Doubletime (DBT), also known as discs overgrown (DCO), is a gene that encodes the double-time protein in fruit flies (Drosophila melanogaster). Michael Young and his team at Rockefeller University Rockefeller University first identified and characterized the gene in 1998.

The DBT-encoded protein is a kinase that phosphorylates the period (PER) protein, which is crucial in controlling the biological clock that regulates circadian rhythms.^[1] Variousmutations in the DBT gene have been observed to cause alterations in the period of locomotor activity in flies, including lengthening, shortening, or complete loss of the period of locomotor activity in flies. In mammals, the homolog of DBT is casein kinase I epsilon, which plays a similar role in regulating the circadian rhythm.

The circadian function of Drosophila and certain vertebrate Casein Kinase I enzymes has been conserved over a long evolutionary timescale, making DBT and its homologs essential targets for research into the molecular mechanisms that underlie circadian rhythm regulation in diverse organisms.^[2]

Discovery

The double-time gene (DBT) was first discovered and characterized in 1998 by Michael Young and his team at Rockefeller University.^[3] Young's research group, headed by Jeffrey Price, published their findings in a paper which characterized three alleles of DBT in fruit flies.^[4] It was reported that two mutant alleles, named short and long (DBT^s and DBT^l, respectively), were able to disrupt the normal cycling of the genes Period (per) and Timeless (TIM).^[3][4]

The team suspected that the delay between the rise in mRNA levels of per and TIM and the rise of PER and TIM protein was due to the effects of another protein.

Young suspected that this protein postponed the intercellular accumulation of PER protein by destroying it. Only when PER was paired with TIM was this breakdown not possible. This work showed that DBT regulated the break-down of PER.^[3][4]

Young named the novel gene double-time (DBT) due to its effect on the normal period of Drosophila. Mutant flies that only expressed DBT^S had an 18-hour period, while those expressing DBT^L had a 28-hour period.^[4] In addition, Young's team also identified a third allele, DBT^P, which caused lethality in pupa while ablating any per or TIM products in larvae.^[4] DBT^P mutants were important because they provided clues as to how the gene product functioned.^[3]

Without functional DBT protein, flies accumulate high levels of PER. These PER proteins do not disintegrate without pairing with TIM proteins. Such mutants expressed higher cytosolic levels of PER than cells in which PER protein was associated with TIM protein. The double-time gene regulates the expression of PER, which in turn controls circadian rhythm.^[3] Young's team later cloned the dbt gene and found that the DBT protein was a kinase that specifically phosphorylated PER proteins. Thus, in dbt^P mutants, PER proteins were not phosphorylated by DBT protein.^[4]

Gene

The gene is located on the right arm of chromosome 3.^[4] The mRNA transcript of dbt is 3.2 kilo-base pairs long and contains four exons and three introns.

Protein

The DBT protein is composed of 440 amino acids.^[5] The protein has an ATP binding site, serine/threonine kinase catalytic domains, and several potential phosphorylation sites, including a site for autophosphorylation.^[5]

Function

Regulation of circadian rhythm

In Drosophila, a molecularly driven clock mechanism works to regulate circadian rhythms such as locomotor activity and eclosion by oscillating the levels of the proteins PER and TIM via positive and negative feedback loops.^[4][6] The double-time gene produces the protein DBT, a kinase that phosphorylates PER to regulate its accumulation in the cytoplasm and its degradation in the nucleus.^[6][7] In the cytoplasm, PER and TIM levels rise during the night, and DBT binds to PER while levels of TIM are still low.^[8] DBT phosphorylates the cytoplasmic PER, which leads to its degradation. Only once TIM accumulates do PER and TIM bind, inhibiting the degradation of PER. This cytoplasmic PER degradation, followed by accumulation, causes a 4-6 hour delay between the levels of per mRNA and the levels of PER protein.^[8] The PER/TIM complex, still bound to DBT, migrates into the nucleus, where it suppresses the transcription of per and tim. TIM is lost from the complex, following which DBT phosphorylates PER, degrading it. This allows for the transcription of the clock and clock-controlled genes (with transcription controlled by circadian mechanisms).^[8][9] The oscillations in the presence of PER and TIM proteins cause oscillations in the expression of their own and other genes; forming is the basis for circadian rhythmicity.^[6]

The transcription of dbt mRNA and the levels of the DBT protein are consistent throughout the day and not controlled by PER/TIM levels—however, the location and concentration of the DBT protein within the cell change throughout the day.^[5] It is consistently present in the nucleus at varying levels, but in the cytoplasm it is predominantly present in the late day and early night, when PER and TIM levels peak.^[5]

Before DBT begins phosphorylating PER, a different protein called NEMO/NLK kinase begins phosphorylating PER at its per-short domain. This phosphorylation stimulates DBT to begin phosphorylating PER at multiple nearby sites. In total, there are about 25-30 phosphorylation sites on PER.^[10] The phosphorylated PER binds to the F-box protein SLIMB, and it is then targeted for degradation through the ubiquitin-proteasome pathway.^[7] Therefore, the phosphorylation of PER by DBT leads to a decrease in PER abundance, which is a necessary step in the function of the organism's internal clock.^[7]

The activity of DBT on PER is aided by the activity of the proteins CKII and SGG, and a rhythmically expressed protein phosphatase antagonizes it. It is possible, but currently unknown, if DBT regulates other functions of PER or other circadian proteins.^[6] There has been no evidence that suggests that DBT binds directly to TIM.^[5] Rather, the only kinase known to phosphorylate TIM directly is the SHAGGY (SGG) kinase protein, but this does not majorly affect TIM stability, suggesting the presence of a different kinase or phosphatase.^[11] DBT does play a role in recruiting other kinases into PER repression complexes. These kinases phosphorylate the transcription factor CLK, which releases the CLK-CYC complex from the E-Box and represses transcription.^[1]

Mutant alleles

There are three primary mutant alleles of DBT: DBT^S, which shortens the organism's free-running period (its internal period in constant light conditions); DBT^L, which lengthens the free-running period; and DBT^P, which causes pupal lethality and eliminates circadian cycling proteins and per- and TIM transcription.^[4] All mutants except for DBT^S produce differential PER degradation that directly corresponds with their phenotypic behavior. DBT^S PER degradation resembles wild-type DBT, suggesting that DBT^S does not affect the clock through this degradation mechanism. It has been suggested that DBT^S works by acting as a repressor or producing a different phosphorylation pattern of the substrate. DBT^S causes early termination of per transcription.^[7]

The DBT^L mutation causes the period of PER and TIM oscillations, as well as animal behavioral activity, to lengthen to about 27 hours. This extended rhythm is caused by a decreased rate of phosphorylation of PER due to lower DBT kinase activity levels. This mutation is caused by a substitution in the protein sequence (Met-80→Ile mutation).

The DBT^S mutation causes a PER/TIM oscillation period of 18–20 hours. There is no current evidence for the mechanism affected by the mutation, but it is caused by a substitution in the protein sequence (Pro-47→ Ser mutation).^[7]

Another DBT mutation is DBT^AR, which causes arrhythmic activities in Drosophila. It is a hypermorphic allele resulting from a His 126→Tyr mutation. Homozygous flies with this mutation are viable but arrhythmic, whereas DBT^AR/+ heterozygotes have extra-long periods of about 29 hours, and their DBT kinase activity is reduced to the lowest rate of all of the DBT alleles.^[7]

Noncircadian roles

Clock gene mutations, including those in Drosophila's DBT, alter the sensitization of drug-induced locomotor activity after repeated exposure to psychostimulants. Drosophila with mutant alleles of DBT failed to display locomotor sensitization in response to repeated cocaine exposure.^[12] Additionally, there is experimental evidence for this gene to function in 13 unique biological processes, including biological regulation, phosphorus metabolic process, the establishment of planar polarity, positive regulation of the biological process, cellular process, single-organism developmental process, response to stimulus, response to an organic substance, sensory organ development, macromolecule modification, growth, cellular component organization or biogenesis, and rhythmic process.^[13] The gene's alternative name, discs overgrown, refers to its role as a cell growth-regulating gene that has strong effects of cell survival and growth control in imaginal discs, an attribute of the larvae fly stage. The protein is necessary in the mechanism linking cell survival during proliferation and growth arrest.^[5]

Noncatalytic role

The DBT protein may play a noncatalytic role in attracting kinases that phosphorylate CLOCK (CLK), an activator of transcription.^[1] DBT has a noncatalytic role in recruiting kinases, some of which have not yet been discovered, into the transcription-translation feedback loop (TTFL).^[14] DBT's catalytic activity is not affiliated with the phosphorylation of CLK or its transcriptional repression. PER phosphorylation by DBT is integral to repressing CLK-dependent transcription. The DBT protein is noncatalytic in recruiting additional kinases that indirectly phosphorylate CLK, thus downregulating transcription. A similar pathway exists in mammals due to the mechanistic conservation of the CKI homolog.^[1] In 2004, in dbt^s and dbt^l mutants, Drosophila cells has reduced CKI-7 activity.^[15]

Mammalian homologs

Casein kinase I

The casein kinase 1 (CK1) family of kinases comprises a highly conserved group of proteins found in organisms ranging from Arabidopsis, to Drosophila, to humans.^[16] Since dbt is a member of this family, it has prompted questions regarding the roles of these related genes in other model systems. Within mammals, there are seven CK1 isoforms, each with distinct roles surrounding protein phosphorylation. CK1ε was found to be the most homologous to dbt, with a similarity of 86%.^[16] This genetic similarity extends to functional homology; for instance, while phosphorylation by dbt in Drosophila targets PER proteins for proteasome degradation, CK1ε phosphorylation marks mammalian PER proteins for degradation by reducing their stability.^[16][17][18] However, although dbt and CK1ε play similar roles in their respective organisms, studies examining the effectiveness of CK1ε in Drosophila have revealed they are not completely functionally interchangeable.^[19] Nonetheless, their functions are highly analogous; for example, CK1ε has been shown to reduce the half-life of mPER1, one of the three mammalian PER homologs.^[16] Furthermore, the nuclear localization of mPER proteins is associated with phosphorylation, underscoring another vital function of the CK1ε protein.^[16] Overall, the genetic similarity between dbt and CK1ε only partly tells the story; their nearly identical roles within the circadian clocks of their respective systems, involving periodic phosphorylation, significantly contribute to regulating the oscillations of these clocks.

The casein kinase 1 (CKI) family of kinases is a highly conserved group of proteins that are found in organisms ranging from Arabidopsis, to Drosophila, to humans.^[16] Because dbt is a member of this family, questions arose about the role of these related genes in other model systems. Within mammals, there are seven CKI isoforms, all with various roles surrounding the phosphorylation of proteins. CKIε was found to be most homologous to dbt, with a similarity of 86%.^[16] Along with this genetic similarity, the proteins are functionally homologous. Just as phosphorylation by dbt in Drosophila targets PER proteins for proteasome degradation, CKIε phosphorylation reduces the stability of mammalian PER proteins, labeling them for degradation.^[16][17][18] However, while dbt and CKIε do play similar roles in their respective organisms, studies looking at the effectiveness of CKIε in Drosophila have shown that they are not completely functionally interchangeable.^[19] Nonetheless, the functions are extremely similar. Specifically, CKIε has been shown to reduce the half-life of mPER1, one of the three mammalian PER homologs.^[16] In addition, nuclear localization of the mPER proteins is related to phosphorylation, adding another essential role to the activity of the CKIε protein.^[16] Overall, the genetic similarity of dbt and CKIε is not the end of the story; the roles they play within the circadian clock in their respective systems are almost identical. Both are involved with periodic phosphorylation, regulating the oscillations of the circadian clocks.

Role of CKIε

Initially, the role of CKIε within the circadian clock of mammals was discovered due to a mutation in hamsters. The tau mutation in the Syrian golden hamster was the first to show a heritable abnormality of circadian rhythms in mammals.^[16] Hamsters with the mutation exhibit a shorter period than the wild-type. Heterozygotes have a period of about 22 hours. In contrast, the period of homozygotes is even shorter, at about 20 hours.^[16] Because of previous research indicating the role of dbt in establishing periods, the tau mutation was found to be at the same locus as the CKIε gene.^[20] Thus, this mutation relates to the mutations dbt^S and dbt^L, which both effect the internal period of the fly. However, the forces driving these changes in the period seem different. It was found that the point mutation resulting in the tau mutant decreased the activity of the CKIε kinase in vitro. In flies, on the other hand, the dbt^L mutation is associated with a decrease in dbt activity and a longer period. This is consistent with another experiment done on hamsters that showed a lengthening of the period caused by CKI inhibition.^[18] To investigate this discrepancy, researchers studied the half-life of PER2 under the influence of wild-type CKIε, CKIεtau, and CKIε (K38A), which is a kinase-inactive mutant.^[18] The results indicated that the tau mutation was actually a gain-of-function mutation, instead of a loss-of-function mutation, that caused the more rapid degradation of the PER proteins. Therefore, the tau mutation in hamsters can be seen as similar to mutations in dbt that change the internal period.

Importance of Rhythmic Phosphorylation

CKIε also plays a role in humans concerning Familial Advanced Sleep Phase Syndrome (FASPS), where individuals exhibit a significantly shorter circadian period compared to the general population. Interestingly, this anomaly doesn't appear to be due to a mutation in the CKIε protein itself, but rather in the binding site for the phosphorylation of the PER2 protein.^[16]

Moreover, kinase activity is implicated in the nuclear localization of PER and other genes pivotal to circadian rhythmicity.^[21] This phosphorylation enables PER to inhibit its own transcription, thereby imposing a delay within the circadian system. Absent the phosphorylation of PER—by dbt in Drosophila or by CKIε in mammals—the circadian oscillations would cease due to the disruption of the feedback loop.

There is a proposition that rhythmic phosphorylation could be a fundamental driver of circadian clocks. Traditionally, the transcription-translation negative feedback loop has been recognized as the source of oscillations and rhythms in biological clocks. However, in vitro experiments showcasing the phosphorylation of the cyanobacterial protein KaiC demonstrated that rhythmic oscillations could persist even in the absence of transcription or translation processes.^[22] Thus, kinases like dbt and CKIε may hold even more crucial roles within circadian clocks beyond merely targeting proteins for degradation.