Phosphorylation of a Central Clock Transcription Factor Is Required for Thermal but Not Photic Entrainment

As an initial attempt to better understand the role(s) of dCLK phosphorylation we sought to map phosphorylation sites using recombinant protein production in cultured Drosophila S2 cells. This simplified cell culture system was successfully used to identify physiologically relevant phosphorylation sites on Drosophila PER [15], [16], [18], [40]. Prior work showed that production of recombinant dCLK in S2 cells leads to significant shifts in electrophoretic mobility that are due to phosphorylation [29]. Thus, we established S2 cell lines stably expressing HA-dClk-V5 under the inducible metallothionein promoter (pMT). Total cell lysates were subjected to immunoprecipitation with anti-V5 antibody, followed by multi-protease digestion, titansphere nanocolumn phosphopeptide enrichment, and tandem mass spectrometry, as previously described [16], [41]. We identified 14 phosphorylation sites on dCLK, all of which are at Ser residues, with the possible exception of Tyr607 (Table 1). Many of the identified phosphorylation sites are in the C-terminal half of dCLK, which contains several Q-rich regions that might function in transcriptional activation (Figure 1A). Phosphorylation was also detected at sites close to the N- and C-terminus of the dCLK protein. Interestingly, no phosphorylation sites were found in any of the known functional domains of dCLK; e.g. bHLH, PAS domains and Q-rich regions (Figure 1A and Table 1).

In preliminary studies we individually mutated each of the identified phosphorylated Ser residues to Ala residues but did not see major effects on dCLK electrophoretic mobility, except for the S859A mutant version of dCLK, which manifested slightly faster electrophoretic mobility. (Figure S1A and B). The transcriptional activities of most single site mutants were somewhat increased (≤2 fold), except for the S924A mutant version of dCLK, which manifested a slight but reproducible decrease (Figure S1C). Overall, our initial studies in S2 cells were not able to identify whether certain individual phospho-sites are particularly significant in regulating dCLK metabolism or activity. While ongoing work is aimed at better understanding the roles of individual phospho-sites, in this study we focused on more global aspects of dCLK phosphorylation by generating a mutant version wherein all the Ser phospho-acceptor sites identified by mass spectrometry were switched to Ala. Since the mass spectrometry data did not unambiguously identify which Ser among amino acids 209–211 is phosphorylated, we switched all 3 Ser to Ala. In addition, although Tyr 607 or Ser 611 is phosphorylated, to focus on Ser phosphorylation, we only mutated Ser 611 to an Ala. By using site-directed mutagenesis, we serially mutated the aforementioned 16 Ser to Ala (dCLK-16A). The electrophoretic mobility of dCLK-16A is indistinguishable from that of λ-phosphatase treated wild-type dCLK and was not altered by λ-phosphatase treatment (Figure 1B), indicating that we mapped most, if not all, the sites on dCLK phosphorylated by endogenous kinases in S2 cells.

dCLK-16A interacts with either CYC or PER proteins to a similar extent as that observed for the wild-type version (dCLK-WT), demonstrating that dCLK-16A is not grossly misfolded (Figure 1C). In addition, our findings suggest that the phosphorylated state of dCLK is not a major signal regulating interactions with core clock partners. Consistent with prior work, we observed increases in non/hypo-phosphorylated isoforms of dCLK when dPER is co-expressed (Figure 1C, compare lane 1 and 6) [31]. With regards to transcriptional activity, dCLK-16A is more potent compared to dCLK-WT in stimulating E-box dependent transcription (Figure 1D), while still maintaining its sensitivity to inhibition by dPER (Figure 1E). Earlier findings showed that hyper-phosphorylated dCLK is less stable and that DBT might contribute to this instability, although the exact role of DBT is not clear [28]–[30]. We compared the stabilities of dCLK-16A and dCLK-WT under a variety of conditions, including overexpressing DBT, but did not detect a significant difference (Figure S2A and B), suggesting we did not map one or more phosphorylation sites critical for regulating dCLK stability and/or the pathway for dCLK degradation in S2 cells is not identical to that in flies (see below). Taken together, the results obtained using well-established S2 cell based assays indicate that dCLK-16A retains key clock-relevant biochemical functions and suggest that global phosphorylation of dCLK reduces its transactivation potential.

To investigate whether the dCLK phosphorylation sites we identified play a physiological role in the Drosophila circadian timing system, we first evaluated the ability of a novel wildtype dClk transgene to rescue behavioral rhythms in the arrhythmic Clk^out genetic background (herein, termed as p{dClk-WT};Clk^out). Clk^out is a newly described arrhythmic null mutant that does not produce dCLK protein (Mahesh et al., submitted). The transgene was constructed with a 13.9 kb genomic fragment that contains the dClk gene, which we modified by introducing a V5 epitope tag at the C-terminus of the dClk open reading frame for enhanced protein surveillance. Flies were exposed to standard entraining conditions of 12 hr light∶12 hr dark cycles [LD; where zeitgeber time 0 (ZT0) is defined as lights-on] at 25°C, followed by several days in constant dark conditions (DD) to measure free-running behavioral periods. In the behavioral analysis, p{dClk-WT};Clk^out flies manifested robust locomotor activity rhythms with normal ∼24 hr periods (Table 2, Mahesh et al., submitted), indicating that the circadian clock system functions properly in these flies.

Next, we sought to generate transgenic flies harboring a dCLK-16A version of the dClk rescue transgene. However, because of technical difficulties in generating a version that also included replacing Ser5 with Ala, we made a dClk version wherein the other 15 Ser residues were switched to Ala, termed dClk-15A. In S2 cells, dCLK-15A behaves similar to dCLK-16A, including no observable effect of phosphatase treatment on electrophoretic mobility and enhanced E-box dependent transcriptional activity (Figure S3). Although phosphorylation of Ser5 might affect dCLK function in a manner that is not revealed in the S2 cell based assays we used, the CLK-15A protein contains the majority of phosphorylation sites and should address if global phosphorylation of dCLK plays an important role in the circadian timing system. Two independent lines of transgenic flies harboring the dClk-15A transgene were obtained and circadian behavior was monitored in the Clk^out genetic background (referred to as p{dClk-15A}, 2M; Clk^out and p{dClk-15A}, 6M; Clk^out). In sharp contrast to flies harboring the control version of dClk, the two independent lines of p{dClk-15A};Clk^out flies manifested generally weaker behavioral rhythms that are approximately 1.5 hr shorter compared to their wild-type counterparts (Table 2).

Under standard conditions of LD at 25°C, D. melanogaster exhibits a bimodal distribution of activity with a “morning” and “evening” bout of activity centered around ZT0 and ZT12, respectively. Although p{dClk-15A};Clk^out flies manifest the typical bimodal distribution of locomotor activity, the onset of both the morning and evening bouts of activity were earlier (Figure 2, compare panels B and C to A), consistent with the shorter free-running periods. The Clk^out flies only showed a “startle” response to the lights-on transition but no rhythmic behavior (Figure 2E). In constant dark conditions, the downswing in evening activity is clearly earlier in p{dClk-15A};Clk^out flies, in agreement with the shorter free-running period (Table 2, Figure 2 and S4). We also examined the locomotor behavior of flies harboring the dClk-WT transgene in a wild-type genetic background, resulting in flies with four copies of the dClk gene (herein referred to as p{dClk-WT};+/+). The circadian period was shortened to approximately 23 hr (Table 2 and Figure 2D), which is well correlated with previous reports demonstrating that increasing the copy number of dClk shortens the circadian period of behavioral rhythms [42], [43].

A hallmark property of circadian rhythms is that the period length is very constant over a wide range of physiologically relevant temperatures, termed temperature compensation [44]. To investigate whether phosphorylation of dCLK might have a role in temperature compensation, we analyzed behavioral rhythms at three standard temperatures (i.e., 18°, 25° and 29°C). Although we noted a decrease in rhythmicity for dClk-15A;Clk^out flies at 29°C, the periods were quite similar over the temperature range tested (Table 2), suggesting that global phosphorylation of dCLK does not play a major role in temperature compensation.

We examined the temporal profiles of dCLK protein by analyzing head extracts prepared from p{dClk-WT};Clk^out and p{dClk-15A};Clk^out flies in LD conditions (Figure 3A and C). dCLK-WT protein undergoes daily changes in phosphorylation that are consistent with earlier results probing endogenously produced dCLK; namely, hypo- to medium- phosphorylated isoforms present during the mid-day/early night (e.g., ZT 8 to ZT 16) and mostly hyper-phosphorylated isoforms present in the late night/early day (e.g., ZT20 to ZT4) (Figure 3A) [28], [29]. However, the mobility of dCLK-15A was similar throughout a daily cycle (Figure 3A), and co-migrated with λ phosphatase treated dCLK-WT (Figure 3B). Thus, similar to results in S2 cells, dCLK-15A exhibits little to no phosphorylation in vivo, suggesting that the phospho-sites we identified by mass spectrometry comprise, at the very least, a major portion of the total phosphorylation sites found on dCLK in flies (it is also possible that one or more of the phospho-sites we mutated are required for phosphorylation at other sites, but this would still result in a mainly hypo-phosphorylated dCLK protein). Intriguingly, the levels of dCLK-15A were substantially higher compared to dCLK-WT throughout a daily cycle. Quantification of immunoblots indicated that the average daily levels of dCLK-15A are about 2.5 times more than those of dCLK-WT (Figure 3C).

To examine whether the high levels of dCLK-15A proteins results from elevated mRNA abundance, we measured dClk transcript levels. As reported previously, although the overall daily abundance of dCLK-WT protein is essentially constant throughout a daily cycle, dClk-WT mRNA levels oscillate with peak amounts attained during the late night-to-early day and reaching trough values around ZT12 [45], [46]. The daily oscillation in dClk-15A mRNA abundance is similar to the wild-type situation and even seemed to have lower peak levels (Figure 3D). These results indicate that in general global phosphorylation of dCLK decreases its stability in vivo, consistent with prior findings using S2 cells [28], [29].

To further examine the status of the clockworks, we measured the daily profiles in per and tim transcripts and protein levels. Both per and tim mRNA levels in p{dClk-15A};Clk^out flies were reproducibly higher, especially during the daily upswing that occurs between ZT4 – 12 (Figure 4A and B). These result further support the notion that dCLK levels are normally rate-limiting for circadian transcription and suggest that despite the increased abundance of dCLK-15A there is sufficient PER to engage in normal repression of dCLK-15A/CYC activity. Indeed, PER protein levels were reproducibly higher in p{dClk-15A};Clk^out flies (Figure 4C and D), consistent with the increased transcript levels. In p{dClk-15A};Clk^out flies, TIM protein levels were slightly but reproducibly increased (Figure 4E and F). The increased daily upswing in tim mRNA levels in p{dClk-15A};Clk^out flies might have a smaller effect on overall TIM protein levels because light induces the rapid degradation of TIM [47], possibly limiting the ability of TIM to accumulate during the early night prior to the start of transcriptional feedback repression. Taken together, we show that the stability of dCLK in flies is strongly increased by blocking phosphorylation at one or more sites. Moreover, augmenting the total abundance of dCLK accelerates the daily accumulation of per/tim transcripts and increases their peak levels, indicative of higher overall dCLK-CYC-mediated transcription. In addition, increased in vivo transcriptional activity of dCLK-15A may also contribute to higher dCLK-CYC-mediated transcription, as is the case in S2 cells (Figure 1D). These results demonstrate that dCLK phosphorylation plays a key role in setting the amplitudes of the per mRNA and protein rhythms, molecular oscillations that are central to the primary TTFL and circadian speed control in Drosophila[16], [48].

Besides light-dark cycles, daily changes in temperature can also synchronize (entrain) circadian rhythms in a wide variety or organisms [49]. Prior work showed that D. melanogaster can entrain to daily cycles of alternating 12 hr ‘warm’/12 hr ‘cold’ cycles that differ by as little as 2–3°C [50]–[52]. To determine if flies expressing dCLK-15A have a defect in entraining to temperature cycle, flies were kept in constant darkness, exposed to 12 hr∶12 hr temperature cycles of 24°C∶29°C (TC) and locomotor activity rhythms analyzed (Table 3 and Figure 5). The daily distribution of activity in p{dClk-15A};Clk^out flies is strikingly different compared to the wild-type control. As previously observed for wildtype strains of D. melanogaster entrained to daily temperature cycles [50]–[52], p{dClk-WT};Clk^out flies exhibit the classic “anticipatory” rise in activity just prior to the low-to-high and high-to-low temperature transitions, similar to what is observed in light-dark cycles around ZT0 and ZT12 (Figure 5 and S5; there is a “startle” response at the transition from low-to-high temperature that is also observed in Clk^out flies, analogous to the transient burst in activity at lights-on in a LD cycle). In sharp contrast, during the beginning of the temperature entrainment regime although p{dClk-15A};Clk^out flies also manifest two activity peaks, they occur much earlier at around the middle of the warm- and cryo-phases (Figure 5B).

Interestingly, while the timing of the “startle” response at the transition from low-to-high temperature remained constant in p{dClk-15A};Clk^out flies, the timing of the major activity peak occurring during the mid-warm phase appeared to progressively advance on subsequent days in TC (Figure 5B). Analysis of individual activity records also confirmed this trend (Figure 5E). The abnormal behavioral pattern under temperature cycles for p{dClk-15A};Clk^out flies was also observed when flies were exposed to a temperature cycle after first treating them with constant light for 6 days to abolish the circadian timing system (Figure S6). Thus, the defective entrainment of p{dClk-15A};Clk^out flies to TC is not dependent on the status of the clock at the time that the temperature entrainment was initiated. Furthermore, although the main activity bout in p{dClk-15A};Clk^out flies advanced on subsequent days during TC, the rate of advancement was clearly greater during free-running conditions following TC (Figure S5), suggesting partial entrainment during TC. Following entrainment to TC, the free-running period of dCLK-15A producing flies is about 1.5 hr shorter compared to the wild type dCLK-WT control (Table 3 and Figure S5). The faster running clock in p{dClk-15A};Clk^out flies during free-running conditions after exposure to TC is consistent with results obtained following entrainment to LD (Table 2). Thus, it appears that when exposed to daily temperature cycles p{dClk-15A};Clk^out flies can adopt some alignment with the entraining conditions, albeit without a normal phase relationship, but that this entrainment is weak and the flies partially free-run at their shorter endogenous periods, leading to progressive advances in their behavioral rhythm relative to the 24 hr entraining regime. Although not as dramatic, the timing of the warm-phase activity bout in p{dClk-WT};+/+ flies also advanced during TC (Figure 5F), whereas this was not the case for p{dClk-WT};Clk^out flies (Figure 5D). In addition, the clock in p{dClk-WT};+/+ runs about 1 hr faster than the control situation, strongly suggesting that augmenting dCLK levels (Figure S7) impairs the ability of the circadian timing system to entrain to daily temperature cycles.

Temperature cycles can entrain behavioral rhythms in Drosophila exposed to constant light (LL) despite the fact that LL normally abolishes circadian rhythms [51], [53], [54]. Intriguingly, constant light exposure rescues the ability of the p{dClk-15A};Clk^out flies to maintain a more stable 24-hr phase relationship with the temperature cycle (Figure 5, panels G–J), further supporting the notion that entrainment to temperature but not light is specifically impaired in these flies. Taken together, these data suggest that in the absence of light, the dCLK phosphorylation program is required for the proper entrainment of behavioral rhythms to daily temperature cycles and reveal an unanticipated role for a central clock transcription factor in modality-specific entrainment.

In p{dClk-WT};Clk^out flies, hypo/intermediate-phosphorylated dCLK isoforms are present throughout the thermo phase in TC (Figure 6A, lane 2 and 3), while hyper-phosphorylated dCLK isoforms are only observed during the latter half of the cryo phase (Figure 6A, lane 5 and 6). This temporal pattern in dCLK phosphorylation is similar to that observed in LD cycles and is consistent with prior work showing that the circadian clock mechanism in Drosophila can be synchronized by daily temperature cycles [50], [51]. As expected and similar to results using LD cycles, dCLK-15A attains higher overall daily levels and does not exhibit significant phosphorylation in p{dClk-15A};Clk^out flies exposed to temperature cycles (Figure 6A).

Since p{dClk-15A};Clk^out flies display altered entrainment to TC cycles that becomes progressively more abnormal with prolonged duration, we tested whether the molecular clock might also exhibit a more defective status with increasing time by measuring the levels of the tim mRNA at both early (e.g., day 3) and later (e.g., day 6) days of exposure to TC. We chose tim mRNA levels as a surrogate marker for clock dynamics because it normally has a robust high amplitude rhythm (Figure 4A, B; [55]), facilitating measuring changes in molecular oscillations over the course of several days. Although the daily average levels in tim mRNA were higher in p{dClk-15A};Clk^out flies on day three of TC compared to the wild-type situation (Figure 6B), consistent with findings in LD (Figure 4B), both genotypes showed similar and robust cycling patterns. However, by day six of TC, the tim mRNA oscillation pattern in p{dClk-15A};Clk^out flies became significantly different from that observed for p{dClk-WT};Clk^out flies (Figure 6C). Most notably, while tim mRNA cycling still manifested high-amplitude cycling in p{dClk-WT};Clk^out flies on day six of TC, tim mRNA levels during the upswing phase were significantly higher in p{dClk-15A};Clk^out flies, resulting in an abnormal cycling pattern. Although we did not establish a causal relationship between the observed loss in normal tim mRNA cycling and the defective behavioral entrainment in p{dClk-15A};Clk^out flies during TC, the results clearly show that prolonged exposure to TC is not only associated with increasingly altered phasing of rhythms at the behavioral level (Figure 5) but also at the molecular level.

As with behavioral rhythms prior work showed that circadian molecular cycles can be synchronized to TC in the presence of constant light [51], [53]. In agreement with the observation that constant light exposure enabled p{dClk-15A};Clk^out flies to more robustly synchronize to temperature cycles (Figure 5), daily rhythms in the levels of tim mRNA for both genotypes were quite similar even after six days in constant light during TC (Figure 6D and E), indicating the clock in p{dClk-15A};Clk^out flies is functioning in a more wild-type manner under these conditions. Taken together, while this molecular analysis is of limited scope, it suggests that constant light exposure facilitates the ability of p{dClk-15A};Clk^out flies to entrain to TC by enhancing normal clock function.

The pMT-dClk-V5, pMT-HA-dClk-V5, pMT-HA-dClk, pAct-per, pAct-per-V5 and pMT-dbt-V5 plasmids were described previously [20], [29], [31]. pMT-dClk15A-V5 and pMT-dClk16A-V5 were generated by serially changing codons for Ser to those of Ala by using a Quick Change site-directed mutagenesis kit (Stratagene). All final constructs were verified by DNA sequencing.

Hygromycin-resistant stable Schneider 2 (S2) cell lines expressing pMT-HA-dClk-V5 were established for dCLK purification. dClk expression was induced by adding 500 µM CuSO₄ to the medium and cells were harvested 24 hr post-induction. 200 ml of culture (3×10⁶ cells/ml) was used and harvested cells were lysed using modified-RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.25% Sodium deoxycholate) with the addition of a protease inhibitor cocktail (Roche) containing 1 mM EDTA, 25 mM NaF, and 1 mM Na₂VO₃. To extracts, anti-V5 antibody (Invitrogen) was added and incubated overnight with gentle rotation at 4°C followed by the addition of Dynabeads Protein A (Invitrogen) with a further overnight incubation. Beads were collected using DynalMPC. dCLK was eluted with modified Laemmli buffer (150 mM Tris-HCl [pH 6.8], 6 mM EDTA, 3% SDS, 30% Glycerol) supplemented with 50 mM reducing agent TCEP (Calbiochem) at 65°C for 20 min. Alkylation was performed by adding 0.5M IAA (iodoacetamide) for 20 min at room temperature in the dark. The eluate was resolved using 8% SDS-PAGE, and all the detectable dCLK bands of differing electrophoretic mobility excised (which under the conditions used was mainly the ‘intermediate’ phosphorylated band), subjected to protease digestion and analyzed by mass spectrometry. Mass spectrometry was performed as described in Schlosser et al. 2005. Data analysis was performed as described previously [41].

S2 cells were obtained from Invitrogen and transfected using effectene following the manufacturer's protocol (Qiagen). Luciferase (luc) reporter assay was performed as described previously [29], [75]. Briefly, S2 cells were placed in 24-well plates and co-transfected with 0–100 ng pMT-dClk-V5 and pMT-dClk-16A-V5 along with 10 ng of perEluc, 30 ng of pAct-β-gal-V5/His as indicated. dPER mediated repression of dCLK dependent transactivation was measured by transfecting 0–20 ng of pAct-dper together with 2 ng of pMT-dClk-V5 or pMT-dClk-16A-V5. One day after transfection, dClk expression was induced with 500 µM CuSO₄ (final in the media), and after another day cells were washed in phosphate buffered saline (PBS), followed by lysis in 300 µl of Reporter Lysis Buffer (Promega). Aliquots of cell extracts were assayed for β-galactosidase and luciferase activities using the Luciferase Assay System and protocols supplied by the manufacturer (Promega).

Clk^out flies were generated in one of our laboratories (P.E.H.) as follows: 5.2 kb deletion of dClk exon 1 and upstream sequences was generated by FLP-mediated recombination between FRT sites in the pBac Clk[f06808] and pBac Clk [f03095] [76], [77]. Flippase (FLP)-induced recombination was induced by a daily 1 h heat-shock at 37°C given to hsFLP;;f06808/f03095 larvae and pupae. Three recombinants were recovered, and each produced a deletion rather than a duplication of intervening dClk sequences. The remaining pBac insert was excised via pBac transposase induced transposition resulting in white-eyed flies harboring the deletion [78]. A DNA fragment containing the deleted sequences was amplified using primers situated upstream of the f03095 insertion site (5′ CGGAATATTGGACAACAAACAG 3′) and downstream of the f06808 insertion site (5′CAGCAGTGGAATCTTAATACAG 3′), and sequenced to confirm the endpoints of the deletion. This new dClk deletion allele was named Clk^out.

To generate transgenic flies that produce wild-type dCLK tagged with V5 at the C-terminus, dClk-containing P[acman] transgene was generated using recombineering-mediated gap repair [79]. To prepare the P[acman] vector, homology arms were amplified from genomic DNA with primers clkLA-f (ATGTGGCGCGCCGCCCCAAAAATCCATAAATGCT) and clkLA-r (GTGTTGGATCCAGGGGTGTTATAGAGAGGGACA) for the left arm and clkRA-f (GTGTGGATCCGCAGAGTGAAACCTGTGCAA) and clkRA-r (ATATATGTGCGGCCGCTCCCGGTTATGAGTTTTTCG) for the right arm via PCR, and cloned as AscI-BamHI and BamHI-NotI fragments into AscI and NotI digested attB-P[acman]-ApR vector (modified to remove the SphI site) to form attB-P[acman]ClkLARA. Recombination-competent SW102 cells harboring BAC clone RP98 5K6 (BACPAC Resource Center, Oakland, Ca, USA), which contains the dClk genomic region, were transformed with the attB-P[acman]ClkLARA vector (linearized with BamHI). Recombinants containing 15.5 kb of genomic sequence beginning ∼8 kb upstream of the dClk translation start and ending ∼2.5 kb downstream of the dClk stop codon were verified by PCR and sequencing and termed attB-P[acman]-Clk. To introduce a V5 epitope tag at the C-terminus of the dClk open reading frame (ORF), a 3′ genomic fragment of dClk (from 351 bp upstream to 1580 bp downstream of the translation stop) was cloned into pGEM-T vector (Promega, Madison, WI). Sequences encoding V5 were introduced in-frame immediately upstream of the dClk stop codon using the Quickchange site directed mutagenesis kit (Stratagene, La Jolla, CA) to create pGEM-T-dClk3′V5. The 3′ dClk genomic fragment in attB-P[acman]- dClk was swapped with the 3′ fragment in pGEM-T-dClk3′V5 using SphI and NotI to form attB-P[acman]- dClkV5. This transgene was inserted into the VK00018 attP site on chromosome 2 via PhiC31-mediated transgenesis [79], [80].

Transformation vector containing a genomic dClk wherein the codons for the 15 identified phospho-serine were switched to those for alanine was generated in multiple stages as follows: A genomic dClk sub-fragment from NheI to SphI site was isolated from P[acman]-dClk-V5 and subcloned into pSP72 vector where the multi-cloning sites were mutagenized to introduce a NheI site, and named this plasmid as pSP72-dClk(NheI/SphI). Next, we obtained a dClk sub-fragment spanning from the NcoI to SphI sites by restriction digestion of pSP72-dClk(NheI/SphI), subcloned the released fragment into pSP72 where the multi-cloning sites were mutagenized to introduce a NcoI site, and named this plasmid as pSP72-dClk(NcoI/SphI). We performed serial site directed mutagenesis with pSP72-dClk(NcoI/SphI) and finally made pSP72-dClk(NCoI/SphI)-S11A wherein codons for the serine residues at amino acids 209, 210, 211, 444, 450, 487, 504, 611, 645, 859, 902 on dCLK were all switched to those for alanine residues [(GenBank accession number NP_001014576↗)]. We purified the dClk(NCoI/SphI)-S11A insert by restriction enzyme digestion of pSP72-dClk(NCoI/SphI)-S11A and replaced the wild-type dClk(NcoI/SphI) insert, generating pSP72-dClk(NheI/SphI)-S11A. Next, a more 3′ genomic dClk sub-fragment from the SphI to NotI sites was subcloned into pSP72 vector where the multi-cloning sites were mutagenized to include NotI and NheI sites, and named this plasmid as pSP72-dClk(SphI/NotI). We performed serial site directed mutagenesis with pSP72-dClk(SphI/NotI) and made pSP72-dClk(SphI/NotI)-S4A wherein codons for the serine residues at amino acids 924, 934, 938, 1018 were switched to those for alanine. Finally, the genomic dClk(SphI/NotI)-S4A fragment was ligated with pSP72-dClk(NheI/SphI)-S11A generating pSP72-dClk(NheI/NotI)-S15A, and then dClk(NheI/NotI)-S15A fragment was switched with wild-type dClk(NheI/NotI) fragment in pacman-dClk-V5 plasmid yielding P[acman]-dClk-15A-V5. Transgenic flies were generated by BestGene Inc. (CA, USA). P[acman]-dClk-15A-V5 transformation vector was injected into flies carrying the VK00018 attP docking site on the second chromosome for site-specific integration [79]. Two independent germ-line transformants bearing the dClk-15A-V5 transgene in a wild-type background were obtained and then crossed into a Clk^out genetic background to yield dClk-15A-V5;Clk^out.

The locomotor activities of individual flies were measured as previously described using the Drosophila Activity Monitoring system from Trikinetics (Waltham, MA). Young adult flies were used for the analysis and exposed to 4 days of 12 h light followed by 12 h dark [where zeitgeber time 0 (ZT0) is defined as the time when the light phase begins] at 25°C and subsequently kept in constant dark conditions (DD) for 7 days. Temperature entrainment (temperature cycle, TC) was performed in constant dark condition and in some cases, in the presence of constant light (>2000lux). Temperature cycles were 12 h of 24°C (cryo phase) followed by 12 h of 29°C (thermal phase) (where ZT0 is defined as the time when the cryo phase begins) for 4 days and subsequently kept at 24°C for 7 days. The locomotor activity data for each individual fly was analyzed using the FaasX software (Fly Activity Analysis Suite for MacOSX), which was generously provided by F. Rouyer (CNRS, France). Periods were calculated for each individual fly using chi-square periodogram analysis and pooled to obtain a group average for each independent transgenic line or genotype. Power is a quantification of the relative strength of the rhythm during DD. Individual flies with a power ≥10 and a ‘width’ value of 2 or more (denotes number of peaks in 30-min increments above the periodogram 95% confidence line) were considered rhythmic. Actogram represents the locomotor activity data throughout the experimental period. Vertical bars in the actogram represent absolute activity levels for each 30 min intervals averaged for each given genotypes of flies. The strength of this measurement can be manipulated by using the function called hash density, which represent the number of times fly need to make beam crossing to be registered as one vertical bar. The hash density of the actogram was varied for better comparison depending on the activity levels of given genotypes of flies.

Protein extracts from S2 cells were prepared as previously described [31]. Briefly, the cells were lysed using modified-RIPA buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 0.25% Sodium deoxycholate) with the addition of protease inhibitor cocktail (GeneDEPOT) and phosphatase inhibitor cocktail (GeneDEPOT). For detection of dCLK recombinant protein, extracts were obtained using RIPA buffer 25 mM Tris-HCl [pH 7.5], 50 mM NaCl, 0.5% Sodium deoxycholate, 0.5% NP40, 0.1% SDS) and were sonicated briefly as previously described [29]. Flies were collected by freezing at the indicated times in light-dark (LD) or temperature cycles (TC) and total fly head extracts prepared using modified-RIPA buffer or RIPA buffer with sonication (for dCLK). Extracts were resolved by 5% polyacrylamide gels or by 3–8% Tris-acetate Criterion gel (Bio-Rad) in some case for dCLK, transferred to PVDF membrane (Immobilon-P, Millipore), and immunoblots were treated with chemiluminescence (ECL, Thermo). Primary antibodies were used at the following dilutions; anti-V5 (Invitrogen), 1∶5000; anti-HA (12CA5, Roche), 1∶2000; anti-OGT (H-300, Santa Cruz), 1∶3000; anti-PER, (Rb1) 1∶3000; anti-TIM (TR3), 1∶3000; anti-dCLK (GP208) 1∶3000. Quantification of band intensity was performed using image J software.

For immunoprecipitation, cell extracts from S2 cells were prepared and 3 µl of anti-HA (12CA5) or anti-V5 antibody was added depending on the target protein sought, and incubated for overnight at 4°C with gentle rotation. The next day, 20 µl of Gammabind-sephase bead (GE healthcare) was added with a further incubation of 3 hr at 4°C. The immune complexes were eluted with 1X SDS-PAGE sample buffer. For λ- phosphatase treatment, the purified immune complexes were resuspended in λ protein phosphatase buffer (50 mM Tris-HCl [pH 7.5], 0.1 mM EDTA, 5 mM DTT, 0.01% Triton X-100, 2 mM MnCl₂, and 0.1 mg/ml bovine serum albumin), divided into two equal aliquots. One aliquot of bead was treated with 200 units of λ protein phosphatase (NEB) and no addition was made to the other aliquot. Both aliquots were incubated for 30 min at 30°C with occasional shaking, and immune complexes analyzed by immunoblotting.

Total RNA was isolated from frozen heads using QIAzol lysis reagent (QIAGEN). 1 µg of total RNA was reverse transcribed with oligo-dT primer using Prime Script reverse transcriptase (TAKARA) and real-time PCR was performed in Corbett Rotor Gene 6000 (Corbett Life Science) using Quantitect SYBR Green PCR kit (Qiagen). Primer sequences used here are as follows; dper forward: 5′-GACCGAATCCCTGCTCAATA-3′; dper reverse: 5′-GTGTCATTGGCGGACTTCTT-3′; tim forward: 5′-CCCTTATACCCGAGGTGGAT-3′; tim reverse: 5′-TGATCGAGTTGCAGTGCTTC-3′; dClk forward: 5′-CAGCCGCAATTCAATCAGTA-3′; dClk reverse: 5′-GCAACTGTGAGTGGCTCTGA-3′. We also included primers for the noncycling mRNA coding for CBP20 as previously described, and sequences are as follows; cbp20 forward: 5′-GTCTGATTCGTGTGGACTGG-3′; cbp20 reverse: 5′-CAACAGTTTGCCATAACCCC-3′. Results were analyzed with software associated with Rotor Gene 6000, and relative mRNA levels were quantitated using the 2^−ΔΔCt method.

The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.