of behavioral rhythms, altered Per rhythms, and delayed Per nuclear localization in circadian pacemaker neurons. In many respects, mutation of these phosphorylation sites phenocopies mutation of the catalytic subunit of CK2. It is proposed that CK2 phosphorylation at these sites triggers Per nuclear localization (Lin, 2005).

Evidence is provided of a function for Per CK2 phosphorylation sites in circadian clock function and behavior in Drosophila. These phosphorylation sites were initially identified in vitro, and site-directed mutagenesis studies focused attention on three key serines in the N terminus required for robust in vitro phosphorylation of Per by CK2 alpha and the CK2 holoenzyme. This mutant form is still phosphorylated by another kinase, CK1epsilon, and is still able to directly interact with CK2, suggesting that these serines represent in vitro phosphorylation targets for CK2. Mutation of these sites in vivo results in a long circadian period, altered Per rhythms, and delayed Per nuclear entry. Overall, these phenotypes significantly overlap with those observed previously in CK2 mutants (Lin, 2005).

These data contribute significantly to the role of phosphorylation in the regulation of core clock components and in turn the effects on circadian behavior. Despite considerable progress in defining clock components and clock kinases, remarkably little is known about the target phosphorylation sites essential for their function, especially in metazoans. Recent work has defined key serines that are important for CK1epsilon regulation of mouse Per nuclear localization and phosphorylation in cultured cell lines. Although critical serines have been defined, these studies were not performed in the context of a functioning circadian clock. Thus, it is not known whether these sites are essential for clock function. Also, because the studies were performed in cultured cells, it is not clear whether these putative phosphorylation sites are important for circadian behavior. In contrast, this study shows functioning of critical serine targets of CK2 in an in vivo functioning circadian system and on circadian behavior (Lin, 2005).

Although these data argue for a role for CK2 in the phosphorylation of Ser151-153, they cannot exclude the effects of CK2 elsewhere on the Per protein or on other proteins in the circadian clock. Several potential CK2 sites have been identified in Per and in other clock proteins using Phosphobase, raising the possibility that CK2 may work through multiple sites on Per. The in vitro conditions (e.g., absence of Tim) may not fully replicate the in vivo situation and thus miss these other important sites. CK2 action also may not occur exclusively through Per. In vitro phosphorylation of Tim by CK2alpha as well as effects on in vivo Tim rhythms have been reported. Thus, CK2 may affect multiple targets to regulate clock function (Lin, 2005).

Consistent with this hypothesis, period lengthening of perS149-151-153A mutants was observed in a Tik mutant background. in which catalytic activity of CK2alpha is reduced by 50%. Under natural conditions, it is proposed that some fraction of these serines is phosphorylated. In the Tik mutant, it is expected that the activity is reduced but not absent both at this cluster and at other clock-relevant CK2 sites. In the S149-151-153A mutants, these residues cannot be phosphorylated, a more severe situation than that seen in wild type or even Tik. In wild type, normal CK2 phosphorylation elsewhere in Per or in other clock components partially compensates for the loss of these phosphorylation sites. Nonetheless, when CK2 is impaired, as in Tik mutants, an additional period lengthening is still observed, caused by the combination of a complete loss of phosphorylation of these key sites by alanine mutation and reduction of clock-relevant CK2 phosphorylation elsewhere (Lin, 2005).

Moreover, these data do not exclude a function for other kinases in the phosphorylation of Ser149-151-153. Serine 149 also represents a potential GSK3/SGG site. Although in vitro data fail to show GSK3 phosphorylation at this site, it is possible that it is a true in vivo target. Given the similarity of perS149-151-153A and CK2 mutant phenotypes, it is proposed that Ser149-151-153 represents one cluster of these in vivo phosphorylation sites that is phosphorylated, at least, by CK2 (Lin, 2005).

Based on these findings, it is proposed that the distinct CK2 sites present in D. melanogaster Per but not in other closely related species, such as D. pseudoobscura, may represent the basis for species-specific aspects of circadian function. Such variation has been proposed to underlie the process of allochronic speciation. Temporal isolation through the use of species-specific circadian programs may serve as a barrier to gene flow and thus facilitate speciation. Indeed, species-specific differences in locomotor activity and mating behavior have been observed between D. melanogaster and other Drosophila species. By using cross-species rescue of per01 behavior, many of these changes have been attributed to variation in the per gene, i.e., the species of per determines the nature of diurnal, circadian, and/or mating behavior (Lin, 2005).

It is proposed that variation in phosphorylation sites may underlie these species differences in behavior. A comparison of different Drosophila Pers reveals that Ser149-151-153 is part of one of the nonconserved modules of Per. This small region within the larger module is well conserved only within the melanogaster subgroup. Of note, this cluster is conserved in D. ananassae, in which the remainder of the module is not as well conserved, consistent with an underlying function. The single change of Ser153 to glutamate, a phosphomimetic residue, is also consistent with a function in phosphorylation (Lin, 2005).

Species specificity of clock function has also been noted at the molecular level in terms of Per nuclear localization. Of note, Per is predominantly cytoplasmic in the putative pacemaker neurons of the silkmoth (Antherea pernyi), beetle, and hawkmoth. It is interesting that the CK2 sites described in this study are also not conserved with Antherea Per, and mutation of these serines alters Per nuclear entry, a process apparently present in melanogaster but not the silkmoth. It is proposed that nuclear entry triggered by CK2 phosphorylation of Per may be a species-specific aspect of the clock program (Lin, 2005).

The F-box protein Slimb controls the levels of clock proteins Period and Timeless

The Drosophila circadian clock is driven by daily fluctuations of the proteins Period and Timeless, which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments as well as Per/Tim nuclear transfer. However, the pathways regulating degradation of phosphorylated Per and Tim are unknown. The product of the slimb (slmb) gene -- a member of the F-box/WD40 protein family of the ubiquitin ligase SCF complex that targets phosphorylated proteins for degradation -- is shown to be an essential component of the Drosophila circadian clock. slmb mutants are behaviorally arrhythmic, and can be rescued by targeted expression of Slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms (Grima, 2002).

To test whether the SCF-mediated ubiquitin proteasome pathway is involved in the control of Per and Tim oscillations, circadian rhythms were examined of flies defective for genes encoding F-box proteins that are known to target phosphorylated substrates for degradation. The slimb (slmb) gene, which encodes an F-box/WD40 protein regulating transcription factors' levels in the wingless and hedgehog signaling pathways was examined. slmb8 mutants that normally die as early larvae were brought to adulthood by providing the slmb gene product throughout development under the control of a heat-shock promoter. The rescued HS-slmb slmb8 adult flies, hereafter referred to as slmbm mutants, were then tested for their locomotor activity rhythms in both light:dark (LD) and constant darkness (DD) conditions. slmbm mutants were completely arrhythmic in DD, whereas the heterozygous genotype displayed wild-type rhythms. The absence of anatomical defects of the PDF-expressing ventral lateral neurons (LNvs), which control the behavioral rhythms, strongly argues against a developmental origin of the mutants' rhythm defect. Furthermore, targeted slmb expression using well characterized LNvs-specific gal4 drivers restores near wild-type activity rhythms, whereas similarly targeted overexpression in a wild-type background lengthens the circadian period, indicating a cell-autonomous role of the slmb gene in circadian rhythmicity. In LD conditions, slmbm mutants did not display the light-off anticipation of activity that characterizes a functional clock, whereas it was observed in the flies expressing slmb under the LNvs-specific gal1118 driver. These experiments identify the F-box/WD40 protein Slmb as an essential component of the Drosophila brain clock (Grima, 2002).

To understand how Slmb might affect the circadian oscillator, slmbm mutants were analyzed for Per and Tim oscillations in the head. In wild-type flies maintained in LD cycles, Per and Tim proteins accumulate and are progressively phosphorylated during night time, with Tim disappearing at the end of the night whereas hyper-phosphorylated Per persists for a few hours in the morning. A similar temporal pattern persists in DD, and is required to sustain behavioral rhythmicity. In contrast, highly phosphorylated Per and Tim are present at all circadian times in slmbm mutants kept in DD, although low-amplitude oscillations of the hypo-phosphorylated forms indicate a weak residual activity of the molecular clock. In agreement with the persistence of weak protein cycling in slmbm heads, levels of per and tim transcripts displayed low-amplitude oscillations. Per immunoreactivity was examined in the LNvs that control behavioral rhythms. At circadian time (CT) 0 and CT 12, which correspond to the peak and trough of Per labelling in w flies at 20°C, slmbm mutants showed low levels of Per immunoreactivity, indicating that the oscillations of the proteins levels are also abolished in the clock cells. To determine whether Slmb acts at the protein level or through a transcriptional control, per was constitutively overexpressed through a transgene. High-molecular-mass Per proteins were observed to accumulate in head extracts of slmbm but not of wild-type flies carrying GMR-gal4 and UAS-per transgenes that drive strong Per expression in the eye. Altogether, these data indicate that Slmb is involved in the control of phosphorylated Per levels (Grima, 2002).

In LD conditions, Per and Tim degradation in the morning is driven by both the circadian cycle and by light. Light-induced Tim degradation involves ubiquitinylation of the protein, and is blocked by proteasome inhibitors. To test whether Slmb is involved in the light-induced degradation pathway of the clock proteins, Per and Tim levels were assayed in slmbm flies kept in LD conditions. In contrast to constant darkness, robust oscillations of Per and Tim amounts were observed in LD, with both proteins accumulating during the night and showing a strong day-time decrease. This shows that light-induced Per and Tim degradation does not occur through the same slmb-dependent mechanism as their circadian-cycle-controlled degradation in constant darkness. In addition, the absence of light-off anticipation in the slmbm activity profiles suggests that the mutants' altered temporal regulation of phosphorylated Per and Tim does not allow rhythmic outputs to be driven, although protein levels clearly cycle (Grima, 2002).

Clock-dependent Per and Tim degradation occurs at the end of the circadian cycle, and relieves the transcriptional repression that the proteins exert on their own genes. Per degradation has also been proposed to take place during the rising phase of the protein levels in the early night, and to be responsible for the shift (of 5 h) between per messenger RNA and Per protein peaks. In order to determine whether Slmb levels vary during a circadian cycle and may therefore affect Per and Tim only during a limited time window, anti-Slmb antibodies were raised and the Slmb protein was followed in head extracts at different circadian times. A strongly reacting protein, as well as a faintly reacting one slightly above, were detected at a relative molecular mass of 45,000 (Mr 45K) in wild-type flies, and did not show any oscillations of their levels over a 24-h time course. Similarly, slmb mRNA did not show any cycling. Slmb therefore appears not to be circadianly regulated, and could therefore act on different steps of the cycle (Grima, 2002).

Both early- and late-night Per degradation steps appear to depend upon Per phosphorylation, which requires the casein kinase I encoded by the double-time (dbt) gene. To find out how Slmb could affect Per and Tim phosphorylation, Tests were performed to see whether Dbt, and Shaggy (Sgg), that has been shown to phosphorylate Tim, are affected in slmbm mutants. No alterations of the level or the mobility of these kinases were detected in slmbm head extracts. Next, whether Slmb could associate with the Per protein was examined, by searching for Per-Slmb interactions in co-immunoprecipitation experiments on head extracts. The Slmb protein was found to be co-precipitated by anti-Per antibodies, and anti-Slmb can precipitate Per in wild-type flies collected at CT 0. Similar results were obtained with pooled extracts. In addition, Slmb co-precipitates with Dbt . Because Per, but not Dbt, is profoundly affected in slmbm mutants, these results support Per rather than Dbt as a Slmb target for ubiquitinylation, and suggest that the three proteins constitute a complex. Slmb was co-immunoprecipitated by anti-Per antibodies in tim0 flies, indicating that Per-Slmb complexes can form in the absence of Tim. Although twice as much extract was used for tim0 flies to compensate for the low Per levels in this genotype, the amount of immunoprecipitated Slmb suggests that the absence of Tim may favor Per-Slmb complexes. These results fit well with Slmb being involved in the control of unbound Per, either during its cytoplasmic accumulation at the beginning of the protein cycle or during its nuclear degradation at the end. To test whether the formation of Per-Slmb complexes is circadianly controlled, co-immunoprecipitations were performed at the beginning of the night when Per is mostly hypo-phosphorylated, or at the end of the night when Per is highly phosphorylated. All time points showed comparable levels of Per-Slmb complexes, and several forms of Per were immunoprecipitated by the anti-Slmb antibodies (compare CT 1 and CT 13). This indicates that differently phosphorylated Per molecules can be committed to Per-Slmb complexes (Grima, 2002).

Possible explanations for the accumulation of highly phosphorylated Per in slmbm mutants would be that partially phosphorylated Per is the relevant Slmb substrate for degradation, or that Slmb targets some Per kinase that is bound to Per. The presence of highly phosphorylated Per in slmbm indicates that Slmb is required for the control of phosphorylated Per accumulation in the early night. Moreover, Slmb overexpression in the LNvs results in a lengthening of the circadian period. In agreement with the behavioral data, Slmb overexpression slows down the oscillations of Per immunoreactivity in these cells, which showed a ~6 hour delay compared to wild-type controls after two days. These data can be explained by high levels of cytoplasmic Slmb inducing too much degradation of cytoplasmic Per, thus further delaying the night accumulation of the protein, whereas high levels of nuclear Slmb would rather precipitate the fall of the Per protein and shorten the circadian period. It is therefore thought that Slmb is, at least, involved in the control of cytoplasmic Per accumulation in the early night (Grima, 2002).

The presence of low-mobility Tim proteins at all circadian times in slmbm mutants indicates that the accumulation of phosphorylated Tim is also Slmb-dependent. Remarkably, the Tim kinase Sgg controls the Slmb-dependent proteolysis of Cubitus interruptus and degradation of Armadillo. The results suggest that phosphorylated Tim could be a Slmb target or that Tim is phosphorylated by a Slmb-dependent kinase. Because Tim is hypo-phosphorylated in per0 flies, it is also possible that the accumulation of hyper-phosphorylated Per in slmbm influences Tim phosphorylation (Grima, 2002).

Although protein degradation is commonly believed to have a major role in the control of the oscillations of clock proteins, the present work is the first to implicate a characterized component of the ubiquitin proteasome pathway. Because cycling of phosphorylated Per proteins also occurs in the mammalian clock, it would be interesting to determine whether the Slmb mammalian homolog ß-Trcp is involved in the control of phosphorylated Per levels. F-box proteins have been shown to be important at the G1/S transition of the cell cycle, by targeting phosphorylated cyclins and inhibitors of cyclin kinases for degradation by the proteasome. This study therefore suggests that the cell-cycle and the circadian-clock machineries share mechanisms to control the oscillations of phosphorylated proteins (Grima, 2002).

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 homolog of vertebrate casein kinase Iepsilon. However, it has not been clear how Dbt regulates the levels of Per. Using a cell culture system, this study shows 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. These findings suggest that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb (Ko, 2002).

Sequential nuclear accumulation of the clock proteins Period and Timeless in the pacemaker neurons of Drosophila melanogaster

In Drosophila, two intersecting molecular loops constitute an autoregulatory mechanism that oscillates with a period close to 24 hr. These loops touch when proteins from one loop, Period (Per) and Timeless (Tim), repress the transcription of their parent genes, period and timeless, by blocking positive transcription factors from the other loop. The arrival of Per and Tim into the nucleus of a clock cell marks the timing of this interaction between the two loops; thus, control of Per:Tim nuclear accumulation is a central component of the molecular model of clock function. If a light pulse occurs early in the night as the heterodimer accumulates in the nucleus of clock cells, Tim is degraded, Per is destabilized, and clock time is delayed. Alternatively, if Tim is degraded during the later part of the night, after peak accumulation, clock time advances. Current models state that the effect of a light pulse depends on the state of the Per:Tim oscillation, which turns on the changing levels of Tim. However, previous studies have shown that light:dark (LD) regimes mimicking seasonal changes cause behavioral adjustments while altering clock gene expression. This should be reflected in the adjustment of Per and Tim dynamics. LD cycles were manipulated to assess the effects of altered day length on Per and Tim dynamics in clock cells within the central brain as well as light-induced resetting of locomotor rhythms (Shafer, 2004).

Nuclear accumulation profiles of Per and Tim respond in qualitatively different ways to changes in day length. The pattern of Tim accumulation during the early night is very similar within the range of photoperiods tested here; Tim levels are negligible at lights-off, cytoplasmic levels peaked by about 6 hr later, and obvious nuclear Tim is evident by 8-10 hr after lights-off. During night lengths longer than 10 hr, Tim levels begin to fall by 12 hr after lights-off. Under LD 10:14, 8:16, and 6:18, Tim profiles indicate that expression is not altered by increased night length. Thus, under night lengths longer than 12 hr, nuclear Tim begins to wane in anticipation of dawn. A dramatic exception to this pattern was discovered in very short night conditions. With only 6 hr of darkness, no Tim immunoreactivity is detected in the small LNv's during the night (Shafer, 2004).

Unlike Tim, nuclear accumulation of Per reflects the environmental photoperiod. For instance, whereas Per immunoreactivity reachs peak values by 8 hr after lights-off under LD 8:16, under LD 16:8 peak values are not reached until 10 hr after lights-off, meaning that Per's accumulation extends into the day. Nuclear Per is clearly increased in the small LNv's under short-night conditions. Such compensatory Per accumulation is apparent in all of the photoperiods examined. The net effect of this adjustment is the attainment of similarly low levels of nuclear Per by the end of the day for all the photoperiods tested. Given the suggestion that the decline in Per levels allows the resumption of per and tim transcription, such adjustment of Per levels would ensure that the relief of repression occurs in anticipation of lights-off, under a wide range of LD cycles. Thus, nuclear accumulation profiles of Per and Tim are different both within and between the photoperiodic regimes examined in this study (Shafer, 2004).

The results cast doubt on the requirement of Tim for Per nuclear entry and stability. For example, in LD 16:8 Per levels continue to rise steeply during the 2 hr after lights-on even as Tim levels are falling. This finding differs from another set of observations, that noted the longevity of Per after it had accumulated in a tim mutant background. Moreover, another study has shown that in conditions of constant light, per mRNA and protein continues to be rhythmic, whereas no rhythmicity is evident in the products of timeless. Furthermore, another study suggests that Per is not required for Tim nuclear transport but rather prevents the export of Tim from the nucleus once it has entered. Thus, the current results are inconsistent with the proposal of an obligate heterodimer for nuclear accumulation and with the suggestion of a requirement for stoichiometric amounts of Tim to avoid degradation (Shafer, 2004).

The data suggest that Tim and Per rhythms need not occur in the same cell. Under the 6 hr night condition, small LNv's did not express detectable levels of Tim, yet they show a measurable oscillation in nuclear Per accumulation. The differences observed between cells suggest that the functional role of these molecules depends on their cellular context. Just as timing mechanisms may differ between peripheral and central circadian clocks, so it is possible that data obtained from whole-head homogenates might not apply to the regulation of behavior, especially in light of the fact that most of the relevant proteins assayed in these experiments likely come from the compound eye. In this regard, it is worth noting the recent demonstration that the small and large LNv's could be differentially regulated based on their respective light input pathways (Shafer, 2004 and references therein).

Tim levels are dramatically truncated in the short night lengths, whereas Per is present in the nucleus throughout the day. Tim's sensitivity to night length makes it a better candidate than Per for involvement in photoperiodic time measurement, and it is inferred that Per is the workhorse clock factor for control of day-to-day rhythmicity. Previous studies established that Per function is dispensable for the photoperiodic induction of diapause in Drosophila. The effect of short-night conditions on Tim accumulation is consistent with the hypothesis that this element of the circadian clock is also involved in photoperiodic time measurement (Shafer, 2004).

Studies on the adult eclosion rhythm of Drosophila under a wide range of photoperiod conditions have shown that the timing of the overt rhythm systematically shifts relative to lights-off while the phase of the phase response curve is locked to the lights-off signal. The same relationship is observed for the locomotor rhythm; the evening activity peak shifts markedly relative to lights-off under LD conditions ranging from 16:8 to 6:18. However, throughout this range of photoperiods the setting of the phase response curve is essentially the same. The mechanism by which a change in photoperiod can shift the relationship of the phase response curves and the overt rhythm has not been addressed. The differing responses of Per and Tim to changes in photoperiod represent an explanation for how some aspects of clock function (i.e., responses to phase-shifting light stimuli) remain unchanged in the face of varying day length, whereas others (such as clock gene mRNA levels and cyclical behavior) are adjusted for such environmental changes (Shafer, 2004).

PER-TIM interactions in living Drosophila cells: An interval timer for the circadian clock

In contrast to current models, fluorescence resonance energy transfer measurements using a single-cell imaging assay with fluorescent forms of Per and Tim showed that these proteins bind rapidly and persist in the cytoplasm while gradually accumulating in discrete foci. After ~6 hours, complexes abruptly dissociated, as Per and Tim independently moved to the nucleus in a narrow time frame. The perL mutation delays nuclear accumulation in vivo and in a cultured cell system, but without affecting rates of Per/Tim assembly or dissociation. This finding points to a previously unrecognized form of temporal regulation that underlies the periodicity of the circadian clock (Meyer, 2006).

In Drosophila, Per and Tim are two essential proteins of the circadian clock that shift from the cytoplasm of clock cells to the nucleus once a day, promoting ~24-hour oscillations of per and tim transcription. They do this in a regulated manner, and the period length of Drosophila's circadian rhythm is in part determined by how long these proteins are held in the cytoplasm before entering the nucleus (Meyer, 2006).

Formation of Per/Tim heterodimers appears to promote the nuclear accumulation of both proteins. In vivo, a 4- to 6-hour delay in Per nuclear accumulation may be influenced by the slow cytoplasmic assembly of Per/Tim heterodimers, such that once formed, the Per/Tim heterodimer is rapidly transferred from the cytoplasm to the nucleus. It is thought that in the nucleus Per physically interacts with Clock and Cycle, transcriptional activators of per and tim, inhibiting Clock/Cycle activity and hence closing a delayed feedback loop that contributes to oscillating RNA and protein levels (Meyer, 2006).

Recently, the proposal that Per and Tim translocate to the nucleus as obligate heterodimers, and even the necessity of Tim for Per's nuclear accumulation, have been questioned. To follow Per and Tim during their passage from the cytoplasm to the nucleus and to determine the role of Per/Tim interaction in the regulation of nuclear accumulation, a single-cell, fluorescent, live-imaging assay was developed using a Drosophila cell line (Schneider's line 2, S2). Although S2 cells do not express several clock genes and are not rhythmic, this cultured cell system has become an important tool for investigating intracellular mechanisms contributing to Drosophila's circadian clock (Meyer, 2006).

C-terminal fusions of Per and Tim were constructed with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), respectively, and these were monitored separately or together in S2 cells. Expression of per-cfp (without Tim) was followed in two cell lines. In one line, Per-CFP production was controlled by a heat shock promoter. These cells were constantly monitored for 10 hours after induction (~100 cells in 10 independent experiments). In the second line, an actin promoter drove per-cfp expression, and 40 cells in two experiments were followed for 10 hours after transfection. Per-CFP was detected only in the cytoplasm of live S2 cells in both studies. In a third study, cells were followed in which tim-yfp was driven by a heat shock promoter in the absence of Per (130 cells in 10 experiments). Tim was retained in the cytoplasm in most but not all cells 10 hours after induction (123 cytoplasmic, 7 nuclear). In contrast, when cotransfected, per-cfp and tim-yfp gave predominantly nuclear fluorescence for both proteins in most cells (209 nuclear out of 265 cells monitored in 39 experiments at 8 hours after induction). The behavior of the proteins in the S2 cell system is therefore concordant with in vivo findings and indicates that the fluorescent tags do not detectably interfere with either cytoplasmic retention of the individually expressed proteins or with interactions that promote nuclear accumulation (Meyer, 2006).

To evaluate the validity of an existing model in which rates of Per/Tim interaction affect the timing of their nuclear translocation, Per-CFP/Tim-YFP fluorescence resonance energy transfer (FRET) measurements were compared dynamically by continuously imaging CFP and YFP in live single cells. Maximum levels of FRET were reached during the earliest stages of Per-CFP and Tim-YFP accumulation (within 30 min of Per and Tim production), indicating that physical interaction followed Per-CFP/Tim-YFP synthesis without a measurable delay. Moreover, high levels of FRET were maintained for several hours preceding the onset of nuclear accumulation of Per and Tim. Unexpectedly, FRET declined rapidly as Per and Tim proteins were transferred from the cytoplasm to the nucleus. As Per and Tim became predominantly nuclear, FRET levels remained low in all subcellular compartments, which were typically monitored for a further 100 min (Meyer, 2006).

Immediately following coinduction, Per and Tim were always diffusely present in the cytoplasm. However, this largely uniform distribution was followed by a gradual accumulation in prominent cytoplasmic foci. These foci remained in the cytoplasm until Per and Tim translocated to the nucleus. Notably, the formation of foci was not observed when either Per or Tim was expressed alone. In addition, when Per and Tim were coexpressed, the foci often disappeared earlier in Per-CFP images than in Tim-YFP images. Thus, formation of these foci may be an important step in the temporal control of nuclear entry (Meyer, 2006).

The abrupt decrease in FRET upon nuclear translocation could reflect either dissociation or a change in conformation of the Per-CFP/Tim-YFP complex. To differentiate between these two possibilities, the rates of nuclear accumulation for Per and Tim were independently measured. If Per and Tim undergo a conformational change but remain physically associated as nuclear translocation occurs, individual rates of Per and Tim nuclear accumulation should be equal (Meyer, 2006).

In a survey of 85 cells, it was found that the onset of nuclear accumulation, determined as the inflexion point of the nuclear accumulation profile for Per-CFP, occurred in a narrow time frame, 340 ± 70 min after heat shock in S2 cells. Consistent with the observation that Per and Tim associate rapidly and that these association kinetics have no influence on the onset of nuclear translocation in the S2 cell system, it was found that the time of onset of nuclear accumulation in these experiments was not correlated with the level of Per-CFPor Tim-YFP expressed in the cytoplasm. To determine whether the kinetics of the nuclear accumulations of Per-CFP and of Tim-YFP were similar, the rate of each protein's nuclear accumulation was calculated as the coefficient of a first-order linear regression. The latter was taken from the steepest slope of the profile of nuclear translocation, scaled to the mean fluorescence in each cell. It was found that the rates of nuclear accumulation of Per-CFP and Tim-YFP were independent. Also, although the rate of accumulation of Per-CFP was positively correlated with the level of Per-CFP, this rate was independent of the level of Tim-YFP produced in the same cell. Similarly, Tim-YFP accumulation rates were correlated with the Tim-YFP level, but not with the Per-CFP level in the same cell (Meyer, 2006).

One issue that is not resolved by measuring these accumulation rates is whether the Per/Tim complex dissociates before or after traveling to the nucleus. Comparisons of Per and Tim nuclear translocations within individual cells reveal that onset of Per nuclear accumulation often precedes that of Tim, as has been reported in vivo. Earlier work has shown that, in the absence of Per, Tim shuttles between the nucleus and cytoplasm through the action of both nuclear localization and nuclear export signals. Possibly, Tim transports Per to the nucleus in a complex, after which the proteins separate, allowing Tim to return to the cytoplasm to transport more Per (Meyer, 2006).

To determine whether this property of Tim contributes to the independent rates of Per and Tim nuclear translocation observed in these studies, leptomycin B was used to block Tim-YFP nuclear export. In the presence of this inhibitor of nuclear export, for cells expressing only Tim-YFP, the protein was constitutively localized to the nucleus in most cells (45 cells out of 50 surveyed). In contrast, in cells expressing only Per-CFP, Per remained in the cytoplasm (50 out of 50 cells) in the presence of the drug. Intriguingly, addition of leptomycin B to cells coexpressing Per-CFP and Tim-YFP suppressed the rapid transfer of Tim-YFP to the nucleus. Instead, both proteins were sequestered in the cytoplasm for several hours before nuclear translocation, as previously observed in the absence of drug. Evidently, even in the presence of leptomycin B, Tim is retained by its interaction with Per. Addition of leptomycin B also failed to modify the divergent profiles of Per and Tim nuclear accumulation; rates of Per and Tim nuclear accumulation remained uncorrelated in a study of these cells. The latter finding indicates that although Tim shuttling between the nucleus and cytoplasm has been confirmed, this mechanism cannot explain the independent rates of Per-CFP and Tim-YFP nuclear accumulation that were observed. The measurements hence favor an alternative mechanism for nuclear translocation wherein most of the cytoplasmically derived complexes dissociate in the cytoplasm as the proteins translocate to the nucleus (Meyer, 2006).

The perL mutation produces a delayed nuclear translocation phenotype in pacemaker cells of the Drosophila brain. This results in long-period behavioral rhythms of ~28 hours. perL involves a single amino acid substitution, and it also depresses the physical interaction of PerL and Tim when the proteins are coexpressed in yeast. The timUL mutation is associated with a distinct single–amino acid substitution that delays Per and Tim nuclear turnover, resulting in a 33-hour behavioral rhythm. In contrast to perL, timUL has no effect on the timing of nuclear translocation in vivo (Meyer, 2006).

The mean onset of Per-CFP nuclear accumulation in cells coexpressing Per-CFP and TimUL-YFP is 299 ± 33 min (20 cells), and it is also independent of Per-CFP and TimUL-YFP levels. Furthermore, no persistent FRET was found when Per-CFP and TimUL-YFP moved to the nucleus: FRET decay was not delayed when compared to the onset of nuclear accumulation. A loss of FRET was observed with TimUL in parallel with nuclear translocation, which suggests that, as for wild-type Tim, TimUL/Per heterodimers dissociate as nuclear translocation proceeds in this mutant. Previous studies have shown that, in timUL mutants, Per is found in high molecular weight complexes late at night when it is presumably nuclear. The possibility cannot be ruled out that, following translocation, Per and Tim form new associations that do not support FRET in the nucleus in both wild-type and TimUL-expressing cells (Meyer, 2006).

S2 cells reproduce the delay in nuclear translocation onset when PerL is expressed in place of Per. In PerL-expressing cells, the mean onset of nuclear accumulation was at 492 ± 97 min after induction, as compared with 340 ± 70 min in Per-expressing cells (25 cells. The onset of Per and Tim nuclear accumulation remained independent of PerL-CFP and Tim-YFP levels. The profiles of nuclear accumulation of these proteins also indicated significant independence in their rates of translocation. FRET decayed as PerL-CFP and Tim-YFP were transferred to the nucleus, and has been seen from Per/Tim combinations, maximum levels of FRET arose without a measurable delay in cells expressing PerL.This result was not predicted by earlier models, which assumed that an altered rate of PerL and Tim physical association chiefly determines the temporal delay found in nuclear accumulation. Because nuclear translocation instead followed a protracted interval of maximum FRET in PerL-expressing cells, a step distinct from Per/Tim assembly appears to trigger nuclear translocation in S2 cells and is likely also responsible for delayed nuclear translocation in vivo (Meyer, 2006).

These studies indicate that cytoplasmically formed Per/Tim complexes are not translocated to the nucleus: FRET disappears in parallel with Per and Tim nuclear accumulation, suggesting a dissociation of the complex, and measurements of Per and Tim nuclear accumulation rates show that, for a given cell, these are different and independent for each protein. Because Per/Tim associations are not sufficient to initiate nuclear accumulation, these results point to a mechanism in which physical interaction precedes an activity that precisely times nuclear translocation of both proteins. In this respect, Per and Tim appear to act as constituents of an intracellular interval timer. A better understanding of this timer might be sought in the discrete cytoplasmic foci observed to routinely precede nuclear translocation. These foci may reflect condensations of cytoplasmic Per/Tim complexes together with additional factors responsible for their posttranslational modifications. Such factors could include the kinases SGG, DBT, and CK2 or the phosphatase PP2A, each known to affect the phosphorylation of Per and Tim and to influence the timing of nuclear translocation in vivo (Meyer, 2006).

Light entrainment of the mammalian circadian clock by a PRKCA-dependent posttranslational mechanism

Light is the most potent stimulus for synchronizing endogenous circadian rhythms with external time. Photic clock resetting in mammals involves cAMP-responsive element binding protein (CREB)-mediated transcriptional activation of Period clock genes in the suprachiasmatic nuclei (SCN). Evidence is provided for an additional photic input pathway to the mammalian circadian clock based on Protein Kinase C alpha (PRKCA). Prkca-deficient mice show an impairment of light-mediated clock resetting. In the SCN of wild-type mice, light exposure evokes a transient interaction between PRKCA and PERIOD 2 (PER2) proteins that affects PER2 stability and nucleocytoplasmic distribution. These posttranslational events, together with CREB-mediated transcriptional regulation, are key factors in the molecular mechanism of photic clock resetting (Jakubcakova, 2007).

The following scenario is suggested through which PRKCA might affect entrainment of the circadian pacemaker. At the onset of (the subjective) night, SCN cells express high levels of PER and CRY that together represent the negative limb of the circadian feedback loop. Providing a light pulse to a mouse at this time of the day results in the formation of a pool of PER2/PRKCA (and possibly PER2/CRY/PRKCA) in the cytoplasm. In this complex, PRKCA stabilizes PER2. This combination of a transient stabilization and cytoplasmic retention of PER2 has the effect of prolonging the negative feedback. Hence, reactivation of the positive limb of the circadian clock (CLOCK and BMAL1) is delayed. At the behavioral level, this prolongation of the negative feedback causes a phase delay on the day following the light pulse. This model would predict that if cytoplasmic retention and stabilization of PER2 were reduced (as is the case in Prkca−/− mice) the magnitude of light-induced phase delays should be smaller, which is what was observed (Jakubcakova, 2007).

PRKCA is not the sole kinase phosphorylating PER proteins, and conversely, PERs are not the sole PRKC targets. The emblematic tau hamster carries a point mutation in the casein kinase 1 epsilon gene (Csnk1e) and is characterized by a shortened period length and increased degradation of PER1 and PER2. By contrast, the stabilization of PER2 by PRKCA has no consequences on the period length, but rather plays a role in photic clock resetting. Glycogen synthase kinase-3β also phosporylates PER2 in vitro. Whether this translates into a circadian phenotype is not known. PRKCA and PRKCG can phosphorylate CLOCK, and kinase inhibitor studies in NIH3T3 cells suggest that CLOCK mediates phase resetting in these cells through the activation of Per1 (Jakubcakova, 2007).

The clock of Neurospora crassa provides a remarkable analogy to the finding of a posttranslational role of a PRKC in photic entrainment. During the dark phase the Neurospora transcription factor 'white-collar-1' (WC1), which regulates expression of the light-inducible clock gene frequency, is associated with PRKC. A light pulse evokes a rapid dissociation of the kinase from WC1, followed by the activation of the frequency gene. Although in mammals, PRKCA binds to the core clock component PER2 as a result of photic stimulation, while in Neurospora, PRKC dissociates from WC1, regulation is posttranslational in both species and brings about clock resetting. Taken together, this work affirms the existence of posttranslational mechanisms controlling light entrainment of the circadian clock in mammals, and thus demonstrates the evolutionary conservation of this type of regulation among all organisms that have a circadian clock (Jakubcakova, 2007).

Post-translational regulation of the Drosophila circadian clock requires protein phosphatase 1 (PP1): PP1 directly dephosphorylates and stabilizes Tim, which promotes accumulation of Per

Phosphorylation is an important timekeeping mechanism in the circadian clock that has been closely studied at the level of the kinases involved but may also be tightly controlled by phosphatase action. This study demonstrates a role for protein phosphatase 1 (PP1) in the regulation of the major timekeeping molecules in the Drosophila clock, Timeless (Tim) and PERIOD (PER). Flies with reduced PP1 activity exhibit a lengthened circadian period, reduced amplitude of behavioral rhythms, and an altered response to light that suggests a defect in the rising phase of clock protein expression. On a molecular level, PP1 directly dephosphorylates Tim and stabilizes it in both S2R+ cells and clock neurons. However, PP1 does not act in a simple antagonistic manner to Shaggy (Sgg), the kinase that phosphorylates Tim, because the behavioral phenotypes produced by inhibiting PP1 in flies are different from those achieved by overexpressing Sgg. PP1 also acts on Per, and Tim regulates the control of Per by PP1, although it does not affect PP2A action on Per. A modified model is proposed for post-translational regulation of the Drosophila clock, in which PP1 is critical for the rhythmic abundance of Tim/Per while PP2A also regulates the nuclear translocation of Tim/Per (Fang, 2007).

Little is known about the potential action of protein phosphatases in the clock. Unlike the large number of protein kinases (~400) in eukaryotes, there are only ~25 protein phosphatases. Of these, protein phosphatase 2A (PP2A) and PP1 together contribute ~90% of the total serine/threonine phosphatase activity in mammalian cells. PP2A dephosphorylates Per, thereby stabilizing Per and promoting its nuclear translocation. This study examined whether PP1 also has a circadian function. PP1 is a ubiquitous eukaryotic enzyme and plays an important role in many cellular processes, including metabolism, cell cycle, muscle relaxation, and synaptic plasticity. In Drosophila, four genes encode a catalytic subunit of PP1 (PP1c) and are named according to their chromosomal loci: 9C (also called flapwing, flw), 13C, 87B, and 96A. PP1c is highly conserved across species, and the four Drosophila PP1c isoforms are ~90% identical to each other at the amino acid level with indistinguishable activities in vitro. Most PP1 targets and associated proteins contain a conserved PP1c-binding motif, [R/K]-X0-1-[V/I]-{P}-[F/W] (where X denotes any residue and {P} any residue except proline, so-called RVxF motif), which is also found in Tim (RAIGF, amino acids 77-81). This prompted a test of Tim as a possible PP1 target. This study shows that PP1 dephosphorylates and stabilizes Tim, which is a prerequisite for the rhythmic abundance of Tim/Per, and thus plays an essential role in the post-translational regulation of the Drosophila clock (Fang, 2007).

PP1 directly dephosphorylates and stabilizes Tim, which promotes accumulation of Per. The reduction in Tim/Per abundance caused by PP1 inhibition, somewhat resembling a response to continuous dim light, generates a long period coupled with a reduced circadian rhythmicity phenotype (Fang, 2007).

Inhibition of PP1 in flies significantly decreases Tim abundance, especially Tim accumulation in the nucleus, but the onset of Tim nuclear entry appears intact. In cell culture experiments as well it was found that neither PP1c nor nuclear inhibitor of PP1 (NIPP1) affects the subcellular localization of Tim. Thus, although PP1 regulates Tim stability, it does not play a major role in trigging the nuclear entry of Tim, suggesting that additional regulation is required to initiate nuclear translocation. This is consistent with the finding that the onset of nuclear accumulation of Tim and Per is not correlated with their protein levels in the cytoplasm. Moreover, Tim protects Per from inhibition of PP1 but not of PP2A, which allows Tim-stabilized Per to undergo further phosphorylation/dephosphorylation. Since dephosphorylation of Per by PP2A promotes nuclear translocation of Per and nuclear expression of Tim appears to depend on Per, it is concluded that, while PP2A primarily targets Per and controls the timing of Tim/Per nuclear translocation, PP1 plays a central role in stabilizing Tim and Per and regulating their rhythmic abundance (Fang, 2007).

NIPP1-overexpressing flies have a specific early- to mid-night defect, and overexpression of NIPP1 produces an additive effect on period lengthening in a timUL background. It is inferred that the destabilizing effect of NIPP1 during the accumulation (rising phase) does not affect the action of the timUL mutation, which increases Tim stability specifically in the nucleus during the late night (falling phase). The regulation of Tim stability may involve different mechanisms at different times of the cycle, which is also implied by the different mechanisms used for light-independent and light-triggered Tim degradation. These data also suggest that opposite effects on Tim stability can have the same effect on circadian period if they occur at different times of day; in this case, longer periods are produced either by decreased Tim stability during the rising phase (as produced by inhibition of PP1) or by increased Tim stability during the falling phase (produced by the timUL mutation) (Fang, 2007).

Although inhibition of PP1 does not lead to a significant mobility shift, direct dephosphorylation of Tim by PP1 is suggested by the in vitro phosphatase and co-IP assays as well as by the 32P metabolic labeling in S2R+ cells. A mobility change of Tim is observed in Sgg-overexpressing flies, in which Tim nuclear entry is advanced but Tim stability is not significantly decreased. Hence, although PP1 dephosphorylates GSK-3β-phosphorylated Tim in vitro, their target sites may not completely overlap; in addition, the functions of PP1 and Sgg in regulating the clock are not simply antagonistic. It is likely that different phosphorylation sites on Tim mediate different cellular processes and are regulated by different mechanisms. A similar idea has been proposed for the regulation of Drosophila and mammalian Per (Fang, 2007).

Based on these findings, a modified model is proposed for the post-translational regulation of the Drosophila clock by multiple phosphorylation events: Once translated, Tim and Per proteins are subject to modifications including phosphorylation, which targets them for proteasome-mediated degradation. PP1 dephosphorylates Tim at one or a small number of 'stability-critical' phosphorylation sites that enable Tim to accumulate in the cytoplasm. The stabilized Tim binds to and stabilizes Per in the cytoplasm. Per is further stabilized by PP2A, which also promotes Per nuclear translocation. Tim nuclear expression is promoted by Sgg phosphorylation, which does not have a major effect on Tim stability, likely because the 'stability-critical' phosphorylation site is protected by PP1. Tim/Per are continually stabilized by PP1 during their nuclear translocation and accumulation, and they then inhibit their own transcription by repressing Clk and Cyc. However, the data do not exclude additional indirect PP1 regulation of Tim/Per as reported for PP5 in the mammalian clock, nor do they rule out the involvement of additional clock target(s) of PP1 (Fang, 2007).

Although PP1 is no longer viewed as a simple housekeeping gene, a steady state of PP1c levels seems critical for an organism, as PP1c is encoded by multiple genes in most eukaryotic species. In flies, overexpression of NIPP1 using stronger and more widespread drivers such as tim-Gal4, elav-Gal4, and actin-Gal4 causes lethality. In addition, it was found that the expression of the Drosophila PP1c isoforms in S2R+ cells is regulated such that the total PP1c transcript level remains stable despite the loss or reduction of one PP1c mRNA. While it is beyond the scope of this study, it would be interesting to explore the mechanism underlying this phenomenon and to determine whether this regulation of PP1c expression exists in other fly cells (Fang, 2007).

The functional diversity of PP1 is exerted via its association with a large variety of regulatory subunits. PP1 regulatory subunits not only confer in vivo substrate specificity by directing PP1c to various subcellular loci for its substrates, but also allow the activity of PP1 to be modulated in response to intracellular signals and extracellular stimuli. It is possible that some adaptor proteins/PP1 regulatory subunits facilitate the interaction between PP1 and Tim documented in this study through co-IP experiments. In addition, although none of the PP1c isoforms is rhythmically expressed in the fly head, the regulatory subunit(s) targeting PP1 to 'clock substrates' may oscillate. The paradigm for the cyclic phosphatase activity concept is PP2A, whose regulatory subunits TWS and WDB are expressed with a robust circadian rhythm and affect Drosophila behavioral rhythms (Fang, 2007).

The mechanisms by which Tim stabilizes Per are not known, but it is possible that they involve phosphorylation. Perhaps most importantly, Per is stable and nuclear in tim01 flies if the kinase DBT is also knocked down, suggesting that, in the absence of Tim, Per is subject to excessive destabilizing phosphorylation. Thus, Tim may stabilize Per either by decreasing phosphorylation by DBT, or by increasing dephosphorylation by a phosphatase such as PP2A or PP1. Since DBT accumulation is not under circadian control and it is found in complexes with Per at all times in vivo, it is likely that the phosphatase activity is dynamic and limiting, regulating the rhythmic abundance of Per. The data suggest that PP1 is the primary phosphatase involved in the stabilizing effect of Tim on Per, as Tim is not more resistant than Per to PP2A inhibition and does not appear to affect dephosphorylation of Per by PP2A. Given that Per does not contain an RVxF-binding motif as found in Tim, it is tempting to speculate that Tim is a target as well as a regulatory subunit of PP1, which may target PP1c to Per and up-regulate local PP1 activity to antagonize the destabilizing action of clock kinases on Per. It is suggested that identification of the circadian-relevant PP1 regulatory subunit(s) will provide profound insight into the post-translational regulation of the clock (Fang, 2007).

This study demonstrates that PP1 plays an essential role in the regulation of the Drosophila clock. PP1 is one of the most conserved eukaryotic proteins, and it often performs similar essential functions in different species. Indeed, studies in the dinoflagellate and the fungus Neurospora have also implied a clock function for PP1. Consistent with the long period phenotype caused by inhibiting PP1 in flies, short pulses of phosphatase inhibitors in dinoflagellates cause phase delays, and PP1 appears to be the dominant phosphatase mediating this circadian function. In Neurospora, PP1 regulates the stability of the clock component FREQUENCY (FRQ). And recently, PP1 was reported to regulate degradation of the mammalian clock protein Per2. Together, multiple studies indicate an evolutionarily conserved role for PP1 in the circadian clock (Fang, 2007).

A small conserved domain of Drosophila PERIOD is important for circadian phosphorylation, nuclear localization, and transcriptional repressor activity

A 27-amino-acid motif has been identified that is conserved between the Drosophila Period protein (Per) and the three mammalian PERs. Characterization of Per lacking this motif (Per Delta) shows that it is important for phosphorylation of Drosophila Per by casein kinase I epsilon (CKI epsilon; Doubletime protein or DBT) and CKII. S2 cell assays indicate that the domain also contributes significantly to Per nuclear localization as well as to Per transcriptional repressor activity. These two phenomena appear linked, since Per Delta transcriptional repressor activity in S2 cells is restored when nuclear localization is facilitated. Two less direct assays of Per Delta activity in flies can be interpreted similarly. The separate assay of nuclear import and export suggests that the domain functions in part to facilitate Per phosphorylation within the cytoplasm, which in turn promotes nuclear entry. As there is evidence that the kinases also function within the nucleus to promote transcriptional repression, it is suggested that there is a subsequent collaboration between phosphorylated Per and the kinases to repress Clk-Cyc activity, probably through the phosphorylation of Clk. This is then followed by additional Per phosphorylation, which occurs within the nucleus and leads to Per degradation (Nawathean, 2007; full text of article).

The phospho-occupancy of an atypical Slimb-binding site on Period that is phosphorylated by Doubletime controls the pace of the clock

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). This study use the Drosophila model system shows 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. It is proposed 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. This study also used 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 (Chiu, 2008).

To better understand the physiological role of phosphorylation in regulating PER stability, Drosophila was used as a model system. Using a range of strategies, including mutational analysis, mass spectrometry and phospho-specific antibodies S47 was identified as a key phospho-determinant regulating the efficiency of SLIMB binding to dPER. By evaluating the behavior of dPER mutants whereby amino acid 47 is constitutively 'nonphosphorylated' (S47A) or 'phosphorylated' (S47D), it was shown that the phospho-occupancy of S47 is a key biochemical throttle adjusting the pace of the clock. However, phosphorylation of S47 occurs within an atypical SLIMB-binding site. Additional DBT-dependent phosphorylated residues, which likely include one or more nearby Ser residues at amino acid 44/45 and possibly others within the first 100 amino acids, collaborate with pS47 to generate a high-affinity SLIMB-binding region on dPER. As such, the affinity of SLIMB for dPER is proportional to the degree of phospho-occupancy within an extended phosphorylation network centered on S47 that as a unit yields a graded response in the affinity of SLIMB-dPER interactions. Attaining a high proportion of dPER molecules that are phosphorylated at S47 and other key sites mediating SLIMB binding is progressive and occurs several hours after DBT stably interacts with dPER via the centrally located dPDBD, likely because DBT 'activity' is counterbalanced by TIM and protein phosphatases. It is proposed that the relatively slow assembly of a high-affinity SLIMB-binding site on dPER is at least partly 'designed' to extend the time that dPER acts as a transcriptional repressor, critical in generating transcriptional feedback loops with daily time frames. Finally, this mass spectrometry analysis identify 'hot spots' for phosphorylation, indicates that the majority of dPER phosphorylation is unrelated to direct effects on stability and sheds new insights into the underpinnings of several previously characterized mutants, including the classic perS allele (Chiu, 2008).

Early studies identified DpSGPhiX1+npS (pS, phosphorylated Ser; Phi, any hydrophobic amino acid; X, any amino acid) as the consensus motif for recognition by the β-TrCP/SLIMB F-box protein (Fuchs. 2004). Phosphorylation at both sites in this six amino acid consensus generally leads to a high-affinity β-TrCP/SLIMB-binding site. Indeed, the three negatively charged residues (Asp/Glu and two phospho-S/T residues) are important binding contacts underlying β-TrCP/substrate interactions (Wu, 2003). Furthermore, it is thought that the presence of an Asp/Glu at position 2 of the canonical binding domain can circumvent the need for a phospho-S/T at that position, as is the case for Wee1A (Watanabe, 2004). However, accumulating evidence indicates that β-TrCP-binding sites can deviate from this consensus. For example, recent work on the Ci/Gli family of transcription factors suggests a novel class of degenerate and weaker β-TrCP-binding sites that extend beyond the standard six-amino-acid binding motif, especially for those missing a Gly at the third position (Smelkinson, 2007). It was suggested that for these extended β-TrCP/SLIMB-binding motifs, significant contributions are made by the local presence of nonpolar residues, such as those found in the motifs for Ci, Gli, and Wee1A. Additional phosphorylation events at nearby regions are also thought to enhance the inherently weak binding affinities of extended β-TrCP-binding sites, enabling a more graded response compared with the standard sequence (Chiu, 2008).

The major SLIMB-dependent phospho-degron identified in this study [44p*Sp*SGpSSGYGG52; where p* = possible phosphorylation] seems to include signature elements found in both the standard and extended β-TrCP-binding motifs. A rather unique feature of the SLIMB-binding domain on dPER is that it includes two SSG repeats. S47 is phosphorylated, and based on mutational analysis and mass spectrometry, it is almost certain that either S44 and/or S45 are phosphorylated. A physiological role for S45 is further indicated by the perSLIH mutant (S45Y) that exhibits long periods, which based on the findings is likely due to reduced dPER-SLIMB interactions. Although changing S48 to Ala phenocopied the S47A mutation, the S48D mutation did not enhance binding to SLIMB, as was the case for S47D. Together with results showing that S48A did not modulate phosphorylation of S47, the data strongly suggest that S48 has a non-phosphorylation-dependent role as a crucial structural element. Mass spectrometry identified two phospho-residues in a dPER peptide from amino acids 40-48. Thus, there might only be two negatively charged residues in the major SLIMB-binding site on dPER. It is possible that the presence of a SSG tandem and a Tyr at position 50 can compensate for the lack of a third acidic residue normally found in β-TrCP-binding sites. It is also highly probable that other, yet to be identified, DBT-dependent phosphorylation sites besides those within the atypical SLIMB-binding site identified in this study contribute to enhancing SLIMB-dPER interactions (Chiu, 2008).

The presence of numerous suboptimal phospho-determinants is thought to generate a graded response in the binding efficiencies of F-box proteins to substrates. The general molecular framework is that progressive increases in the phospho-occupancy of multiple phosphorylated residues eventually reaches a threshold value that drives sufficient F-box protein/substrate interactions to yield desired outcomes. As such, regulating the kinetics of phosphorylation within the phospho-network mediating F-box recognition is a key determinant in the timing of substrate degradation. In the case of animal PER proteins, they undergo progressive increases in global phosphorylation that occur over an ~10-h time frame, whereby highly phosphorylated isoforms are associated with a rapid decline in levels (Chiu, 2008).

What accounts for the hours-long kinetics underlying the gradual increases in phosphorylation of S47 and likely many other DBT-dependent sites on dPER? Based on the in vitro ability of DBT to phosphorylate S47 despite phosphatase treatment of dPER, it is not believed that hierarchical phosphorylation based on prior priming is a major component in regulating the timing of when S47 is phosphorylated in vivo. Rather, the findings strongly suggest that the gradual build-up in the phospho-occupancy of S47 and other sites is at least partly based on a dynamic balance between DBT-mediated phosphorylation and the opposing activities of TIM and protein phosphatases. In agreement, blocking phosphatase activity strongly enhanced the abundance of phosphorylated S47. Recent evidence suggests that the ability of TIM to stabilize dPER might be by acting as a bridge that facilitates the targeting of protein phosphatase activity toward dPER (Fang, 2007). Indeed, it is likely that the strong protective function of TIM on dPER partially overrides the destabilizing effects of the S47D mutant as it attains peak levels comparable with those of wild-type dPER. Following this line of reasoning, it is suggested that a major reason for the advanced dper RNA and protein cycles in the S47D mutant is that as TIM levels decline in the late night the 'released' dPER(S47D) protein is no longer protected (or less so) and undergoes accelerated nuclear clearance, leading to an earlier disengagement from transcriptional repression, which advances the subsequent dper RNA and protein cycles. Likewise, while this manuscript was under review a recent report showed that the CKIepsilon tau mutation, which shortens rhythms in mice, has an 'asymmetrical' effect on PER protein stability, preferentially accelerating nuclear clearance and hence advancing the molecular oscillations underpinning the clockworks (Meng, 2008). Thus, although differential phosphorylation plays a major role in setting the intrinsic stabilites of PER proteins, additional variables, such as phase-specific protein-protein interactions, are critical in the 'readout' from these phospho-signals (Chiu, 2008).

Many of the DBT-dependent phosphorylation sites that were identified using mass spectrometry do not lie within optimal CKI sites, suggesting that inefficient phosphorylation by DBT might also contribute to the overall rate of progressive increases in dPER phosphorylation. It is also possible that the strong binding of DBT to the centrally located dPDBD, while increasing the local concentration of DBT, could function as a slow 'time-release capsule' whereby the disengagement of DBT is first required prior to phosphorylation of dPER residues at more distantly located sites (Chiu, 2008).

Although the phosphorylation requirements and in vivo significance of regions on mPER1 and mPER2 that interact with β-TrCP are not known, it is likely to also be based on noncanonical β-TrCP-binding sites. In addition, hyperphosphorylation of mammalian PER proteins requires a centrally located CKI-binding site. Therefore, mammalian PER proteins, especially mPER1 and mPER2, are likely to be targeted by β-TrCP to the 26S proteasome in a manner similar to that described here for dPER. This type of mechanism might also apply to other clock proteins such as Frequency (Frq) in Neurospora that undergoes daily changes in phosphorylation and stability that are remarkably similar to those observed for PER proteins. In addition, the phosphorylated state of FRQ is regulated by casein kinases, protein phosphatases, and the rapid degradation of highly phosphorylated isoforms is mediated by the F-box protein FWD1, a homolog of β-TrCP (Chiu, 2008).

An interesting feature of the distribution in phosphorylation sites on dPER that were identified using mass spectrometry is that they seem to concentrate in clusters, suggesting the presence of'phospho-modules' with different functions. Most of these clusters appear anchored by proline-directed phosphorylation sites, which are phosphorylated by endogenous kinases expressed in S2 cells. Of note, one such cluster is located in the dPER 'short domain' (T585-T610). Mutations in this region result in animals with short periods. In fact, the mutated residues of two classic per mutants that have short periods, perS (S589N, 19-h period) and perT (G593D, 16-h period), are right in the heart of this cluster. S589 is phosphorylated in a DBT-dependent manner; and G593, when mutated, may affect phosphorylation at nearby S589 and/or S596. Although the perS mutants was isolated more than 35 years ago, the current results provide the first biochemical understanding for the short period phenotype, suggesting that phosphorylation events in the 'short domain,' some of which are DBT-dependent, may collaboratively function to slow down the clock. It is now becoming apparent that phosphorylation at different sites on PER proteins can result in differential effects on the pace of the clockworks, whereby some lead to faster clocks while others slow it down. The presence of phosphorylated residues with opposing outcomes on the speed of the clock can explain why mutations in CKIepsilon/delta/DBT can yield a variety of period-altering phenotypes from short to long, despite the fact that overall enzymatic activity is generally reduced (Chiu, 2008).

A rather unanticipated finding is that the majority of dPER phosphorylation is unrelated to direct effects on stability. This is supported by the lack of detectable SLIMB binding to a dPER fragment only missing the first 100 amino acids despite extensive phosphorylation as inferred from being the region underlying the majority of phosphorylation-dependent electrophoretic mobility shifts and confirmed by mass spectrometric analysis. Other lines of evidence also imply that a significant amount of multiphosphorylation is not linked to direct effects on PER stability. For example, abolishing phosphorylation at many centrally located sites on mPER3 does not attenuate CKI-mediated in vitro interactions with β-TrCP. Also, a trans-dominant version of CKII reduced global hyperphosphorylation of dPER without major effects on its levels (Chiu, 2008).

Thus, there are likely to be at least two functionally distinct DBT-dependent phosphorylation programs regulating different aspects of PER metabolism and activity: one that controls β-TrCP/SLIMB binding, and another that integrates with other kinases, such as CKII, to modulate nuclear entry/accumulation and/or ability to function as a transcriptional repressor. Indeed, mass spectrometric analysis of dPER identified numerous phosphorylation sites in a putative nuclear localization site and within the CCID mediating dPER inhibition of CLK-mediated transcription. Variants of dPER missing the major DBT docking site are hypophosphorylated and weak repressors. However, the relationship between hyperphosphorylation and repressor potency is not clear, since the DBT docking site on dPER also functions as a molecular scaffold for DBT and perhaps CKII-mediated inhibition of CLK-dependent transcription. Nonetheless, it is clear that the DBT docking site is a critical nexus for coordinating multiple phosphorylation programs. A challenge is to examine the functions of the newly identified phosphorylation sites and dissect the mechanisms by which they regulate dPER metabolism and activity (Chiu, 2008).

Timeless is an important mediator of CK2 effects on circadian clock function in vivo

Circadian oscillations in clock components are central to generation of self-sustained 24-h periodicity. In the Drosophila molecular clock, accumulation, phosphorylation, and degradation of Period and Timeless proteins govern period length. Yet little is known about the kinases that phosphorylate Tim in vivo. It has been shown previously that the protein kinase CK2 phosphorylates Tim in vitro. This study identified a role for CK2 in Tim regulation in vivo. Induction of a dominant-negative CK2α, CK2αTik (Tik), increases Tim protein and tim transcript levels, reduces oscillation amplitude, and results in persistent cytoplasmic Tim localization. Exposure to light and subsequent Tim degradation results in an increase in the fraction of the transcriptional repressor Per that is nuclear and suppression of per and tim RNA levels. Tim protein, but not tim transcript, levels are elevated in Tik mutants in a per01 background. In contrast, Tik effects on Per are undetectable in a tim01 background, suggesting that Tim is required for CK2 effects on Per. To identify potential CK2 target sites, Tim phosphorylation rhythms were assayed in a deletion mutant that removes a conserved serine-rich domain. It was found that Tim protein does not show robust rhythmic changes in mobility by Western blotting, a hallmark of rhythmic phosphorylation. The period lengthening effects in Tik heterozygotes are reduced in a timUL mutant that disrupts a putative CK2 phosphorylation site. Together, these data indicate that Tim is an important mediator of CK2 effects on circadian rhythms (Meissner, 2008).

Evidence is presented that CK2 operates through Tim to control circadian clock function in vivo. Expression of the dominant-negative CK2α allele, Tik, elevates trough levels of Tim protein and RNA during constant darkness and alters Tim subcellular localization. Tik effects on Per are undetectable in a tim01 mutant, whereas Tik effects on Tim are evident in a per01 background, suggesting direct Tim effects. Behavioral period effects of Tik are reduced in the timUL mutant that disrupts a putative CK2 site. The effects on Tim metabolism as well as the genetic requirement of tim for CK2 effects indicate CK2 primarily operates through Tim to regulate the circadian clock (Meissner, 2008).

One potential model consistent with these data is that CK2 regulates Tim abundance and in turn, promotes negative feedback. Elevated trough Tim levels are accompanied by elevated trough tim transcript levels in DD, suggesting impaired turnover, negative feedback, and/or tim transcriptional regulation. Effects on Tim are evident in per01 flies but are not accompanied by changes in tim transcript levels, suggesting a direct effect on Tim protein, perhaps by regulating Tim stability, although a translational effect cannot be ruled out. Importantly, the per01 data argue strongly against a direct effect of CK2 on CLK/CYC-driven transcription of tim. The ability of light to degrade Tim in Tik-expressing flies implies that Tim levels can be regulated by two distinct pathways, a light/CRY-dependent pathway and a CK2-dependent pathway. During light/dark entrainment, light robustly degrades Tim in Tik-expressing flies. This degradation is accompanied by a sharp suppression in per and tim transcript levels, suggesting that it is excessive Tim levels, rather than direct Per effects, that abrogate negative feedback in Tik-expressing flies. per and tim RNA oscillations remain phase delayed during LD in homozygous Tik-expressing flies, suggesting that Tik also affects the timing of Per repression (Meissner, 2008).

Some CK2 effects on Per may be mediated by CK2 effects on Tim. CK2 effects on Per levels require Tim. CK2 is localized to the cytoplasm where Per/Tim dimers are likely present. CK2 regulates Per and Tim nuclear entry, a process that likely depends on the Per/Tim dimer. Although effects on Per require Tim, Per is likely a direct in vivo CK2 substrate. CK2 robustly and specifically phosphorylates Per in vitro and in-vitro-defined sites have clear in vivo functions. Tik mutants can also strongly effect Per mobility on Western blots; Per mobility is highly dependent on phosphorylation. These data are most consistent with the idea that CK2 targets the Per/Tim dimer in the cytoplasm and phosphorylates Per to promote nuclear entry. Additional experiments will be required to test the dimer hypothesis. Based on S2 cell experiments, it has been proposed that CK2 phosphorylation of Per promotes its intrinsic repressor activity independent of its effects on nuclear localization. The observation that light-induced Tim degradation results in a suppression of per and tim RNA in Tik-expressing flies suggests that elevated Tim levels block Per repression. Nonetheless, remaining alterations in transcript levels suggest that the freed Per repressor may not be entirely functional, consistent with additional CK2 effects on Per (Meissner, 2008).

Tim may be an in vivo CK2 substrate. CK2 has been shown to phosphorylate Tim in vitro. In vivo, Tik increases Tim levels in the absence of Per, suggesting CK2 effects on Tim may be direct. Importantly, increases in Tim protein are not accompanied by increases in transcript, indicating CK2 regulates Tim posttranscriptionally. The notion is favored that CK2 acts to phosphorylate Tim in vivo, consistent with published in vitro phosphorylation experiments (Meissner, 2008).

One potential argument against the hypothesis that CK2 phosphorylates Tim is the finding that low-mobility Tim accumulates to high levels in timGAL4-62; UAS-Tik homozygotes compared with wild-type controls. Interestingly, this result is similar to the observation that in Dbtg mutants, low mobility forms of Per accumulate that are even lower in mobility than those in wild-type, although Per is widely established as a DBT substrate. In both cases, phosphorylation-induced mobility changes cannot be attributed solely to a single kinase. It is also possible that elevated Tim levels may render low mobility forms more visible in Tik-expressing flies. Thus, the presence of low-mobility forms does not exclude CK2 as an in vivo kinase for Tim (Meissner, 2008).

Although no phosphorylation sites have been identified in Tim, deletion of a small Tim serine-rich domain (Tim 260-292) reduces or eliminates significant circadian mobility changes. This domain is conserved among insects and may contain multiple phosphorylation sites that are responsible for regulating period length and rhythms in Tim levels and mobility. CK2 may be one of several kinases that phosphorylates Tim serine-rich domain. In addition, although this domain is critical for shifts in Tim mobility, there are likely phosphorylation sites for CK2 outside of this region. When the bacterial expression construct used previously to generate Tim protein for CK2 in vitro phosphorylation assay was sequenced, it was discovered that the construct lacks amino acids 260-292. The possibility cannot be excluded that loss of rhythmic mobility changes may be secondary to reduced Tim levels in serine-rich domain mutants or may not be linked to CK2. Nonetheless, the in vivo data and sequence conservation raise the possibility that the serine-rich domain may be a kinase substrate (Meissner, 2008).

The Tim serine-rich domain contains four predicted CK2 sites. One of these sites, Ser279, is potentially altered in timUL mutants by the Glu283Lys point mutation. CK2 preferentially phosphorylates serine or threonine residues located 2-5 residues N-terminal to acidic amino acid residues such as glutamate or aspartate. The placement of basic residues, such as lysine as in the timUL mutant, near CK2 sites inhibits CK2 phosphorylation of the target Ser/Thr residue. timUL and Tik show allele-specific genetic interactions such that in timUL mutants, Tik period lengthening is partially suppressed. These results are consistent with the hypothesis that timUL is a CK2 site mutant. This prediction suggests that Ser279 plays a very important role in regulating circadian period length in flies. It will be of interest to test the hypothesis that CK2 promotes Tim degradation (Meissner, 2008).

It has been claimed that timUL shows a late-night specific defect principally based on the phase response curve to light and persistent nuclear Tim levels. CK2, however, is mostly restricted to the cytoplasm and thus would be predicted to act on cytoplasmic Per and/or Tim proteins during the early night. Although it is true that timUL shows profound late-night defects, on closer examination, there are alterations in the phase response curve to light during the early night. In addition, Tim protein was not examined in pacemaker neurons during the early night in timUL, so the possibility that there is also an early-night defect in timUL cannot be ruled out. Therefore it is possible that timUL also displays early-night defects, as is proposed for CK2 effects. Second, it is possible that phosphorylation of Tim by CK2 in the cytoplasm could influence the function of phospho-Tim in the nucleus. Third, although CK2 is principally observed in the cytoplasm, the possibility that small but functional levels of CK2 are present in the nucleus cannot be ruled out. Regardless, the allele-specific, nonadditive genetic interaction between Tik and timUL argue that timUL is important for CK2 effects on circadian behavior (Meissner, 2008).

The data are consistent with the speculation that Tik expression reduces CK2 phosphorylation of Tim. This results in retention of Per in the cytoplasm, thus reducing Per-mediated repression. Light-mediated degradation of Tim can then liberate Per (pending some additional events) to enter the nucleus and repress CLK/CYC. Although additional experiments will be needed to test this model, the data demonstrate that it is likely that CK2 effects through Tim will play an important role in circadian clock function (Meissner, 2008).

Activating PER repressor through a DBT-directed phosphorylation switch

Protein phosphorylation plays an essential role in the generation of circadian rhythms, regulating the stability, activity, and subcellular localization of certain proteins that constitute the biological clock. This study examines the role of the protein kinase Doubletime (DBT), a Drosophila ortholog of human casein kinase I (CKI) epsilon/delta. An enzymatically active DBT protein is shown to directly phosphorylate the Drosophila clock protein Period (PER). DBT-dependent phosphorylation sites are identified within PER, and their functional significance is assessed in a cultured cell system and in vivo. The perS mutation, which is associated with short-period (19-h) circadian rhythms, alters a key phosphorylation target within PER. Inspection of this and neighboring sequence variants indicates that several DBT-directed phosphorylations regulate PER activity in an integrated fashion: Alternative phosphorylations of two adjoining sequence motifs appear to be associated with switch-like changes in PER stability and repressor function (Kivimae, 2008).

Initial studies of dbt mutations suggested that Per is the primary target of Dbt in the Drosophila circadian clock. Loss of dbt expression led to hypophosphorylation and overaccumulation of Per in vivo. Mutations of dbt that shorten or lengthen behavioral rhythms led to correspondingly earlier or later degradation of Per in fly heads. The sequence of dbt suggested that it encodes an ortholog of the CKI family of protein kinases in mammals. Direct evidence for Dbt kinase activity and Per phosphorylation has been hampered by the absence of specific enzymatic activity in bacterially produced recombinant Dbt preparations, even though highly homologous mammalian CKIɛ (86% primary sequence identity in the kinase domain) has been recovered in an active form from bacterial expression systems. It is possible that for Drosophila Dbt, protein folding is more sensitive to the cellular environment than the mammalian enzyme. Alternatively, Dbt may require posttranslational modification(s) and/or the presence of a stimulating activity or cofactor(s) to produce an active conformation that is not available in bacterial cells. Nevertheless, this report has shown that recombinant Dbt can be expressed and recovered in an enzymatically active state from an insect cell line. However, even this insect-derived activity is short-lived (Kivimae, 2008).

Recombinant Dbt in vitro preferentially phosphorylates Per protein. No significant phosphorylation was observed when Tim, Sgg, and Cyc were used as a substrate in vitro. Although no direct Dbt phosphorylation of the N-terminal half of Clk was observed, recent studies by others have shown that Dbt is required for the generation of a highly phosphorylated and unstable form of Clk in vivo. Furthermore, to achieve this highly phosphorylated state of Clk, it has been suggested that Per must be present and function as a bridge to physically deliver Dbt to Clk. Clk appears to be present in flies in multiple isoforms, including hypophosphorylated, intermediate-, and highly phosphorylated states. The last form is only observed during the late part of the Clk accumulation cycle and may promote Clk degradation. The lack of in vitro phosphorylation of Clk by Dbt in the current studies may reflect a Dbt phosphorylation target in the C-terminal half of Clk, or a requirement for additional kinases to 'prime' a Dbt target, or a dependence on Per for Dbt-directed phosphorylation of Clk (Kivimae, 2008).

Casein and Per are phosphorylated by enzymatically active recombinant Dbt, and mutations of dbt that are known to affect the behavioral rhythm also have an effect on kinase activity. The short- and long-period mutations dbtS and dbtL both reduce the kinase activity of Dbt. Importantly, when the dbtS mutation was introduced specifically into the Drosophila kinase, the effect was far less severe than a previously reported orthologous substitution; using casein as a substrate, the mutation produced a 15% decrease in activity when composing the fly kinase, but decreased enzyme activity by approximately 55% when introduced into Xenopus CKIδ ;. Vertebrate CKI and Dbt include divergent C-terminal protein segments, and the former enzyme cannot restore Dbt function in transgenic Drosophila. These results suggest that any analysis of Dbt mutations in a heterologous system should be applied with caution. The comparatively subtle effect of dbtS on Dbt activity versus its strong effect on period length could reflect a qualitative change in the mutant enzyme's activity. An assessment of potential differences of this sort might come from further mapping studies of the target specificities of DbtS versus Dbt on Per substrates in vitro (Kivimae, 2008).

Three distinct regions of Per are preferentially phosphorylated by Dbt in vitro. In the C-terminal region of Per, two phosphorylation targets, serine 1,134 and threonine 1,219, have been identified. No prior genetic or biochemical studies have suggested a specific role for this Per interval, and the cultured cell assays indicate that both phosphorylation targets can be removed without a detectable effect on Per's function as a Clk repressor. Near the N-terminus of Per, a region of 20 amino acids (residues 149-169) contains five serines that are candidate phosphorylation sites for Dbt. This interval is located upstream of the PAS domain of Per. A region important for Dbt/Per binding in vitro has been mapped to the first 300 amino acids of Per. However, it has not yet been determined whether there is a closer physical correspondence of the binding and phosphorylation target sequences, or whether elimination of potential Dbt targets in this Per interval affects Per/Dbt binding. As in the case of cultured cell studies of serine 1,134 and threonine 1,219, elimination of all phosphorylation targets between aa 149-169, produced no detectable effect on Per-dependent repression in cultured cells (Kivimae, 2008).

Interestingly, two of the putative CKI sites, serine 151 and serine 153, are substrates of casein kinase 2. Transgenic flies carrying S151A and/or S153A mutations showed period lengthening and delayed Per nuclear localization. It will be important in future studies to distinguish CK2 and Dbt phosphorylation targets in this region of Per and to determine their individual and/or interactive effects on Per function in vivo (Kivimae, 2008).

Of most interest in this study are phosphorylation sites identified in the third Dbt target region of Per, between residues 580-645. This is a genetically well-studied interval previously implicated in the regulation of behavioral rhythms. Mutations of a portion of this region, the per-short domain, are predominantly associated with short-period behavior. The first mutation to be mapped to this region, perS, eliminates a candidate phosphorylation site at serine 589, and S2 cell assays indicate that blocking phosphorylation of this site enhances Per's activity as a repressor of Clk. Earlier work has also shown that perS leads to premature nuclear degradation of Per, and the current studies of transgenic Drosophila producing a Per protein truncated by 17 amino acids including serine 589 (perΔS), similarly indicate a role for this sequence in regulating Per stability and function as a repressor. Thus, the wild-type per-short domain appears to promote Per stability while reducing its activity as a transcriptional repressor (Kivimae, 2008).

It is noted that earlier work has also suggested that the mutation perS enhances Dbt activity. If serine 589 is no longer an available Dbt target in perS, by what mechanism might Dbt function be elevated? Although mutating serine 589 enhances Per's activity as a repressor, this response is partially suppressed by a further mutation of some phosphorylation sites in the perSD region. As well, little or no repressor activity is detected if all phosphorylation targets are removed from perSD, even in the presence of the perS mutation. Together, these results suggest that the phosphorylation state of serine 589 may influence Dbt activity on downstream targets within perSD that are required for Per's function as a Clk repressor. In such a model (see Activity of the per-Short and perSD Domains Regulated by DBT Phosphorylation), dephosphorylation of the per-short domain would promote Dbt-directed phosphorylation of perSD, enhancing Per's activity as a repressor and also destabilizing the protein. Reciprocally, phosphorylation of serine 589 would depress activity of Dbt with respect to perSD, providing a more stable, but less active, Per protein. In this view, a fully dephosphorylated state would provide the most stable protein, but would provide an inactive form of Per, consistent with findings in cultured cells and transgenic flies, and with studies of Dbt-deficient Drosophila. Further in vivo evidence comes from transgenic flies expressing Per that contains a deletion of a conserved segment (aa 516-568) downstream of the PAS domain. This mutant Per protein displays increased stability and low-amplitude oscillations. More importantly, high levels of a hypophosphorylated form of Per are produced by these truncated proteins, which constitutively accumulate in the nucleus. This protein appears to have little or no activity as a transcriptional repressor (Kivimae, 2008).

Earlier studies of Per repression in S2 cells identified a region of Per, distinct from those reported in this study, that is also required for Per's function as a repressor of Clk-mediated transcription (Clk-CYC inhibition domain [CCID]], aa 764-1034). A novel, bipartite nuclear localization signal (NLS) is found in this region that influences nuclear localization of Per in S2 cells. Recent mapping studies of CCID led to the identification of a smaller, Per-Dbt binding domain (PDBD; aa 755-809) that upon deletion increases Per stability, decreases Per phosphorylation, and severely impairs Per function as a transcriptional repressor (Kim, 2007). A related deletion of Per (aa 768-792) produced similar defects, and impaired nuclear accumulation of Per in cultured cells (Nawathean, 2007). Studies of the latter deletion in transgenic flies showed that a hypophosphorylated form of Per is constitutively expressed at high levels and is a very poor repressor. However, repressor function was restored by NLS addition, suggesting that dysfunction of the protein was likely due to its inability to localize to the nucleus (Kivimae, 2008).

The arrangement of four of the phosphorylation targets included within the perSD domain (serines 604, 607, and 613, and threonine 610) resembles a CKI phosphorylation motif found in human Per2 that has been implicated in familial advanced sleep phase syndrome (FASPS). In the human Per protein, there are five serines spaced in an arrangement such that every third amino acid residue is a serine. These five serines are thought to be progressively phosphorylated by CKIɛ/δ, and in certain FASPS subjects, the first serine is mutated to glycine. It has been proposed that this mutation inhibits the phosphorylation of the remaining serines, thus leading to altered hPer2 levels or activity in FASPS subjects. In fact, more-recent studies of this mutation indicate that it may alter Per2′s repressor function because lower levels of mper2 RNA are found in transgenic mice expressing the similarly mutated mPer2 protein. Cultured cell studies have also suggested that the mutation affects mPer2 stability. Accordingly, a role for CKI-directed phosphorylation in the regulation of Per's stability and activity as a transcriptional repressor may be conserved from flies to mammals (Kivimae, 2008).

Drosophila and vertebrate casein kinase Idelta exhibits evolutionary conservation of circadian function

Mutations lowering the kinase activity of Drosophila Doubletime(DBT) and vertebrate casein kinase Iepsilon/delta (CKIepsilon/delta) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckIshort-period mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period(PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKIepsilon/delta. Moreover, expression of either Drosophila DBT or the vertebrate CKIdelta kinase carrying the Drosophila dbtS or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKIdelta carrying the dbtL mutation does not lengthen circadian rhythms, while Drosophila DBTL does. Different effects of the dbtS and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKIdelta and of the functions affected by their period-shortening mutations (Fan, 2009).

Dbt is essential for circadian molecular oscillations because it introduces time delays into the negative feedback exerted by Per. These phosphorylation-dependent delays are thought to be mediated via several mechanisms. For instance, Dbt is thought to destabilize Per in the cytoplasm, while Tim (Per's dimerization partner) is thought to prevent this destabilization and trigger the movement of both proteins to the nucleus, perhaps indirectly. The destabilization of Per in the cytoplasm delays its nuclear accumulation, while the stabilization of Per in the nucleus by Tim delays the Dbt-dependent decrease of nuclear Per. Because Dbt controls the timing of Per's nuclear accumulation so that it does not occur until after the per/tim mRNA levels have peaked, molecular rhythms of per and tim mRNA are possible. Additional regulation has been proposed to occur at the level of Per's capacity to negatively regulate its transcription factor target, Clk/Cyc (Fan, 2009).

Although Dbt's orthologs CKIδ and CKIɛ are involved in the mammalian circadian clock, the extent of evolutionary conservation of their circadian mechanisms has not been clear. Cellular and biochemical analysis argues for a significant degree of conservation. It has been shown that both the original long-period mutation (dbtL) and the short-period dbt mutation (dbtS) reduce the enzymatic activity of Drosophila Dbt and a Xenopus CKI ortholog of Dbt on casein (Preuss, 2004), and this study has shown that Drosophila DbtS and DbtL also exhibit reduced activity on Per. In the current work, deletion of the Dbt C terminus was shown to increase the kinase activity of Dbt and its capacity to target Per for degradation. The latter result is consistent with interaction studies, as it is the N-terminal catalytic domain rather than the C-terminal domain that interacts with Per (Preuss, 2004). Finally, it was shown in this study that in the presence of a general phosphatase inhibitor Dbt produces forms that migrate more slowly on SDS-PAGE, in a manner that requires its kinase activity. These results suggest that Dbt is autophosphorylated. Inhibitory autophosphorylation of the C-terminal domain is a common feature of both vertebrate CKIɛ and -δ. All of these biochemical results demonstrate a high degree of evolutionary conservation between Drosophila Dbt and vertebrate CKIɛ/δ (Fan, 2009).

in vivo analysis herein of vertebrate CKIδ and Drosophila Dbt further establishes the evolutionary conservation of these kinases and the clock mechanisms in which they participate. Mutations that shorten the circadian period in the context of Dbt produced corresponding shortening in the context of CKI in flies, and the tau mutation produced almost identical period shortening in both the mammalian and the Drosophila clocks, in the context of either the fly or the vertebrate enzyme. The data for the tau mutation are particularly complete for analysis of evolutionary conservation, as this mutation has been tested in all possible combinations of organism (fly or mammal) and kinase (Dbt or CKI) except one (DbtTau in mammals). These results argue against the possibility that reduced kinase activity produces only long periods in flies and short periods in mammals. In contrast, the CKIδ FASPS mutation, which has a small effect on period, produces opposite effects on period in flies and mammals. The mutations analyzed in this study have much stronger effects on period and clearly show similar effects in both vertebrates and flies. These findings strongly support the proposed evolutionary conservation of CKIδ and Dbt protein kinases and of at least some of the circadian processes in which they are involved (Fan, 2009).

The reproduction of the period alteration caused by a mutation in CKIɛ when introduced into CKIδ or Drosophila Dbt and tested in a transgenic fly is strong evidence for the high amount of functional conservation between ckIɛ and ckIδ, both of which are proposed regulators of the vertebrate clocks. However, CKIδ and -ɛ are not completely interchangeable, since similar overexpression experiments with CKIɛ in flies have produced a very different set of results from the ones presented in this study for CKIδ. A catalytically active CKIɛ produces a dominant negative effect on circadian rhythms in Drosophila, with relatively constant expression of hypophosphorylated Per at all times of day, while overexpression of a catalytically inactive form of CKIɛ produces only a mild lengthening of circadian period (Sekine, 2008). Taken together with the current results, this result suggests that vertebrate CKIδ is better able to interact with fly clock proteins than is CKIɛ, and in a manner more comparable to Drosophila Dbt in the same expression protocol. The difference between the finding of Sekine (2008) and the current results, as well as those of Xu, (2005) (who also employed overexpression of CKIδ in flies and did not observe dominant negative phenotypes), may be due to the divergent C-terminal domains of CKIδ and -ɛ (Fan, 2009).

This study has shown that flies expressing the dominant negative form of Dbt have very long periods or are arrhythmic, and this result argues that general reductions in Dbt's kinase activity produce long-period rhythms that grade to arrhythmicity. While the lower activity of DbtL is predicted to (and in fact does) lengthen circadian period, the short-period Per oscillation produced by DbtS and tau mutations is not readily explained by their lower kinase activity in vitro. One possible explanation is that the short-period mutants affect something besides kinase activity - an interaction with a regulator, for example (Fan, 2009).

Another possible explanation is that reduced activity of short-period CKI enzymes in vitro may not translate into lower phosphorylation of Per in vivo because other kinases provide compensatory phosphorylation. In fact, progressive phosphorylation of Per, at least as assessed by a reduction in Per's electrophoretic mobility, occurs more rapidly in dbtS flies or cells than in wild-type or dbtL flies or cells, as is also shown in this study for tim-GAL4>UAS-DbtS flies, so the in vivo phosphorylation profile indicates more rapid phosphorylation in the dbtS mutant. Likewise, in the tau mutant, the phosphorylation of Per is only slightly delayed and ultimately appears to be as complete as in wild-type hamsters, despite the strong reduction in kinase activity caused by the tau mutation in vitro. In mammals, at least two kinases (CKIɛ and CKIδ) associate with Per and phosphorylate it, while in flies casein kinase II phosphorylates Per together with Dbt (Fan, 2009).

While the phosphorylation profiles of Per in tim-GAL4>UAS-DbtS, -CKIS, and -DbtL resemble the phosphorylation profiles of the original dbt mutants, the Per phosphorylation profiles of tim-GAL4>UAS-DbtTau and -CKITau do not resemble the profile that has been previously reported for the endogenous mutant; in particular, the oscillation of Per phosphorylation is notably blunted by overexpression of the DbtTau mutant kinases. It is possible the overexpression of the DbtTau has a stronger effect on the phosphorylation profile of Per in flies than in mammals because the tau mutant effects are partially masked in mammals by compensatory activity of CKIδ, with which CKIɛ may be partially redundant (Meng, 2008). Nevertheless, the period of the circadian clock is dramatically shortened by overexpression of both DbtS and DbtTau mutant kinases, and the circadian period of the tau mutation in flies is similar to that for the hamster tau mutant, suggesting that the alterations in the Dbt/CKI kinase are still altering period as they do in the original mutants (Fan, 2009).

Another possible reason for the phenotypes of the short-period mutants is that phosphorylation of specific sites in Per affects multiple, specific aspects of its regulation with opposite effects on period length. Phosphorylation of Drosophila Per at multiple sites, only some of which affect stability, has recently been demonstrated (Chiu, 2008; Kivimae, 2008). Along this line of thinking, it has been proposed that the ckIɛTau mutation is a gain-of-function mutation that enhances phosphorylation of Per at specific sites—an enhancement that is missed in global analysis of multisite substrates like Per. Other explanations have been offered for the tau mutant that include lowered phosphorylation at all sites, but with cytoplasmic destabilization produced by phosphorylation at some sites and increased nuclear retention (and stabilization) produced by phosphorylation at other sites. If these hypotheses for site-specific effects are correct, the dispersed phosphorylation profile detected for Drosophila Per in CKITau- and DbtTau-expressing flies is indicative of general changes in phosphorylation, most of which are not relevant to the period shortening, gain-of-function phenotype produced at a subset of sites. While both the dbtS and tau mutations produce short circadian periods, their effects on the phosphorylation program of Per are different, as reflected in their different effects on circadian changes in Per electrophoretic mobility (Fan, 2009).

Why does CKIL overexpression not lengthen the period of the Drosophila rhythm, while DbtL overexpression does? It is likely that the dbtL mutation compromises CKIδ function and reduces its ability to compete with endogenous wild-type Dbt for interactions with Per. In fact, CKIδ may have generally less ability to compete with endogenous Dbt than transgenic Dbt, as overexpression of CKIS also has weaker effects than overexpression of the corresponding DbtS protein. Not all mutations that reduce the kinase activity of vertebrate CKI produce short periods, or no effect like CKIL. This study shows that the D/N mutation, which like the K/R mutation is predicted to have a very specific effect on the catalytic properties of the enzyme and not other aspects of its function, produced variable period lengthening. The variability is most likely a consequence of chromosomal position effects at the P-element insertion site on the transgene expression levels. The stronger effects of period-lengthening mutations in the context of fly Dbt than in the vertebrate CKI suggest that there may be differences in the way they affect the fly and vertebrate CKI orthologs, with a consequence that fly Dbt can be mutated more readily to produce a long period. These differences argue against the idea that lack of long periods is produced by a difference in circadian targets, as the lack of long periods correlates with the vertebrate enzyme rather than the species in which the enzyme is expressed ( i.e., DbtL can produce long periods in flies, while CKIL cannot) (Fan, 2009).

The involvement of protein kinases with circadian clocks spans a phylogeny that is larger than the one separating vertebrates and fruit flies. Casein kinases I and II are also involved in the bread mold (Neurospora) clock and target both the FRQ transcriptional repressor and the WCC transcriptional activator in a mechanism reminiscent of the one involving Dbt. Recently, a kinase involved in DNA replication control was also shown to target Neurospora FRQ, with implications for the interplay between circadian rhythms and cell cycle control in higher eukaryotes. The core circadian oscillator mechanism in cyanobacteria involves rhythmic phosphorylation and dephosphorylation of the KaiC protein. The evolutionary conservation of kinase function has led to a synergy between research in different organisms—for instance, with work in Drosophila identifying a circadian role for CKI that has now been shown in diverse phyla and work in Neurospora showing a kinase-targeting role for FRQ that was subsequently shown for Per as well. Further elucidation of the evolutionarily conserved processes regulated by phosphorylation will reveal general mechanisms at the core of the circadian mechanism (Fan, 2009).

Kinetics of doubletime kinase-dependent degradation of the Drosophila period protein

Robust circadian oscillations of the proteins Period (Per) and Timeless (Tim) are hallmarks of a functional clock in the fruit fly Drosophila melanogaster. Early morning phosphorylation of Per by the kinase Doubletime (Dbt) and subsequent Per turnover is an essential step in the functioning of the Drosophila circadian clock. Using time-lapse fluorescence microscopy, Per stability in the presence of Dbt and its short, long, arrhythmic, and inactive mutants were studied in S2 cells. Robust Per degradation was observed in a Dbt allele-specific manner. With the exception of doubletime-short (DBTS), all mutants produce differential Per degradation profiles that show direct correspondence with their respective Drosophila behavioral phenotypes. The kinetics of Per degradation with DBTS in cell culture resembles that with wild-type Dbt and posits that, in flies DBTS likely does not modulate the clock by simply affecting Per degradation kinetics. For all the other tested Dbt alleles, the study provides a simple model in which the changes in Drosophila behavioral rhythms can be explained solely by changes in the rate of Per degradation (Syed, 2011).

These studies following the temporal pattern of fluorescently labeled Per and Dbt indicate that the onset of Per degradation is very tightly regulated in S2 cells and Per abundance starts to diminish typically within 3-5 h after protein induction with only ~10% of the substrate remaining at the end of the degradation process. In contrast, when TIM is coexpressed with Per, only a small fraction of the substrate is degraded because of Dbt activity while most of the Per remains bound to Tim and protected from degradation. The data show that the undegraded Per, Tim, and Dbt translocate to the nucleus ~5.5 h postinduction, suggesting that Dbt does not substantially modify timing of Per/Tim entry into the nucleus. Although the average timing of nuclear entry is unaffected by Dbt, presence of the kinase appears to reduce the translocation stochasticity observed in the cell population. It has been reported that Per/Tim nuclear entry events uniformly distribute over an interval of ~5 h. In the present studies, over 70% of cells coexpressing Dbt show Per/Tim nuclear entry within a narrower temporal window of ~3 h. This reduction in the variation of nuclear translocation is consistent with the reconstitution of an interval timer that more closely resembles the one found in vivo. Indeed, immunostaining in pacemaker neurons shows an ~2 h variation in the appearance of nuclear Per, when compared among multiple wild-type fly brains. Additionally, the Per/Tim/Dbt nuclear entry data reveal that Dbt can translocate to the nucleus 1-2 h prior to Per. These data further refine a model that was based on in vivo results with lower temporal resolution indicating that Per/Dbt nuclear accumulation occurs concurrently (Syed, 2011).

The behavioral phenotypes of a number of dbt mutants have been described previously. However, a detailed molecular description of how these mutations on dbt ultimately give rise to behavioral changes in Drosophila has been missing. This study was a thorough cell-based study aimed at elucidating the effects of Dbt mutations on Per turnover kinetics. To quantify the data, a multiple parameter-based hypothesis was formulated and the onset of Per turnover, the degradation half-life, and the fraction of the substrate that is degraded in individual cells were quantitated. Analysis of the data shows that Per half-life ranges between ~1.2 and 6.5 h, depending on the allele of the kinase that is coexpressed with the substrate. In the presence of wild-type, short-period, and long-period alleles of Dbt, Per stability appears to be independent of the substrate concentration. However, in the cells expressing only endogenous Dbt or overexpressing the catalytically compromised kinases Dbtar and DbtK38R, Per stability varies strongly with its abundance. Crucially, the substrate abundance remains steady for hours in the cells expressing high levels of Per, perhaps due to multimerization of the protein with other cellular components. The data do not reveal identity of these components but they are speculated to be Per itself or other endogenous proteins that titrate the substrate and hinder its interaction with low concentration of endogenous kinase molecules. These findings also provide a possible explanation for why several others may have concluded that singly overexpressed Per is stable in S2 cells. Because these studies sample bulk population of cells, it is likely that their measurement signal is dominated by cells expressing maximal levels of Per (equivalent of Per > 500 A.U.), the regime where single-cell measurements show that the protein is indeed in a more stable form (Syed, 2011).

Most importantly, the data permit direct comparison between kinetic parameters determined in cell culture to behavioral measurements in vivo. In particular, a plot of Per half-life in the presence of the five different alleles of Dbt and the period of daily activity from animals carrying those forms of Dbt shows a remarkable correspondence between the two quantities. A simple linear relationship emerges from this comparison with one noticeable outlier. The emergent model from that comparison suggests that alterations in the period of circadian rhythms that are observed in animals carrying the variants of Dbt can be explained mostly by the resultant changes in the rate of Per degradation (Syed, 2011).

Two previous studies addressing DbtS activity in Drosophila concluded that the mutation causes increased kinase activity. However, the first study did not reveal a discernible difference in Per phosphorylation or degradation when dbt was replaced by dbtS, and data from the second study were derived from the human short-period mutant and substrate to argue about the Drosophila system. Since the latter work, it has been demonstrated that expression of mammalian casein kinase I in the fly does not rescue Drosophila dbt mutants, indicating that comparisons of mutant forms of the two proteins across these systems would be difficult. The current detailed measurements on Per degradation with DbtS show negligible changes on substrate stability due to the Pro-47 -> Ser mutation on the kinase (Syed, 2011).

In summary, these data indicate that several mutations of the kinase Dbt affect a common feature in the circadian clock in altering length of the period. Most of the mutations appear to modulate activity rhythm simply by changing Per stability. Deviation of the DbtS mutation from the proposed model raises the possibility that it likely affects period length through a mechanism other than changing the rate of Per turnover in vivo. It has been conjectured that DbtS might modulate Per activity as a repressor by producing a qualitatively different phosphorylation pattern of the substrate. Additionally, data has suggested that dbtS causes early termination of per transcription, consistent with the previous finding that Per starts to decrease ~6 h earlier in dbtS mutants compared with wild-type animals. Another possibility is that DbtS activity is modified by co-factors in vivo. These cofactors are missing in S2 cells but are presumably present in the nuclei of clock neurons. Regardless of the actual mechanism through which DbtS shortens period length, the current results suggest it is different from that of all the other variants of Dbt examined in this study (Syed, 2011).

NEMO/NLK phosphorylates PERIOD to initiate a time-delay phosphorylation circuit that sets circadian clock speed

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, a time-delay circuit based on hierarchical phosphorylation was identified that controls the daily downswing in Per abundance. Phosphorylation by the Nemo/Nlk kinase at the 'per-short' phospho cluster domain on Per stimulates phosphorylation by Doubletime (Dbt/Ck1delta/epsilon) 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/β-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. These findings identify Nemo as a clock kinase and demonstrate that long-range interactions between functionally distinct phospho-clusters collaborate to set clock speed (Chiu, 2011).

This study shows that the per-short domain functions as a discrete hierarchical phospho-cluster that delays Dbt-mediated phosphorylation at the Slimb recognition site on Per, providing new insights into how clock protein phosphorylation contributes to circadian timing mechanisms. The cumulative effect of this delay circuit is to slow down the pace of the clock by ~8 hr. It is proposed that Dbt functions in a stepwise manner to phosphorylate clusters on Per that have distinct biochemical functions and effects on the rate of Per degradation, e.g., elements such as the per-short phospho-cluster that delays Per degradation and those such as the Slimb-binding site and global phosphorylation that enhance instability. Nmo plays a major role in the relative timing of Dbt activity at these different elements because it stimulates multisite phosphorylation at the per-short delay cluster by Dbt, which slows down the ability of Dbt to phosphorylate instability elements. Thus, a large portion of the phosphorylation events dictating when in a daily cycle Per is targeted for rapid degradation is not directly linked to binding ofSlimb per se. The current findings demonstrate that presumptive long-range interactions between distinct positively and negatively acting phospho-clusters collaborate to set clock speed and helps to explain why mutations in clock protein phosphorylation sites and/or the kinases that phosphorylate them can yield both fast and slow clocks (Chiu, 2011).

A proposed mechanism for the function of the per-short domain is supported by the congruence between in vitro biochemical studies based on purified recombinant Per protein from cultured S2 cells and in vivo changes in the pace of behavioral rhythms using transgenic models. This suggests that a primary biochemical effect of the per-short domain on clock speed in the fly is via modulating the rate of Dbt-mediated phosphorylation at the Slimb phospho-degron on Per. The physiological role of T583 phosphorylation is not clear, as mutating this site does not have detectable effects on the binding of Per to Slimb in S2 cells. In this regard, it is interesting to note that the original per-short domain was identified as encompassing aa 585-601 of Per (Baylies, 1992). Thus, it is likely that the 8 hr per-short delay circuit is governed by the dynamics underlying the phosphorylated status of three sites (i.e., S596, S589, and S585) (Chiu, 2011).

At present, it is not clear how phosphorylation in the per-short cluster slows down subsequent phosphorylation by Dbt at Ser47 and other sites. Inactivating the per-short cluster leads to increases in the rate of Dbt-mediated phosphorylation at not only the N terminus, but also the C terminus of Per, suggesting that it is a major control center for regulating the relative efficiency of Dbt phosphorylation at many sites on Per. It is suggested that the per-short phospho- cluster acts as a transient 'temporal trap' for Dbt. Once the sites in the per-short domain are phosphorylated by Dbt, this somehow allows it to continue its normal rate of phosphorylation at other phospho-clusters. Although speculative, progressive increases in phosphorylation at some of these other phospho-clusters might generate time-dependent local/overall conformational changes in Per, possibly via electrostatic repulsion, eventually leading to a more open Per structure that is more accessible to phosphorylation by Dbt at the Slimb-binding site and/or a more efficient substrate for degradation. Thus, the rapid degradation of Per during the early day is likely due to a combination of synchronous increases in the phospho-occupancy of Ser47 and overall phosphorylation of Per. Other factors such as protein phosphatases and the action of Timeless also play major roles in regulating the speed of the Per phosphorylation program (Chiu, 2011).

How might phosphorylation at S596 enhance phosphorylation at S589 and S585 by Dbt? Phosphorylation by the CK1 kinase family is generally enhanced by priming. However, phosphorylation at the per-short domain by Dbt does not follow the consensus priming-dependent recognition motif for the CK1 family of kinases (i.e., S/Tp-X-X-S/T, wherein S/Tp refers to the primed site, X is any amino acid, and the italicized residues the CK1 target site, as the S596 priming site is located C terminal to the Dbt sites. Thus, it is likely that phosphorylation of S596 by Nmo stimulates Dbt phosphorylation at the per-short region in a nonpriming-dependent manner (Chiu, 2011).

Ongoing studies are aimed at understanding the biochemical events underlying the ability of phosphorylation at S596 to enhance phosphorylation by Dbt in the per-short region. The discovery of a delay phospho-circuit also sheds light on why mutations in different phosphorylation sites on Per or Frq proteins, although affecting stability, can speed up or slow down the clock. The current findings also offer a logical explanation for why mutations that lower the kinase activity of CKI, which overall is expected to slow down the rate of PER degradation, can yield fast clocks. For example, although other mechanisms have been offered to explain the short-period phenotypes that are observed for the CKI3tau mutation in hamsters and a CKIdelta mutation associated with familial advanced sleep phase syndrome (FASPS) in humans, it is possible that phosphorylation at a per-short type delay cluster is preferentially compromised by the mutant kinase, which could appear as a substrate-specific gain-of-function mutation (Chiu, 2011).

Negatively acting phospho-clusters are likely to be a general feature of the timing mechanisms regulating the daily abundance cycles of clock proteins such as Pers in animals and Frq in Neurospora. However, other regulatory modules that operate in a phase-specific manner must participate to generate an ~24 hr oscillator. Most conspicuously, clock speed is intimately linked to the Per and Frq abundance cycles necessitating daily phases of de novo synthesis to replenish the pools of previously degraded proteins.Asrecently shown, the transcriptional negative feedback aspect of Per regulating Clk-Cyc-mediated transcription is also a component of the period-setting mechanism in Drosophila. Therefore, the ~24 hr Per abundance cycle is based on a combination of 'time constraints' that are generated using different regulatory modules. It is proposed that the per short- based timer mainly functions once Per has accumulated and begins participating in transcriptional repression, controlling Per abundance once it is disengaged from the dynamics of its cognate mRNA by setting in motion a series of sequential phosphorylation events that are calibrated to stimulate Per degradation in the nucleus at the appropriate time in a daily cycle, enabling the next round of circadian gene expression. In this context, it is interesting to note that a prior study analyzing the per-short domain suggested that it functions with a nearby 'perSD' domain to increase the transcriptional repressor function of Per. It is possible that the same phosphorylation events leading to Per degradation also function to increase its potency within the repressor complex (Chiu, 2011).

These studies also identify Nemo as a clock kinase. Nemo is the founding member of the evolutionarily conserved Nemo-like kinase (Nlk) family of proline-directed serine/threonine kinases closely related to mitogen-activated protein kinases (MAPK). It was originally characterized in Drosophila as required for planar cell polarity during eye development and is now known to function in many pathways. Nmo/Nlk is localized in the nucleus and is another factor in the circadian clock that also functions in the Wnt/Wg-signaling pathway, such as CKI3/Dbt, β-TrCP/Slimb, and GSK-3β/Sgg. It will be of interest to determine whether Nlk functions in the mammalian clock. Intriguingly, the phosphorylation sites on Per are largely clustered, and several of them have the same spatial arrangement as the per-short cluster, with a predicted pro-directed kinase site at the C-terminal end of the phospho-cluster. This suggests that Nmo and/or other pro-directed kinases serve as control points to activate spatially and perhaps functionally distinct phospho-clusters. Indeed, it has recently been shown that phosphorylation at Ser661 of Per by an as yet unidentified pro-directed kinase primes further phosphorylation by Sgg at Ser657 to regulate the timing of Per nuclear entry in key pacemaker neurons (Chiu, 2011).

In summary, a central aspect of circadian clocks is the presence of one or more clock proteins that provide a dual function by behaving as phospho-based timers and linking its timer role to gene expression by operating in a phase-specific manner to recruit repressor complexes that inhibit central clock transcription factors. These studies suggest that a major part of the timing mechanism underlying these phospho-clock proteins is based on spatially and functionally discrete phospho-clusters that interact to impose calibrated and sequentially ordered biochemical time constraints. In the case of Per, the per-short phospho-cluster functions as a central timing module by slowing down the ability of Dbt to phosphorylate instability elements regulating Per degradation and, hence, when Per repressor activity is terminated and the next round of circadian gene expression begins (Chiu, 2011).

NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator

The Drosophila circadian oscillator is comprised of transcriptional feedback loops that are activated by Clock (Clc) and Cycle (Cyc) and repressed by Period (Per) and Timeless (Tim). The timing of Clk-Cyc activation and Per-Tim repression is regulated posttranslationally, in part through rhythmic phosphorylation of Clk, Per, and Tim. Although kinases that control Per and Tim levels and subcellular localization have been identified, additional kinases are predicted to target Per, Tim, and/or Clk to promote time-specific transcriptional repression. A screen was carried out for kinases that alter circadian behavior via clock cell-directed RNA interference (RNAi) and the proline-directed kinase nemo (nmo) was identified as a novel component of the circadian oscillator. Both nmo RNAi knockdown and a nmo hypomorphic mutant shorten circadian period, whereas nmo overexpression lengthens circadian period. Clk levels increase when nmo expression is knocked down in clock cells, whereas Clk levels decrease and Per and Tim accumulation are delayed when nmo is overexpressed in clock cells. These data suggest that nmo slows the pace of the circadian oscillator by altering Clk, Per, and Tim expression, thereby contributing to the generation of an ~24 hr circadian period (Yu, 2011).

This study has identified nmo as a new component of the Drosophila circadian oscillator. The short-period behavioral rhythms of nmoP1/Df and nmo RNAi flies indicates that Nmo acts to slow the pace of the circadian oscillator, consistent with the lengthening of circadian period when nmo is overexpressed in clock cells. A nmo P[lacZ] enhancer trap line reveal nmo expression in the sLNv and other brain clock cells, consistent with the short-period behavioral rhythms that result from expressing nmo RNAi in LNvs. Nmo is present in complexes with Per, Tim, and Clk when Clk-Cyc transcription is repressed and alters Per, Tim, and Clk levels and/or phosphorylation state. These results suggest that Nmo acts within Per-Tim-Clk regulatory complexes to lengthen circadian period. These results are likely relevant to the mammalian circadian oscillator because mPer and Clock are also highly phosphorylated when transcription is repressed and a single nmo ortholog, Nemo-like kinase (NLK), is present in mice and humans (Yu, 2011).

Cullin-3 controls Timeless oscillations in the Drosophila circadian clock

Eukaryotic circadian clocks rely on transcriptional feedback loops. In Drosophila, the Period and Timeless proteins accumulate during the night, inhibit the activity of the Clock (Clk)/Cycle (Cyc) transcriptional complex, and are degraded in the early morning. The control of Per and Tim oscillations largely depends on post-translational mechanisms. They involve both light-dependent and light-independent pathways that rely on the phosphorylation, ubiquitination, and proteasomal degradation of the clock proteins. Slmb, which is part of a CULLIN-1-based E3 ubiquitin ligase complex, is required for the circadian degradation of phosphorylated Per. This study shows that Cullin-3 (Cul-3) is required for the circadian control of Per and Tim oscillations. Expression of either Cul-3 RNAi or dominant negative forms of Cul-3 in the clock neurons alters locomotor behavior and dampens Per and Tim oscillations in light-dark cycles. In constant conditions, Cul-3 deregulation induces behavioral arrhythmicity and rapidly abolishes Tim cycling, with slower effects on Per. Cul-3 affects Tim accumulation more strongly in the absence of Per and forms protein complexes with hypo-phosphorylated Tim. In contrast, Slmb affects Tim more strongly in the presence of Per and preferentially associates with phosphorylated Tim. Cul-3 and Slmb show additive effects on Tim and Per, suggesting different roles for the two ubiquitination complexes on Per and Tim cycling. This work thus shows that Cul-3 is a new component of the Drosophila clock, which plays an important role in the control of Tim oscillations (Grima, 2012).

The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila.

In the Drosophila circadian clock, the Clock/Cycle complex activates the period and timeless genes that negatively feedback on Clock/Cycle activity. The 24-h pace of this cycle depends on the stability of the clock proteins. RING-domain E3 ubiquitin ligases have been shown to destabilize Period or Timeless. This study identifies a clock function for the circadian trip (ctrip) gene, which encodes a HECT-domain E3 ubiquitin ligase. ctrip expression in the brain is mostly restricted to clock neurons and its downregulation leads to long-period activity rhythms in constant darkness. This altered behaviour is associated with high Clock levels and persistence of phosphorylated Period during the subjective day. The control of Clock protein levels does not require PERIOD. Thus, Ctrip seems to regulate the pace of the oscillator by controlling the stability of both the activator and the repressor of the feedback loop (Lamaze, 2011).

Clk and Per thus seem to be the main targets of Ctrip. The ubiquitin ligase might act independently on Clk and Per, with the two proteins possibly competing for Ctrip binding. Alternatively, a Ctrip-mediated effect on Clk could affect Per stability. Such a mechanism might provide an efficient way to counterbalance changes in Clk levels. For example, it could help to keep the pace of the oscillator more resistant to variations in Clk levels, which might be induced by physiological stress or environmental changes (Lamaze, 2011).

In mammals, trip12 has recently been shown to be part of the ubiquitin fusion degradation (UFD) pathway, in which poly-ubiquitin is added to the N-terminus of the target protein as a degradation signal. Putative UFD pathway components are present in Drosophila, but no role for N-terminal ubiquitination has been shown. The current results raise the possibility that the UFD pathway is involved in tuning the speed of the circadian oscillator by controlling the stability of both Clk and Per (Lamaze, 2011).

A role for O-GlcNAcylation in setting circadian clock speed

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 (~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. This study identified 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 Per protein 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 Per is predominantly located in the cytoplasm. Intriguingly, the timing of when Per translocates from the cytoplasm to the nucleus is advanced or delayed in flies, wherein ogt expression is reduced or increased, respectively. These results suggest that O-GlcNAcylation of Per contributes to setting the correct pace of the clock by delaying the timing of Per nuclear entry. In addition, OGT stabilizes Per, suggesting that O-GlcNAcylation has multiple roles in circadian timing systems (E. Y. Kim, 2012).

Circadian clocks operate through negative feedback loops wherein positive elements activate negative elements, which in turn repress the activity of the positive elements until the levels of the negative elements decline, enabling another round of activation by the positive elements. To construct such a type of oscillating system, a lag is required before the negative elements can act to inhibit the positive elements. In Drosophila, the delayed nuclear entry/accumulation of Per contributes to the time delay in feedback repression. A complex web of kinases and phosphatases regulates when in a daily cycle Per participates in repressing Clk-Cyc-mediated transcription by regulating its stability, timing of nuclear entry, and duration in the nucleus. While Dbt is the major kinase driving daily cycles in Per levels, other kinases such as CK2 and GSK-3β/Sgg appear to have preferential effects on regulating the translocation of Per from the cytoplasm to the nucleus. The association of Tim with Per in the cytoplasm not only protects Per from Dbt-mediated degradation, but also enhances, yet is not obligatory for, Per nuclear entry (E. Y. Kim, 2012).

Thus, the regulation of when in a daily cycle Per translocates from the cytoplasm to the nucleus involves numerous factors. This study shows that the timing of Per nuclear entry is more complex than previously thought and identifies O-GlcNAcylation as a critical post-translational modification in setting clock speed (E. Y. Kim, 2012).

This study shows that Per is a direct target of OGT and is modified by O-GlcNAcylation. This modification occurs in a temporally regulated manner, first detected in the early night, peaking in the middle of the night, and declining/disappearing thereafter for the remainder of the Per daily life cycle. At present, it is not clear how O-GlcNAcylation of Per is temporally regulated. Based on studies indicating that expression of either ogt or oga is not under circadian regulation, some other factor(s) might favor O-GlcNAcylation of Per during the first half of the night. For example, uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), the donor substrate of OGT, is generated from glucose via the hexosamine biosynthetic pathway, indicating that the extent of protein O-GlcNAcylation can be sensitive to nutrient availability. Thus, it is possible that metabolic cues are affecting the activity of OGT in clock cells, leading to rhythmic activity of OGT (E. Y. Kim, 2012).

The physiological significance of O-GlcNAc modification in circadian clock systems was demonstrated by showing that the period of daily activity rhythms is sensitive to the levels of ogt expression in clock-specific cells. Remarkably, the periodicity of behavioral rhythms in ogt knockdown flies is shortened, whereas longer periods are observed in flies overexpressing ogt. This strongly suggests that O-GlcNAcylation of Per is a key variable in setting clock speed. The timing in Per nuclear entry was identified as a key event in the clockworks that is altered by changes in ogt expression in a manner consistent with the changes in overt behavioral rhythms. For example, inhibiting endogenous ogt expression in clock cells advanced the time of Per nuclear entry, likely underlying the shorter behavioral rhythms (E. Y. Kim, 2012).

Alterations in the timing of Per nuclear entry by genetically manipulating ogt levels might also explain the differences in per mRNA abundance cycles. For example, the more rapid nuclear entry of Per in ogt knockdown flies could contribute to the earlier decline in per mRNA levels and its inability to attain maximal peak values. Clearly, the earlier nuclear entry of Per when ogt levels are reduced is not due to an increase in the abundance of Per. Rather, it appears that Per stability is increased by more extensive O-GlyNAcylation. Experiments in S2 cells suggest that OGT has a primary effect on stabilizing Per against DBT-mediated degradation. How this occurs is presently not clear. It is also not established whether OGT-mediated changes in the levels of Per contribute to altering the timing of Per nuclear entry. However, it should be noted that other factors, such as Dbt and Tim, regulate both the stability of Per and its nuclear entry time, so multiple effects of O-GlyNAcylation on both Per levels and nuclear translocation are not unanticipated. Because O-GlyNAcylation of Per is mainly observed when it resides in the cytoplasm, it is proposed that this modification acts as an interval timer to prevent the premature nuclear entry of Per. While future work will be required to better understand the mechanism for how O-GlcNAcylation regulates Per nuclear entry, it is possible that O-GlcNAcylation of Per might attenuate phosphorylation at sites that enhance its translocation to the nucleus. Indeed, there are numerous reports illustrating complex interplays between phosphorylation and O-GlcNAcylation (E. Y. Kim, 2012).

It is intriguing that in plants, Spindly (Spy), which has significant similarity to animal OGT, functions in the same pathways with GIGANTE (GI). Like GI, SPY affects circadian rhythms in Arabidopsis. In loss of spy function mutants, the circadian period in cotyledon movement rhythm is lengthened, whereas overexpressing spy shortens the rhythm. Recent work using cardiomyocytes showed diurnal variations in total protein O-GlcNAcylation and identified Bmal1 as an O-GlcNAcmodified protein in mammals. Together with these results, this implies that O-GlcNAc modification has a conserved role in regulating circadian clock pace (E. Y. Kim, 2012).

Noncanonical FK506-binding protein BDBT binds Dbt to enhance its circadian function and forms foci at night

The kinase Doubletime is a master regulator of the Drosophila circadian clock, yet the mechanisms regulating its activity remain unclear. A proteomic analysis of Doubletime interactors led to the identification of an unstudied protein designated CG17282. RNAi-mediated knockdown of CG17282 produced behavioral arrhythmicity and long periods and high levels of hypophosphorylated nuclear Period and phosphorylated Doubletime. Overexpression of Doubletime in flies suppresses these phenotypes and overexpression of CG17282 in S2 cells enhances Doubletime-dependent Period degradation, indicating that CG17282 stimulates Doubletime's circadian function. In photoreceptors, CG17282 accumulates rhythmically in Period- and Doubletime-dependent cytosolic foci. Finally, structural analyses demonstrated CG17282 is a noncanonical FK506-binding protein with an inactive peptide prolyl-isomerase domain that binds Doubletime and tetratricopeptide repeats that may promote assembly of larger protein complexes. CG17282 was names Bride Of Doubletime and was established as a mediator of Doubletime's effects on Period, most likely in cytosolic foci that regulate Period nuclear accumulation (Fan, 2013).

While FKBPs were originally identified as mediators of the immunosuppressive effects of FK506 on calcineurin and rapamycin on the Target of Rapamycin (TOR), subsequent work has suggested their involvement in a wide range of signaling processes, including ones involved in neurodegeneration and cancer. In many cases, their function derives from their catalysis of cis-trans conversions of peptide bonds involving prolines. However, BDBT lacks the necessary catalytic residues, as do several other noncanonical FKBPs. One of these noncanonical FKBPs (FKBP38) has been proposed to interact with TOR to suppress its activity, while interactions between FKBP38 and the small GTP-binding protein RHEB relieve this repression and activate TOR. Intriguingly, TOR and RHEB have recently been show to modulate the circadian clock of Drosophila (Fan, 2013).

However, FKBP proteins have also been implicated in regulation of nuclear localization and protein stability. For instance, the noncanonical FKBP-like protein (FKBPL) has been implicated in the nuclear import of steroid hormone receptors in complexes with HSP90 proteins. An interesting possibility is that BDBT is involved in regulating the import of Per/Dbt complexes to the nucleus, and that at least some of this regulation is negative, as Per exhibits increased nuclear accumulation in BDBT knockdown flies. This hypothesis is consistent with structural work, which uncovered a resemblance between BDBT and the HSP90-binding protein FKBP51. The HSP90-binding site in FKBP51 localizes to its TPR domain and all but one of the residues that account for HSP90 binding are conserved in BDBT in spite of the low sequence homology with BDBT. Since the N-terminal, PPIase-like domain of BDBT binds to Dbt in HEK293 cells, it is possible that BDBT assembles a Dbt/Per/HSP90 complex, with Dbt bound to the PPIase-like domain, HSP90 to the TPR domain, and Per bound to Dbt (Fan, 2013).

FKBPs have also been implicated in the regulation of the stabilities of proteins with which they form a complex. A role in enhancement of Per's phosphorylation-dependent proteolysis is particularly attractive for BDBT, as it would explain the RNAi knockdown phenotype in head extracts (elevated levels of hypophosphorylated Per) and the enhancement of Dbt-dependent degradation of Per in S2 cells. The cytosolic BDBT foci in Drosophila photoreceptors accumulate at a time (ZT13-19) when Per transitions from a destabilized cytosolic form to a stabilized nuclear form, and the data supporting the involvement of BDBT in enhancement of Per proteolysis suggest that BDBT may be a negative regulator of this transition (i.e., BDBT antagonizes Per accumulation and nuclear localization). The BDBT foci are intriguing in light of the finding by Neyer of Per/Tim cytosolic foci, which form prior to accumulation of Per and Tim in S2 cell nuclei (Meyer, 2006). It was proposed that processes in these foci trigger the nuclear accumulation of both Per and Tim. Since the suggestion from this work is that BDBT foci antagonize nuclear accumulation of Per and no obvious Per foci were observed that colocalize with the BDBT foci, it is possible that BDBT antagonizes focal accumulation of Per or immediately triggers the degradation of Per in these foci. In this scenario, in contrast to the situation in S2 cells, Per might accumulate in foci in vivo only when BDBT is not present or active in the foci, and Per's presence in foci in vivo may therefore be difficult to detect because it rapidly accumulates and localizes to nuclei when BDBT is not active in the foci. It is also possible that focal accumulation of BDBT negatively regulates BDBT activity toward Dbt, since highest levels of BDBT foci are detected at ZT19, when Per is rapidly accumulating in nuclei and therefore any BDBT inhibition of Per nuclear accumulation might be inhibited (Fan, 2013).

Because the mammalian orthologs of Dbt (CKIepsilon and CKIdelta) are also essential for the molecular mechanism of the mammalian circadian clock, it is possible that the mechanism in which bdbt participates is conserved in mammals. While this manuscript was in preparation, FKBP and FKBP-like proteins were reported to form complexes with mammalian CKIepsilon and CKIdelta (Kategaya, 2012), although neither these proteins nor any other protein in the mammalian genome is an ortholog of BDBT. The lack of homology between the PPIase-like region in BDBT, which mediates binding to DBT, and the ones found in vertebrates, was initially surprising. However, the binding experiments indicate that the BDBT binding site in DBT spans its well-conserved N-terminal kinase domain and the poorly conserved C-terminal tail. Thus, it seems likely that the binding modes between BDBT and Dbt on one hand and the ones between the vertebrate homologs of BDBT and CKIepsilon and CKIdelta differ substantially. While it is not known if this interaction has any role in the mammalian circadian clock, these results offer the tantalizing prospect that this class of proteins and their roles are conserved in the mechanisms of the mammalian and Drosophila clocks (Fan, 2013).

Casein kinase 1 promotes synchrony of the circadian clock network

Casein kinase 1, known as Doubletime (Dbt) in Drosophila, is a critical component of the circadian clock that phosphorylates and promotes degradation of the Period (Per) protein. However, other functions of Dbd in circadian regulation are not clear, in part because severe reduction of dbt causes pre-adult lethality. This study reports the molecular and behavioral phenotype of a viable dbtEY02910 loss-of-function mutant. It was found that Dbt protein levels are dramatically reduced in adult dbtEY02910 flies, and the majority of mutant flies display arrhythmic behavior, with a few showing weak, long period (approximately 32h) rhythms. Peak phosphorylation of Per is delayed, and both hyper- and hypo-phosphorylated forms of the Per and Clock proteins are present throughout the day. In addition, molecular oscillations of the circadian clock are dampened. In the central brain, Per and Tim expression is heterogeneous and decoupled in the canonical clock neurons of the dbtEY02910 mutants. An interaction is also reported between dbt and the signaling pathway involving Pigment Dispersing Factor (PDF), a synchronizing peptide in the clock network. These data thus demonstrate that overall reduction of Dbt causes long and arrhythmic behavior and reveal an unexpected role of Dbt in promoting synchrony of the circadian clock network (Zheng, 2014).

The MAP kinase p38 is part of Drosophila melanogaster's circadian clock

All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. This study demonstrates by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, it was found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining approximately 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed the Western Blot results and point to p38 as a potential 'clock kinase' phosphorylating Period. Taken together, these findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways (Dusik, 2014 - Open access: 25144774).

p38 MAP Kinase regulates circadian rhythms in Drosophila

The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. This study found that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. This study also found that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, p38Kb was shown to suppress the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, manipulating p38K signaling in free-running conditions was found to alter the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways (Vrailas-Mortimer, 2014).

Drosophila peptidyl-prolyl isomerase Pin1 modulates circadian rhythms via regulating levels of PERIOD

In animal circadian clock machinery, the phosphorylation program of Period (Per) leads to the spatio-temporal regulation of diverse Per functions, which are crucial for the maintenance of approximately 24-hr circadian rhythmicity. The peptidyl-prolyl isomerase PIN1 modulates the diverse functions of its substrates by inducing conformational changes upon recognizing specific phosphorylated residues. This study shows that overexpression of Drosophila pin1, dodo (dod), lengthens the locomotor behavioral period. Using Drosophila S2 cells, it was demonstrated that Dod associates preferentially with phosphorylated species of Per, which delays the phosphorylation-dependent degradation of Per. Consistent with this, Per protein levels are higher in flies overexpressing dod. Taken together, it is suggested that Dod plays a role in the maintenance of circadian period by regulating Per metabolism (Kang, 2015).

Identification of light-sensitive phosphorylation sites on PERIOD that regulate the pace of circadian rhythms in Drosophila

The main components regulating the pace of circadian clocks in animals are Period (Per) proteins, transcriptional regulators that by means of complex multi-site phosphorylation programs undergo daily changes in levels and nuclear accumulation. This study investigated the function of two phosphorylation sites at Ser826 and Ser828 located in a putative nuclear localization signal (NLS) on the Drosophila melanogaster Per protein. These sites are phosphorylated by Doubletime (Dbt; Drosophila homolog of CK1delta/), the key circadian kinase regulating the daily changes in Per stability and phosphorylation. Mutant flies where phosphorylation at Ser826/Ser828 is blocked manifest behavioral rhythms with periods slightly longer then 1 hour and altered temperature compensation properties. Intriguingly, although phosphorylation at these sites does not influence Per stability, timing of nuclear entry or transcriptional autoinhibition, the phospho-occupancy at Ser826/Ser828 is rapidly stimulated by light and blocked by Timeless (Tim), the major photosensitive clock component in Drosophila and a crucial binding partner of Per. These findings identify the first phosphorylation sites on core clock proteins that are acutely regulated by photic cues and suggest that some phospho-sites on Per proteins can modulate the pace of downstream behavioral rhythms without altering central aspects of the clock mechanism (Chiu, 2015).

period: Biological Overview | Evolutionary Homologs | Regulation | Targets of Activity and Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

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