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Darkness And Flame: The Dark Side Activation Code Generator


timeless REGULATION Protein Interactions and Post-transcriptional Regulationtimeless was cloned by chromosomal walking and subsequently used, in yeast, to identify Per as a physical partner. Timeless and Period interact, and both are required for production of circadian rhythms. The tim gene encodes a protein of 1389 amino acids, and possibly another protein of 1122 amino acids. The arrhythmic mutation tim01 is a 64-base pair deletion that truncates TIM to 749 amino acids. Absence of sequence similarity to the Per dimerization motif (PAS) indicates that direct interaction between Per andTim would require a heterotypic protein association (Meyers, 1995).Tim was isolated based on its ability to physically interact with the Per protein. A restricted segment of Tim binds directly to PAS, a part of the Per dimerization domain. PerL, a mutation in Per that causes a temperature-sensitive lengthening of circadian period and a temperature-sensitive delay in Per nuclear entry, exhibits a temperature-sensitive defect in binding to Tim (Gekakis, 1995).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 Drosophilaclock gene period (per) were measured. Photic stimuli perturb the timing of the Per protein andmessenger RNA cycles in a manner consistent with the direction and magnitude of the phase shift.The recently identified clock protein Timeless interacts with Per in vivo, andthis association is rapidly decreased by light. This disruption of the Per-Tim complex in thecytoplasm is accompanied by a delay in Per phosphorylation and nuclear entry and disruption inthe nucleus by an advance in Per phosphorylation and disappearance. These results suggest amechanism for how a unidirectional environmental signal elicits a bidirectional clock response (Lee, 1996). Many circadian features of the Tim cycle resemble those of the Per cycle. However, Tim israpidly degraded in the early morning or in response to light, releasing Per from the complex. ThePer-Tim complex is a functional unit of the Drosophila circadian clock, and Tim degradation maybe the initial response of the clock to light (Zeng, 1996). Drosophila Clock protein (dClock) is a transcription factor that is required for the expression of the circadianclock genes period (per) and timeless (tim). dClock undergoes circadian fluctuations in abundance, is phosphorylated throughout a daily cycle, andinteracts 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 Timare 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 totallevels of Tim and Per in head extracts peak at ZT16 and ZT20, respectively. This suggests that during thenight dClock is present in limiting amounts compared to Per and Tim. Despite the higher levels ofimmunoprecipitated dClock between ZT4 and ZT8 compared to values obtained between ZT12 and ZT16, very little, ifany, Per and Tim are detected. A likely explanation forthis 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 canstill interact with dClock. Because Tim is apparently located exclusively in thecytoplasm in the absence of Per, this result could suggest that thenuclear localization of dClock also requires Per or a functional oscillator. Alternatively, low levels of Tim might beable to enter the nucleus in the absence of Per. In contrast, several attempts to visualize a specific interaction betweenPer and dClock in the absence of Tim were unsuccessful. There are atleast two nonmutually exclusive reasons that might account for thr inability to detect Per in dClock-containingimmune 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 thedetection limit, and (2) the interaction of Per with dClock requires Tim, possibly via formation of the Per-Timcomplex 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 anLD cycle. Head extracts were incubated with antibodies against either Per or Tim, and immune complexes probed fordClock, Per, and Tim. At ZT20 almost identical levels of dClock copurify with antibodies directedagainst either Per or Tim. Equivalent amounts of Per were also present in both immunepellets, but 1.6-fold more Tim is immunoprecipitated with antibodies toTim, as compared to those directed against Per. These results are almost identicalwith a previous study showing that (1) in head extracts prepared from flies collected at ZT20, 80% of thetotal 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 Timproteins that interact with dClock are in the form of a heterodimeric Per-Tim complex. During the early day, only lowlevels 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 earlyday (Lee, 1998). How might a trimeric complex containing Per, Tim, and dClock be assembled? Presumably the HLH domain ofdClock does not participate in mediating protein-protein interactions in this putative trimeric complex, because neitherPer nor Tim seems to have a similar dimerization region. The only other regions that have been shown to mediateprotein-protein interactions are the PAS domain found in Per and dClock and a not so well characterized region in Tim thatspans 400 amino acids and interacts with the PAS domain of Per. It is tempting tospeculate that one or both of these domains has the capacity to engage in at least trimeric formation. Although these studiesdo 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 intranscriptional autoinhibition by physically interacting with dClock or a dClock-containing complex. Nevertheless, in the absence of Per or Tim, thelevels of dClock are constitutively low, indicating that Per and Tim also act as positive elements in the feedback loop by stimulating the production ofdClock. Although Per and Tim inhibit dClock activity, Per and Tim arerequired for the high-level production of dClock protein and mRNA. Thus, Per andTim appear to be the main "motor" of the Drosophila circadian oscillator, driving both positive and negative elements ofthe transcriptional-translational feedback loop. These observations suggest an explanation for the previously unexplainedfinding that the levels of Per mRNA in per mutant flies are approximately half as high as those obtained at peak times inwild-type flies. In contrast, mutations that abolish Neurospora FRQ activity result in high levels of frqRNA, suggesting that the frq-based circadian oscillator in Neurospora is based on a more simple negativetranscriptional 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 ofnegative factors leading to the stimulation of gene expression. In addition to regulating the transcriptional activity of thedClock-CYC complex, Per and Tim might also interact with other transcription factors that are not involved in thecircadian oscillator and as such molecularly couple the timekeeping mechanism to downstream effector pathways (Lee, 1998). The cyclic expression of the Period (Per) and Timeless (Tim)proteins is critical for the molecular circadian feedback loop inDrosophila. The entrainment by light of the circadian clock ismediated by a reduction in Tim levels. To elucidate the mechanism ofthis process, the sensitivity of Tim regulation by light was testedin an in vitro assay with inhibitors of candidate proteolyticpathways. The data suggest that Tim is degraded through aubiquitin-proteasome mechanism. In addition, in cultures fromthird-instar larvae, Tim degradation is blocked specifically byinhibitors of proteasome activity. Degradation appears to bepreceded by tyrosine phosphorylation. Finally, Tim is ubiquitinatedin response to light in cultured cells (Naidoo, 1999).An in vitro assay was developed to investigatethe nature of the Tim light response.Flies were entrained to a 12 hour light/12 hour dark cycle andprepared head extracts from flies collected at either ZT (zeitgebertime) 20 or immediately after a 1-hour light pulse delivered atZT 19 (ZT0 = lights on; ZT12 = lights off).These extracts were incubated with Tim protein immunoprecipitatedfrom fly heads. After a 1-hour incubation at room temperature, Tim levels were assayed by protein immunoblots. Addition of the pulsedextract reduces the Tim signal. Unpulsedhead extract has no effect on the level of Tim, indicating that thereduction is light-specific. This light-induced reduction is alsoobserved in tim0 flies (which lack Tim protein) andis, in fact, routinely higher in these flies, which may suggest somedown-regulation by the clock in wild-type flies. Animmunoprecipitated Per substrate is not degraded by addition of thepulsed extract (Naidoo, 1999).In order to determine the nature of the proteolytic activity, several general classes of protease inhibitors were assayed. Inhibitors ofserine proteases [phenylmethylsulfonyl fluoride (PMSF) and aprotinin]and aspartate proteases (pepstatin) are not very effective inblocking Tim degradation. However,degradation is inhibited by the proteasomal inhibitorsacetyl-leu-leu-norleucinal (ALLN), cbz-leu-leu-norvalinal(ZL2NVaH or MG115) and cbz-leu-leu-leucinal(ZL3H or MG132). These peptidealdehydes strongly inhibit the chymotryptic activity of theeukaryotic 26S proteasome. Timdegradation is also blocked by bestatin, a metalloproteaseinhibitor, and by leupeptin, which inhibits cysteine proteases andhas some effects on other proteolytic systems, including theproteasome. The precise mechanism of actionin this case is not known. Consistent with a role for the proteasome,depletion of ubiquitin from the extract blocks Timdegradation (Naidoo, 1999). Although the in vitroassay indicates a mechanism for Tim's response to light, itsusefulness is limited by its variability.To verify the findings of this assay, an in vivosystem was developed. Thus, a primary culture assay was used to test the effect of two proteasomal inhibitors (lactacystinand MG115) on the Tim light response. Lactacystin, a microbialmetabolite, is the most specific one known; a naturally occuring inhibitorof the proteasome. It spontaneouslyhydrolyzes into clastolactacystin B lactone, which is the activespecies that reacts with the proteasome, inhibiting its chymotrypticand tryptic peptidase-like activity. MG115 is a potent synthetic peptide aldehydeinhibitor. For the assay, the centralnervous system (CNS) of third-instar larvae was dissected and maintained in culture medium for1 hour. Some samples were exposed to a pulse of light for20 min and were fixed at the end of the hour. Dark controlsamples were also incubated for an hour in the dark. Tim expression was then examined in thelateral neurons (clock cells), which were located by costaining withan antibody to pigment-dispersing hormone. Strong Tim staining is seen in lateral neurons ofunpulsed tissue, but little to no Tim in CNS tissue that has receiveda light pulse. The effect of inhibitors was tested by adding them to theculture medium at the start of the incubation. Tissue treated withlactacystin and MG115 before the light pulse revealed robust Timstaining in the lateral neurons. The strong inhibition by MG115 isconsistent with a report that this is a much more effective inhibitorof proteolysis in intact cells than it is of in vitro hydrolysis ofmacromolecular substrates. The 100%block by lactacystin may reflect variable permeability or instabilityof the lactone metabolite (Naidoo, 1999). Proteasomes are multicatalytic, multisubunit proteolytic complexeswith highly conserved structures; they play a key role in avariety of cellular processes, including the cell cycle,transcriptional regulation, removal of abnormal proteins from thecell, antigen presentation, and even in theturnover of a mammalian circadian-regulated protein. The Tim response to light is blockedspecifically, in two different assays, by several inhibitors of theproteasome; this is important, given that lactacystin, which wasthought to affect only the proteasome, has been shown to also act on a second multisubunit enzyme. Because the newly identified enzyme isinsensitive to ALLN, it cannot account for the Tim response. For the ubiquitin-proteasome system, prolineglutamate serine threonine (PEST) regions sometimes serve as putativedegradation/phosphorylation signals in the target molecule. The Tim protein sequence reveals the presenceof seven PEST regions concentrated near the NH2 and COOHtermini (Naidoo, 1999). Most cellular proteins that are degraded by theproteasome are ubiquitinated and then targeted to the proteasome. To determine whether Tim is ubiquitinated,which would also demonstrate that it is a direct target of theproteasome, a cell culture system was used. Tim and ahemagglutinin (HA)-tagged ubiquitin octamer were expressed under heat shock control in Drosophila S2 cells. After a30-min heat shock, cells were either maintained in the dark ortreated with light for 2 hours, after which the cells were lysedand immunoprecipitates of Tim were probed with an antibody to HA. Tim was found to be ubiquitinated in response to light. The effect is specific for Tim, because Peris not ubiquitinated with or without light treatment. Extended light treatment also degrades Tim inthese cells, and this degradation is inhibited by the proteasomeinhibitor MG115. Although these data implicate a ubiquitin-proteasomal mechanism,they do not preclude a role for other proteolyic systems (Naidoo, 1999). To investigatea possible role for phosphorylation in the degradation of Tim, the effect of several kinase inhibitors in the in vivoprimary culture assay were examined. The tyrosine kinase inhibitor genisteinblocks the degradation of Tim in the lateral neurons after a pulseof light, whereas the serine-threonine inhibitors staurosporin andcalphostin C and the MEK inhibitor PD98059 do not. These results suggest that tyrosine kinaseactivity precedes degradation of Tim. The concentrations of genisteinthat were effective in this assay suggest ac-src-like kinase activity, althoughthe concentration dependence must be interpreted with caution,because it could be a measure of permeability or drug stability (Naidoo, 1999).To determine whether the tyrosine phosphorylation occurs on Timitself, protein immunoblots of Tim immunoprecipitates were probed withan antibody to phosphotyrosine. After 20 min of light treatmentat ZT19, Tim could be detected with the antibody tophosphotyrosine. Tim in the 'dark'samples is sometimes detected with this antibody but notconsistently, which suggests that tyrosine phosphorylation of Tim isincreased by light. The mobility of the Tim band in the light-treatedsample is also reduced, presumably because of phosphorylation (Naidoo, 1999). Together, these data indicate that the Tim response to lightinvolves tyrosine phosphorylation and ubiquitination, followed byproteasomal degradation. What then is the role of the proteasome pathway infree-running behavioral rhythms? Are the mechanisms that degrade Timin response to light the same as those that degrade it in constantdarkness? If this is the case, light may serve only to further activate aprocess that is already under way. It is proposed that cyclic turnover ofTim under free-running conditions is mediated by phosphorylation,which targets it for degradation, perhaps by the proteasome. Tim isprogressively phosphorylated throughout the night, and maximallyphosphorylated forms are found just before the rapid decline ofprotein levels. From this point on, untilthe middle of the day, Tim levels remain low because of the lowlevels of RNA. As the repression of transcription is released, mostlikely because of the decrease in Per levels, RNA accumulates andprotein also starts to accumulate, albeit slowly, because it is stillsubject to phosphorylation and degradation. When the rate of Timsynthesis exceeds the rate of phosphorylation/degradation, higherlevels of protein are observed, but as the phosphorylation programcontinues and RNA levels are reduced (because of negative feedback),levels of the protein drop off. Light could enhance Tim degradationby increasing Tim phosphorylation and/or by increasing proteolytic activityin some manner. This model would predict that the presenceof light accelerates the falling phase of the protein and delays therising phase, both through the same mechanism (Naidoo, 1999).Phosphorylation is an important feature of pacemaker organization in Drosophila. Genetic and biochemical evidence suggestsinvolvement 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 morninghours, 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


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