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Mol Cell Biol. Jan 2004; 24(2): 584–594.
PMCID: PMC343819

Direct Association between Mouse PERIOD and CKIepsilon Is Critical for a Functioning Circadian Clock

Abstract

The mPER1 and mPER2 proteins have important roles in the circadian clock mechanism, whereas mPER3 is expendable. Here we examine the posttranslational regulation of mPER3 in vivo in mouse liver and compare it to the other mPER proteins to define the salient features required for clock function. Like mPER1 and mPER2, mPER3 is phosphorylated, changes cellular location, and interacts with other clock proteins in a time-dependent manner. Consistent with behavioral data from mPer2/3 and mPer1/3 double-mutant mice, either mPER1 or mPER2 alone can sustain rhythmic posttranslational events. However, mPER3 is unable to sustain molecular rhythmicity in mPer1/2 double-mutant mice. Indeed, mPER3 is always cytoplasmic and is not phosphorylated in the livers of mPer1-deficient mice, suggesting that mPER3 is regulated by mPER1 at a posttranslational level. In vitro studies with chimeric proteins suggest that the inability of mPER3 to support circadian clock function results in part from lack of direct and stable interaction with casein kinase Iepsilon (CKIepsilon). We thus propose that the CKIepsilon-binding domain is critical not only for mPER phosphorylation but also for a functioning circadian clock.

Circadian rhythms have been observed in organisms from cyanobacteria to humans (11). These rhythms are under the direct influence of environmental cues, most notably the day-night cycles and by a genetically determined, endogenous clock (2, 6). In mammals, a master clock located in the suprachiasmatic nuclei of the anterior hypothalamus controls peripheral clocks through neuronal and humoral connections (19).

A major area of interest in circadian biology is to understand the time-keeping mechanism at the molecular level. Transcriptional feedback loops constitute a central feature of most, if not all, circadian clocks (2, 6, 18). In the mouse, two basic-helix-loop-helix-PAS-containing transcription factors, CLOCK and BMAL1, form heterodimers that bind to E-box enhancer sequences and drive the rhythmic transcription of three Period genes (mPer1, mPer2, and mPer3) and two Cryptochrome genes (mCry1 and mCry2) (9, 12). As mPER and mCRY are translated in the cytoplasm, they form mPER-mCRY complexes, which then translocate into the nucleus to inhibit their own transcription by directly interacting with the CLOCK-BMAL1 complex (14, 15, 17, 21). Based on in vitro and in vivo studies, it appears that the CRY proteins play a major role in this inhibition (8, 10, 14, 21).

At the posttranslational level, the mPER1 and mPER2 proteins are temporally phosphorylated in a circadian manner (15). Casein kinase Iepsilon (CKIepsilon) and CKIδ are important kinases for PER and possibly other clock proteins in mammals (4, 7, 15, 16). Phosphorylation of clock proteins may affect their stability, cellular location, and interactions with one another (1, 13, 15, 22, 25, 26).

Recently, mutant mice with targeted disruption of all three mPer genes have been generated and characterized (3, 5, 20, 27, 28). Studies of these mutant mice reveal that mPER1 and mPER2 have critical roles in the mammalian circadian clock, whereas mPER3 is dispensable for a functional clock. The molecular basis for the difference in clock-relevant functions among the mPER proteins is unknown, however.

We therefore sought to determine why mPER3 cannot sustain circadian clock function by comparing its posttranslational regulation with that of mPER1 and mPER2 in wild-type mice and in mice with various mPer mutations. In this way, the functions of each mPER protein could be inferred by correlating biochemical defects with the disruption of specific mPer gene(s). Our results show that mPER1 and mPER2 are redundant for the rhythmic, posttranslational regulation of clock proteins, including the phosphorylation program, the rhythmic assembly of clock protein complexes, and nuclear translocation. In vitro studies with chimeric proteins show that the inability of mPER3 to support circadian clock function results in part from lack of direct and stable interaction with CKIepsilon. The data suggest that the CKIepsilon-binding domain (CKBD) is an essential feature for mPER1 and mPER2 function in the circadian clock mechanism.

MATERIALS AND METHODS

Mouse strains and tissue collection.

The homozygous mPer single-mutant (mPer1ldc-, mPer2ldc-, and mPer3-deficient) and mPer double-mutant (mPer1/2-, mPer1/3-, and mPer2/3-deficient) mice used in the present study were described previously (3, 20). The wild-type mice were of the same SV129 genetic background. BALB/c mice were used in the experiments depicted in Fig. 1B to D, ,2,2, ,4,4, and and5.5. Mice were entrained to a 24-h light-dark cycle (12-h light and 12-h dark) and sacrificed on the first day in constant darkness. Tissues were dissected and frozen on dry ice.

FIG. 1.
Rhythmic posttranslational modification of mPER3. (A) Characterization of antisera against mPER3. Equal amounts of in vitro translated mPER1, mPER2, mPER3, and reticulolycyte lysate were resolved by SDS-PAGE, along with liver extracts from wild-type (wt) ...
FIG. 2.
mPER3 interacts with other clock proteins in a circadian manner. Liver extracts from CT06 and CT18 were immunoprecipitated with antibodies to CLOCK, BMAL1, mPER1, mPER2, mCRY1, and mCRY2. The immune complexes were Western blotted for CLOCK, mPER1, mPER2, ...
FIG. 4.
Circadian oscillation of mPer3 mRNA. mPer1, mPer3, and Bmal1 RNA rhythms in liver were determined by RNase protection assay at 3-h intervals on the first day in constant darkness (DD). Each value was normalized to β-actin and converted to the ...
FIG. 5.
The mPER1-mPER3 complex is more abundant than the mPER2-mPER3 complex. Liver extracts from CT15 and CT18 were subjected to sequential IP reactions. Antibody to mPER2 was first added, and immune complexes were separated from supernatant. The supernatant ...

Recombinant plasmids.

mPER1, mPER2, mPER3, mCRY1, and mCRY2 plasmids were described previously (15). To generate CKIepsilon expression plasmid, the open reading frame of mouse CKIepsilon (AB028736) was cloned into KpnI and XhoI sites of pcDNA3.1/myc-His(A) vector (Invitrogen). For chimeric mPER2 (chPER2) and mPER3 (chPER3) plasmids, the CKBD of mPER2 (amino acids 555 to 754) (1) was replaced with that of mPER3 (amino acids 509 to 710) (29) and vice versa. To replace the endogenous CKBD with the heterologous CKBD, NotI and XhoI were introduced into the N terminus of CKBD and the C terminus of CKBD, respectively. This resulted in the introduction of four exogenous amino acids in the chimeric PER proteins. The chimeric full-length cDNAs were cloned into KpnI and XhoI sites of pcDNA3.1/V5-His(A) vector.

Generation of anti-mPER3, anti-mCRY1, and anti-mCRY2 antibodies.

We used PCR to amplify DNA sequences that encodes amino acids 4 to 227 for mPER3 (GenBank accession no. AF050182), amino acids 486 to 606 for mCRY1 (AB000777), and amino acids 504 to 592 for mCRY2 (AB003433). The PCR product for mPER3 was cloned into BamHI and HindIII sites of PET23b, and those for mCRY1 and mCRY2 were cloned into BamHI and EcoRI sites of PET23b (Novagen). Antibodies were produced as described previously (15). Representative antisera to each clock protein were affinity purified and designated P3-28R and P3-28GP (R, raised in rats; GP, raised in guinea pigs) for mPER3, C1-R and C1-GP for mCRY1, and C2-R and C2-GP for mCRY2. Antibodies to mCRY1 and mCRY2 were tested by using in vitro translated proteins and liver extracts prepared from wild-type and mCRY-deficient mice (24). The anti-mCRY1 and mCRY2 antibodies reacted with a band present in liver extracts from wild-type mice but not in liver extracts from mCRY-deficient mice (data not shown). Similar results were obtained with anti-mCRY antibodies from Alpha Diagnostic International.

RNase protection assay.

RNase protection assays were performed as described previously. The antisense probes for Bmal1 and mPer1 were described previously (15). To make antisense probe for mPer3, we used PCR to amplify nucleotide sequences 462 to 678 of the mPer3 open reading frame (GenBank accession no. AF050182). The PCR product was cloned into EcoRI and HindIII sites of the pCR II-TOPO vector (Invitrogen). The plasmid was linearized with EcoRI and in vitro transcribed by Sp6 RNA polymerase.

In vitro translation.

In vitro translated proteins were produced in the presence of l-[35S]methionine as described previously (15).

Nuclear and cytoplasmic extracts from liver.

Subcellular fractionation of liver extracts was performed as described previously (15). Fractionation was evaluated by examining distribution of RNA polymerase II between cytoplasmic and nuclear fractions as described previously (15). More than 95% of RNA polymerase was found in nuclear fractions in all cases (data not shown).

IP and phosphatase treatment.

For frozen liver tissue, immunoprecipitation (IP) was performed as described previously (15), and the immune complexes were analyzed by Western blotting as described below. The antibodies used for IP were P3-28GP for mPER3, C1-GP for mCRY1, and C2-GP for mCRY2. Other antibodies used for IP were described previously (15). For transfected NIH 3T3 cells, IP was performed with the following modifications. NIH 3T3 cells were washed with phosphate-buffered saline and harvested 24 h posttransfection. The cells were homogenized in an Eppendorf tube with a minihomogenizer (Kontes) at 4°C in 5 volumes of extraction buffer (EB; 20 mM HEPES [pH 7.5], 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.05% Triton X-100, 10 μg of aprotinin/μl, 5 μg of leupeptin/μl, and 1 μg of pepstatin A/μl) and centrifuged at 12,000 × g for 10 min, and the supernatant was transferred to a new tube. Protein concentration was measured by Coomassie protein assay reagent (Pierce), and equal amounts of total protein were used for each experiment. IP was performed as described previously by using the clarified supernatant (15). Anti-hemagglutinin (HA) and V5 antibodies were purchased from Invitrogen. The antibody to immunoprecipitate CKIepsilon was CKIepsilon-GP (15). For in vitro-translated proteins, reticulocyte lysates containing known amounts of target proteins were mixed in different combinations as indicated in figure legends and incubated in the presence of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 10 μg of aprotinin/μl, 5 μg of leupeptin/μl, 1 μg/μl of pepstatin A, and 1 mM dithiothreitol) at room temperature for 30 min. Subsequently, 300 μl of EB was added. The samples were precleared and subjected to IP as described previously (15). For phosphatase treatment of mPER3, liver extract was subjected to IP with anti-mPER3 antibody (P3-28GP) to obtain mPER3-containing immune complexes. The immune complexes were divided into three aliquots. Then, 200 U of lambda protein phosphatase (λPP; New England Biolabs) was added to one aliquot. To a second aliquot, 40 mM vanadate was added before the addition of λPP. No addition was made to the last aliquot. All three aliquots were incubated at room temperature for 20 min and analyzed by Western blotting as described below.

WB.

For frozen tissues, Western blotting (WB) was performed as described previously (15). To visualize mPER3, 6% sodium dodecyl sulfate (SDS)-polyacrylamide gels were used. The primary antibodies used for mPER3, mCRY1, and mCRY2 were P3-28R, C1-R, and C2-R, respectively. Other primary antibodies and a secondary antibody were as described previously (15). For transfected cells, cells were homogenized as described above, and equal amounts (~5 μg) of total protein were analyzed by Western blotting as described previously (15).

Cell culture and transfection.

NIH 3T3 cells were grown and transfected as described previously (14). Then, 6-cm dishes were used for straight WB, and 10-cm dishes were used for IP experiments.

In vitro kinase assay.

mPERs, chPERs, and CΚIepsilon were synthesized in vitro as described above. Each of the native and chimeric PERs was mixed with CKIepsilon or the same volume of unprogrammed reticulocyte lysate. The molar ratio of PER to CKIepsilon was 1:3. These mixtures were incubated in the presence of protease inhibitors (see above) on ice for 30 min. Subsequently, 100 μl of EB and 0.1 mM ATP were added, and these mixtures were further incubated at room temperature with gentle shaking. The reactions were terminated at 60 min after the addition of ATP for experiments depicted in Fig. Fig.8E,8E, upper panel. For results shown in Fig. Fig.6E,6E, lower panel, small aliquots from the mixtures were taken and mixed with 2× sample buffer at the indicated times after the addition of ATP. These samples were resolved by SDS-polyacrylamide gel electrophoresis (PAGE; 6% gels) and visualized by autoradiography.

FIG. 6.
mPER3 cannot participate in rhythmic assembly of clock protein complexes. (A) mPER complexes in wild-type (wt) and mPer double-mutant mice. Liver extracts from wild-type and mPer double-mutant mice collected at CT06 and CT18 were immunoprecipitated with ...
FIG. 8.
CKBD of mPER is important for interaction with CKIepsilon and efficient phosphorylation. (A) Diagram of CKBD swapping between mPER2 and mPER3. Amino acids 510 to 709 of mPER3 were exchanged for amino acids 551 to 750 of mPER2. (B) The apparent sizes ...

RESULTS

Rhythmic phosphorylation and nuclear accumulation of mPER3.

We first examined the posttranslational regulation of mPER3 in the livers of wild-type mice. We characterized mPER3 protein in vivo by using novel antisera raised against a fragment of mPER3. One antisera (P3-28R) recognized in vitro-translated mPER3 by WB analysis but did not cross-react with equivalent amounts of mPER1 or mPER2 (Fig. (Fig.1A).1A). Furthermore, the antibody reacted with a band whose size ranges between 135 to 150 kDa, which is present only in liver extracts from wild-type mice, but not mPer3-deficient mice.

We next examined the expression profile of mPER3 at 3-h intervals on the first day in constant darkness (DD). Like mPER1 and mPER2, mPER3 exhibited a circadian rhythm in abundance and electrophoretic mobility (Fig. (Fig.1B)1B) (15). Rapidly migrating forms start to accumulate at circadian time 06 (CT06). Both abundance and size increase with time, peaking between CT12 and CT15. The mRNA peak of mPER3 in liver occurs at CT09, indicating a delay between mRNA peak and protein peak, as observed for the other mPERs (Fig. (Fig.4)4) (15). The electrophoretic mobility shift was due mainly to phosphorylation, as treatment with λPP altered the mobility of the slowly migrating forms to more rapidly migrating forms (Fig. (Fig.1C1C).

We showed previously that mPER1 and mPER2 translocate into the nucleus in a time-dependent manner, and hyperphosphorylated forms of mPER1 and mPER2 are detected predominantly in the nucleus (15). mPER3 also showed a striking circadian rhythm in the nuclear accumulation in liver, with hyperphosphorylated mPER3 found primarily in the nucleus (Fig. (Fig.1D1D).

mPER3 associates with other clock proteins in a time-dependent manner.

Our previous studies showed that seven clock proteins (CLOCK, BMAL1, mPER1, mPER2, mCRY1, mCRY2, and CKIepsilon) associate as a multimeric complex in a time-dependent manner in liver (15). To test whether mPER3 is also part of this complex, we used co-IP to examine interactions of mPER3 with other clock proteins before (CT06) and during (CT18) negative feedback. Hyperphosphorylated mPER3 was copurified along with each of the seven clock proteins examined at CT18 (Fig. (Fig.22 and data not shown).

mPER1 regulates the rhythmic posttranslational modifications of mPER3.

The robust oscillations in mPER1 and mPER2 abundance and phosphorylation allow these rhythms to be used to assess the functional state of the circadian clock. Consistent with the loss of locomotor rhythmicity in mPer1/2 double-mutant mice, the double mutants lacked rhythmic posttranslational modification of mPER3 (Fig. (Fig.3A).3A). In mPer1/3 and mPer2/3 double-mutant mice, in which molecular and behavioral rhythmicity persist for several days in DD (3), the remaining PER gene product (mPER2 and mPER1, respectively) underwent rhythmic changes in abundance and phosphorylation, as in wild-type mice, except the rhythm phases appeared advanced compared to those in wild-type mice (Fig. (Fig.3A).3A). Thus, either mPER1 or mPER2 is sufficient for its own circadian changes in phosphorylation and abundance, whereas mPER3 is not.

FIG.3.
mPER3 is posttranslationally regulated by mPER1. (A) mPER profile in mPer double-mutant mice. Liver extracts from wild-type (wt), mPer1/2, mPer1/3, and mPer2/3 double-mutant mice collected at indicated times were subjected to Western blot for mPER1, mPER2, ...

The lack of rhythmic posttranslational modifications of mPER3 in mPer1/mPer2 double-mutant mice could be secondary to a broken circadian clock; that is, the posttranslational modification of mPER3 could simply be dependent upon a functional circadian clock. Alternatively, there may be a specific dependence of mPER3 on one of the other two mPER proteins for its posttranslational regulation. To dissect these possibilities, we examined the posttranslational regulation of mPER proteins in homozygous single-mutant mice. In mPer2ldc or mPer3-deficient mice, mPER1 exhibited robust oscillations in phosphorylation and abundance, as in wild-type mice (Fig. (Fig.3B).3B). Similarly, circadian rhythms of mPER2 phosphorylation and abundance were not significantly altered in mPer1ldc or mPer3-deficient mice (Fig. (Fig.3B).3B). mPER3 rhythms also appeared normal in mPer2ldc mice (Fig. (Fig.3B3B).

Remarkably, however, mPER3 did not show temporal changes in phosphorylation and abundance in mPer1ldc mutant mice (Fig. (Fig.3B).3B). Instead, mPER3 abundance was constant, and the protein remained hypophosphorylated across the circadian cycle in the absence of mPER1. The loss of rhythmicity in mPER3 was not due to altered transcriptional regulation of mPer3 because mPer3 mRNA levels manifested robust oscillations in mPer1ldc and mPer2ldc mutant mice, as in wild-type mice (Fig. (Fig.3C).3C). Examination of the subcellular localization of mPER3 revealed that the protein was trapped in the cytoplasm in the absence of mPER1 (Fig. (Fig.3D).3D). Immunodepletion experiments of wild-type livers confirmed that the majority of hyperphosphorylated mPER3 is complexed with mPER1 (Fig. (Fig.55).

mPER3 cannot participate in the rhythmic assembly of clock protein complexes.

We next examined the ability of mPER3 to direct the rhythmic assembly of non-PER clock proteins by using the mPer double-mutant mice. As previously indicated (Fig. (Fig.2),2), mPER proteins associated with mCRY1, mCRY2, and CKIepsilon more at CT18 than at CT06 in wild-type mice (Fig. (Fig.6A,6A, lanes 1 to 6). The same interaction pattern was maintained in mPer1/3 and mPer2/3 double-mutant mice; associations between the single remaining mPER member and mCRY1, mCRY2, and CKIepsilon were most apparent at CT18 (Fig.6A, lanes 9 to 12). However, the interactions between mPER3 and mCRY1, mCRY2, and kinase in mPer1/2 double-mutant mice were greatly reduced and did not appear to be rhythmic (Fig. (Fig.6A,6A, lanes 7 and 8), suggesting that mPER3 association with these clock proteins requires mPER1 and/or mPER2. There appeared to be a decrease in mPER2 abundance in the absence of mPER1 in some experiments (Fig. (Fig.3D3D and and6A),6A), but this was not a consistent finding (Fig. (Fig.3A3A and B).

We also examined whether the remaining mPER protein in the double-mutant mice can induce rhythmic interactions between the major transcriptional inhibitors (mCRY1 and mCRY2) and the transcriptional activators (CLOCK-BMAL1). As expected, the interactions between CLOCK and the mCRY proteins were much greater at CT18 than at CT06 in wild-type mice (Fig. (Fig.6B).6B). The same rhythmic interactions were maintained in mPer1/3 and mPer 2/3 double-mutant mice, but these interactions were not detectable at either time in the mPer1/2 double-mutant mice (Fig. (Fig.6B).6B). The absence of these interactions is not due to low levels of the mCRY proteins in the mutants, since the mCRYs were readily detectable in the mPer1/2 double-mutant mice and in the other mutant and wild-type mice (Fig. (Fig.6B,6B, right panels).

Taken together, our data indicate that either mPER1 or mPER2 alone is sufficient for rhythmic posttranslational regulation of clock proteins and maintaining a functioning clock for a few circadian cycles. In contrast, mPER3 is not sufficient to support rhythmic molecular oscillations for a functioning circadian clock, implying that mPER3 is mechanistically different from mPER1 and mPER2 in clock-relevant functions.

In vitro binding assays reveal a lack of stable interaction between mPER3 and CKIepsilon.

Our previous studies suggested that the interactions between mPER proteins and other clock proteins in the cytoplasm trigger nuclear translocation of mPER-containing complexes and subsequent inhibition of CLOCK-BMAL1 transcription (15). Therefore, it is possible that the inability of mPER3 to sustain the circadian clock results from the lack of interactions with CKIepsilon and/or the mCRY proteins in the cytoplasm. We thus examined affinities of each mPER protein for CKIepsilon and the mCRY proteins.

First, the physical interaction between each mPER protein and CKIepsilon were examined in transiently transfected cells. When the kinase and each mPER protein were coexpressed in NIH 3T3 cells, CKIepsilon was able to coprecipitate with mPER1 and mPER2, but not mPER3, a finding consistent with studies reported by Akashi et al. (Fig. (Fig.7A).7A). CKIepsilon-induced phosphorylation of mPER3 was enhanced when mPER1, but not mPER2, was coexpressed in NIH 3T3 cells (Fig. (Fig.7B,7B, lane 5 versus lane 6). In agreement with our in vivo studies (Fig. (Fig.3),3), the data suggest that mPER3 requires mPER1 for stable interaction with CKIepsilon and efficient phosphorylation.

FIG.7.
Quantitative difference of mPER protein interactions with CKIepsilon and mCRY1. (A) mPER3 does not stably associate with CKIepsilon in NIH 3T3 cells. NIH 3T3 cells were transfected with CKIepsilon-Myc and mPER1-V5, mPER2-V5, or mPER3-V5. The cell ...

To determine the affinities of the mPER proteins for CKIepsilon quantitatively, we performed binding assays with in vitro translated proteins. Each of the mPER proteins and kinase were mixed at various molar ratios and then subjected to IP with an anti-CKIepsilon antibody. Subsequently, the affinity of the mPER for CKIepsilon was measured by quantifying the amount of copurified mPER.

The amounts of mPER2 bound to CKIepsilon were about twofold higher than those of mPER1 (Fig. (Fig.7C).7C). Consistent with the results in Fig. Fig.7A,7A, we could not detect stable interaction between mPER3 and CKIepsilon despite numerous attempts (Fig. (Fig.7C).7C). These data demonstrate that mPER3 is intrinsically defective in its ability to bind to CKIepsilon.

A set of similar experiments was conducted to examine interactions between the mPER and mCRY proteins. As previously reported (14), all three mPER proteins associated with mCRY1 in transiently transfected NIH 3T3 cells (Fig. (Fig.7D).7D). The relative binding affinity of mPER2 for mCRY1 appeared to be greater than that of mPER1 or mPER3. Similar results were obtained when mCRY2 was cotransfected with each of the mPER proteins (data not shown). The affinities of the mPER proteins for mCRY1 were quantitatively determined with in vitro binding assays (Fig. (Fig.7E).7E). The rank order of mPER affinity for mCRY1 was mPER2 > mPER1 > mPER3. At all molar ratios, the amounts of mPER3 bound to mCRY1 were two- to threefold less than those of mPER2.

Although the affinity of mPER3 for mCRY1 is lower than that of mPER1 or mPER2, the interactions between mPER3 and the mCRY proteins are readily observed in cultured cells (Fig. (Fig.7D)7D) (14). Thus, mPER3 is not intrinsically defective in binding to the mCRY proteins. Furthermore, mPER3 is more abundant than mPER1 in liver extracts, since immunodepletion of mPER1 removed less than 50% of mPER3 from liver extracts (Fig. (Fig.55 and data not shown). Higher levels of expression would mitigate the impact of a slightly reduced affinity. We therefore conclude that the inability of mPER3 to maintain molecular or behavioral rhythms is due primarily to a deficiency in interaction with CKIepsilon. The reduced affinity of mPER3 for the mCRY proteins may also contribute, but to a lesser degree.

The CKBD of mPER is essential for kinase interaction and phosphorylation.

If the direct association of mPER with CKIepsilon is essential for clock-relevant functions of mPER, exchanging the CKBDs between mPER2 and mPER3 should cause predictable alterations in chimeric protein function. We thus generated the chimeric chPER2 and chPER3 proteins containing each other's CKBD (Fig. (Fig.8A).8A). The apparent sizes of chPER2 and chPER3 were similar to their wild-type counterparts (Fig. (Fig.8B8B).

To determine whether the CKBD alters the functions of the hybrid proteins, we performed in vitro binding assays between the chimeric proteins and CKIepsilon. As expected, the amount of chPER3 bound to CKIepsilon greatly increased compared to that of native mPER3 (Fig. (Fig.8C).8C). In contrast, the binding of chPER2 to CKIepsilon was barely detectable. When the interactions were examined in transiently transfected NIH 3T3 cells, mPER2 and chPER3 were copurified with CKIepsilon, whereas mPER3 and chPER2 were not (Fig. (Fig.8D).8D). These data indicate that the CKBD determines the binding affinity of mPER proteins for CKIepsilon. Even although mPER3 and chPER2 did not appreciably bind CKIepsilon in cell culture, the proteins were highly phosphorylated, suggesting endogenous kinase activity not involving CKIepsilon.

Next, we examined the efficiency of chPER phosphorylation by CKIepsilon. Wild-type and chimeric proteins and CKIepsilon were synthesized in vitro, and each of the PER proteins was mixed with CKIepsilon to compare phosphorylation efficiency of the chimeric and native mPERs by CKIepsilon in the absence of other interacting proteins. mPER2 and chPER3 were more readily phosphorylated by CKIepsilon than were their counterparts, chPER2 and mPER3, respectively (Fig. (Fig.8E8E).

Taken together, these data suggest that the CKBD of mPERs not only determines kinase binding affinity but also phosphorylation efficiency. Since it has been suggested that mPER phosphorylation plays important roles in regulating interactions with other clock proteins, nuclear translocation, and degradation, our data suggest that the mPER CKBD is essential for clock-relevant functions of mPER1 and mPER2.

DISCUSSION

As in other organisms, a negative feedback loop constitutes the backbone of the molecular circadian clock in mammals (18). The mPER-mCRY complex plays an essential role in the negative feedback loop by exerting inhibitory activity on CLOCK-BMAL1-driven transcription in a time-dependent manner (14, 15). Although mCRYs play a major role in the inhibition, the timing of this inhibition seems to be regulated by mPERs (15). The present study suggests that the CKBD of an mPER protein dictates its clock-relevant functions.

Although mPer3 has been placed outside of the core feedback loop based on previous genetic studies (3, 20), we show here that mPER3 is in fact part of the transcriptional inhibitory complex and is regulated in a manner similar to the regulation of mPER1 and mPER2. Like mPER1 and mPER2, mPER3 is rhythmically phosphorylated and translocated into the nucleus, and it interacts rhythmically with other clock proteins. Among clock proteins, mPER3 is most strongly associated with mPER1. In addition, in the absence of mPER1 (mPer1ldc and mPer1/2 double-mutant mice) mPER3 phosphorylation is abolished. Thus, mPER3 is regulated by mPER1 at a posttranslational level. Consistent with these observations, phosphorylation of mPER3 by CKIepsilon is enhanced in the presence of mPER1 in NIH 3T3 cells (Fig. (Fig.7B),7B), and the nuclear translocation of mPER3 is promoted by mPER1 in NIH 3T3 cells (14). The presence of mPER3 in circadian clock protein complexes is likely mediated by interaction with mPER1.

mPER1 and mPER2 in mPer2/3 and mPer1/3 double-mutant mice, respectively, show apparently normal phosphorylation, interaction with other clock proteins, and nuclear translocation, suggesting that the single remaining mPER can support the molecular clock. The levels of the remaining mPER protein in the mutant mice were comparable to those of the mPER proteins in wild-type mice, suggesting that circadian changes of mPER abundance in liver were not significantly altered by disruption of mPer1/3 or mPer2/3 genes.

Unlike mPER1 or mPER2, mPER3 alone cannot support the molecular clock even for one cycle. mPER3 in mPer1/2 double mutant mice is apparently not phosphorylated and interacts very weakly with other clock proteins. In addition, mPER3 in the double-mutant mice is always cytoplasmic. Our data suggest that the lack of physical association of mPER3 with CKIepsilon might explain the functional inadequacy of mPER3 in terms of supporting the circadian clock. In fact, CKIepsilon has been implicated in the nuclear translocation of the mPER proteins (1, 22).

Our results suggest that stable interactions between CKIepsilon and mPERs are important for effective phosphorylation and likely lie upstream of nuclear translocation of mPERs. If mPER3 alone cannot support the circadian clock due to the lack of stable interaction with CKIepsilon, then replacement of CKBD of mPER3 with that of mPER2 should rescue the inability of mPER3 to support the clock. Our study indeed shows that chPER3 physically interacts with CKIepsilon and is efficiently phosphorylated in vitro. Taken together, our data strongly suggest that the CKBD of mPERs is essential for stable interaction with CKIepsilon, effective phosphorylation by CKIepsilon, and possibly other clock-relevant functions, such as nuclear translocation. The importance of the CKBD for a functioning circadian clock is supported by a human genetic study showing that a mutation in the CKBD of hPER2 is associated with a severe sleeping disorder known as familial advanced sleep phase syndrome (23).

Acknowledgments

We thank Jason DeBruyne for helpful comments during the preparation of the manuscript.

This study was supported by NIH grant NS39303.

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