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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