period
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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 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).