The first circadian clock protein to be characterized biochemically is dPER from Drosophila melanogaster
). It was shown to undergo progressive changes in phosphorylation over a significant portion of the day, from hypo- to hyperphosphorylated species. Daily changes in abundance are temporally linked to the phosphorylated state of dPER, whereby the appearance of hyperphosphorylated species precedes rapid decreases in overall levels. More-recent work established that DBT is a major kinase controlling the phosphorylation and stability of dPER (35
) and that hyperphosphorylated variants of dPER are targeted to the 26S proteasome/ubiquitin pathway by the F-box protein SLIMB (26
). A highly shared mechanism operates in the mammalian system for the mPERs, whereby the mammalian homologs of DBT (CK1
and the δ variant) and SLIMB (β-TrCP1 and possibly β-TrCP2) play major roles in the temporal regulation of mPERs' phosphorylation and stability (37
). Herein, we identify the major or sole DBT interaction domain on dPER (termed dPDBD) that is required in vivo for dPER hyperphosphorylation, temporal instability, and repressor function. Similar results were also obtained by others using a slightly smaller internal deletion (51a
). Moreover, our findings suggest that the dPDBD plays distinct roles in dPER degradation and repressor function and that dPER has a mode of action in transcriptional inhibition that is more indirect than previously thought (Fig. ).
FIG. 8. Model for the multiple roles of DBT in regulating the levels and activities of dPER and dCLK. (A) Role for DBT in the hyperphosphorylation and degradation of dPER. DBT stably associates with dPER via the dPDBD (dark green stripe within the dPER oval), (more ...)
Earlier reports suggested that the amino-terminal half of dPER and possibly just the first 385 aa are sufficient to stably bind DBT (35
). However, we show that in flies the absence of the dPDBD abolishes any detectable interaction between dPER and DBT (Fig. ). Based on the effects of DBT binding to dPER(Δ) in S2 cells, which are less dramatic than those seen for flies (compare Fig. and ), it is possible that the use of high-level expression systems (35
) or a highly stable dPER-β-Gal fusion (36
) in the earlier studies exaggerated weak interactions. Thus, while it is likely that there are other DBT binding regions on dPER (especially those that might be involved only in transient interactions), our findings indicate that in vivo the DBT-dependent hyperphosphorylation and degradation of dPER have an absolute requirement for the dPDBD, highlighting the tight association between hyperphosphorylation and enhanced degradation. These data are well accommodated by prior work showing that hyperphosphorylated isoforms of dPER are preferentially targeted by SLIMB (26
). Presumably, the ability of DBT to promote the hyperphosphorylation of dPER requires a stable and perhaps long-term association with the substrate, consistent with the observation that DBT is bound to dPER during a significant portion of the daily cycle (36
). The tight interaction between dPER and DBT is also highlighted by the redistribution of predominately nucleus-localized DBT to the cytoplasm in pacemaker cells only during times in a daily cycle when dPER is present in the cytoplasm (36
). Although the ability of CK1 to phosphorylate dPER in an in vitro reconstituted assay (Fig. and data not shown) suggests that DBT and dPER directly interact, our data do not rule out the possibility that the stable interaction between DBT and dPER observed in vivo is stimulated or dependent on other factors that either directly or indirectly interact with the dPDBD.
Likewise, in the mammalian clockworks a region beginning around the middle of mPER1 and mPER2 and extending about 200 aa mediates their stable association with CK1
). Here too, removal of major CK1
binding domains (CKBD) on mPER1 or -2 attenuates their CK1
-driven instability and hyperphosphorylation (1
), although the physiological relevance of these regions has not been tested in animals. In contrast to mPER1 and -2, mPER3 displays no or weak interactions with CK1
). Rather, it is thought that the association of mPER3 with mPER1 enables mPER1 to act as a bridge and bring CK1
into a favorable proximity to phosphorylate mPER3 (43
). There is only limited knowledge of the structural features underlying CK1
docking regions, with arguably the best characterized being the F-X-X-X-F motif present in the mammalian NFAT1 transcription factor (52
). mPER1 and -2 but not -3 have this signature motif in their CKBD regions, and alteration of this motif greatly attenuates binding to CK1
). Intriguingly, when we did a BLAST analysis of our 57-aa region encompassing the dPDBD against the mPER1 and mPER2 protein sequences, it aligned within a subset of their CKBD that included the F-X-X-X-F motifs, but the alignment with mPER3 was noticeably dispersed (Fig. ). While this finding is highly suggestive, the F-X-X-X-F motif is not found in the dPDBD, and future work will be required to more precisely define the key structural features.
FIG. 9. Alignment of aa 755 to 809 from dPER against mPER1, mPER2, and mPER3. Pairwise alignment of dPER aa 755 to 809 against mPER1 and mPER2 (A) or mPER3 (B) was done using ClustalW software. Asterisks indicate amino acid identity, colons indicate that conserved (more ...)
Although the dPDBD promotes DBT-dependent hyperphosphorylation of dPER, it is unlikely to be a major site directly modified by phosphorylation. When the majority of Ser and Thr residues in this region (those conserved with dPER from other Drosophila
species) are converted to Ala residues, the modified dPER undergoes progressive phosphorylation and degradation kinetics indistinguishable from those of the wild-type control version (Fig. ). This also seems to be the case for mammalian PERs. For example, a more detailed analysis showed that deletion of either of two small regions on mPER2 (aa 582 to 606 or aa 731 to 756) greatly reduced the ability of CK1
to stably interact and promote degradation of mPER2 (22
). However, recent work mapping phosphorylation sites on mPER2 did not identify either aa 582 to 606 or aa 731 to 756 as areas modified by phosphorylation (66
Thus, although there might be structural features that differ between the DBT/CK1
docking sites on mammalian and fly PERs, there are remarkable similarities. Most notable is the presence of one or a few small regions that are not major targets for phosphorylation but serve to stably bind DBT/CK1
. Once stably bound, DBT/CK1
either directly or indirectly (e.g., via other kinases) stimulates progressive phosphorylation at other sites until a threshold of multiphosphorylation is attained that enhances degradation by SLIMB/β-TrCP-mediated pathways (Fig. ). Obviously, time-of-day-dependent variations in phosphorylation that regulate the levels of key transcriptional repressors constitute one mechanism that can control the timing and relative potency of repressor function, giving rise to cyclical gene expression.
In addition to regulating stability, DBT has been implicated in modulating the subcellular localization of dPER (e.g., references 7
, and 54
), a function also ascribed to CK1
in the case of mPERs (e.g., reference 67
). We did not observe differences in the subcellular distributions between dPER(Δ) and wild-type dPER expressed in S2 cells, and whole-mount staining of adult Drosophila
heads showed strong staining of dPER(Δ) in nuclei of dPER-expressing cells (data not shown), consistent with the exclusively nuclear staining of dPER in key pacemaker cells of mutants with abolished or severely reduced dbt
). These results are in agreement with our biochemical studies showing that similar amounts of dCLK, which is the limiting component (5
) and exclusively localized in the nucleus of both S2 cells (34
) and fly clock cells (32
), copurifies with dPER(Δ) and wild-type dPER (Fig. and ). Nonetheless, recent work by Nawathean and colleagues using an internal deletion smaller than ours suggests that the DBT binding domain enhances the nuclear localization of dPER (51a
). The reason for this apparent discrepancy is not clear, but we note that by far the majority of recombinant dPER is found in the cytoplasm when expressed in S2 cells (e.g., references 10
, and 51
). It is possible that in our S2 cell system the overall proportion of cytoplasmic dPER is higher, making it difficult to detect subtle differences in nuclear staining. Nonetheless, similar to the results obtained by Nawathean et al., when we placed a nuclear localization signal (NLS) on dPER and dPER(Δ), both proteins were highly localized in the nucleus with increased repressor function; however, dPER(Δ) was still approximately twofold less efficient in blocking dCLK-dependent transcription (data not shown). Although we cannot exclude possible effects on subcellular distribution, our results indicate that the strongly attenuated repressor function of dPER(Δ) is not explained simply by a lack of interaction with dCLK.
Why is the binding of dPER(Δ) to dCLK not sufficient for robust inhibition of transactivation activity? A possible answer is based on prior work suggesting that hyperphosphorylated dPER might be a more potent repressor of dCLK-CYC transcriptional activity (51
), rendering the mainly hypophosphorylated dPER(Δ) deficient in this capacity. Another non-mutually exclusive possibility is based on our recent findings indicating that dPER is required for the DBT-dependent hyperphosphorylation of dCLK during the night/early day, events that promote the rapid degradation of dCLK and possibly reduce its transactivation activity (34
). Presumably, dPER can act as a bridge to enhance the phosphorylation of dCLK by DBT (73
) (Fig. ). Indeed, in per0
flies DBT is located largely in the nucleus of pacemaker neurons (36
), yet hyperphosphorylation of dCLK is not observed (34
) (Fig. ). Our findings greatly strengthen this proposal by showing that dPER(Δ), which can bind dCLK but is impaired in its association with DBT, does not support the hyperphosphorylation of dCLK (Fig. ). A scenario where PER acts as a molecular bridge by which DBT can phosphorylate other clock proteins is similar to that proposed for mPER1 in stimulating CK1
phosphorylation of mPER3 (43
) and mCRYs (21
), suggesting that PERs might have a general role in targeting DBT/CK1
to numerous clock components. Even more impressive, a similar mechanism also occurs in the Neurospora
clock, whereby the binding of CK1a (a CK1 homolog) to FREQUENCY is critical for inhibiting the transcriptional activity of the WHITE COLLAR COMPLEX (31
). Thus, there is remarkable conservation of posttranslational regulatory pathways operating in widely divergent clocks.
Despite the reasonable possibilities that the dPDBD modulates dPER repressor function by regulating dPER phosphorylation and/or serving as a conduit to facilitate DBT-dependent phosphorylation of dCLK, there is circumstantial evidence from S2 cells and flies suggesting that neither scenario is obligatory for dPER to exhibit repressor capabilities. For example, in our S2 cell culture system, dPER(Δ) displays little repressor function compared to wild-type dPER even in the absence of exogenous DBT (Fig. ), conditions under which only the hypophosphorylated isoforms of dPER(Δ) and wild-type dPER are observed (Fig. ) and in which more dCLK stably interacts with dPER(Δ) (Fig. ). Furthermore, in mutant flies with highly impaired DBT activities/levels, evidence suggests that dPER can still inhibit dCLK-CYC-driven transcription (14
), though the relative strength of this repression is not known. These results raise the possibility that the dPDBD acts as a docking site for other factors besides DBT that play a more direct role in inhibiting dCLK-CYC-dependent transactivation, e.g., activities involved in chromatin remodeling (8
) (Fig. ). Indeed, the DBT-mediated phosphorylation of dCLK might be involved only in regulating dCLK stability whereas repression of its activity is carried out by other factors. However, it is important to note that in the aforementioned examples based on results obtained with cultured S2 cells or dbt
-impaired mutants, the elevated levels of dPER might override stimulatory effects of phosphorylation and/or DBT on the ability of dPER to function as a transcriptional inhibitor. This reasoning might also explain why recombinant dPER was able to block dCLK-CYC binding to E-box containing DNA elements in an in vitro assay (41
). Taken together, we speculate that dPER “alone” has low intrinsic repressor capabilities that are enhanced by the dPDBD-dependent assembly of DBT and/or other factors that more directly modulate the ability of dCLK-CYC to stimulate transcription.
Although future work will be required to determine if phosphorylation regulates the transactivation potential of dCLK, our findings highlight a clear qualitative difference in the roles of the dPDBD with regards to dPER instability and repressor function. Namely, whereas differences in the SLIMB-mediated degradation of wild-type dPER and dPER(Δ) are observed only under conditions of DBT-dependent hyperphosphorylation (Fig. ), this is not the case for transcriptional inhibition (Fig. ).
A more secondary mode of action for dPER as a transcriptional repressor is reminiscent of how mPERs are thought to function in the negative limb of the mammalian clockworks, where the mCRYs are the major inhibitors. Although mPERs exhibit some repressor capacity in cultured cell assays and they are found in complexes with mammalian CLK-BMAL1, it is likely that their main function in transcriptional regulation is to control the timing of mCRYs nuclear localization (42
). This theme of roles as indirect repressors of circadian gene expression is also observed for FREQUENCY in Neurospora
) and KaiC in cyanobacteria (64
). A strikingly common feature of these indirect repressors is that they undergo complex daily changes in phosphorylation that are central to clock progression. Indeed, a daily rhythm in the cyanobacterial clock protein KaiC can be recapitulated in vitro using ATP and recombinant versions of the three critical clock proteins (KaiA, KaiB, and KaiC) from this system (50
). While it is likely that the “minimal” circadian phosphorylation programs in eukaryotes are more complex, the bacterial work elegantly suggests that the most basic and ancient building block of circadian clocks is a biochemical oscillator based on time-of-day-specific phosphorylation of one or more key clock proteins. This central biochemical oscillator can be viewed as a semiautonomous subsystem from which temporal coordinates are largely transduced by phase-specific interactions with a variety of regulatory factors that subsequently control the activities of major transcription factors, leading to rhythmic gene expression and ultimately to daily changes in physiology and behavior.