Gene name - discs overgrown
Synonyms - double-time
Cytological map position - 100B2
Function - protein kinase
Symbol - dco
FlyBase ID: FBgn0002413
Genetic map position -
Classification - casein kinase I delta/epsilon
Cellular location - cytoplasmic
|Recent literature||Means, J.C., et al. (2015). Drosophila Spaghetti and Doubletime link the circadian clock and light to caspases,
apoptosis and tauopathy. PLoS Genet 11: e1005171. PubMed ID: 25951229
While circadian dysfunction and neurodegeneration are correlated, the mechanism for this is not understood. This study shows that the knock-down of a regulator (spag) of the circadian kinase Dbt in circadian cells lowers Dbt levels abnormally, lengthens circadian rhythms and causes expression of activated initiator caspase (Dronc) in the optic lobes during the middle of the day or after light pulses at night. Likewise, reduced Dbt activity lengthened circadian period and caused expression of activated Dronc, and a loss-of-function mutation in Clk also lead to expression of activated Dronc in a light-dependent manner. Genetic epistasis experiments placed Dbt downstream of Spag in the pathway, and Spag-dependent reductions of Dbt were shown to require the proteasome. Importantly, activated Dronc expression due to reduced Spag or Dbt activity occurred in cells that did not express the spag RNAi or dominant negative Dbt and required PDF neuropeptide signaling from the same neurons that support behavioral rhythms. Furthermore, reduction of Dbt or Spag activity lead to Dronc-dependent Drosophila Tau cleavage and enhanced neurodegeneration produced by human Tau in a fly eye model for tauopathy. Aging flies with lowered Dbt or Spag function showed markers of cell death as well as behavioral deficits and shortened lifespans, and even old wild type flies exhibited Dbt modification and activated caspase at particular times of day. These results suggest that circadian clock defects confer sensitivity to expression of activated Dronc in response to prolonged light. This study has established a link between the circadian clock factors, light, cell death pathways and Tau toxicity.
Fan, J. Y., Means, J. C., Bjes, E. S. and Price, J. L. (2015) Drosophila DBT autophosphorylation of its C terminal domain antagonized by SPAG and involved in UV-induced apoptosis Mol Cell Biol [Epub ahead of print] PubMed ID: 25939385
|Venkatesan, A., Fan, J. Y., Nauman, C. and Price, J. L. (2015). A Doubletime nuclear localization signal mediates an interaction with Bride of Doubletime to promote circadian function. J Biol Rhythms [Epub ahead of print]. PubMed ID: 26082158
Doubletime (DBT) has an essential circadian role in Drosophila melanogaster because it phosphorylates Period (Per). To determine if DBT antagonism can produce distinct effects in the cytosol and nucleus, forms of a dominant negative DBTK/R with these 2 alternative localizations were produced. DBT has a putative nuclear localization signal (NLS), and mutation of this signal confers cytosolic localization of DBT in the lateral neurons of Drosophila clock cells in the brain. By contrast, addition of a strong NLS domain (e.g., SV40 NLS) to DBT's C terminus leads to more nuclear localization. Expression of DBTK/R with the mutated NLS (DBTK/R NLS-) using a timGAL4 driver does not alter the circadian period of locomotor activity. By contrast, expression of DBTK/R with the strong NLS (DBTK/R stNLS) lengthens period more strongly than DBTK/R, with damped oscillations of Per phosphorylation and localization. Both DBTK/R and DBTWT without the NLS fail to interact with Bride of Doubletime (BDBT) protein, which is related to FK506-binding proteins and shown to interact with DBT to enhance its circadian function. This result suggests that the DBTK/R NLS- has lost its dominant negative property because it does not form normal clock protein complexes. DBTWT proteins with the same changes (NLS- and stNLS) also produce equivalent changes in localization that do not produce opposite period phenotypes. Additionally, the lack of a dominant negative for the DBTK/R NLS- is not due to failure to localize to nuclei. Finally, bdbt RNAi increases the cytosolic localization of DBTK/R but not of DBTWT, suggesting a role for BDBT in DBT kinase-dependent nuclear localization of DBT.
|Chiu, J. C. and Edery, I. (2015). Identification of light-sensitive phosphorylation sites on PERIOD that regulate the pace of circadian rhythms in Drosophila. Mol Cell Biol [Epub ahead of print]. PubMed ID: 26711257
The main components regulating the pace of circadian clocks in animals are Period (Per) proteins, transcriptional regulators that by means of complex multi-site phosphorylation programs undergo daily changes in levels and nuclear accumulation. This study investigated the function of two phosphorylation sites at Ser826 and Ser828 located in a putative nuclear localization signal (NLS) on the Drosophila melanogaster Per protein. These sites are phosphorylated by Doubletime (Dbt; Drosophila homolog of CK1delta/), the key circadian kinase regulating the daily changes in Per stability and phosphorylation. Mutant flies where phosphorylation at Ser826/Ser828 is blocked manifest behavioral rhythms with periods slightly longer then 1 hour and altered temperature compensation properties. Intriguingly, although phosphorylation at these sites does not influence Per stability, timing of nuclear entry or transcriptional autoinhibition, the phospho-occupancy at Ser826/Ser828 is rapidly stimulated by light and blocked by Timeless (Tim), the major photosensitive clock component in Drosophila and a crucial binding partner of Per. These findings identify the first phosphorylation sites on core clock proteins that are acutely regulated by photic cues and suggest that some phospho-sites on Per proteins can modulate the pace of downstream behavioral rhythms without altering central aspects of the clock mechanism.
Mutations in double-time effect circadian rhythms in Drosophila. dbt contributes to circadian rhythmicity by determining the stability of Period (Per) protein. It has been proposed that Dbt promotes molecular cycles of per and timeless (tim) expression by ensuring the instability of monomeric Per proteins, thus allowing Per accumulation only in conjunction with high titers of Tim (Price, 1998). Cloning of dbt has revealed that Dbt protein is closely related to a family of protein kinases (Kloss, 1998). The most closely related kinases are the delta and epsilon isoforms of human casein kinase I (Fish, 1995).
Double-time turns out to be identical to the discs overgrown (dco) cell growth regulating gene of Drosophila. In contrast to the weak double-time alleles, which appear to affect only the circadian rhythm, discs overgrown alleles show strong effects on cell survival and growth control in imaginal discs. The Discs overgrown protein is a crucial component in the mechanism that links cell survival during proliferation to growth arrest in imaginal discs. Since the amino acid sequences and three-dimensional structures of Casein kinase I delta/epsilon enzymes are highly conserved, the results suggest that these proteins may also function in controlling cell growth and survival in other organisms. Analysis of phenotypes of various discs overgrown mutants, including heteroallelic combinations of strong and null alleles, suggests that this gene is required for inhibition of apoptosis during cell proliferation as well as for growth arrest in imaginal discs (Zilian, 1999).
This overview will treat the involvement of Double-time in the photoperiod response. Information about the role of dbt/discs overgrown in the regulation of imaginal disc survival, proliferation and growth arrest can be found in the Effects of mutation section. The current understanding of the molecular regulation of circadian rhythmicity in Drosophila comes from integrating genetics and molecular biology. Null mutations in either of two genes, per and tim, abolish behavioral rhythmicity, while alleles encoding proteins with missense mutations have been recovered at both loci and show either short- or long-period behavioral rhythms. The RNA and protein products of these genes oscillate with a circadian rhythm in wild-type flies. These molecular rhythms are abolished by null mutations of either gene, and the periods of all molecular rhythms are correspondingly altered in each long- and short-period mutant, indicating a regulatory interaction between these genes. Production of these molecular cycles appears to depend on the rhythmic formation and nuclear localization of a complex containing the Per and Tim proteins. A physical interaction of Per and Tim is required for nuclear localization of either protein, and nuclear activity of these proteins coordinately regulates per and tim transcription through a negative feedback loop. A complex of two transcription factors, Clock and Cycle, positively regulates both per and tim transcription, and this positive regulation is suppressed by nuclear Per/Tim proteins. A model for the Drosophila clock has been proposed in which delayed formation of Per/Tim complexes ensures separate phases of per/tim transcription and nuclear function of the encoded proteins. Entrainment of this oscillator is regulated through the Tim protein, which is rapidly eliminated from the nucleus and cytoplasm of pacemaker cells when Drosophila are exposed to daylight (Price, 1998 and references).
Although some key features of the Drosophila clock have been identified, the involvement of additional, essential factors is suspected from prior work. Per fails to accumulate in the absence of Tim even in the presence of high per RNA levels, pointing to the existence of an activity that destabilizes cytoplasmic Per monomers. Both Per and Tim (Edery, 1994 and Zeng, 1996) as well as Clock (Lee, 1998), are phosphorylated with a circadian rhythm, which is an indication of the presence of unidentified kinases. Per, in particular, becomes progressively phosphorylated over many hours, and the timing of its phosphorylation is changed in period-altering mutants (Edery, 1994 ), suggesting either circadian regulation of Per phosphorylation or a role in establishing rhythmicity. double-time alleles have been found that either shorten or lengthen the periods of behavioral and molecular rhythms. A strongly hypomorphic dbt allele has been isolated that is associated with pupal lethality and blocks circadian oscillations of per and tim gene products in larvae. Dbt is therefore a central clock component alongside Per and Tim. dbt period-altering alleles alter the kinetics of Per phosphorylation and degradation. The hypomorphic allele constitutively produces unusually high levels of Per proteins that are hypophosphorylated. Thus, a normal function of Dbt appears to be regulation of Per accumulation. Evidence is presented that dbt contributes to circadian rhythmicity by determining the stability of Per. It is proposed that DBT activity in wild-type flies promotes molecular cycles of per and tim expression by ensuring the instability of monomeric Per proteins, thus allowing Per accumulation only in conjunction with high titers of Tim (Price, 1998 and references).
What is the effect of dbt mutation on Per and Tim levels in the brain? It was reasoned that the strongly hypomorphic P-element insertion allele dbtP might show the most dramatic effects on clock gene cycling. Although dbtP embryos take longer to develop into third instar larvae than do their heterozygous siblings, the foraging motility of these larvae and their touch sensitivity appear normal. Behavioral studies have demonstrated that a circadian clock is active in Drosophila larvae. It has recently been shown that a specific group of central brain cells is likely to compose the larval pacemaker (Kaneko, 1997). In each hemisphere of the third instar larval brain, four to five cells coexpress Per and Tim with circadian oscillations that are in phase with the oscillations of these proteins in adult pacemaker cells. The only detectable staining in larval brain hemispheres for pigment-dispersing hormone (PDH), a marker for adult lateral neurons (Helfrich-Forster, 1995), is found in the cell bodies and axons of these Per-Tim expressing cells (Kaneko, 1997). Therefore, the Per-Tim-PDH coexpressing larval brain cells can be considered larval lateral neurons (lvLNs). In pero larvae, Tim is constitutively cytoplasmic in lvLNs; in tim01 larvae, Per is undetectable in these cells by immunocytochemistry. Both of these mutant phenotypes are characteristic of adult clock cells. Per and Tim oscillations can also be seen in two groups of cells at the anterior of the third instar larval brain, but in one of these groups, the oscillations are out of phase with the lvLNs and may be regulated by activity of the lvLNs. Since there are no good markers to distinguish clearly between these two anterior cell types, for analysis, focus was placed on the lvLNs (Price, 1998).
LvLNs are indeed present in dbtP larvae. PDH staining is detected in the cytoplasm of four cells in each hemisphere in all dbtP larval brains examined at different times in alternating light-dark (LD) and constant darkness (DD) cycles. The axons of these dbtP lvLNs fasciculate and head to the anterior of the brain as in wild type. However, dbtP lvLNs are found slightly more peripherally than in wild type, as seen in the staining patterns of Per and Tim. This probably indicates a subtle developmental effect of dbt on the architecture of the brain, which might be expected given that the dbtP mutation causes lethality before completion of pupal development (Price, 1998).
Although regulation of Tim's light sensitivity and nuclear localization are not affected by dbtP mutation, oscillations of Tim protein and TIM mRNA cease in dbtP larval brains in constant darkness. When wild-type larvae are transferred to constant darkness, Tim continues to oscillate robustly with only night time Tim accumulation in the lvLNs. In contrast, in dbtP larvae transferred to constant darkness (DD), Tim is weakly detected in lvLNs in the first subjective morning at CT5 (CT, circadian time, indicates time in DD), disappears by CT10, and is undetectable thereafter in the lvLNs. Tim is always detected in dbtP in the anterior larval brain cells; these cells serve as a positive control for the procedure. For the lvLNs, the differential effects of dbtP on Tim in LD and DD are not due to selecting larvae from slightly different developmental stages since identical results were derived from larvae that had been synchronized developmentally. In wild-type larvae, TIM mRNA shows robust oscillations in the lvLNs in both LD and DD. TIM mRNA levels oscillate in the lvLNs of dbtP mutants in an LD cycle, indicating that Per/Tim complexes can still negatively regulate tim gene expression in dbtP larvae and that this regulation can be blocked by light-dependent degradation of Tim. When dbtP larvae are transferred to DD, TIM mRNA is weakly detected in lvLNs on the first subjective morning (CT2) but is undetectable thereafter in these cells. Thus, the effects of the dbtP mutation on levels of Tim protein probably reflect more direct effects of dbtP on TIM mRNA levels (Price, 1998).
Per protein levels oscillate in wild-type lvLNs in an LD cycle, reaching peak levels at ZT23. In DD, Per continues to cycle in wild-type lvLNs and is detected at CT1 but not CT13. Per proteins produced by dbtP larvae show three significant differences from wild type. (1) Per is constitutively expressed in lvLNs in LD and DD cycles; (2) the intensity of staining in the lvLNs is stronger in dbtP than in wild-type larvae; (3) the pattern of expression in dbtP is widened to include regions of the brain not significantly stained in a wild-type background. The elevated level of Per in dbtP was confirmed by Western blotting using extracts from dissected larval brains collected in LD. The latter results show that Per protein accumulation is dramatically increased by the dbtP mutation, and the high levels of accumulated Per protein do not show significant differences between ZT12 and ZT24 in LD cycles in dbtP. In addition, the electrophoretic mobility of Per proteins is relatively high and uniform in dbtP larvae, in contrast to the broad spectrum of lower Per protein mobilities observed in wild-type larvae and adult heads. As the spectrum of protein mobilities in wild-type Drosophila reflects Per protein phosphorylation (Edery, 1994), the results suggest that Per is hypophosphorylated in dbtP mutants (Price, 1998).
To determine whether the high levels of Per protein found by Western blotting of dissected dbtP larval brains reflects altered PER mRNA levels, RNase protection was used to detect per RNA in these tissues at ZT14-16 (time of expected peak PER mRNA accumulation in wild-type Drosophila). PER mRNA is expressed at similar levels in wild-type and dbtP larval brains. Thus, the aberrant accumulation of Per proteins in dbtP mutants does not reflect increased per transcription or per RNA stability but must be downstream of these events (Price, 1998).
The pattern of Per expression in dbtP is similar to a Per beta-galactosidase fusion protein, Per-SG, expressed from the per promoter. In adults, per-SG RNA oscillates, but Per-SG protein does not. In fact, the Per-SG protein accumulates over progressive cycles, suggesting that it is a stable protein. Per-SG, detected with an antibody against beta-galactosidase, is expressed in larvae in the lvLNs and other cell clusters in the brain hemispheres, as well as cells adjacent and close to the ventral ganglion midline. The presence of the noncycling Per-SG fusion protein therefore marks cells in which the per promoter is active, or has been active, during development. Comparable patterns were seen with two independent SG lines. Per in wild-type larvae has also been detected at very low levels in these cells. Thus, the pattern of staining for Per seen in dbtP reflects the normal spatial expression of per, but this pattern is only easily visible with a stable fusion protein or in a dbtP background (Price, 1998).
Per is detected at high levels in dbtP larval brains in DD as in LD. The persistence of Per proteins in lvLNs in DD is presumably occurring in the presence of very low levels of the Tim protein, which fall below immunocytochemical detection in these cells in DD. In dbt+ larvae and adults, Per accumulation is suppressed in the absence of Tim. It therefore seems likely that Per in dbtP has become less dependent on Tim for its accumulation than in wild type, especially since Per is detectable in brain cells where Tim is not detected in wild-type or dbtP larvae. Ideally this possibility would have been tested using tim01; dbtP larvae. However, it has not been possible to obtain third instar larvae from two different tim01; dbtP/TM6 lines. This may indicate a shift in the lethal phase of dbtP by the tim01 mutation. An alternative to tim01 was possible: constant light (LL) in a wild-type background produces a tim01 phenocopy through light-dependent degradation of Tim. In wild-type larvae, Per accumulation is suppressed in response to LL as previously seen for adults. In contrast, in dbtP larvae raised in LL, Per continues to be strongly detected in the lvLNs and the other Per-expressing cells. The persistence of Per in DD and LL indicates that Per proteins can accumulate in dbtP mutants even with very low levels of Tim (Price, 1998).
Where does Dbt act in the cell? In tim01 mutants, which block Per nuclear translocation, Per is unstable indicating the presence of a cytoplasmic activity that destabilizes Per monomers. In wild-type adults, constant light suppresses Tim, which subsequently results in very low levels of Per, and the same holds true for wild-type third instar larvae when raised in constant light. However, in dbtP mutants, similar high levels of Per accumulate under LD, DD, and LL conditions. Since the dbtP allele allows comparable Per accumulation with either high or low levels of Tim, it is concluded that dbtP allows Tim-independent Per accumulation and that DBT is a component of the cytoplasmic activity that destabilizes Per monomers in wild-type and tim01 flies. Consistent with this conclusion, predominantly cytoplasmic accumulation for the expanded Per pattern is seen in dbtP larval brains. tim does not appear to be expressed in this expanded pattern in wild-type larvae, so expanded accumulation of Per monomers in dbtP, but not wild-type, larval brains indicates novel cytoplasmic stability for Per in the mutant. There is also evidence that Dbt influences stability of nuclear Per proteins. Per is detected immunocytochemically in nuclei of mutant dbt lvLNs prior to Per's disappearance, suggesting that the differing kinetics of Per degradation in wild type and dbt mutants reflect different rates of Per elimination from the nucleus. Increased nuclear stability of Per monomers is apparent from the analyses of dbtP lvLNs: Per is lost from wild type but persists in dbtP nuclei after exposure to light has removed most Tim proteins. Thus, dbt may affect the stability of both cytoplasmic and nuclear Per monomers. This does not mean that Dbt must function in both subcellular compartments, since a posttranslational modification generated in the cytoplasm could have delayed effects in the nucleus. Thus it is proposed that Dbt activity in wild-type flies promotes molecular cycles of per and tim expression by ensuring the instability of monomeric Per proteins, thus allowing Per accumulation only in conjunction with high titers of Tim (Price, 1998).
A model is presented for the functioning of Dbt in photoperiod response. Dbt function promotes phosphorylation of cytoplasmic Per monomers. Once modified, Per proteins turn over rapidly. Physical association of Per and Tim stabilizes Per either because residual, unphosphorylated Per proteins are incorporated into Per/Tim dimers and these are no longer subject to modification by Dbt, or phosphorylated Per proteins are stabilized by association with Tim. Because monomeric Per proteins also stably accumulate in nuclei in dbtP mutants, but not in wild-type Drosophila (Price, 1998), the latter alternative is favored. The model indicates that instability of phosphorylated Per monomers delays Per/Tim heterodimerization until PER and TIM mRNA levels are high. Thus, phosphorylation promotes a delay between phases of per /tim transcription and Per/Tim complex function, which establishes molecular oscillations of RNA and protein. Edery (1994) has shown that Per is phosphorylated over many hours and that the most highly phosphorylated forms of the protein occur at times of night when Per proteins are predominantly nuclear. This suggests that Dbt may act in both the cytoplasm and the nucleus or that additional kinases continue to phosphorylate Per in the nucleus. The finding that dbtP mutants accumulate high levels of nuclear Per and that dbtL mutants show delayed degradation of hyperphosphorylated, monomeric forms of Per (Price, 1998) suggests that Dbt activity can influence stability of nuclear Per proteins after they have dissociated from TIM. However, this could be due to the production of a phosphorylated form of Per by cytoplasmic Dbt, with subsequent transfer of phosphorylated Per to nuclei as a Per/Tim complex. It is not yet known whether all monomeric Per proteins are phosphorylated in response to Dbt, so that Per/Tim complexes stabilize previously phosphorylated Per proteins, or whether only Per proteins that have escaped phosphorylation as a monomer contribute to Per/Tim complexes. Future immunocytochemical studies of DBT will allow subcellular localization of the protein and resolution of some of these issues (Kloss, 1998).
Exons - 4
PROSITE searches have identified an ATP-binding site between amino acids 15-38 and a serine/threonine kinase catalytic domain between amino acids 124 and 136. BLAST searches reveal that DBT is most closely related to human casein kinase I epsilon, being 86% identical at the amino acid level over the length of the kinase domain. Significant homology to other kinases ends with amino acid 292 (Kloss, 1998).
Genomic and cDNA sequence analysis has shown that the transcribed portion of discs overgrown (dco) consists of four exons and three introns. The first intron frequently remains unspliced despite the use of two closely spaced alternative 5' donor splice sites. Surprisingly, all introns reside in the region corresponding to the untranslated leader, whereas the last exon contains the entire coding region. The longest open reading frame encodes a protein of 440 amino acids, of which the 300 N-terminal amino acids have a high level of sequence identity with the catalytic domain of members of the CKI family. The most closely related isoforms are human CKIepsilon and delta, with 86% sequence identity (both isoforms) and 92% or 91% similarity, respectively, through the kinase domain. Even when compared to CKI isoforms of lower eukaryotes, such as HRR25 of budding yeast (78% similarity) or Cki1 and Cki2 of fission yeast (68% similarity), the catalytic domain of Dco is highly conserved. The ultimate evidence for the extreme structural conservation of the catalytic domain of CKI proteins, at least among members of the CKIdelta/epsilon subfamily, comes from a comparison of their 3-D structures at 2 Angstrom resolution, which shows that the yeast and human isoforms are nearly identical. This allows for the interpretation of the effects of dco mutations in the catalytic domain in terms of these known 3-D structures from yeast and humans (Zilian, 1999 and references therein).
The 140 amino-acid C-terminal domain of Dco is not conserved among CKId/e subfamily members. However, these domains do share the feature of consisting predominantly of regions that are abnormally rich in a few amino acids. In addition, they encompass several potential phosphorylation sites, including a site for autophosphorylation and a short arg-rich stretch, possibly serving as a nuclear localization signal. A potential SH3- binding motif in the C-terminal domain of the CKIdelta/epsilon subfamily appears to be conserved in Dco (Zilian, 1999 and references therein).
date revised: 15 October 99
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