discs overgrown: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - discs overgrown

Synonyms - double-time

Cytological map position - 100B2

Function - protein kinase

Keywords - photoperiod response and imaginal disc survival, proliferation and growth arrest, Fat signaling pathway

Symbol - dco

FlyBase ID: FBgn0002413

Genetic map position -

Classification - casein kinase I delta/epsilon

Cellular location - cytoplasmic

NCBI link: Entrez Gene

dco orthologs: Biolitmine
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
Drosophila DBT and vertebrate CKI/δ phosphorylate Per to produce circadian rhythms. While the C termini of these orthologs are not conserved in amino acid sequence, they inhibit activity and become autophosphorylated in the fly and vertebrate kinases. This study identified sites of C terminal autophosphorylation. Mutation of 6 serines and threonines in the C terminus (DBTC/ala) prevented autophosphorylation-dependent DBT turnover and electrophoretic mobility shifts in S2 cells. Unlike the effect of autophosphorylation on CKIdelta, DBT autophosphorylation in S2 cells did not reduce its in vitro activity. Moreover, overexpression of DBTC/ala did not affect circadian behavior differently from DBTWT, and neither exhibited daily electrophoretic mobility shifts, suggesting that DBT autophosphorylation is not required for clock function. While DBTWT protected S2 cells and larvae from UV-induced apoptosis and was phosphorylated and degraded by the proteasome, DBTC/ala did not protect and was not degraded. Finally, it was shown that the HSP-90 cochaperone spaghetti (SPAG) antagonizes DBT autophosphorylation in S2 cells. These results suggest that DBT autophosphorylation regulates cell death and suggest a potential mechanism by which the circadian clock might affect apoptosis.

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.
Top, D., O'Neil, J. L., Merz, G. E., Dusad, K., Crane, B. R. and Young, M. W. (2018). CK1/Doubletime activity delays transcription activation in the circadian clock. Elife 7. PubMed ID: 29611807
In the Drosophila circadian clock, Period (PER) and Timeless (TIM) proteins inhibit Clock-mediated transcription of per and tim genes until PER is degraded by Doubletime/CK1 (DBT)-mediated phosphorylation, establishing a negative feedback loop. Multiple regulatory delays within this feedback loop ensure ~24 hr periodicity. Of these delays, the mechanisms that regulate delayed PER degradation (and Clock reactivation) remain unclear. This study shows that phosphorylation of certain DBT target sites within a central region of PER affect PER inhibition of Clock and the stability of the PER/TIM complex. The results indicate that phosphorylation of PER residue S589 stabilizes and activates PER inhibitory function in the presence of TIM, but promotes PER degradation in its absence. The role of DBT in regulating PER activity, stabilization and degradation ensures that these events are chronologically and biochemically linked, and contributes to the timing of an essential delay that influences the period of the circadian clock.
Venkatesan, A., Fan, J. Y., Bouyain, S. and Price, J. L. (2019). The circadian tau mutation in Casein Kinase 1 is part of a larger domain that can be mutated to shorten circadian period. Int J Mol Sci 20(4). PubMed ID: 30769795
Drosophila Double-time (DBT) phosphorylates the circadian protein Period (PER). The period-altering mutation tau, identified in hamster casein kinase I (CKIepsilon) and created in Drosophila DBT, has been shown to shorten the circadian period in flies, as it does in hamsters. Since CKI often phosphorylates downstream of previously phosphorylated residues and the tau amino acid binds a negatively charged ion in X-ray crystal structures, this amino acid has been suggested to contribute to a phosphate recognition site for the substrate. Alternatively, the tau amino acid may affect a nuclear localization signal (NLS) with which it interacts. This study mutated the residues that were close to or part of the phosphate recognition site or NLS. Flies expressing DBT with mutations of amino acids close to or part of either of these motifs produced a shortening of period, suggesting that a domain, including the phosphate recognition site or the NLS, can be mutated to produce the short period phenotype. Mutation of residues affecting internally placed residues produced a longer period, suggesting that a specific domain on the surface of the kinase might generate an interaction with a substrate or regulator, with short periods produced when the interaction is disrupted.
Wang, J., Fan, J. Y., Zhao, Z., Dissel, S. and Price, J. (2022). DBT affects sleep in both circadian and non-circadian neurons. PLoS Genet 18(2): e1010035. PubMed ID: 35139068
Sleep is a very important behavior observed in almost all animals. Importantly, sleep is subject to both circadian and homeostatic regulation. The circadian rhythm determines the daily alternation of the sleep-wake cycle, while homeostasis mediates the rise and dissipation of sleep pressure during the wake and sleep period. As an important kinase, Dbt plays a central role in both circadian rhythms and development. This study investigated the sleep patterns of several ethyl methanesulfonate-induced dbt mutants and discusses the possible reasons why different sleep phenotypes were shown in these mutants. In order to reduce DBT in all neurons in which it is expressed, CRISPR-Cas9 was used to produce flies that expressed GAL4 in frame with the dbt gene at its endogenous locus, and knock-down of DBT with this construct produced elevated sleep during the day and reduced sleep at night. Loss of sleep at night is mediated by dbt loss during the sleep/wake cycle in the adult, while the increased sleep during the day is produced by reductions in dbt during development and not by reductions in the adult. Additionally, using targeted RNA interference, this study uncovered the contribution of dbt on sleep in different subsets of neurons in which dbt is normally expressed. Reduction of dbt in circadian neurons produced less sleep at night, while lower expression of dbt in noncircadian neurons produced increased sleep during the day. Importantly, independently of the types of neurons where dbt affects sleep, this study demonstrated that the PER protein is involved in DBT mediated sleep regulation.
Thakkar, N., Giesecke, A., Bazalova, O., Martinek, J., Smykal, V., Stanewsky, R. and Dolezel, D. (2022). Evolution of casein kinase 1 and functional analysis of new doubletime mutants in Drosophila. Front Physiol 13: 1062632. PubMed ID: 36589447
Circadian clocks are timing devices that rhythmically adjust organism's behavior, physiology, and metabolism to the 24-h day-night cycle. Eukaryotic circadian clocks rely on several interlocked transcription-translation feedback loops, where protein stability is the key part of the delay between transcription and the appearance of the mature proteins within the feedback loops. In bilaterian animals, including mammals and insects, the circadian clock depends on a homologous set of proteins. Despite mostly conserved clock components among the fruit fly Drosophila and mammals, several lineage-specific differences exist. This study has systematically explored the evolution and sequence variability of insect DBT proteins and their vertebrate homologs casein kinase 1 delta (CKIδ) and epsilon (CKIε), dated the origin and separation of CKIδ from CKIε, and identified at least three additional independent duplications of the CKIδ/ε gene in Petromyzon, Danio, and Xenopus. Conserved regions were identified in DBT specific to Diptera, and a subset of those were functionally tested in D. melanogaster. Replacement of Lysine K224 with acidic residues strongly impacts the free-running period even in heterozygous flies, whereas homozygous mutants are not viable. K224D mutants have a temperature compensation defect with longer free-running periods at higher temperatures, which is exactly the opposite trend of what was reported for corresponding mammalian mutants. All DBTs of dipteran insects contain the NKRQK motif at positions 220-224. The occurrence of this motif perfectly correlates with the presence of BRIDE OF DOUBLETIME, BDBT, in Diptera. BDBT is a non-canonical FK506-binding protein that physically interacts with Drosophila DBT. The phylogeny of FK506-binding proteins suggests that BDBT is either absent or highly modified in non-dipteran insects. In addition to in silico analysis of DBT/CKIδ/ε evolution and diversity, four novel casein kinase 1 genes specific to the Drosophila genus were identified.
Nolan, R. B., Bontrager, C., Bowser, A., Corley, A., Fiedler, H., Flathers, C., Francis, L., Le, A., Mahmoudjafari, S., Nim, T., Muolo, C. E., Shores, B., Viermann, C., Waldren, A., Zatezalo, C., Fan, J. Y. and Price, J. L. (2023). Visual and circadian regulation of Drosophila BDBT and BDBT effects on DBT and PER localization. iScience 26(4): 106343. PubMed ID: 36994075
BRIDE OF DOUBLETIME (BDBT) interacts with the circadian kinase DOUBLETIME (DBT) and accumulates in eye foci during the dark of a light:dark cycle. BDBT foci are shown in this study to be broadly expressed in constant dark and low in constant light. Analysis of circadian photoreceptor cry and visual photoreceptor ninaE mutants showed that disappearance of eye BDBT foci requires both the CRYPTOCHROME and the RHODOPSIN-1 pathways. The arr1 and arr2 mutants, which affect rhodopsin quenching, eliminated BDBT foci under dark conditions. arr1 and arr2 mutants also caused increased nuclear PER protein. The changes in BDBT foci do not result from altered BDBT levels in the eye but from changes in its immunodetection. Knockdown of BDBT specifically in the eye produced constitutively nuclear PER and constitutively cytosolic DBT. The results show that BDBT is necessary for co-transport of DBT and PER into the nucleus and suggest that this process is regulated by a light-dependent mechanism.
Philpott, J. M., Freeberg, A. M., Park, J., Lee, K., Ricci, C. G., Hunt, S. R., Narasimamurthy, R., Segal, D. H., Robles, R., Cai, Y., Tripathi, S., McCammon, J. A., Virshup, D. M., Chiu, J. C., Lee, C. and Partch, C. L. (2023). PERIOD phosphorylation leads to feedback inhibition of CK1 activity to control circadian period. Mol Cell 83(10): 1677-1692.e1678. PubMed ID: 37207626
PERIOD (PER) and Casein Kinase 1&delta regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1Δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. This study shows that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. This study found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.
Khatib, L., Subasi, B. S., Fishman, B., Kapun, M. and Tauber, E. (2023). Unveiling Subtle Geographical Clines: Phenotypic Effects and Dynamics of Circadian Clock Gene Polymorphisms. Biology (Basel) 12(6). PubMed ID: 37372143
Understanding of the gene regulatory network that constitutes the circadian clock has greatly increased in recent decades, notably due to the use of Drosophila as a model system. In contrast, the analysis of natural genetic variation that enables the robust function of the clock under a broad range of environments has developed more slowly. The current study analyzed comprehensive genome sequencing data from wild European populations of Drosophila, which were densely sampled through time and space. Hundreds of single nucleotide polymorphisms (SNPs) were identified in nine genes associated with the clock, 276 of which exhibited a latitudinal cline in their allele frequencies. While the effect sizes of these clinal patterns were small, indicating subtle adaptations driven by natural selection, they provided important insights into the genetic dynamics of circadian rhythms in natural populations. Nine SNPs in different genes were chosen and their impact on circadian and seasonal phenotypes was assessed by reconstructing outbred populations fixed for either of the SNP alleles, from inbred DGRP strains. The circadian free-running period of the locomotor activity rhythm was affected by an SNP in doubletime (dbt) and eyes absent (Eya). The SNPs in Clock (Clk), Shaggy (Sgg), period (per), and timeless (tim) affected the acrophase. the time period in a cycle during which the cycle crests or peaks. The alleles of the SNP in Eya conferred different levels of diapause and the chill coma recovery response.

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).

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

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

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

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

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

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

Reciprocal action of Casein Kinase Iepsilon on core planar polarity proteins regulates clustering and asymmetric localisation

The conserved core planar polarity pathway is essential for coordinating polarised cell behaviours and the formation of polarised structures such as cilia and hairs. Core planar polarity proteins localise asymmetrically to opposite cell ends and form intercellular complexes that link the polarity of neighbouring cells. This asymmetric segregation is regulated by phosphorylation through poorly understood mechanisms. This study shows that loss of phosphorylation of the core protein Strabismus in the Drosophila pupal wing increases its stability and promotes its clustering at intercellular junctions, and that Prickle negatively regulates Strabismus phosphorylation. Additionally, loss of phosphorylation of Dishevelled - which normally localises to opposite cell edges to Strabismus - reduces its stability at junctions. Moreover, both phosphorylation events are independently mediated by Casein Kinase Iepsilon. It is concluded that Casein Kinase Iepsilon phosphorylation acts as a switch, promoting Strabismus mobility and Dishevelled immobility, thus enhancing sorting of these proteins to opposite cell edges (Strutt, 2019).

Phosphorylation is a widespread means of controlling protein activity, regulating protein-protein interactions, protein stability and conformation. The activity of most signalling pathways is regulated by phosphorylation of pathway components. This includes the 'core' planar polarity pathway: however, compared to other signalling pathways, the molecular mechanisms are poorly understood (Strutt, 2019).

The core planar polarity proteins (hereafter, the 'core proteins') regulate the production of polarised structures or polarised cell behaviours in the plane of a tissue. This includes polarised production of cilia and of stereocilia bundles in the inner ear, and the coordinated polarisation of tissue movements necessary for convergence and extension of the body axis. In Drosophila, the core pathway controls the production of polarised hairs and bristles on many adult tissues, for example the trichomes that emerge from the distal edge of each cell in the adult wing (Strutt, 2019).

The core pathway specifies polarised structures via the asymmetric localisation of pathway components. In the Drosophila pupal wing, the seven-pass transmembrane protein Frizzled (Fz), and the cytoplasmic proteins Dishevelled (Dsh) and Diego (Dgo) localise to distal cell ends, where the trichome will emerge. The four-pass transmembrane protein Strabismus (Stbm, also known as Van Gogh [Vang]) and Prickle (Pk) localise to proximal cell ends, and the atypical cadherin Flamingo (Fmi, also known as Starry Night [Stan]) localises to both proximal and distal cell ends (see Planar polarity and the cloud model of core protein localisation). Fmi mediates homophilic adhesion that is important for coupling polarity between cells (Strutt, 2019).

The overall direction of polarisation is determined by tissue-specific global cues. Polarity is then thought to be refined and amplified by feedback interactions between the core proteins. Mathematical modelling has suggested that feedback may involve destabilisation of complexes of opposite orientation and/or stabilisation of complexes in the same orientation. This can lead to sorting of complexes such that they all align in the same direction (Strutt, 2019).

With regard to possible stabilising mechanisms, core protein asymmetry is associated with clustering of proteins into punctate membrane subdomains and reduced core protein turnover. Based on a detailed study of core protein organisation in puncta, it was recently proposed that core proteins form a non-stoichiometric 'cloud' around a Fmi-Fz nucleus. Feedback interactions lead to sorting of complexes, and multiple protein-protein interactions are thought to promote a phase transition into higher order 'signalosome-like' structures, where arrays of complexes of the same orientation are stabilised. Interestingly, Stbm stoichiometry was found to be much higher than that of the other core proteins. The reasons for this are unclear, but could relate to a role for Stbm in promoting higher order structures. Furthermore, Pk may stabilise Stbm by promoting complex clustering (Strutt, 2019).

Mechanisms of destabilisation may include competitive binding between core proteins. More specifically, Pk (a 'proximal' complex component) is known to destabilise Fz and/or Dsh ('distal' components) in the same cell. In addition, Pk has been suggested to destabilise complexes containing Stbm and Fmi. However, knowledge of additional molecular mechanisms by which core proteins might become destabilised or clustered together is very poor, and post-translational modifications such as phosphorylation are likely to be a key element (Strutt, 2019).

Indeed, core protein phosphorylation is essential for feedback amplification of asymmetry. In particular, reduced activity of Casein Kinase Iε (CKIε, also known as Discs Overgrown [Dco] or Doubletime [Dbt] in flies) causes planar polarity defects and a reduction in core protein asymmetry. Interestingly, CKIε has been implicated in phosphorylation of both Stbm and Dsh. CKIε was first found to bind to and phosphorylate the vertebrate Dsh homologue (Dvl) in canonical Wnt signalling. In planar polarity in flies, Dsh phosphorylation correlates with its recruitment to cellular junctions by Fz, where it is incorporated into stable complexes, and decreased Dsh phosphorylation is seen in &dco; mutants (Strutt, 2019).

The exact phosphorylation sites for CKIε in Dsh/Dvl are not well defined, but a mutation of a serine/threonine-rich region upstream of the PDZ domain affects Dvl recruitment to membranes in Xenopus. Moreover, mutation of one of these residues (S236 in fly Dsh) blocks phosphorylation of Dsh by Dco in vitro. However, a transgene in which these residues were mutated largely rescued the planar polarity defects of dsh mutants in the adult fly wing (Strutt, 2019).

More recently, CKIε has been implicated in phosphorylating Stbm and its vertebrate homologue Vangl2. In particular, Wnt gradients were proposed to lead to a gradient of Vangl2 phosphorylation and asymmetry in the vertebrate limb. CKIε promotes Stbm/Vangl2 phosphorylation in cell culture. Two clusters of conserved serine and threonine residues were identified as CKIε phosphorylation sites. Mutation of some or all of these residues leads to a loss of Stbm/Vangl2 phosphorylation in cell culture, and defects in planar polarisation (Strutt, 2019).

The fact that CKIε has been implicated in phosphorylating both Stbm/Vangl2 and Dsh/Dvl in cell culture leads to the question of whether both proteins are bona fide targets in vivo. For instance, both Fz and Dsh/Dvl have been proposed to promote Stbm/Vangl2 phosphorylation by CKIε. Thus, it is possible that only Stbm/Vangl2 are direct targets of CKIε and that Stbm/Vangl2 phosphorylation has a secondary effect on Fz-Dsh/Dvl behaviour. Moreover, mechanistic insight into how these phosphorylation events affect core protein sorting and asymmetry is lacking (Strutt, 2019).

This study demonstrates that CKIε has independent and reciprocal actions on Dsh and Stbm during planar polarity signalling in Drosophila. This study used phosphorylation site mutations in Stbm to show that lack of Stbm phosphorylation leads to its clustering in 'mixed' puncta that contain complexes in both orientations. CKIε-dependent phosphorylation increases Stbm turnover at junctions, and thus promotes complex sorting, while phosphorylation of Dsh decreases its turnover. Pk negatively regulates Stbm phosphorylation and increases Stbm stability. These results support a direct role for Dco in phosphorylating both Stbm and Dsh in vivo in planar polarity signalling (Strutt, 2019).

This paper describes a dual role for CKIε/Dco kinase in regulating planar polarity in the fly pupal wing. In the first case, Dco promotes phosphorylation of Stbm. Stbm phosphorylation acts as a switch, changing Stbm from a stable immobile form that can enter junctional complexes, to an unstable mobile form that can redistribute within cells. Inhibiting Stbm phosphorylation causes an increase in Stbm stability at junctions that prevents sorting of complexes: thus complexes are 'locked' in an unsorted state. In contrast, hyperphosphorylation of Stbm destabilises Stbm, allowing it to leave junctions, hence permitting complex sorting. A second role for Dco is to mediate Dsh phosphorylation, which increases Dsh localisation at junctions. Significantly, the effects of Dco on Dsh are independent of Stbm and vice versa (Strutt, 2019).

In the 'cloud model', it is envisaged that multiple binding interactions drive a phase transition from a loosely packed, disordered association of core proteins in non-puncta, towards a highly cross-linked array of complexes within puncta that are all aligned in the same orientation. Stbm is well-placed to be a key component driving such a clustering mechanism, as not only can it multimerise with itself, but it also has a high stoichiometry within junctions. Also consistent with a role for Stbm in complex clustering is the observation that Stbm phosphorylation site mutants act as dominant negatives, recruiting wild-type Stbm into non-polarised puncta. Phosphorylation may inhibit a clustering mechanism, due to an increase in negative charge (Strutt, 2019).

Interestingly, excess clustering of unphosphorylated Stbm in unsorted complexes is also expected to lead to destabilising feedback interactions with the other core components. When Stbm is unphosphorylated, the increase in Stbm stability is sufficient for Stbm to 'win' over Fmi and Fz. Thus, there is an overall increase in Stbm stability in phosphomutant Stbm puncta, that is accompanied by decreased stability of Fmi and Fz (Strutt, 2019).

Pk both promotes Stbm stability and reduces its phosphorylation. A role for Pk in increasing Stbm stability is not surprising, as overexpression of Pk is known to cause excess clustering of the core proteins. A number of mechanisms can be envisioned by which Pk could affect Stbm phosphorylation. A previous study provided evidence that Pk has two roles: firstly, it acts via Dsh to destabilise Fz in the same cell (see Model for how Pk and phosphorylation of Stbm regulate complex sorting and clustering); secondly, it acts via Stbm to stabilise Fz in adjacent cells. In the first case, Pk would promote sorting of complexes, and one possibility is that Stbm is inaccessible to the kinase in sorted complexes, and thus Pk is indirectly reducing Stbm phosphorylation by promoting sorting. Arguing against this, loss of fz or dsh also abolishes core protein asymmetry, but no hyperphosphorylation is seen. The boundary FRAP experiments instead support Pk acting directly in the same cell to stabilise Stbm. A mechanism is therefore proposed whereby direct binding of Pk to Stbm protects Stbm from phosphorylation (Strutt, 2019).

Interestingly, Stbm has a significantly higher stoichiometry within junctions than Pk. One possibility is that Stbm forms multimers, and that association of Pk with these multimers causes a conformational change that reduces accessibility to kinase-binding sites. Alternatively, Pk might recruit a phosphatase (albeit no candidates for such a phosphatase are known). The reduced negative charge might then allow Stbm to form higher order structures, which promotes clustering of the entire core protein complex into puncta (Strutt, 2019).

Puncta formation in both wild-type and phosphomutants is also dependent on Dsh. Dsh is another a good candidate for promoting clustering as it too can multimerise, and thus puncta formation may be dependent on clustering on both sides of the complex. Moreover, direct interactions between Stbm and Dsh may promote clustering of unsorted complexes in the absence of phosphorylation (Strutt, 2019).

Feedback models for core protein asymmetry suggest that particular components of the core pathway signal to other components to either stabilise or destabilise them. An attractive model would be that Fz or Dsh recruits a kinase which phosphorylates Stbm and destabilises complexes of the opposite orientation. Consistent with this, a proportion of Dco localises to apicolateral junctions in pupal wings. However, no change was seen in Stbm phosphorylation in fz or dsh mutants, nor are Fz and Dsh required for the hyperphosphorylation of Stbm seen in pk mutants. Therefore, it is concluded that Stbm phosphorylation is more likely to be constitutive. Such constitutive phosphorylation would be sufficient to keep Stbm mobile and allow complex sorting; and Pk would then counterbalance this and promote complex stability. The balance between Stbm phosphorylation/complex mobility and Pk binding (leading to reduced Stbm phosphorylation) would resolve over time towards a more stable state as complexes segregate to opposite cell edges (Strutt, 2019).

It is noted that in normal development, Stbm downregulates Pk levels. This suggests Pk levels are finely tuned, in order to prevent unrestrained clustering (as seen when Pk is overexpressed) (Strutt, 2019).

Evidence is also provided that Dco regulates Dsh phosphorylation and junctional levels independently of Stbm. These findings are consistent with previous observations that Dsh phosphorylation correlates with its recruitment by Fz into junctional complexes. The mechanism by which Dsh phosphorylation acts in planar polarity remains to be elucidated, but the data show that &dco; overexpression phenotypes are suppressed by reduced dsh gene dosage, and that Dsh phosphomutants have reduced core protein asymmetry in pupal wings. Furthermore, a small but significant decrease in Dsh stability at junctions is observed in Dsh phosphomutants. Overall, these data are consistent with a model in which phosphorylation of Dsh promotes its stable association at junctions (Strutt, 2019).

In summary, it is proposed that Dco regulates the asymmetric localisation of the core proteins by reciprocal actions on Stbm and Dsh. Dco regulates Stbm phosphorylation and turnover and causes it to leave junctions, while phosphorylation of Dsh by Dco promotes its junctional association (Strutt, 2019).

Fan, J. Y., Agyekum, B., Venkatesan, A., Hall, D. R., Keightley, A., Bjes, E. S., Bouyain, S. and Price, J. L. (2013). Noncanonical FK506-binding protein BDBT binds DBT to enhance its circadian function and forms foci at night. Neuron 80(4): 984-996. PubMed ID: 24210908

Strutt, H. and Strutt, D. (2020). DAnkrd49 and Bdbt act via Casein kinase Iepsilon to regulate planar polarity in Drosophila. PLoS Genet 16(8): e1008820. PubMed ID: 32750048

DAnkrd49 and Bdbt act via Casein kinase Iepsilon to regulate planar polarity in Drosophila

The core planar polarity proteins are essential mediators of tissue morphogenesis, controlling both the polarised production of cellular structures and polarised tissue movements. During development the core proteins promote planar polarisation by becoming asymmetrically localised to opposite cell edges within epithelial tissues, forming intercellular protein complexes that coordinate polarity between adjacent cells. This study describes a novel protein complex that regulates the asymmetric localisation of the core proteins in the Drosophila pupal wing. DAnkrd49 (an ankyrin repeat protein) and Bride of Doubletime (Bdbt, a non-canonical FK506 binding protein family member) physically interact, and regulate each other's levels in vivo. Loss of either protein results in a reduction in core protein asymmetry and disruption of the placement of trichomes at the distal edge of pupal wing cells. Post-translational modifications are thought to be important for the regulation of core protein behaviour and their sorting to opposite cell edges. Consistent with this, it was found that loss of DAnkrd49 or Bdbt leads to reduced phosphorylation of the core protein Dishevelled and to decreased Dishevelled levels both at cell junctions and in the cytoplasm. Bdbt has previously been shown to regulate activity of the kinase Discs Overgrown (Dco, also known as Doubletime or Casein Kinase Iε), and Dco itself has been implicated in regulating planar polarity by phosphorylating Dsh as well as the core protein Strabismus. This study demonstrates that DAnkrd49 and Bdbt act as dominant suppressors of Dco activity. These findings support a model whereby Bdbt and DAnkrd49 act together to modulate the activity of Dco during planar polarity establishment (Strutt, 2020).

Planar polarity describes the phenomenon whereby cells coordinate their polarity in the plane of a tissue: for example the hairs on the skin point in the same direction, cilia coordinate their beating, and cells coordinate their movements during tissue morphogenesis. Understanding the mechanisms by which this coordinated polarisation occurs is of prime importance, as disruption of polarity can have diverse consequences, including neural tube closure defects, hydrocephalus and defects in neuronal migration (Strutt, 2020).

The fly wing is a well-characterised model system in which to study planar polarity. Each cell within the adult wing produces a single hair, or trichome, which points towards the distal end of the wing. Furthermore, viable mutations that cause characteristic swirling of the trichomes have been identified, and the genes associated with these mutations were subsequently found to be highly conserved, and to regulate planar polarity throughout the animal kingdom (Strutt, 2020).

The core planar polarity proteins (hereafter known as the core proteins) are the best characterised group of proteins that regulate planar polarity. In the pupal wing, the core proteins adopt asymmetric subcellular localisations prior to trichome emergence, and in their absence trichomes emerge from the centre of the cell rather than at the distal cell edge. The core proteins comprise the atypical cadherin Flamingo (Fmi, also known as Starry Night [Stan]), the transmembrane proteins Frizzled (Fz) and Strabismus (Stbm, also known as Van Gogh [Vang]), and three cytoplasmic proteins Dishevelled (Dsh), Prickle (Pk) and Diego (Dgo). Fmi localises to proximal and distal cell edges in the pupal wing, but is excluded from lateral cell edges, while Fz, Dsh and Dgo localise to distal cell edges and Stbm and Pk to proximal cell edges. These proteins form intercellular complexes at cell junctions, that couple neighbouring cells and allow them to coordinate their polarity (Strutt, 2020).

The mechanisms by which the core proteins become asymmetrically localised are poorly understood. The overall direction of polarity is thought to be determined by tissue-specific 'global' cues: these may include gradients of morphogens or Fat/Dachsous cadherin activity, or other cues such as mechanical tension. In the wing these global cues may directly regulate core protein localisation or act indirectly via effects on growth and tissue morphogenesis. Global cues are thought to lead to subtle biases in core protein localisation within cells that are subsequently amplified by feedback between the core proteins, in which positive (stabilising) interactions between complexes of the same orientation are coupled with negative (destabilising) interactions between complexes of opposite orientation. In mathematical models, such feedback interactions have been demonstrated to be sufficient to amplify weak biases in protein localisation, leading to sorting of complexes and robust asymmetry (Strutt, 2020).

Experimental evidence for feedback is only beginning to be elucidated. Cell culture experiments have demonstrated competitive binding between several of the core proteins, which may be important for feedback. Furthermore, it has recently been shown that the core protein Pk acts through Dsh to destabilise Fz in the same cell, while stabilising Fz across cell junctions via Stbm (Strutt, 2020).

In order for feedback to operate, cells must utilise the general cellular machinery: for example active endocytosis is necessary for Pk to destabilise Fz. Furthermore, post-translational modifications of the core proteins are likely to be key mediators of feedback. For example loss of ubiquitination pathway components and some protein kinases have been shown to disrupt planar polarity. In flies, a Cullin-3/Diablo/Kelch ubiquitin ligase complex regulates Dsh levels at cell junctions, while the de-ubiquitinase Fat Facets regulates Fmi levels. Stbm also negatively regulates Pk levels, and ubiquitination of Pk by Cullin-1/SkpA/Slimb promotes internalisation of Fmi-Stbm-Pk complexes. Similarly, in vertebrates, the Stbm homologue Vangl2 may promote local degradation of Pk via ubiquitination by Smurf E3 ubiquitin ligases. Furthermore, Drosophila Fz phosphorylation is mediated by atypical Protein Kinase C, and Dsh is a target of phosphorylation by the Discs Overgrown (Dco, also known as Doubletime [Dbt] or Casein Kinase Iε [CKIε]) and Abelson kinases. Dco/CKIε has also been implicated in phosphorylation of Stbm in both flies and vertebrates (Strutt, 2020).

This study describes the identification of two new regulators of planar polarity in the Drosophila wing. Bride of Doubletime (Bdbt) and DAnkrd49 were shown to be binding partners that regulate each other's levels. Loss of either protein disrupts asymmetric localisation of the core proteins. Furthermore, they regulate overall levels of Dsh and Dsh phosphorylation in the pupal wing, and evidence is provided that they act by modulating the activity of the kinase Dco (Strutt, 2020).

This study present evidence for a novel protein complex regulating planar polarity, consisting of the ankyrin repeat protein DAnkrd49 and the non-canonical FKBP family member Bdbt. DAnkrd49 and Bdbt physically interact in vitro and regulate each other's levels in vivo. This suggests that they may act in a complex in which each is required to stabilise the other, a model supported by the phenotypic similarity in mutant clones. Their activity is required for core protein asymmetry in the pupal wing, and for normal levels and phosphorylation of the cytoplasmic core protein Dsh (Strutt, 2020).

Previous work on circadian rhythms has established a physical interaction between Bdbt and the kinase Dco, and that Dco activity is regulated by Bdbt (Fan, 2013). Notably, Dco has previously been implicated in promoting both Dsh and Stbm phosphorylation in planar polarity signalling. Thus, the data are consistent with both DAnkrd49 and Bdbt acting to regulate Dco activity in planar polarity. Firstly, loss of function clones of dco are seen, and DAnkrd49/Bdbt share a common phenotype: in their absence reduced overall levels of Dsh and reduced levels of Dsh phosphorylation. Secondly, strong genetic interactions are seen between dco and DAnkrd49/Bdbt. Although a physical interaction has been observed between Dco and Bdbt, no direct interaction was seen between Dco and DAnkrd49. Therefore, a simple model is that the role of DAnkrd49 is to stabilise Bdbt, while Bdbt directly regulates Dco activity. Thus this study defines a regulatory cascade, whereby DAnkrd49 and Bdbt promote Dco activity, which in turn regulates phosphorylation of core proteins and asymmetric localisation (Strutt, 2020).

An alternative model is that DAnkrd49 and Bdbt act to stabilise Dsh, independently of Dco. Proteins of the FKBP family are known to regulate the stability of target proteins. In canonical FKBP family members this stabilisation is a result of PPIase activity, which assists protein folding; whereas other family members stabilise their target proteins by direct binding. The catalytic sites for PPIase are not conserved in Bdbt. A binding interaction between Dsh and either DAnkrd49 or Bdbt was not seen, arguing that Bdbt is unlikely to directly stabilise Dsh (Strutt, 2020).

In addition to defects in planar polarity, other pleiotropic defects are seen in DAnkrd49 clones and Bdbt clones, including cell size defects, reduced proliferation and poor viability. These could plausibly be explained by regulation of Dco activity by DAnkrd49 and Bdbt. Dco acts in multiple signalling pathways. It phosphorylates the tumour suppressor Fat, which regulates cell growth and survival via the Hippo signalling pathway. Interestingly, a hypomorphic mutation in dco causes tissue overgrowth and increased activity of the caspase inhibitor DIAP1, phenocopying loss of Hippo signalling pathway components, while cells completely lacking Dco activity have reduced expression of the caspase inhibitor DIAP1 and reduced proliferation. Dco may also act in additional signalling pathways, and these are thought to include Hedgehog signalling and canonical Wnt signalling. Hence, the multitude of signalling pathways regulated by Dco may explain the complex phenotypes seen in the absence of DAnkrd49 and Bdbt. Alternatively, it is possible that DAnkrd49 and Bdbt may regulate other downstream targets in addition to Dco (Strutt, 2020).

As Dco has also been implicated in phosphorylating Stbm, loss of DAnkrd49 and Bdbt is also expected to reduce Stbm phosphorylation in pupal wings. However this could not be verified directly, as Stbm mobility in SDS-PAGE is only marginally increased when Dco activity is reduced, and is not noticeably altered in extracts from animals with reduced activity of DAnkrd49 or Bdbt. Nevertheless, recent work has shown that phosphorylation of both Stbm and Dsh by Dco is functionally important in establishing correct planar polarity, making the regulation of Dco activity of great interest (Strutt, 2020).

Interestingly, protein interactions studies in human cell lines have identified a STRING network between Ankrd49, CKIδ/CKIε and FKBP family members. Although the FKBP proteins identified in these studies are not the closest orthologues to Drosophila Bdbt, this nevertheless suggests conservation of a regulatory cascade of Ankrd49/FKBP promoting Dco activity, which in turn might phosphorylate core planar polarity proteins. Furthermore, the DISEASES online resource, which integrates results from text mining, manually curated disease-gene associations and genome-wide association studies, has linked Ankrd49 with brachydactyly subtypes. Brachydactyly is a key feature of Robinow syndrome, a disease closely associated with core planar polarity mutations; thus understanding this regulatory cascade may be of importance for human health (Strutt, 2020).


Transcript length - 3.2 kb

Exons - 4


Amino Acids - 440

Structural Domains

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).

discs overgrown: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 3 December 2023

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