2004, 24(2):584. DOI:
Mol. Cell. Biol.
Choogon Lee, David R. Weaver and Steven M. Reppert
Is Critical for a Functioning
Direct Association between Mouse PERIOD
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MOLECULAR AND CELLULAR BIOLOGY, Jan. 2004, p. 584–594
0270-7306/04/$08.00?0 DOI: 10.1128/MCB.24.2.584–594.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 2
Direct Association between Mouse PERIOD and CKIε Is Critical for a
Functioning Circadian Clock
Choogon Lee,† David R. Weaver, and Steven M. Reppert*
Department of Neurobiology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Received 4 September 2003/Returned for modification 19 September 2003/Accepted 7 October 2003
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 I? (CKI?).
We thus propose that the CKI?-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 suprachias-
matic 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. Transcrip-
tional 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 trans-
lated 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 pro-
teins are temporally phosphorylated in a circadian manner
(15). Casein kinase Iε (CKIε) and CKI? 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 sus-
tain 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 corre-
lating biochemical defects with the disruption of specific mPer
gene(s). Our results show that mPER1 and mPER2 are redun-
dant for the rhythmic, posttranslational regulation of clock
proteins, including the phosphorylation program, the rhythmic
assembly of clock protein complexes, and nuclear transloca-
tion. 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 CKIε.
The data suggest that the CKIε-binding domain (CKBD) is an
essential feature for mPER1 and mPER2 function in the cir-
cadian 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 back-
ground. BALB/c mice were used in the experiments depicted in Fig. 1B to D, 2,
4, and 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.
Recombinant plasmids. mPER1, mPER2, mPER3, mCRY1, and mCRY2
plasmids were described previously (15). To generate CKIε expression plasmid,
the open reading frame of mouse CKIε (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)
* Corresponding author. Mailing address: Department of Neurobi-
ology, LRB-728, University of Massachusetts Medical School, 364
Plantation St., Worcester, MA 01605. Phone: (508) 856-6148. Fax:
(508) 856-6266. E-mail: Steven.Reppert@umassmed.edu.
† Present address: Department of Biological Sciences, College of
Medicine, Florida State University, Tallahassee, FL 32306.
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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 desig-
nated 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 nu-
clear 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 ?g of aprotinin/?l, 5 ?g of leupeptin/?l, and 1
?g of pepstatin A/?l) and centrifuged at 12,000 ? g for 10 min, and the super-
natant 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 CKIε was
CKIε-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 ?g of aprotinin/?l, 5 ?g
of leupeptin/?l, 1 ?g/?l of pepstatin A, and 1 mM dithiothreitol) at room
temperature for 30 min. Subsequently, 300 ?l of EB was added. The samples
were precleared and subjected to IP as described previously (15). For phospha-
tase treatment of mPER3, liver extract was subjected to IP with anti-mPER3
antibody (P3-28GP) to obtain mPER3-containing immune complexes. The im-
mune complexes were divided into three aliquots. Then, 200 U of lambda protein
phosphatase (?PP; New England Biolabs) was added to one aliquot. To a second
aliquot, 40 mM vanadate was added before the addition of ?PP. 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)-poly-
acrylamide gels were used. The primary antibodies used for mPER3, mCRY1,
and mCRY2 were P3-28R, C1-R, and C2-R, respectively. Other primary anti-
bodies and a secondary antibody were as described previously (15). For trans-
fected cells, cells were homogenized as described above, and equal amounts (?5
?g) of total protein were analyzed by Western blotting as described previously
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) and
mPer3-deficient mice. The proteins were transferred onto nitrocellulose membrane and Western blotted. Representative Western blots with one
of our strongest antisera (P3-28R) are shown. The antibody only recognized in vitro-translated mPER3 and not equal amounts of mPER1 or
mPER2. Anti-mPER1 (PER1-1-R) and -mPER2 (PER2-1-R) antibodies only recognized in vitro-translated mPER1 and mPER2, respectively
(data not shown). Similar sized bands were detected only in liver extracts from wild-type mice and not mPer3-deficient mice. (B) Circadian rhythms
of mPER3. Liver extracts prepared from mice collected at 3-h intervals were Western blotted with P3-28R and anti-?-actin antibodies. Even
loading of total protein was assured by Western blot with anti-?-actin antibody (data not shown). (C) Electrophoretic mobility shift of mPER3 is
due to phosphorylation. Liver extract from CT15 was subjected to IP with anti-mPER3 antibody (P3-28GP). The immune complexes were split into
three aliquots and incubated in the presence (?) or absence (?) of ?PP or with both ?PP and 40 mM vanadate (??). (D) mPER3 exhibits nuclear
accumulation in a circadian manner. Mouse liver extracts were collected at the indicated times, fractionated into cytoplasmic and nuclear portions,
and blotted for mPER3. Both cytoplasmic and nuclear fractions for each time point were derived from the same number of cells.
VOL. 24, 2004 DIFFERENTIAL ROLES OF mPER PROTEINS585
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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 C?Iε were synthesized in vitro as
described above. Each of the native and chimeric PERs was mixed with CKIε or
the same volume of unprogrammed reticulocyte lysate. The molar ratio of PER
to CKIε was 1:3. These mixtures were incubated in the presence of protease
inhibitors (see above) on ice for 30 min. Subsequently, 100 ?l of EB and 0.1 mM
ATP were added, and these mixtures were further incubated at room tempera-
ture with gentle shaking. The reactions were terminated at 60 min after the
addition of ATP for experiments depicted in Fig. 8E, upper panel. For results
shown in Fig. 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.
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. 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
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, mPER3, mCRY1, and mCRY2. Underlined lanes indicate the primary target proteins of IP.
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, and mPER3.
The black arrow indicates a nonspecific band. The results from one of two independent experiments are shown. Similar results were obtained from
the other experiment (data not shown). (B) mPER profile in wild-type (wt) and mPer single-mutant mice. Liver extracts prepared from wild-type
and mPer1ldc, mPer2ldc, and mPer3-deficient mice collected at the indicated times were subjected to Western blotting for mPER1, mPER2, and
mPER3. (C) mPer3 RNA levels in mPer mutant mice. RNA levels were analyzed at 6-h intervals. One of two independent experiments is shown.
Duplicate values varied by no more than 20% from each other. (D) mPER3 is mostly cytoplasmic in mPer1ldcmice. Liver extracts from mPer1ldc
and mPer2ldcmice collected at CT14 and CT18 were fractionated and analyzed as in Fig. 1D. C and N, cytoplasmic and nuclear fractions,
586 LEE ET AL.MOL. CELL. BIOL.
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VOL. 24, 2004DIFFERENTIAL ROLES OF mPER PROTEINS 587
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intervals on the first day in constant darkness (DD). Like
mPER1 and mPER2, mPER3 exhibited a circadian rhythm in
abundance and electrophoretic mobility (Fig. 1B) (15). Rap-
idly 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. 4) (15).
The electrophoretic mobility shift was due mainly to phosphor-
ylation, as treatment with ?PP altered the mobility of the
slowly migrating forms to more rapidly migrating forms (Fig.
We showed previously that mPER1 and mPER2 translocate
into the nucleus in a time-dependent manner, and hyperphos-
phorylated forms of mPER1 and mPER2 are detected pre-
dominantly 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
mPER3 associates with other clock proteins in a time-de-
pendent manner. Our previous studies showed that seven clock
proteins (CLOCK, BMAL1, mPER1, mPER2, mCRY1,
mCRY2, and CKIε) 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 inter-
actions of mPER3 with other clock proteins before (CT06) and
mPER3 was copurified along with each of the seven clock
proteins examined at CT18 (Fig. 2 and data not shown).
mPER1 regulates the rhythmic posttranslational modifica-
tions 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 post-
translational modification of mPER3 (Fig. 3A). In mPer1/3 and
mPer2/3 double-mutant mice, in which molecular and behav-
ioral rhythmicity persist for several days in DD (3), the remain-
ing PER gene product (mPER2 and mPER1, respectively)
underwent rhythmic changes in abundance and phosphoryla-
tion, as in wild-type mice, except the rhythm phases appeared
advanced compared to those in wild-type mice (Fig. 3A). Thus,
either mPER1 or mPER2 is sufficient for its own circadian
changes in phosphorylation and abundance, whereas mPER3
The lack of rhythmic posttranslational modifications of
mPER3 in mPer1/mPer2 double-mutant mice could be second-
ary 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 spe-
cific 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
mPer2ldcor mPer3-deficient mice, mPER1 exhibited robust
oscillations in phosphorylation and abundance, as in wild-type
mice (Fig. 3B). Similarly, circadian rhythms of mPER2 phos-
phorylation and abundance were not significantly altered in
mPer1ldcor mPer3-deficient mice (Fig. 3B). mPER3 rhythms
also appeared normal in mPer2ldcmice (Fig. 3B).
Remarkably, however, mPER3 did not show temporal
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 ?-actin and converted to the percentage of
maximal value for each gene. Mean values and standard errors from
three separate experiments are shown. A representative autoradio-
gram is shown at the bottom of the figure.
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 was transferred to a new tube and subjected to a second IP
with anti-mPER1 antibody. Immune complexes and supernatant from
both IP reactions were analyzed by WB for mPER1, mPER2, and
mPER3. Two arrowheads indicate nonspecific bands that confirm
equal loading of total protein. The majority of mPER3 was copurified
with mPER1, indicating that mPER1 and not mPER2 is the major
binding partner for mPER3.
588 LEE ET AL.MOL. CELL. BIOL.
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changes in phosphorylation and abundance in mPer1ldcmutant
mice (Fig. 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 oscilla-
tions in mPer1ldcand mPer2ldcmutant mice, as in wild-type
mice (Fig. 3C). Examination of the subcellular localization of
mPER3 revealed that the protein was trapped in the cytoplasm
in the absence of mPER1 (Fig. 3D). Immunodepletion exper-
iments of wild-type livers confirmed that the majority of hy-
perphosphorylated mPER3 is complexed with mPER1 (Fig. 5).
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. 2), mPER proteins associated with mCRY1,
mCRY2, and CKIε more at CT18 than at CT06 in wild-type
mice (Fig. 6A, lanes 1 to 6). The same interaction pattern was
maintained in mPer1/3 and mPer2/3 double-mutant mice; as-
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
antibodies to mPER1, mPER2, and mPER3 (P1, P2, or P3). The immune complexes were Western blotted for mPERs, mCRYs, and CKIε.
(B) Interaction between CLOCK-BMAL1 and mCRY is abolished in mPer1/2 double-mutant mice. Liver extracts from wild-type (wt) and mPer
double-mutant mice collected at CT06 and CT18 were subjected to IP with anti-CLOCK antibody. The immune complexes were Western blotted
for CLOCK, mCRY1, and mCRY2 (left panels). Straight liver extracts were Western blotted for mCRY1 and mCRY2 (right panels) to show that
mCRYs are readily detectable in mice of each genotype.
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590 LEE ET AL.MOL. CELL. BIOL.
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sociations between the single remaining mPER member and
mCRY1, mCRY2, and CKIε 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. 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 abun-
dance in the absence of mPER1 in some experiments (Fig. 3D
and 6A), but this was not a consistent finding (Fig. 3A and B).
We also examined whether the remaining mPER protein in
the double-mutant mice can induce rhythmic interactions be-
tween 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. 6B). The same rhythmic interactions were main-
tained 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. 6B). The absence of these interac-
tions 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. 6B, right panels).
Taken together, our data indicate that either mPER1 or
mPER2 alone is sufficient for rhythmic posttranslational reg-
ulation 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 dif-
ferent from mPER1 and mPER2 in clock-relevant functions.
In vitro binding assays reveal a lack of stable interaction
between mPER3 and CKI?. 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 CKIε and/or the
mCRY proteins in the cytoplasm. We thus examined affinities
of each mPER protein for CKIε and the mCRY proteins.
First, the physical interaction between each mPER protein
and CKIε were examined in transiently transfected cells. When
the kinase and each mPER protein were coexpressed in NIH
3T3 cells, CKIε was able to coprecipitate with mPER1 and
mPER2, but not mPER3, a finding consistent with studies
reported by Akashi et al. (Fig. 7A). CKIε-induced phosphor-
ylation of mPER3 was enhanced when mPER1, but not
mPER2, was coexpressed in NIH 3T3 cells (Fig. 7B, lane 5
versus lane 6). In agreement with our in vivo studies (Fig. 3),
the data suggest that mPER3 requires mPER1 for stable in-
teraction with CKIε and efficient phosphorylation.
To determine the affinities of the mPER proteins for CKIε
quantitatively, we performed binding assays with in vitro trans-
lated proteins. Each of the mPER proteins and kinase were
mixed at various molar ratios and then subjected to IP with an
anti-CKIε antibody. Subsequently, the affinity of the mPER for
CKIε was measured by quantifying the amount of copurified
The amounts of mPER2 bound to CKIε were about twofold
higher than those of mPER1 (Fig. 7C). Consistent with the
results in Fig. 7A, we could not detect stable interaction be-
tween mPER3 and CKIε despite numerous attempts (Fig. 7C).
These data demonstrate that mPER3 is intrinsically defective
in its ability to bind to CKIε.
A set of similar experiments was conducted to examine in-
teractions between the mPER and mCRY proteins. As previ-
ously reported (14), all three mPER proteins associated with
mCRY1 in transiently transfected NIH 3T3 cells (Fig. 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. 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. 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 immunodeple-
tion of mPER1 removed less than 50% of mPER3 from liver
extracts (Fig. 5 and data not shown). Higher levels of expres-
sion would mitigate the impact of a slightly reduced affinity.
We therefore conclude that the inability of mPER3 to main-
tain molecular or behavioral rhythms is due primarily to a
deficiency in interaction with CKIε. The reduced affinity of
FIG. 7. Quantitative difference of mPER protein interactions with CKIε and mCRY1. (A) mPER3 does not stably associate with CKIε in NIH
3T3 cells. NIH 3T3 cells were transfected with CKIε-Myc and mPER1-V5, mPER2-V5, or mPER3-V5. The cell lysates were subjected to IP with
anti-V5 antibody and the immune complexes were Western blotted with anti-V5 and Myc antibodies. CKIε-Myc was comparably expressed in the
three cell lysates (left panel). (B) CKIε-induced phosphorylation of mPER3 is enhanced by mPER1 but not by mPER2. CKIε-Myc and various
combinations of mPER were expressed in NIH 3T3 cells. The cell lysates were subjected to Western blotting with anti-V5, HA, and Myc antibodies.
The arrow indicates hyperphosphorylated mPER3. (C) Binding of mPER with CKIε. mPERs and CKIε were synthesized in vitro in the presence
of L-[35S]methionine. Each of the mPER proteins and CKIε were mixed at a molar ratio of 1:1, 1:2, or 1:3. The mixtures were subjected to IP with
CKIε antibody. The immune complexes were resolved by SDS-PAGE and visualized by autoradiography or quantified by Fuji phosphorimager. All
IP reactions recovered more than 95% of input primary antigens (data not shown). A representative autoradiogram is shown (upper panel).
Coimmunoprecipitated mPER was quantified from three independent experiments. Each value was converted to the percentage of the maximal
value of the experiment. Mean values with standard errors are shown (lower panel). (D) NIH 3T3 cells were transfected with mCRY1-HA and
mPER1-V5, mPER2-V5, or mPER3-V5. The cell lysates were subjected to IP with anti-HA antibody, and the immune complexes were Western
blotted with anti-HA and V5 antibodies. The left panel shows the amounts of mPER proteins present in the three cell lysates. (E) Experiments
with mPER proteins and mCRY1 were performed as in panel C to determine mPER affinity for mCRY1.
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FIG. 8. CKBD of mPER is important for interaction with CKIε 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 of chPERs
are the same as their wild-type counterparts. mPER2, mPER3, chPER2, and chPER3 were synthesized in vitro in the presence of L-[35S]methi-
onine, and they were resolved by SDS-PAGE. (C) chPER3 binds to CKIε more efficiently than mPER3, whereas chPER2 binds to CKIε much less
efficiently than mPER2. The PERs and CKIε were synthesized in vitro in the presence of L-[35S]methionine. Each of the PERs was mixed with CKIε
at a molar ratio of 1:3. The mixtures were subjected to IP with CKIε antibody, and the immune complexes were resolved by SDS-PAGE and
visualized by autoradiography. CKIε antibody immunoprecipitated ?95% of input CKIε in all cases (data not shown). When CKIε was absent from
the mixtures, mPER2 and mPER3 were not significantly immunoprecipitated by anti-CKIε antibody (last lane). Slow-migrating forms were visible
for copurified PER proteins (lanes 3, 4, and 8) compared to input PERs (lanes 1, 2, and 6). (D) Results similar to those in panel C were obtained
with transiently expressed proteins in NIH 3T3 cells. Native and chimeric PERs were coexpressed with CKIε in NIH 3T3 cells. The cell lysates were
subjected to IP with anti-CKIε antibody, and the immune complexes were Western blotted with anti-V5 and Myc antibodies (right panel). The left
panel indicates the amounts of mPERs and CKIε present in the cell lysates. (E) Phosphorylation of chPER3 is enhanced, but that of chPER2 is
attenuated compared to their wild-type counterparts in vitro. In vitro-translated PER proteins were incubated with or without CKIε for 60 min.
at room temperature (upper panel) or incubated with CKIε for the indicated times (lower panel). The mixtures were then resolved by SDS-PAGE
and visualized by autoradiography. Representative autoradiograms are shown.
592 LEE ET AL.MOL. CELL. BIOL.
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mPER3 for the mCRY proteins may also contribute, but to a
The CKBD of mPER is essential for kinase interaction and
phosphorylation. If the direct association of mPER with CKIε
is essential for clock-relevant functions of mPER, exchanging
the CKBDs between mPER2 and mPER3 should cause pre-
dictable alterations in chimeric protein function. We thus gen-
erated the chimeric chPER2 and chPER3 proteins containing
each other’s CKBD (Fig. 8A). The apparent sizes of chPER2
and chPER3 were similar to their wild-type counterparts (Fig.
To determine whether the CKBD alters the functions of the
hybrid proteins, we performed in vitro binding assays between
the chimeric proteins and CKIε. As expected, the amount of
chPER3 bound to CKIε greatly increased compared to that of
native mPER3 (Fig. 8C). In contrast, the binding of chPER2 to
CKIε was barely detectable. When the interactions were ex-
amined in transiently transfected NIH 3T3 cells, mPER2 and
chPER3 were copurified with CKIε, whereas mPER3 and
chPER2 were not (Fig. 8D). These data indicate that the
CKBD determines the binding affinity of mPER proteins for
CKIε. Even although mPER3 and chPER2 did not appreciably
bind CKIε in cell culture, the proteins were highly phosphor-
ylated, suggesting endogenous kinase activity not involving
Next, we examined the efficiency of chPER phosphorylation
by CKIε. Wild-type and chimeric proteins and CKIε were syn-
thesized in vitro, and each of the PER proteins was mixed with
CKIε to compare phosphorylation efficiency of the chimeric
and native mPERs by CKIε in the absence of other interacting
proteins. mPER2 and chPER3 were more readily phosphory-
lated by CKIε than were their counterparts, chPER2 and
mPER3, respectively (Fig. 8E).
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 in-
teractions with other clock proteins, nuclear translocation, and
degradation, our data suggest that the mPER CKBD is essen-
tial for clock-relevant functions of mPER1 and mPER2.
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 feed-
back 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 (mPer1ldcand
mPer1/2 double-mutant mice) mPER3 phosphorylation is
abolished. Thus, mPER3 is regulated by mPER1 at a post-
translational level. Consistent with these observations, phos-
phorylation of mPER3 by CKIε is enhanced in the presence of
mPER1 in NIH 3T3 cells (Fig. 7B), and the nuclear translo-
cation of mPER3 is promoted by mPER1 in NIH 3T3 cells
(14). The presence of mPER3 in circadian clock protein com-
plexes 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 transloca-
tion, suggesting that the single remaining mPER can support
the molecular clock. The levels of the remaining mPER pro-
tein 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 in-
teracts 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 CKIε might explain the functional inadequacy of mPER3
in terms of supporting the circadian clock. In fact, CKIε has
been implicated in the nuclear translocation of the mPER
proteins (1, 22).
Our results suggest that stable interactions between CKIε
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 CKIε, 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 CKIε and is effi-
ciently phosphorylated in vitro. Taken together, our data
strongly suggest that the CKBD of mPERs is essential for
stable interaction with CKIε, effective phosphorylation by
CKIε, and possibly other clock-relevant functions, such as nu-
clear translocation. The importance of the CKBD for a func-
tioning 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).
We thank Jason DeBruyne for helpful comments during the prep-
aration of the manuscript.
This study was supported by NIH grant NS39303.
1. Akashi, M., Y. Tsuchiya, T. Yoshino, and E. Nishida. 2002. Control of
intracellular dynamics of mammalian period proteins by casein kinase I?
(CKI?) and CKI? in cultured cells. Mol. Cell. Biol. 22:1693–1703.
2. Allada, R., P. Emery, J. S. Takahashi, and M. Rosbash. 2001. Stopping time:
the genetics of fly and mouse circadian clocks. Annu. Rev. Neurosci. 24:
3. Bae, K., X. Jin, E. S. Maywood, M. H. Hastings, S. M. Reppert, and D. R.
Weaver. 2001. Differential functions of mPer1, mPer2, and mPer3 in the SCN
circadian clock. Neuron 30:525–536.
4. Camacho, F., M. Cilio, Y. Guo, D. M. Virshup, K. Patel, O. Khorkova, S.
Styren, B. Morse, Z. Yao, and G. A. Keesler. 2001. Human casein kinase I?
VOL. 24, 2004DIFFERENTIAL ROLES OF mPER PROTEINS593
on June 12, 2014 by guest
phosphorylation of human circadian clock proteins period 1 and 2. FEBS
5. Cermakian, N., L. Monaco, M. P. Pando, A. Dierich, and P. Sassone-Corsi.
2001. Altered behavioral rhythms and clock gene expression in mice with a
targeted mutation in the Period1 gene. EMBO J. 20:3967–3974.
6. Dunlap, J. C. 1999. Molecular bases for circadian clocks. Cell 96:271–290.
7. Eide, E. J., E. L. Vielhaber, W. A. Hinz, and D. M. Virshup. 2002. The
circadian regulatory proteins BMAL1 and cryptochromes are substrates of
casein kinase Iε. J. Biol. Chem. 277:17248–17254.
8. Etchegaray, J. P., C. Lee, P. A. Wade, and S. M. Reppert. 2003. Rhythmic
histone acetylation underlies transcription in the mammalian circadian clock.
9. Gekakis, N., D. Staknis, H. B. Nguyen, F. C. Davis, L. D. Wilsbacher, D. P.
King, J. S. Takahashi, and C. J. Weitz. 1998. Role of the CLOCK protein in
the mammalian circadian mechanism. Science 280:1564–1569.
10. Griffin, E. A., Jr., D. Staknis, and C. J. Weitz. 1999. Light-independent role
of CRY1 and CRY2 in the mammalian circadian clock. Science 286:768–771.
11. Harmer, S. L., S. Panda, and S. A. Kay. 2001. Molecular bases of circadian
rhythms. Annu. Rev. Cell Dev. Biol. 17:215–253.
12. Hogenesch, J. B., Y. Z. Gu, S. Jain, and C. A. Bradfield. 1998. The basic-
helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes
with circadian and hypoxia factors. Proc. Natl. Acad. Sci. USA 95:5474–5479.
13. Keesler, G. A., F. Camacho, Y. Guo, D. Virshup, C. Mondadori, and Z. Yao.
2000. Phosphorylation and destabilization of human period I clock protein
by human casein kinase I epsilon. Neuroreport 11:951–955.
14. Kume, K., M. J. Zylka, S. Sriram, L. P. Shearman, D. R. Weaver, X. Jin, E. S.
Maywood, M. H. Hastings, and S. M. Reppert. 1999. mCRY1 and mCRY2
are essential components of the negative limb of the circadian clock feedback
loop. Cell 98:193–205.
15. Lee, C., J. P. Etchegaray, F. R. Cagampang, A. S. Loudon, and S. M.
Reppert. 2001. Posttranslational mechanisms regulate the mammalian circa-
dian clock. Cell 107:855–867.
16. Lowrey, P. L., K. Shimomura, M. P. Antoch, S. Yamazaki, P. D. Zemenides,
M. R. Ralph, M. Menaker, and J. S. Takahashi. 2000. Positional syntenic
cloning and functional characterization of the mammalian circadian muta-
tion tau. Science 288:483–492.
17. Okamura, H., S. Miyake, Y. Sumi, S. Yamaguchi, A. Yasui, M. Muijtjens,
J. H. Hoeijmakers, and G. T. van der Horst. 1999. Photic induction of mPer1
and mPer2 in cry-deficient mice lacking a biological clock. Science 286:2531–
18. Reppert, S. M., and D. R. Weaver. 2002. Coordination of circadian timing in
mammals. Nature 418:935–941.
19. Reppert, S. M., and D. R. Weaver. 2001. Molecular analysis of mammalian
circadian rhythms. Annu. Rev. Physiol. 63:647–676.
20. Shearman, L. P., X. Jin, C. Lee, S. M. Reppert, and D. R. Weaver. 2000.
Targeted disruption of the mPer3 gene: subtle effects on circadian clock
function. Mol. Cell. Biol. 20:6269–6275.
21. Shearman, L. P., S. Sriram, D. R. Weaver, E. S. Maywood, I. Chaves, B.
Zheng, K. Kume, C. C. Lee, G. T. van der Horst, M. H. Hastings, and S. M.
Reppert. 2000. Interacting molecular loops in the mammalian circadian
clock. Science 288:1013–1019.
22. Takano, A., K. Shimizu, S. Kani, R. M. Buijs, M. Okada, and K. Nagai. 2000.
Cloning and characterization of rat casein kinase 1epsilon. FEBS Lett. 477:
23. Toh, K. L., C. R. Jones, Y. He, E. J. Eide, W. A. Hinz, D. M. Virshup, L. J.
Ptacek, and Y. H. Fu. 2001. An hPer2 phosphorylation site mutation in
familial advanced sleep phase syndrome. Science 291:1040–1043.
24. van der Horst, G. T., M. Muijtjens, K. Kobayashi, R. Takano, S. Kanno, M.
Takao, J. de Wit, A. Verkerk, A. P. Eker, D. van Leenen, R. Buijs, D.
Bootsma, J. H. Hoeijmakers, and A. Yasui. 1999. Mammalian Cry1 and Cry2
are essential for maintenance of circadian rhythms. Nature 398:627–630.
25. Vielhaber, E., E. Eide, A. Rivers, Z. H. Gao, and D. M. Virshup. 2000.
Nuclear entry of the circadian regulator mPER1 is controlled by mammalian
casein kinase I epsilon. Mol. Cell. Biol. 20:4888–4899.
26. Yagita, K., F. Tamanini, M. Yasuda, J. H. Hoeijmakers, G. T. van der Horst,
and H. Okamura. 2002. Nucleocytoplasmic shuttling and mCRY-dependent
inhibition of ubiquitylation of the mPER2 clock protein. EMBO J. 21:1301–
27. Zheng, B., U. Albrecht, K. Kaasik, M. Sage, W. Lu, S. Vaishnav, Q. Li, Z. S.
Sun, G. Eichele, A. Bradley, and C. C. Lee. 2001. Nonredundant roles of the
mPer1 and mPer2 genes in the mammalian circadian clock. Cell 105:683–694.
28. Zheng, B., D. W. Larkin, U. Albrecht, Z. S. Sun, M. Sage, G. Eichele, C. C.
Lee, and A. Bradley. 1999. The mPer2 gene encodes a functional component
of the mammalian circadian clock. Nature 400:169–173.
29. Zylka, M. J., L. P. Shearman, D. R. Weaver, and S. M. Reppert. 1998. Three
period homologs in mammals: differential light responses in the suprachias-
matic circadian clock and oscillating transcripts outside of brain. Neuron
594LEE ET AL.MOL. CELL. BIOL.
on June 12, 2014 by guest