CKI␧/␦-dependent phosphorylation is atemperature-insensitive, period-determining
process in the mammalian circadian clock
Yasushi Isojimaa,b,1, Masato Nakajimac,1, Hideki Ukaic,1, Hiroshi Fujishimac, Rikuhiro G. Yamadac, Koh-hei Masumotoc,
Reiko Kiuchia,b, Mayumi Ishidac, Maki Ukai-Tadenumac, Yoichi Minamic, Ryotaku Kitoc, Kazuki Nakaod,
Wataru Kishimotoc, Seung-Hee Yooe,f,g, Kazuhiro Shimomuraf,h, Toshifumi Takaoi, Atsuko Takanoj, Toshio Kojimaa,
Katsuya Nagaij, Yoshiyuki Sakakia, Joseph S. Takahashie,f,g,h,2, and Hiroki R. Uedac,k,l,2
aComparative Systems Biology Team, Genomic Science Center and bDevelopmental Systems Modeling Team, Advanced Computational Sciences Department,

RIKEN, 1-7-22, Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; cLaboratory for Systems Biology, kFunctional Genomics Unit, and dAnimal Resource Unit,
RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan; eHoward Hughes Medical Institute, fDepartmentof Neurobiology and Physiology, and hCenter for Functional Genomics, Northwestern University, 2205 Tech Drive, Evanston, IL 60208; gDepartment of
Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390-9111; iLaboratory of Protein Profiling and Functional Proteomics,
and jLaboratory of Proteins Involved in Homeostatic Integration, Institute for Protein Research, Osaka University, Yamadaoka 3-2, Suita, Osaka 565-0871,
Japan; and lDepartment of Bioscience, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
contributed by Joseph S. Takahashi, August 4, 2009 (sent for review June 14, 2009)

A striking feature of the circadian clock is its flexible yet robust
response to various environmental conditions. To analyze thebiochemical processes underlying this flexible-yet-robust characteristic, we examined the effects of 1,260 pharmacologically active
compounds in mouse and human clock cell lines. Compounds thatmarkedly (>10 s.d.) lengthened the period in both cell lines, also
lengthened it in central clock tissues and peripheral clock cells.
Most compounds inhibited casein kinase I␧ (CKI␧) or CKI␦ phosphorylation of the PER2 protein. Manipulation of CKI␧/␦dependent phosphorylation by these compounds lengthened theperiod of the mammalian clock from circadian (24 h) to circabidian(48 h), revealing its high sensitivity to chemical perturbation. The
degradation rate of PER2, which is regulated by CKI␧/␦-dependent
phosphorylation, was temperature-insensitive in living clock cells,
yet sensitive to chemical perturbations. This temperature-insensitivity was preserved in the CKI␧/␦-dependent phosphorylation ofa synthetic peptide in vitro. Thus, CKI␧/␦-dependent phosphorylation is likely a temperature-insensitive period-determining process in the mammalian circadian clock.
chemical biological approach 兩 temperature compensationT

he circadian clock is a molecular mechanism underlying
endogenous, self-sustained oscillations with a period of ⬇24h, manifested in diverse physiological and metabolic processes(1–3). The most striking feature of circadian clock is its flexible
yet robust response to various environmental conditions. For
example, circadian periodicity varies with light intensity (4–6)
while remaining robust over a wide range of temperatures(‘‘temperature compensation’’) (1, 3, 7–9). This flexible-yetrobust characteristic is evolutionarily conserved in organisms
ranging from photosynthetic bacteria to warm-blooded mammals (3, 10–12), and has interested researchers from a broad
range of disciplines. However, despite many genetic and molecular studies (13–22), the detailed biochemical mechanism underlying this characteristic remains poorly elucidated (3).
The simplest explanation for this flexible-yet-robust property
is that the key period-determining reactions are insensitive totemperature but responsive to other environmental conditions.
Indeed, Pittendrigh proposed the existence of a temperatureinsensitive component in the clock system in 1954 (7), and in
1968, he and his colleagues demonstrated that both the wave
form and the period of circadian oscillations are invariant withtemperature (23). However, the idea of a temperatureinsensitive biochemical reaction is counterintuitive, as elementary chemical processes are highly temperature-sensitive. One
15744 –15749 兩 PNAS 兩 September 15, 2009 兩 vol. 106 兩 no. 37

exception is the cyanobacterial clock, in which temperatureinsensitive enzymatic reactions are observed (24, 25). However,the cyanobacterial clock is quite distinct from other clock
systems, and this biochemical mechanism has not been demonstrated in other clocks.
Recently, a chemical-biological approach was proposed tohelp elucidate the basic processes underlying circadian clocks(26), and high-throughput screening of a large chemical compound library was performed (27). In this report, to analyze
systematically the fundamental processes involved in determining the period length of mammalian clocks, we tested 1,260
pharmacologically active compounds for their effect on period
length in mouse and human clock cell lines, and found 10
compounds that most markedly lengthened the period of bothclock cell lines affected both the central and peripheral circadianclocks. Most compounds inhibited CKI␧ or CKI␦ activity, suggesting that CKI␧/␦-dependent phosphorylation is an important
period-determining process in the mammalian circadian clock.
Surprisingly, the degradation rate of endogenous PER2, whichis regulated by CKI␧-dependent phosphorylation (28) and probably by CKI␦-dependent phosphorylation, was temperatureinsensitive in the living clock cells, and the temperatureinsensitivity was preserved even for the in vitro CKI␧/␦dependent phosphorylation of a synthetic peptide derived from
PER2. These results suggest that this period-determining process is flexible in response to chemical perturbation yet robust in
the face of temperature perturbations. Based on these findings,
we propose that CKI␧/␦-dependent phosphorylation is a temperature-insensitive period-determining process in the mammalian circadian clock.
Results
Ten Compounds Markedly Lengthened Period Length. We examined
1,260 pharmacologically active chemical compounds from the

Author contributions: Y.I., M.N., H.U., T.T., K. Nagai, and H.R.U. designed research; Y.I.,
M.N., H.U., H.F., K.-h.M., R. Kiuchi, M.I., M.U.-T., Y.M., R. Kito, K. Nakao, W.K., S.-H.Y., K.S.,
A.T., T.K., Y.S., and J.S.T. performed research; W.K. contributed new analytic tools; R.G.Y.
analyzed data; and Y.I., M.N., H.U., and H.R.U. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
1Y.I., M.N., and H.U. contributed equally to this work.
2To

whom correspondence may be addressed. E-mail: uedah-tky@umin.ac.jp or
j-takahashi@northwestern.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/
0908733106/DCSupplemental.

www.pnas.org兾cgi兾doi兾10.1073兾pnas.0908733106

Potent Compounds Inhibit CKI␧/␦ Enzymatic Activity. Most of the
proposed targets of the 10 potent compounds on the list in Table
S3 have not been identified as components of the mammalian
circadian clock, although one of the 10 is an inhibitor of CK2,
recently found as a component of the mammalian circadian clock(29, 30). The potent protein kinase inhibitors SB202190,
SP600125, and roscovitine were previously reported to inhibit
CKI in addition to their primary targets (31, 32). Since evenspecific protein kinase inhibitors also usually affect other kinases(31, 33), we hypothesized that the seven protein kinase inhibitorsthat we identified suppress CKI activity in addition to theirprimary targets.
To examine this hypothesis, we performed the siRNA knockdown of CKI genes, as well as the genes related to the 10 potent
compounds (Fig. 1 A–C and Fig. S2 B–G). The knockdown of
CKI␧ or CKI␦ exhibited the greatest period-lengthening effects(Fig. 1B), more than 28 h in CT, whereas knockdown of othergenes showed no or only a slight effect (Fig. S2 B–G). Moreover,the combinatorial knockdown of CKI␧ and CKI␦ additively
lengthened the period of circadian oscillations to over 30 h in CT(Fig. 1C), supporting the idea that CKI␧/␦ play one of the mostimportant roles in the period-determination processes of mammalian circadian clocks. These results suggest that at least someof our compounds were not acting via their primary target, but
through inhibition of CKI␧/␦.
The inhibitory effect of the potent compounds on CKI␧/␦
activity was confirmed by the in vitro CKI␧ or CKI␦ kinase assay.
Importantly, nine of the 10 compounds [i.e., not 17 ␣ hydroxyprogesterone (17-OHP)] strongly inhibited the catalytic
domain of wild-type CKI␧, lacking the C-terminal regulatory
domain (⌬CKI␧) as effectively as or better than IC261, a specificinhibitor of CKI (Fig. 1D). SP600125 and TG003, which dramatically extended the circadian period and potently inhibited⌬CKI␧ activity, strongly inhibited both activity of the ⌬CKI␧ andcatalytic domain of CKI␦ (⌬CKI␦), with lower IC50 (⬍ 0.55 ␮M)than the IC50 of IC261 (about 4 ␮M), whereas 17-OHP did notinhibit both of ⌬CKI␧ and ⌬CKI␦ (Fig. 1E). Since double
knockdown of the CKI␧ and CKI␦ genes using siRNA had
additive effects on period lengthening (Fig. 1C), and thesechemical compounds, except 17-OHP, inhibited CKI␧ and CKI␦
similarly, they probably acted on both CKIs to affect the period
length of circadian oscillations.
We also examined the effect of potent compounds on thestability of the mPER2 protein, which was regulated by CKI␧(28) (and probably by CKI␦) in non-clock cell, 293T cells. Fig. S3
A and B show that the two putative CKI inhibitors significantly
enhanced the stability and slowed the degradation rate of
LUC::mPER2 (P ⬍ 0.01, one-way ANOVA), whereas 17-OHP
did not significantly affect LUC::mPER2 stability (P ⫽ 0.18,
one-way ANOVA). These results were supported by immunoblot
experiments (Fig. S4). This degradation rate of overexpressed
LUC::mPER2 was not affected without the co-overexpression of
CKI␧(tau) (Fig. S3C; P ⫽ 0.193 for SP600125 and P ⫽ 0.728 for
TG003; two-way ANOVA), presumably because relative expression levels of LUC::mPER2 in 293T cells compared with CKI␧/␦
Isojima et al.

Fig. 1. Effects of knockdowns of CSNK1s on the period length and effect ofpotent compounds on the CKI␧/␦ activity in vitro. (A–C) Graphs indicate
relationship between gene knockdown effects and period length in U2OShPer2-Luc cells. The x-axis indicates the expression level of genes relative to thesamples transfected with control siRNA. The y-axis indicates the period length,
described in circadian time (CT), with the control samples assigned as 24 h.
Each symbol represents the mean ⫾ SEM of independent experiments (n ⱖ 3).
(A) The effect of CRY2 knockdown as a positive control. (B) Gene knockdownsof CKI␧/␦. CSNK1E, CKI␧; CSNK1D, CKI␦. (C) Effect of the double knockdown of
CSNK1E and CSNK1D. The gene expression level represents the total amountof CKI␦ and CKI␧. (D) ⌬CKI␧ phosphorylation activity for a synthetic mPER2
peptide in the presence of chemical compounds [25, 50, or 100 ␮M for the 10compounds, 100 ␮M for IC261] was measured using a modified IMAP assay
with 100 ␮M ATP. The average results for each condition are shown as therelative activity compared to the control condition (DMSO). Error bars denote
1 s.d. (E) Dose-dependent effects of SP600125 and TG003 on ⌬CKI␧/␦ phosphorylation activity. ⌬CKI␧/␦ phosphorylation activity was measured in thepresence of compounds at the indicated concentrations. Each symbol indicates the activity relative to the control condition in two independent experiments. The lines are approximate functions using the equation: y ⫽ 100e-ax.
The IC50s for CKI␧ calculated from the equations were 4.0, 0.55, and 0.22 ␮M
for IC261, TG003, and SP600125, respectively, and for CKI␦ were 4.1, 0.40, and0.13 ␮M.

were much higher that in MEFs. We used CKI␧(tau) for theseassays because the degradation rate with co-overexpression of
CKI␧(tau) was faster than that of CKI␧(wt), which enable us tomeasure the PER2 degradation rate precisely. We next confirmed that the stability of LUC-fused mPER1 protein(LUC::mPER1), the closest relative of mPER2, was also decreased with co-expression of CKI␧(tau) as well as
LUC::mPER2,
whereasLUC-fused
mBMA L1(LUC::mBMAL1) and native LUC were not (Fig. S5). These
results suggest that the inhibitory effect of the potent compounds
on CKI␧/␦ activity is the primary mechanism by which theylengthened the period of clock cells. In contrast, 17-OHP
apparently functions via a different mechanism.
Flexibility and Robustness of a Period Determination Process. To

explore how important this process is for determining the period
PNAS 兩 September 15, 2009 兩 vol. 106 兩 no. 37 兩 15745

CELL BIOLOGYLOPAC1280 library for their effect on circadian period length inmammalian clock cell lines, NIH 3T3-mPer2-Luc and U2OShPer2-Luc (Table S1 and Table S2 and SI Text). We identified 10,
all with period-lengthening effects, that substantially altered theperiod in both cell lines (⬎ ⫹10 s.d. or ⬍ ⫺10 s.d., ⬇2.0–3.0 h)(Fig. S1 A and D and Tables S3 and S4). These compounds,
labeled ‘‘potent,’’ also lengthened the period of primary culturesof mouse embryonic fibroblasts (MEFs, as peripheral clock cells)(Fig. S1 B and E) and slice cultures of suprachiasmatic nucleus(SCN; as central clock tissue) (Fig. S1 C and E). These results
supported the idea that the potent compounds alter canonicalclock processes.

Fig. 2. Flexibility and robustness of a period-determination process of the
mammalian circadian clock. (A) Dose-dependent effects of SP600125 andTG003 on period length in U2OS-hPer2-Luc cells. The period length is indicatedboth in real-time (right axis) and in circadian time (left axis). For circadian time,
the average period length in two independent control experiments was
assigned as 24.0 h. The two lines in each graph correspond to two independent
experiments. Each value represents the mean ⫾ SEM. At the concentrations
without data points, the cells behaved arrhythmically. (B and C) Dosedependent effect of SP600125 on the period length and degradation rate ofmPER2::LUC in mPer2Luc MEFs. A pair of plates with cultured mPer2Luc MEFs, towhich 0 to 10 ␮M SP600125 was applied, were prepared. One was used tomeasure mPER2::LUC decay and the other to determine the period. (B) Decay
of mPer2::LUC bioluminescence in mPer2Luc MEFs. The degradation ofmPer2::LUC protein was monitored after the administration of CHX to MEFs.
The time-course data of each sample were normalized to approximate functions in which time point 0 was 100%. Each value represents the mean ⫾ SEM.
of the normalized data. The lines represent approximated curves in which y ⫽100 at time ⫽ 0 and y ⫽ 50 at the averaged half-life time. The colors in orderfrom gray to blue to red represent the concentration of SP600125 with 0.25%
DMSO (n ⫽ 6). (C) Correlation between the period length and degradation
rate of mPER::LUC in mPer2Luc MEFs with the administration of SP600125. Eachvalue represents the mean ⫾ SEM (n ⫽ 6). (D) Temperature dependency of
decay of the mPER2::LUC bioluminescence in mPer2Luc MEFs. The degradationof mPER2::LUC protein was monitored after the addition of CHX to MEFs. The
time-course data of each sample were normalized to an approximate functionin which time point 0 was 100%. Each value represents the mean ⫾ SEM of thenormalized data. The lines represent an approximated curve in which y ⫽ 100
at time ⫽ 0 and y ⫽ 50 at the averaged half-life time. The blue dots and line
indicate the data at 27 °C; green, 32 °C; and magenta, 37 °C (n ⫽ 23). (E)Temperature compensation in the half-lives of the mPer2::LUC protein. The
graph indicates the mean ⫾ SEM. The gray broken line indicates the approximated line described by the equation: y ⫽ 53.69 ⫹ 0.333x, and the Q10 value
between 27 and 37 °C calculated from the equation is 0.950. (F) Temperaturecompensation in the period length of mPer2Luc MEFs. The graph indicates themean ⫾ SEM. The gray broken line indicates the approximated line describedby the equation: y ⫽ 19.02 ⫹ 0.097x, and the Q10 value between 27 and 37 °C
calculated from the equation is 0.957.

length of the mammalian circadian clock, we examined theeffects of these two compounds on the circadian period length
at higher concentrations (50–500 ␮M). Interestingly, the higher
concentrations approximately doubled the period length (to53.98 and 52.50 h at 100 ␮M for TG003, and 48.63 and 49.74 h
at 500 ␮M for SP600125) relative to the control U2OS-hPer2Luc cells (26.89 and 27.02 h) (Fig. 2A). Therefore, these results
suggest that a period-determining process mediated by CKI␧/␦15746 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0908733106

dependent phosphorylation is remarkably flexible to chemical
perturbation, since a single compound lengthened the period
from circadian (⬇24 h) to almost circabidian (⬇48 h).
To further confirm this flexibility, we next investigated thesensitivity of this process to chemical perturbation in living clock
cells by using mPer2Luc MEFs. We observed that the period
length of the circadian oscillation in MEFs correlated well withthe mPER2::LUC stability under the administration of a potent
compound (Fig. 2 B and C). Given this strong correlation, we
concluded that CKI␧/␦ activity on the PER2 protein is one of themost important period-determining processes in the mammalian
circadian oscillator.
If important period-determination processes were highly sensitive to temperature, it would be very difficult to maintain thetemperature compensation over the entire circadian period.
Thus, we next investigated the temperature dependency of thisprocess in living clock cells by using mPer2Luc MEFs. We foundthat the degradation rate of mPER2::LUC and the period length
were completely temperature-insensitive in the MEFs (Fig. 2
D–F). The observed temperature-insensitivity seemed to reflect
a molecular property of the endogenous mPER2 protein, because the degradation rate of LUC is sensitive to temperature in
mammalian cells (Fig. 4 A and B). These results imply that theperiod-determination process, which was sensitive to chemical
perturbation, was remarkably robust against physical perturbation such as temperature differences.
CKI␧/␦ Activity Is Insensitive to Temperature. To examine thebiochemical foundation underlying the observed temperatureinsensitivity, we analyzed the phosphorylation activity of CKI␧and CKI␦ in vitro. To facilitate this analysis, we designed asynthetic peptide substrate derived from the putative ␤TrCPbinding region of mouse PER2 (referred to here as ␤TrCPpeptide) (Fig. S6A), because this region is important for phosphorylation-dependent degradation of PER2 in the mammalianclock system (34). We first used just the catalytic domain of
wild-type CKI␧, lacking the C-terminal regulatory domain[⌬CKI␧(wt)] to prevent potential confusion that could result
from the autophosphorylation of this regulatory domain and thesubsequent repression of CKI␧ kinase activity. This use of thecatalytic domain was also justified by evidence that CKI␧ is kept
in a dephosphorylated, active state in vivo (35).
Under this experimental setup, we successfully recapitulated,
at least partially, the enhanced phosphorylation seen with a⌬CKI␧ tau mutation [⌬CKI␧(tau)] (36) in comparison with
⌬CKI␧(wt) at 35 °C (Fig. 3A, red). Consistent with our otherfindings, ⌬CKI␧(wt) and ⌬CKI␧(tau) phosphorylated the peptide substrate at similar rates whether at 25 °C (Fig. 3A, blue) or
35 °C, indicating a strong temperature-insensitivity (Q10 ⫽ 1.0)
[Fig. 3D, ⌬CKI␧(wt) and ⌬CKI␧(tau)]. Similar temperatureinsensitivity were also observed by using catalytic domain of
wild-type CKI␦ lacking the C-terminal regulatory domain[⌬CKI␦(wt)] (Q10 ⫽ 1.2) [Fig. 3D, ⌬CKI␦].
The temperature-insensitivity of the CKI␧ reaction depended
substantially on the phosphorylation state of CKI␧ itself, because
the Q10 of the autophosphorylated full-length construct (preincubated with ATP) was greater than for the isolated catalytic
domain (Fig. 3 B and D, CKI␧, preincubated). We also confirmed an earlier report that preincubating full-length CKI␧ with
ATP repressed its enzymatic activity (⬇15-fold and 8-fold at 25 °and 35 °C, respectively) (37); this was probably due to the
autophosphorylation of the C-terminal regulatory domain (Fig.
S6 B and C). The effects of autophosphorylation on enzymatic
activity were also recapitulated by a mutant in which the eight
autophosphorylated serine residues in the C-terminal regulatory
domain (37) were replaced with glutamate [Fig. 3D, CKI␧(D8)].
Using this autophosphorylation mutant, we observed both aslightly enhanced temperature sensitivity [Q10 ⫽ 1.3, an increase
Isojima et al.

of 0.3 compared with ⌬CKI␧(wt)] and a repression of enzymatic
activity (⬇6-fold and ⬇5-fold at 25 ° and 35 °C, respectively). We
confirmed these effects on the time-course of phosphorylation
by full-length CKI␧ that was not preincubated with ATP: As the
incubation time increased (and probably as autophosphorylation
increased), the enzymatic activity slowed (slopes of the curves
decreased), and the temperature sensitivity (i.e., the differencein slope between 25 ° and 35 °C) increased (Fig. 3C, CKI␧). We
further confirmed these effects on the time course of phosphorylation using dephosphorylated full-length CKI␧ without preincubation (Fig. S6 B and C) and found that the enzymatic activity
slowed substantially and the temperature sensitivity increased
between the first hour and the next 3 h (Fig. 3C, CKI␧,dephosphorylated). The effect of autophosphorylation on thetemperature dependency was more prominent with CKI␧ thanwith the closely related CKI␦; the repression of enzymatic
activity was, however, also observed for CKI␦ (Fig. 3D, ⌬CKI␦
and CKI␦, preincubation).
PER2 phosphorylation is a complicated process involving
multiple phosphorylation sites (38). To explore the temperaturedependency of CKI␧ with another substrate, we designed asynthetic peptide substrate derived from the region near thefunctional amino acids in PER2, which is responsible for familial
advanced sleep phase syndrome (FASPS) (referred to here as
FASPS-peptide) (Fig. S6A). We chose this site because a serine
at position 662 of human PER2 (amino acid 659 of mPER2) was
identified as a mutation for FASPS (39). We found that thephosphorylation of FASPS-peptide by ⌬CKI␧(wt) was also
temperature-insensitive, with a Q10 ⫽ 1.2, whereas the phosphorylation by ⌬CKI␧(tau) was more temperature-sensitive(Q10 ⫽ 1.6) (Fig. S6D). These results with the FASPS- and␤TrCP-peptides suggest that CKI-dependent phosphorylation
can be temperature-insensitive, depending on both the substrate
and the state of the enzyme.
Since CKI␧/␦ is involved in a variety of other biologicalprocesses, we next investigated the temperature dependency of
its activity for two clock-unrelated substrates: casein, a convenIsojima et al.

tional protein substrate, and CK1tide, a commercially available
peptide substrate for CKI␧/␦, which was a peptide substrate
phosphorylated at two residues N-terminal of CKI␧/␦ phosphorylation site. Enhanced temperature sensitivity was observed forthese substrates (Q10 ⫽ 1.6 for casein and 1.4 for CK1tide) (Fig.
3D), although it was still less than expected for canonicalenzymatic reactions (Q10 ⫽ 2–3). As with ␤TrCP-peptide,enhanced temperature sensitivity due to autophosphorylation
was also observed for these substrates (Q10 ⫽ 2.4 for casein, an
increase of 0.8; and Q10 ⫽ 2.0 for CK1tide, an increase of 0.6)
(Fig. 3D), confirming that the temperature insensitivity depends
substantially on the substrate as well as on the state of the
enzyme (see also SI Text).
Although we demonstrated that the enzymatic activity of
CKI␧ was temperature-insensitive for two synthetic peptide
substrates (␤TrCP-peptide and FASPS-peptide) derived from
mPER2 as described above, it was still unclear if this temperature
insensitivity would hold for the full-length mPER2 protein,which may have multiple phosphorylation sites outside theputative ␤TrCP-binding region and FASPS site. We therefore
examined the degradation rate of mPER2::LUC, which wasoverexpressed in mouse clock cells (NIH 3T3) and compared it
to that of LUC. As expected, the degradation rate of LUC was
highly sensitive to temperature between 27 ° and 32 °C (Q5 ⫽2.08 and 2.10, extrapolated Q10 ⫽ 4.33 and 4.41, without or with
⌬CKI␧, respectively; Fig. 4 A, B, and E). The Q5 represent therelative change of a physical property as a consequence ofincreasing the temperature by 5 °C. On the other hand, thedegradation rate of mPER2::LUC was less sensitive to temperature (Q5 ⫽ 1.51, extrapolated Q10 ⫽ 2.28; Fig. 4 C and E).
Importantly, enhancing the enzymatic reaction between mPER2
and CKI␧ by additionally expressing the catalytic domain of
CKI␧ (⌬CKI␧) in NIH 3T3 cells recapitulated the temperature
insensitivity of the mPER2::LUC degradation rate (Q5 ⫽ 1.22,
extrapolated Q10 ⫽ 1.49; Fig. 4 D and E). Based on these results
together with our finding in mPer2Luc MEFs (Fig. 2 D–F), we
concluded that CKI␧/␦-dependent phosphorylation of the
PNAS 兩 September 15, 2009 兩 vol. 106 兩 no. 37 兩 15747

CELL BIOLOGY

Fig. 3. Temperature insensitivity of the CKI␧/␦ phosphorylation activity. (A) Temperature dependency of the ⌬CKI␧(wt) (circles) and ⌬CKI␧ (tau) (squares)
phosphorylation activity for the ␤TrCP-peptide substrate. Assays were performed at 25 °C (blue) and 35 °C (red). (B) Phosphorylation activity of full-length
CKI␧(wt) autophosphorylated by preincubation with ATP. Assays were performed at 25 °C (blue) and 35 °C (red). (C) Temperature dependency of the full-length
CKI␧ phosphorylation activity for the ␤TrCP-peptide substrate. Phosphorylation activity of full-length CKI␧(wt) (CKI␧, solid line), and CKI␧(wt) dephosphorylatedwith ␭ protein phosphatase (CKI␧, dephosphorylated) (broken line). Assays were performed at 25 °C (blue) and 35 °C (red). (D) Summary for temperaturedependency of CKI␧/␦ enzymatic activities.

which is regulated by CKI␧- or CKI␦-dependent phosphorylation, was temperature-insensitive in the living clock cells (Fig. 2
D and F). We also found that temperature insensitivity waspreserved even in the CKI␧/␦-dependent phosphorylation of asynthetic peptide derived from mPER2 in vitro (Fig. 3). Moreover, the expression of exogenous full-length mPER2 protein
and the catalytic domain of CKI␧ in NIH 3T3 cells recapitulated
the temperature-insensitive reaction (Fig. 4). Based on theseexperimental data, we propose that CKI␧/␦-dependent phosphor ylation is likely a temperature-insensitive perioddetermining process in the mammalian circadian clock. In
particular, we note that this enzymatic reaction is a periodaccelerating reaction in mammalian circadian clocks, because
enhancing this reaction by genetic mutation leads to shorteningof the circadian period (36, 40), and because pharmacologicalinhibition of this reaction led to lengthening of the circadianperiod of human clock cells from a circadian (⬇24 h) to almost
circabidian (⬇48 h) period.
A Period-Accelerating and Temperature-Insensitive Reaction Is Consistent with a General Theory of Temperature Compensation. The

Fig. 4. Expression of full-length mPER2 and a catalytic domain of CKI␧ in NIH
3T3 cells recapitulates the temperature-insensitive reaction. (A–D) Temperature dependency of decay of LUC (A and B) or mPer2::LUC (C and D) bioluminescence in NIH 3T3 cells. NIH 3T3 cells, transfected with reporter vector(pMU2-Luc or pMU2-mPer2::Luc) and an expression vector for ⌬CKI␧ (B and D)or empty vector (A and C), were used. The degradation of LUC or mPer2::LUC
was monitored after the administration of CHX to cells. The time-course dataof each sample were normalized to approximate functions in which time point
0 was 100%. Each value represents the mean ⫾ SEM of the normalized data.
The lines represent an approximated curve in which y ⫽ 100 at time ⫽ 0 andy ⫽ 50 at the averaged half-life time. Blue dots and line indicate the data at
27 °C; and green, 32 °C (n ⫽ 6 –11). (E) Q5 values (the ratio between 27 and
32 °C) of the degradation rate of LUC or mPER2::LUC with or without coexpression of ⌬CKI␧. The half-lives of LUC or mPER2::LUC in each sample were
calculated as described in the SI Materials and Methods.

mPER2 protein, which can determine the period length ofcircadian oscillations in clock cells, is likely temperatureinsensitive.
Discussion
A Period-Determining and Temperature-Insensitive Process. We discovered a highly flexible period-lengthening response in the
mammalian clock: A human clock cell’s circadian period could
be stretched into a circabidian period (Fig. 2 A and E) throughchemical manipulation, probably of the CKI activity, by
SP600125 and TG003. We found that the knock-down of
CSNK1E and CSNK1D had strong period-lengthening effects,
but that of other kinases had much weaker effects, if any (Fig.
1 A–C and Fig. S2 B–G). Although we cannot completely exclude
the possibility that our identified compounds inhibit otherenzymes involved in mammalian circadian oscillations, we reason, as a first-order approximation, that these compounds
lengthen the mammalian circadian oscillation primarily by inhibiting the CKI␧ and CKI␦ enzymatic activity (see also SI Text).
We also found that this chemically f lexible perioddetermination process is highly robust against temperature
differences, because the degradation of endogenous mPER2,
15748 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0908733106

observed period-accelerating and temperature-insensitive reaction is consistent with the previously proposed general theory forthe temperature compensation of circadian clocks (41). In 1957,
Sweeney and Hastings proposed that the temperature insensitivity of the primary period-determining reaction might beachieved through a balance between multiple, counteracting
chemical reactions (8). This balance idea, which was mathematically formulated and generalized by Ruoff in 1992 into thebalance theory (41), describes a system in which there is abalance between period-accelerating and period-decelerating
reactions that yields the temperature-compensated circadianoscillation. In detail, if the period-accelerating and perioddecelerating reactions were equally sensitive to temperature, thecircadian oscillation would get faster with an increase in temperature. To reconcile this issue, the balance theory proposesthat the period-accelerating reaction(s) are more temperatureinsensitive than the period-decelerating reaction(s), and, hence,these two sets of enzymatic reactions are balanced to maintaina constant circadian oscillation. In an extreme and ideal case, thebalance theory predicts that the period-accelerating reaction
would be temperature-insensitive (41), which was suggested bythis study.
A Period-Accelerating and Temperature-Insensitive Reaction Is an
Evolutionarily Conserved Design Principle from Cyanobacteria to
Mammals. This observed temperature insensitivity of an enzy-matic reaction is the second example of such a characteristic ina circadian system. A similar temperature insensitivity wasrecently observed in the evolutionarily distinct cyanobacterial
clock system (Q10 ⫽ ⬇1.3 for autophosphorylation of the
cyanobacteria clock protein KaiC) (24). This remarkable similarity between two very different systems implies that temperature-insensitive enzymatic reactions might represent an evolutionarily conserved design principle. We do not insist here that
all individual period-determining reactions in the mammalian
circadian clock are temperature-insensitive, as is observed in the
cyanobacteria clock. Rather, we propose that there are sometemperature-insensitive period-determining enzymatic reactions in the mammalian circadian clock, which is consistent withthe balance theory. Since biochemical reactions have been
believed to be highly sensitive to temperature differences for along time, our results on the existence of temperature-insensitive
reaction in vitro and in cellulo, with possible implications fortemperature compensation of circadian clocks, suggest the surprising capability of CKI␧/␦-dependent phosphorylation. Therefore, remaining challenges are to obtain the atomic resolution
model of this temperature-insensitive reaction, as well as to
Isojima et al.

Materials and Methods
Analysis of the Effects of Compounds on Period Length in Cultured Cells. NIH
3T3-mPer2-Luc cells and U2OS-hPer2-Luc cells were cultured on 24-well(PerkinElmer), 96-well, or 384-well (Falcon) culture plates in medium supplemented with 200 ␮M luciferin (Promega) and a chemical compound withstimulation with 10 nM forskolin (Nacalai Tesque). The time-course bioluminescence data were analyzed as reported previously (42).

assay, the full-length CKI␧ and CKI␦, the catalytic domain of CKI␧, CKI␦, and thetau-mutant of CKI␧ [⌬CKI␧, ⌬CKI␦, ⌬CKI␧ (tau)], and a mutant in which theeight autophosphorylated serine residues in the C-terminal regulatory domain were replaced with glutamate [CKI␧(D8)] purified from bacterial lysate
were used as the enzymes in this assay.
Analysis of Temperature Sensitivity of mPER2 or LUC Degradation in NIH 3T3
Cells. NIH 3T3 cells were transfected with reporter vector (pMU2-mPer2::Luc or
pMU2-Luc) and pMU2-⌬CKI␧(wt) or empty pMU2 using FuGene6. The transfected cells were harvested by trypsin and plated on 24-well culture plates 24 hafter transfection, and the degradation of mPER2::LUC or LUC was measured
as described above for mPer2Luc MEFs.

Measurement of CKI␧ and CKI␦ Enzymatic Activity. CKI activity was measured
by the IMAP assay using the IMAP Screening Express kit (Molecular Devices),
according to the manufacturer’s protocol. For the P81 phosphocellulose paper

ACKNOWLEDGMENTS. We thank K. Inoue, Y. Takamiya, S. Yoshimura, K. Torii,
N. Esumi, and other staff members of the Animal Resource Unit, RIKEN CDB,
for the care of transgenic animals; H. Omori and Y. Uehara, Research Group for
Super Analyzer Development Technology, ASI, RIKEN, for help with theconstruction of the CCD-Tron system; A. Taya for technical support; J.E. Baggs
for technical information about gene knockdown studies using siRNA; Y.
Koyama for valuable comments; J.B. Hogenesch and E.A. Kuld for valuablecomments and critical reading of the manuscript; C. Johnson and M. Rosbash
for information about the historical work on temperature-compensation andfor suggestions for experiments; and S. Onami, the Developmental Systems
Modeling Team, ASI, RIKEN, for total support. This research was supported by
the National Project on Protein Structural and Functional Analyses (RIKEN),
Grant-in-Aid for Scientific Research on ‘‘Development of Basic Technology toControl Biological Systems Using Chemical Compounds’’ from the New Energy
and Industrial Technology Organization (NEDO) (to H.R.U.), Genome Network
Project grant (to H.R.U.) from the Japanese Ministry of Education, Culture,
Sports, Science and Technology, an intramural Grant-in-Aid from the RIKEN
Center for Developmental Biology (CDB), Director’s Fund from CDB (to H.R.U.),and intramural budget from the Genomic Science Center (to T.K.), and CDB(to H.R.U.), RIKEN; and National Institutes of Health Grant P50 MH074924 (toJ.S.T.). J.S.T. is an Investigator in the Howard Hughes Medical Institute.

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Analysis of the Temperature Sensitivity of mPER2 Degradation in the mPer2Luc
MEFs. The mPer2Luc MEFs cultures were maintained on 24-well culture plate at
27, 32, or 37 °C for 4 days, and the bioluminescence from the cells wasmonitored by the PMT-Tron system until the oscillations of the bioluminescence from mPER2::LUC triggered by the temperature change were diminished. The cells were then treated with 400 ␮g/mL CHX. The bioluminescence
was recorded every 30 min for 5 h by the PMT-Tron system. The time course of
the bioluminescence in each well was normalized and analyzed as described
for the degradation rate of the LUC::mPER2 protein in 293T cells (SI Text).

Isojima et al.

PNAS 兩 September 15, 2009 兩 vol. 106 兩 no. 37 兩 15749

CELL BIOLOGY

demonstrate the physiological relevance of this temperatureinsensitive reaction in circadian temperature compensation. Ourin vitro temperature-insensitive reaction system, consisting of ashort peptide, ␤TrCP-peptide, and a monomeric enzyme, CKI␧or CKI␦, would be an ideal experimental platform, which will
provide atomic and molecular information required to test thephysiological relevance of this temperature-insensitive reaction
in circadian temperature compensation.

Table S3. Ten potent chemical compounds that markedly (> 10 s.d.) altered theperiod length in both mouse NIH3T3 and human U2OS clock cells
Abbreviateda
Name
Roscovitine

TG003

e

SB202190

e

PD169316e

SU5416

DRB

e

g

c

FunctiondPotent, selective inhibitor of cyclin-dependent kinases (Cdc2, Cdk2

(Z)-1-(3-Ethyl-5-methoxy2,3-dihydrobenzothiazol-2ylidene)-propan-2-one

Potent, specific, and reversible Cdc2-like kinase (Clk) inhibitor.
Competes with ATP. Highly potent for Clk1 and Clk4, less for Clk2,
and not effective for Clk3 (25)4-[4-(4-Fluorphenyl)-5-(4pyridinyl)-1H-imidazol2-yl]phenol

Highly selective, potent and cell permeable inhibitor of p38 MAP

and Cdk5, but not Cdk4 or Cdk6) (24)kinase alpha and beta (26)Potent, cell-permeable, and selective p38 MAP kinase inhibitor (27)

1,3-Dihydro-3-[(3,5dimethyl-1H-pyrrol-2-yl)
methylene]-2H-indol-2-one

Potent and selective vascular endothelial growth factor (VEGF)

5,6-Dichlorobenzimidazole riboside

Inhibitor of Casein Kinase 2 (29) and CK2-dependent RNA
synthesis9-Chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinazolin-5amine
1,3,5-tris(4-hydroxyphenyl)-4- propyl-1H-pyrazoleg17-OHPf

Structure(R)-2-(1-Ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine

Anthrapyrazolone; 1,9Pyrazoloanthronee

CGS-15943b

4-(4-Fluorophenyl)-2-(4nitrophenyl)-5-(4-pyridyl)1H-imidazolee

SP600125

PPT

e

Name

17alpha-Hydroxyprogesterone

receptor protein tyrosine kinase 1/2 inhibitor (28)

Selective c-Jun N-terminal kinase (c-JNK) inhibitor. Effective for all
family members of JNK (26)Highly potent, non-selective adenosine receptor antagonist thatbinds to human A1, A2, or A3 subtypes but not to rat A3 (30, 31)
Specific estrogen receptor alpha (ERα) agonist. No inhibition to ERβ(32)
Metabolite of progesterone. It binds to membrane progesterone
receptor (33) as well as nuclear progesterone receptor (34)

The abbreviated name (i.e. the one used in this manuscript) (a), name (b), structure (c) and short summary of its function (d) areindicated. These compounds primarily target protein kinase (e), GPCR (f) and steroid hormone receptor (g).

Table S4 u The "effective" chemical compounds that significantly (> 3 s.d. or < - 3 s.d.) alter the period-length ofboth mouse and human clock cells.S
Name aStructure Name b

Function c

Amsacrinehydrochloride

4-(9-Acridinylamino)-N(methanesulfonyl)-m-anisidinehydrochloride

DNA topoisomerase II inhibitor (35)

Ketoconazole

cis-1-Acetyl-4-[4-[[2-(2,4-dichlorophenyl)-2(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4yl]methoxy]phenyl]-piperazine

Potent inhibitor of cytochrome P450c17 enzyme; antifungal agent (36)
GABA-C receptor antagonist; powerful, nonspecific CNS stimulant isolated
from Anamirta cocculin (37)

Picrotoxin
SB 216763

3-(2,4-Dichlorophenyl)-4-(1-methyl-1H-indol3-yl)-1H-pyrrole-2,5-dione

Potent, selective, cell permeable inhibitor of glycogen synthetase kinase-3(GSK-3) (38)

BP 897N-[4-(4-(2-methoxyphenyl)piperazinyl)butyl]2-naphthamide

Partially selective D3 dopamine receptor agonist (39)9-Cp-Ade

9-cyclopentyladenine

Cell-permeable, non-competitive adenylyl cyclase inhibitor; targets the P-site
domain (40)2-Chloroadenosine

6-Amino-2-chloropurine riboside

Adenosine receptor agonist with selectivity for A1 over A2 (41)

Cyproterone acetate

6-Chloro-1beta,2beta-dihydro-17-hydroxy3'H-cyclopropa(1,2)-pregna-1,4,6-triene3,20-dione acetate

Androgen antagonist; synthetic steroid (42)

Chloro-IB-MECA

2-Chloro-N6-(3-iodobenzyl)-adenosine-5'-Nmethyluronamide

A3 adenosine receptor agonist (43)

(R,R)-cis-Diethyl
tetrahydro-2,8chrysenediol(5R, 11R)-5,11-Diethyl-5,6,11,12-tetrahydro2,8-chrysenediol

Potent estrogen receptor beta antagonist; potent partial agonist at estrogen
receptor alpha (44)cis-(Z)-Flupenthixol
dihydrochloride
IB-MECAIIK7
Phenamilmethanesulfonate
SB206553
hydrochloride(Z)-4-[3-[2-(Trifluoromethyl)-9H-thioxanthen9-ylidene]propyl]-1-piperazine-ethanol
dihydrochloride
1-Deoxy-1-[6-[[(3-iodophenyl)
methyl]amino]-9H-purin-9-yl]-N-methyl-betaD-ribofuranuronamide
N-Butanoyl 2-(9-methoxy-6H-isoindolo[2,1a]indol-11-yl)ethanamine
3,5-Diamino-6-chloro-N[imino(phenylamino)methyl]pyrazinecarboxamide methansulfonate
N-3-Pyridinyl3,5-dihydro-5-methyl-benzo[1,2-b:4,5b']dipyrrole-1(2H)-carboxamidehydrochloride

Dopamine receptor antagonist; antipsychotic (45)Selective A3 adenosine receptor agonist (43)Melatonin receptor agonist (46)
Irreversible inhibitor of amiloride-sensitive Na
amiloride (47)+channels; derivative ofPotent 5-HT2C/5-HT2B serotonin receptor antagonist (48)

Trequinsinhydrochloride

HL 725

Phosphodiesterase III (PDE III) inhibitor (49)

CGP 57380

N3-(4-fluorophenyl)-1h-pyrazolo[3,4d]pyrimidine-3,4-diamine

Cell-permeable, selective mitogen-activated protein kinase-interacting kinase
1 (MNK1) inhibitor (50)

(R)-(+)-WIN 55,212-2
mesylate(R)-(+)-[2,3-Dihydro-5-methyl3[(morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4benzoxazinyl]-(1-naphthalenyl)methanone mesylate

High affinity cannabinoid receptor agonist (51)

The alias (Name) (a), structure name (b), and shortly summarized functions of the compounds (c) are listed.
The upper four compounds colored gray are those that shortened the period, and the others that lengthened
the period. See also Table S3, in which "potent effective" compounds that markedly (> 10 s.d.) alter the periodlength of both clock cells are also listed.

Supporting Information
Isojima et al. 10.1073/pnas.0908733106
SI Text
Detailed Procedures of the Screenings of Chemical Compounds That
Affect the Period Length Both in Mouse and Human Clock Cells. As

described in the main text, we examined 1,260 pharmacologically
active chemical compounds from the LOPAC1280 library (SigmaAldrich) (1). We first screened this library with relatively highconcentrations (25 ␮M for NIH 3T3-mPer2-Luc and 50 ␮M for
U2OS-hPer2-Luc) to reduce false negatives. We found 253
compounds for NIH 3T3-mPer2-Luc cells and 294 compounds
for U2OS-hPer2-Luc cells that either altered the period length
by more than 3 s.d. from the negative control or induced
apparent arrhythmicity. The intersection of these data yielded
166 compounds: Six and 40 compounds that shortened andlengthened, respectively, the period length of both cell types, and120 compounds that induced apparent arrhythmicity (including
compounds that were potentially cytotoxic or could affect the
LUC readout) in both cell types (Table S1).
To confirm the efficacy of the 166 compounds selected above,and to exclude potentially cytotoxic or LUC-readout altering
compounds, we conducted dose-dependency assays using thetwo clock cell lines. Three concentrations of each compound
were tested for period-altering effects of approximately one
order of magnitude from the concentration used in the primary
assay (25, 8.3, and 2.8 ␮M for NIH 3T3-mPer2-Luc; and 50, 16.7,
and 5.6 ␮M for U2OS-hPer2-Luc). If the lowest concentration of
a compound induced an apparent arrhythmicity in the cells, two
additional decreases in concentration were tested (0.93 and 0.31
␮M for NIH 3T3-mPer2-Luc; 1.9 and 0.62 ␮M for U2OS-hPer2Luc). This experiment identified nine compounds that significantly shortened (⬍-3 s.d.) and 30 that significantly lengthened(⬎⫹3 s.d.) the period length of NIH 3T3-mPer2-Luc cells, andseven and 90 compounds that shortened and lengthened, respectively, the period length of U2OS-hPer2-Luc cells (Table
S2). After integrating these data across the cell types, we found
four and 24 compounds that respectively shortened and lengthened the period of both cell lines. We designated these 28
compounds as ‘‘effective’’ compounds, and we believe they will
be a useful chemical resource for circadian biology (Table S3 andTable S4). We further examined these compounds to find thosewith the ‘‘potent’’ effects (⬎⫹10 s.d. or ⬍-10 s.d., ⬇2.0–3.0 h)
as described in the main text.
Chemical Landscape of Period Determination in Mammalian Circadian
Clocks. As described in the main text, a chemical-biologicalapproach was recently proposed to help elucidate the basic
processes underlying circadian clocks (2). Working on a relatively small scale, previous studies revealed that several protein
kinase inhibitors [such as IC261 (3, 4), CKI-7 (5), lithium
chloride (6, 7), SP600125 (4, 8), and SB203580 (9)], adenylate
cyclase inhibitors [such as THFA (9-tetrahydro-2-furyl-denine),2⬘5⬘-dideoxyadonosine, and 9-cyclopentyladenine (4)], and proteasome inhibitors [such as MG132 and lactacystin (3)], canlengthen the period of mammalian circadian clocks by 2% to⬇40%. Moreover, high-throughput screening of a large chemicalcompound library (1) supported using a chemical-biologicalapproach to probe the fundamental processes of the mammalian
circadian clock.
This study presents a chemical-biological approach to controlling, analyzing, and exploring the period-determination processes in the mammalian circadian clock. Using this approach, we
identified 28 chemical compounds that significantly (⬎3 s.d.)altered the period length of both human and mouse clock cell
Isojima et al. www.pnas.org/cgi/content/short/0908733106

lines (Table S3 and Table S4). Among them, 10 ‘‘potent’’compounds markedly (⬎10 s.d.) lengthened the period (Fig. S1
A and D), and lengthened it in both peripheral clock cells(MEFs) and central clock tissues (SCN) (Fig. S1 B, C, and E).
These results suggest that our protocol, which featured a highthroughput chemical screening strategy and multiple cell lines ofdifferent origin [NIH 3T3 and U2OS cells were derived from
NIH/Swiss mouse embryo cultures (10) and from human osteocarcinoma cells (11), respectively], and which applied a stringent
threshold for ‘‘potency,’’ enabled the identification of exceptionally potent chemical probes that target fundamental processes inmammalian circadian clocks. By probing this ‘‘chemical landscape,’’ we uncovered ‘‘potent,’’ ‘‘effective,’’ and even ‘‘ineffective’’ compounds, which will serve as a useful guide for drug
researchers seeking cures for rhythm disorders.
So far, this chemical landscape features at least two salient
landmarks: One is the period-determining process targeted by
17-OHP and the other is the period-determining process targeted by CKI␧/␦ activity. Since progesterone and progestins,
synthetic progesterone derivatives such as 17-hydroxyprogesterone caproate and medroxyprogesterone, are widely available asclinical drugs (12), the 17-OHP ‘‘landmark’’ serves as a good
vantage point for putative therapeutics (Fig. S1) to mitigate
rhythm disorders such as advanced sleep phase syndrome(ASPS) (i.e., via an agonist like 17-OHP). While guiding drug
discovery, the landmarks on this revealing chemical landscapewill also be useful for further exploration of the fundamental
properties of mammalian circadian clocks.
Validity of Relatively Higher-Dose Applications of Potent Effective
Compounds in Vitro and in Cellulo. In this chemical-biological study,

we discovered a highly flexible period-lengthening response in
the mammalian clock: A human clock cell’s circadian period
could be stretched into a circabidian period (Fig. 2 A) throughchemical manipulation, probably of the CKI activity, by
SP600125 and TG003. The half-maximum concentrations ofthese compounds for their period-lengthening effect in cellulo
were ⬇22 and 63 ␮M, respectively. In contrast, the IC50s of
SP600125 and TG003 for the CKI␧ kinase activity with 20 ␮M
ATP in vitro were ⬇0.22 and 0.55 ␮M, respectively (Fig. 1E). If
these compounds function as competitive inhibitors for ATP,
this apparent discrepancy can be largely explained by the difference in ATP concentration, which is ⬇1 mM inside cells(13–15). This competitive-inhibition hypothesis is experimentally supported by our observations that higher amounts ofinhibitors were required in assays with a high concentration of
ATP (100 ␮M ATP; Fig. 1D) than with a lower concentration (20␮M ATP; Fig. 1E). Given that the Michaelis constant for ATP
with CKI␧ is ⬇20 ␮M, the ratio of the IC50 with 1 mM ATP (incellulo) to that with 20 ␮M ATP (in vitro kinase assay) is 25.5,
and hence the IC50s of SP600125 and TG003 with 1 mM ATP
should be ⬇5.6 and 14.5 ␮M, respectively. Although there is stilla 4-fold difference between the half-maximum concentrations incellulo and the estimated IC50s with 1 mM ATP, these values arewithin the same order of magnitude. The difference might beowing to the dynamics of these compounds in cellulo, such as theefficiency of their penetration into cells, nonspecific buffering byother proteins within cells, and excretion from cells. Anotherpossibility is that these potent compounds act on other targets inaddition to CKI␧ and CKI␦, such as their ‘‘primary’’ targets,
which might be strongly involved in the period-determining
processes of mammalian circadian clocks. However, the latter1 of 12possibility was not supported by the results of our siRNA
experiments, in which we knocked down kinases including the‘‘primary’’ targets of the potent compounds (Fig. S2).
Temperature-Insensitivity of CKI Phosphorylation Depended on the
Substrate Recognition. CKIs recognize the motif ‘‘Ser/Thr-x-x-

Ser/Thr,’’ and phosphorylate the Ser/Thr residue at the Cterminal end. Negative charge at the N-terminal end of the motif
was thought to be the ‘‘priming signal’’ of the CKI-dependent
phosphorylation, because phosphorylation of the Ser/Thr residue at the N-terminal end or replacement of the residue to theacidic amino acid (Glu or Asp) remarkably improved the efficiency of the CKI-dependent phosphorylation (16). As describedin the main text, phosphorylation of ␤TrCP peptide or FASPSpeptide by ⌬CKI␧(wt) were relatively temperature-insensitive(␤TrCP; Q10 ⫽ 1.0, FASPS; Q10 ⫽ 1.2), although that of CK1tide
was more temperature-sensitive (CK1tide; Q10 ⫽ 1.4) (Fig. 3). It
should be noted that the CK1tide was ‘‘primed,’’ but the former
two substrates were not. Moreover, the tau mutation (R178C) of
CKI␧, which are thought to affect substrate recognition of CKI␧,
resulted to become temperature-sensitive in the phosphoryaltionof the FASPS-peptide (Q10 ⫽ 1.6). These results implied that
substrate recognition by CKI␧/␦ was a crucial process for defining temperature-dependency of CKI␧/␦-dependent phosphorylation. We also noted that priming phosphorylation at a Ser-662
residue of hPER2, associating with FASPS, was regulating
protein stability and nuclear translocation or transcriptional
repressor activity, although a kinase responsible for the priming
phosphorylation have not been identified (17).
SI Materials and Methods
Establishment of a High-Throughput Screening System Using a Cooled
CCD-Camera (CCD-Tron System). To perform high-throughput

screening of a chemical compound library, we developed ascreening system that uses a high-performance CCD camera,
based on a monitoring system using photomultiplier tubes(PMT) described in a previous report (PMT-Tron system) (18).
The round turntable (Mashinax or NEXSYS Corp. ) enables theobservation of up to 12 culture plates at once, and thus 4,608
samples can be screened simultaneously if 384-well culture plates
are used. The computer-controlled turntable rotates and sequentially sets each of the plates under the CCD camera every
5 min. The bioluminescence from the culture plate is imaged by
the high-performance CCD camera (VersArray XP or PIXIS;Roper Scientific). The intensity of each well is calculated fromthe obtained image by a custom-made computer program
Establishment of Stable Transfectants of NIH 3T3 Cells Expressing themPer2-Luc Reporter. The mouse Per2 promoter-driven reporter

plasmid (mPer2-Luc) (19) and the pIREShyg3 plasmid (BDBiosciences) were cotransfected into NIH 3T3 cells with FuGene6 transfection reagent (Roche). NIH 3T3 clones expressing
mPer2-Luc (NIH 3T3-mPer2-Luc) were selected using 200
␮g/mL hygromycin B (Invitrogen).
Establishment of Stable Transfectants of U2OS Cells Expressing thehPer2-Luc Reporter. A 334-bp fragment of the human Per2 genepromoter, 502- to 169-bp upstream from exon 1, was amplified
by PCR with PrimeSTAR DNA polymerase (TaKaRa) and thefollowing primers:
F: 5⬘-GGGGTACCGAGGGCGTAGTGAATGGAAG-3⬘
R: 5⬘-CCCAAGCTTAGCTGCACGTATCCCCTCAG-3⬘
This promoter fragment was subcloned into the reporter
vector, pGL4.14 (Promega) with the restriction enzymes, KpnIand HindIII (TaKaRa). This hPer2 promoter-driven reporter
plasmid (hPer2-Luc) was transfected into U2OS cells (purchasedfrom American Type of Culture Collections; ATCC), and stable
Isojima et al. www.pnas.org/cgi/content/short/0908733106

transfectants (U2OS-hPer2-Luc) were selected as described for
the NIH 3T3 cells.
Preparation of Embryonic Fibroblasts from mPer2Luc Mice. The
mPer2Luc mice (20) were carefully kept and handled according to
the RIKEN Regulations for Animal Experiments. Embryos ofmPer2Luc mice at E13 ⬇15 were excised and washed several times
with Hanks’ balanced salt solution. After the placenta andinternal organs were completely removed, the embryos were cut
into small pieces, and the cells were dissociated by incubation in
0.05% trypsin solution with 0.53 mM EDTA for 30 min at 37 °C.
The dissociated cells (mouse embryonic fibroblasts frommPer2Luc mice; mPer2Luc MEFs) were suspended and cultured in
the growth medium (DMEM supplemented with 10% FBS).
Preparation of SCN Slices from mPer2Luc Mice. The brains were

removed from quickly decapitated, young (older than 4 weeks ofage) mPer2Luc mice, then 300-␮m-thick coronal sections containing the SCN were made using a vibratome type linearslicer(PRO7; Dosaka EM Corp) in ice cold Hanks’ balanced salt
solution (Invitrogen). The slices were then placed on a culture
membrane (MilliCell-CM; Millipore) and set on a dish with 1.2
mL culture medium [DMEM supplemented with 1.2 g/L
NaHCO3 (Nacalai Tesque), 15 mM HEPES (Dojindo), 20 mg/L
kanamycin (Invitrogen), 5 ␮g/mL insulin (Sigma), 20 nM putrescine (Sigma), 100 ␮g/mL apo-transferrin (Sigma), 20 nM
progesterone (Sigma), 30 nM sodium selenite (Sigma), onefiftieth part B-27 supplement (Invitrogen), and 100 ␮M luciferin]
containing the chemical compounds at the concentrations indicated in Fig. S1C.
Analysis of the Effects of Compounds on Period Length in Cultured
Cells and Slices. NIH 3T3-mPer2-Luc cells, U2OS-hPer2-Luc cells,

and mPe2Luc MEFs were cultured on 24-well (PerkinElmer),
96-well, or 384-well (Falcon) culture plates, and 300 ␮m-thickcoronal slices containing the SCN from mPe2Luc mice on aculture membrane (MilliCell-CM; Millipore) in medium supplemented with 200 ␮M luciferin (Promega) and a chemicalcompound with (cells) or without (slices) stimulation with 10 nM
forskolin (Nacalai Tesque). The culture plates were placed in ahigh-sensitivity bioluminescence detection system (LM-2400;
Hamamatsu Photonics, for slices; PMT-Tron for MEFs; or
CCD-Tron, for others), and the bioluminescence of each well
was measured at 30 °C (cells) or 37 °C (slices) (Fig. S1 D and E).
The time-course bioluminescence data were analyzed as reported previously (18).
Rhythmicity and Period Length Analysis of Real-Time Bioluminescence
Data. The rhythmicity and period length were determined fromthe bioluminescence data of NIH 3T3-mPer2-Luc cells, U2OShPer2-Luc cells, MEFs, and SCN slices, as previously reported(18). Although this method could not be used for samples witha period length longer than 36 h, we modified the analysis
method to examine the long periods of circadian clocks in thepresence of high concentrations of potent period-lengthening
compounds (TG003 and SP600125) (Fig. 2 A). The data obtained
36 h after the addition of forskolin and compound, instead of
21 h, were used for analysis to eliminate the first cycle, anddetrending was performed not to detect the periodicity longer
than 72 h, instead of 42 h in usual method. These modifications
enabled us to examine periodicities up to 56 h.
Gene Knockdown Studies Using siRNA in U2OS-hPer2-Luc Cells.

U2OS-hPer2-Luc cells were plated at 3.0 ⫻ 104 cells per well in
24-well multiwell plates, 24 h before the transfection. The
predesigned Silencer Select siRNAs (Ambion) were transfectedwith Lipofectamine 2000 reagent (Invitrogen) as described in the
manufacturer’s protocol. At least three siRNA clones for each
2 of 12gene were examined. The siRNA was used at 5 to 20 pmol/well,and the total amount of transfected siRNA was adjusted to 20
pmol/well using Silencer Select negative control #2 siRNA(Ambion).
A pair of culture plates was prepared for each transfection
condition. The medium on one plate was changed to therecording medium containing 10 nM forskolin and 100 ␮M
luciferin 24 h after the transfection, placed into the PMT-Tronsystem, and the period length was measured as described above.
The total RNA of the cells on the other plate was purified with
TRIzol reagent (Invitrogen), and the cDNA was reversetranscribed using SuperScript II or III (Invitrogen) with random
primers (Invitrogen), as described in the manufacturer’s protocol. The gene expression levels were quantified with the QuantiFast SYBR PCR kit (Qiagen) with a LightCycler 480 (Roche
Applied Science) or PRISM 7900 HT (PerkinElmer), according
to the manufacturers’ protocols.
The ID numbers of the predesigned Silencer Select siRNAs
used in these studies were as follows:
Cry2. s3537; CSNK1E: s57, s58, and s59; CSNK1D: s3627, s3628,
and s3629; MAPK8: s11152, s11153, and s11154; MAPK9: s11158,
s11159, and s11160; CLK1: s3162, s3163, s3164; CLK2: s3165,
s3166, and s3167; CLK4: s32986, s32987, and s32988; CDK2:
s204, s205, and s206; CDK5: s2825, s2826, and s2827; CDC2:
s463, s464, and s465: MAPK14: s3585, s3586, and s3587;
MAPK11: s11155, s11156, and s11157; MAPK12: s12467, s12468,
and s12469; CSNK2A1: s3636, s3637, and s3638; CSNK2A2:
s3639, s3640, and s3641; ADORA1: s1085, s1086, and s1087;
ADORA2A: s1088, s1089, and s1090; ADORA2B: s1091, s1092,
and s1093. In addition, the following custom designed Silencer
Select siRNAs were used:
CLK1: s229161 and s229162; CLK2: s229159 and s229160;
CLK4: s229157 and s221958.
Expression and Purification of mCKI␧/␦ Protein. Mouse CKI␧ ORFs,

corresponding to amino acids 2–416 (full-length CKI␧) and
2–319 (⌬CKI␧), and CKI␦ ORFs, corresponding to amino acids
2–415 (full-length CKI␦) and 2–317 (⌬CKI␦), were cloned into
the pGEX-6P-1 vector (GE Healthcare) and then introduced
into Escherichia coli Rosetta2 (Novagen). A CKI␧ ORF in whicheight serine residues identified as autophosphorylation aminoacids (21) in the C-terminal region were replaced with glutamate, referred to as CKI␧ (D8), was also cloned and introduced
into E. coli. Cells were grown at 25 °C until they reached an
OD600 absorbance of about 0.5. After the addition of isopropyl
␤-D-1-thiogalactopyranoside (IPTG) to a final concentration of
0.1 mM, the cells were further grown at 25 °C for 24 h. The cells
were collected and resuspended in extraction buffer [50 mM
Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, 10%
glycerol, and complete protease inhibitor mixture (Roche)] andhomogenized by sonication. The homogenate was spun at
38,000⫻ g, and the supernatant was applied to a Glutathione
Sepharose 4B column (GE Healthcare). The column was washedwith five column volumes of the buffer. PreScission Protease(GE Healthcare) was then applied to the column to remove the
GST-tag, according to the manufacturer’s protocol. The proteins
were eluted with two column volumes of elution buffer (50 mM
Tris-HCl, pH 7.5, 300 mM NaCl, 1 mM DTT, 1 mM EDTA, and
10% glycerol), and the eluent was diluted and applied to a
HiTrap SP HP column (GE Healthcare). The proteins were theneluted with 600 mM NaCl. The protein concentration wasdetermined by the Bradford method, using the Bio-Rad Laboratories protein assay kit with BSA (Bio-Rad Laboratories) as astandard. The purified proteins used in this study are shown in
Fig. S6 B and C.
Isojima et al. www.pnas.org/cgi/content/short/0908733106

Measurement of CKI Enzymatic Activity. ⌬CKI␧ protein (0.5 ␮M)
was added to the reaction buffer (25 mM Tris-HCl, pH 7.5, 7.5
mM MgCl2, 1 mM DTT, 0.1 mg/mL BSA, and 200 nM synthetic
peptide substrate) with different concentrations of chemicals(25, 50, or 100 ␮M for the potent compounds; 100 ␮M for
IC261), preincubated at 30 °C for 5 min, and then the reaction
was started by adding a one-fourth volume of 400 ␮M ATP.
Aliquots (1 ␮L) of the reaction mixture were withdrawn in
triplicate at 0 and 1 h of the reaction and mixed with 30 ␮L IMAP
binding buffer in 384-well glass-bottomed plates (Olympus).
After incubating for at least 1 h at ambient temperature, thefluorescent polarization was measured using a single-molecule
fluorescence detection (SMFD) system (MF20; Olympus).
The measurement of CKI kinase activity using a P81 phosphocellulose paper assay was performed as follows. Peptides
consisting of 34 aa (RKKKPHSGSSGYGSLGSNGSHEHLMSQTSSSDSN), referred to as ␤TrCP-peptide (22),and 29 aa (RKKKTEVSAHLSSLTLPGKAESVVSLTSQ), referred to as FASPS-peptide, were synthesized and purchasedfrom Bio-Synthesis Inc. CK1tide (KRRRAL[pS]VASLPGL;
Upstate), and ␣-casein from bovine milk (Sigma) were used as
the substrate.
⌬CKI␧/␦ proteins (0.2 ␮M for ␤TrCP-peptide and FASPSpeptide, 0.02 ␮M for ␣-casein, 0.01 ␮M for CK1tide) in thereaction buffer (25 mM Tris, pH 7.5, 7.5 mM MgCl2, 1 mM DTT,
0.1 mg/mL BSA, and 200 ␮M peptide substrate or 40 ␮M casein)
were preincubated for 5 min. The reaction was started by adding
a one-fourth volume of 2 mM (Fig. S6D), 400 ␮M (Fig. 1D), or80 ␮M (Fig. 1E) ATP with 0.5–1 ␮Ci [␥-32P] ATP per reaction.
In assays for autophosphorylated CKI␧/␦ (0.4 ␮M for ␤TrCPpeptide, 0.04 ␮M for ␣-casein, and CK1tide), the enzyme waspreincubated with buffer (25 mM Tris, pH 7.5, 7.5 mM MgCl2,
1 mM DTT, 0.1 mg/mL BSA, 500 ␮M ATP) at 30 °C for 1 h, andthe reaction was started by adding substrate. The preincubationand incubation steps were performed at 25 ° or 35 °C. Twoaliquots (10 ␮L) of the reaction mixture were withdrawn at eachtime point and spotted onto P81 phosphocellulose paper (Whatman) in the case of peptide substrates, or mixed with SDS-PAGE
buffer for casein. The P81 phosphocellulose papers were washedfive times with 75 mM orthophosphate and once with 100%
EtOH, then placed in scintillation vials with scintillation mixture(AQUASOL-2; PerkinElmer). The incorporation of Pi wasquantified using a scintillation counter (LSC-6100; Aloka) or byautoradiography after SDS-PAGE (BAS-2500; Fuji Photo Film).
Analysis of the Degradation Rate of mPER2 Protein in 293T, NIH 3T3,
or mPer2Luc MEF Cells. The intensity of the LUC::mPER2 from293T cells, mPER2::LUC from NIH 3T3 cells, or mPer2Luc
MEFs was normalized so that the average luminescence detected
at t ⫽ 0 was defined as ’100⬘. To calculate the half-life of
LUC-fused protein, the normalized results were fitted with theequation,y ⫽ ae⫺kt ⫹ 共100 ⫺ a兲y, normalized bioluminescence intensity; t, time after addition of
cycloheximide (CHX); a, k, variables. e, Napier’s constant.
And the half-life t1/2 was calculated as follows:
t1/2 ⫽ ⫺ln冉冊

共a ⫺ 50兲 1.
akAnalysis of the Temperature Sensitivity of mPER2 Degradation in themPer2Luc MEFs. The cultures were maintained at 27 °, 32 °, or 37 °C

for 4 days, and the bioluminescence from the cells was monitoredby the PMT-Tron system until the oscillations of the bioluminescence from mPER2::LUC triggered by the temperaturechange were diminished. The cells were then treated with 400
3 of 12␮g/mL CHX. The bioluminescence was recorded every 30 min
for 5 h by the PMT-Tron system. The time course of thebioluminescence in each well was normalized and analyzed as
described for the degradation rate of the LUC::mPER2 protein
in 293T cells.

cated times after adding 200 ␮g/mL CHX. The LUC activity wasmeasured using the Dual-Glo bioluminescence detection kit(Promega) following the manufacturer’s protocol. The degradation rate was calculated as described above.
Western Blot of mPER2 Protein Expressed in 293T Cells. 293T cells,

Construction of Expression Vectors for a Reporter Assay to Examine
Clock-Related Protein Stability. To investigate the stability ofclock-related proteins (PER2, PER1, and BMAL1) in cultured
293T cells, we constructed an expression plasmid bearing thefirefly Luciferase protein fused to the N-terminal end of clockrelated proteins (LUC::mPER2, LUC::mPER1, and
LUC::mBMAL1) under the CMV promoter (CMV-Luc::mPer2,
CMV-Luc::mPer1, and CMV-Luc::mBmal1). We also constructed an expression vector bearing the firefly luciferase
protein fused to the C-terminal end of the mouse Per2 protein(mPER2::LUC) using a custom expression vector pMU2 (18) for
studies in cultured NIH 3T3 cells (pMU2-mPer2::Luc). The
cDNAs of these clock-related proteins were kindly provided by
Dr. H. Tei (Mitsubisi Kagaku Institute for Life Sciences, Japan).
A control expression vector expressing the LUC protein under
the CMV promoter (CMV-Luc) was also constructed.
The coding sequence of mCKI␧(wt) was subcloned into thepMU2 expression vector as previously described [pMU2mCKI␧(wt)]. An expression vector for the mCKI␧(tau) mutant(in which Arg-178 was replaced with Cys) was constructed byinverse PCR using pMU2-mCKI␧(wt) as a template. Inverse
PCR was performed using the following PCR primers.
F: 5⬘-TGCTATGCCTCTATCAACACCCAC-3⬘
R: 5⬘-GGCAGTGCCGGTCAGGTTCTTG-3⬘ (the 5⬘ end
was phosphorylated).
Finally, self-ligation of the PCR product was performed. The
resultant vector was named pMU2-mCKI␧(tau).
An expression vector bearing only the catalytic domain of
mCKI␧ (⌬mCKI␧) was also constructed. The coding sequence ofthe regulatory domain of mCKI␧, corresponding to amino acids
320 to 416, was deleted from pMU2-mCKI␧(wt) by inverse PCR.
The resultant vector was named pMU2-⌬mCKI␧(wt).
The mCKI␧(wt), mCKI␧(tau), or ⌬mCKI␧(wt) gene was fusedin-frame with 1⫻ Flag Tag at the N terminus, and regulated by
the CMV promoter.

grown in DMEM supplemented with 10% FBS and antibiotics(25 U/mL penicillin, 25 mg/mL streptomycin; Invitrogen), wereplated at 4 ⫻ 105 cells per dish in 35-mm dishes. Twenty-fourhours later, the cells were transfected with FuGene6 (Roche),according to the manufacturer’s instructions. The cells weretransfected with 2 ␮g pcDNA3-Myc-mPer2 (a gift from Dr. E.
Nishida, Kyoto University) (23), 2 ␮g pMU2-mCKI␧(tau), or
empty plasmid pMU2, and 2 ␮g pMU2-Luc. After 24 h, the cells
transfected with the mCKI␧(tau) expression vector were harvested, combined, and then replated onto the same number of
35-mm dishes to normalize for variations in the transfection
efficiencies due to different plates. After 48 h, the cells weretreated with or without 100 ␮M TG003 or SP600125 in 0.2%
DMSO (Nacalai Tesque). The treated cells were harvested at thetimes indicated in Fig. S4, lysed with the extraction reagent [50mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS, Halt Protease
Inhibitor Mixture, EDTA-free (Pierce)], and sonicated. The
protein concentration of the samples was measured by the
Bradford method using a Bio-Rad Laboratories protein assay kit(Bio-Rad Laboratories). Equal amounts of protein (0.25 mg)
were then mixed with NuPAGE LDS sample buffer (Invitrogen),and the samples were separated by electrophoresis and transferred to PVDF membranes using the NuPAGE electrophoresis
system (Invitrogen), as described in the manufacturer’s protocol.
Myc-mPER2 protein was detected with an anti-Myc monoclonal
antibody (clone A-14; Santa Cruz Biotechnology.) and HRPconjugated anti-rabbit IgG secondary antibody (GE Healthcare). An anti-tubulin␣ monoclonal antibody (clone DM1A;LAB VISION) and anti-mouse IgG secondary antibody (GEHealthcare) were used to detect tubulin␣ protein. The immunoreactivities were visualized and quantified using the ECL Plus
Western Blotting Detection system (GE Healthcare) and LAS1000 (Fujifilm), according to the manufacturers’ instructions.
Analysis of Temperature Sensitivity of mPER2 or LUC Degradation in
NIH 3T3 Cells. NIH 3T3 cells cultured on 100-mm plates werefected with expression vectors for LUC::mPER2, CKI␧(tau), andRenilla Luciferase were subcultured on 96- or 384-well culture
plates. The stability of the LUC::mPER2 protein was measured
after a 24-h incubation with a chemical compound, and thedegradation rate of LUC::mPER2 was determined at the indi-transfected with 30 ␮g reporter vector (pMU2-mPer2::Luc or
pMU2-Luc) and 6 ␮g pMU2-⌬CKI␧(wt) or empty pMU2 using
60 ␮L FuGene6. The transfected cells were dissociated by trypsin
and plated on 24-well culture plates 24 h after transfection, andthe degradation of mPER2::LUC or LUC was measured as
described above for mPer2Luc MEFs.

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D

SB202190

SU5416SP600125

PPT

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

TG003

PD169316

DRB

CGS-15943

17-OHP

Bioluminescence

40
36
32
28
24

20

50

0
10

0
70

0
10

0
60

0

2

0
48

96

0

48

0 10 20 30 40 50

0 10 20 30 40 50

B

Luc

mPer2
Roscovitine

MEF

SU5416

SB202190

SP600125(A.U.)

0 10 20 30 40 50

Bioluminescence

NIH3T30 10 20 30 40 50

96 144

0
0

48

0

96

NIH3T3

U2OS
20

80

2
12

0
100

0
16

0
70

2

0
0

48

96

0

96 144

U2OS

60

2

48

PD169316

20

PPT

40

U2OS

60

SB202190

Concentration (µM)

48

96 144

0
0

48

96

0

48

96 14436

0 5 10 15 20 25

0 5 10 15 20 25

TG003

PD16931640

0

5 10 15 20 25

0 5 10 15 20 25

0 5 10 15 20 25

CGS-15943

17-OHP

DRB

20

36

32

28

24
0 5 10 15 20 25

0 5 10 15 20 25

0 5 10 15 20 25

0 5 10 15 20 25

18

60

4
12

0
60

2
12

0
70

2
48

SCN

SU5416SP600125

PPT

32

32

Period Length (h, real)

28

24

24
0 20 40 60 80 100 0 20 40 60 80 100

TG003

PD169316

DRB

0 5 10 15 20 25

0 20 40 60 80 100

CGS-15943

17-OHP

32

32

28

28

24
5 10 15 20 25

0

5 10 15 20 25

0 5 10 15 20 25

10
0

96 14448

0 5 10 15 20 25

96

NIH3T3

U2OS
24

60

2
16

0
60

4
14

10
60

2

2

0
48

96

0

0 5 10 15 20 25

48

96 14448

96

0

NIH3T3

U2OS

60

2
16

0
50

0
8

0
50

0

0

2.8 µM
8.3 µM
25.0 µM

96

96 144

U2OS

14

48

48

17-OHP60

0

96 144

10
0

24

2

48

U2OS

60

NIH3T3

Concentration (µM)

0

CGS-15943

PPT

24
0

48

30

0

Bioluminescence28

0 5 10 15 20 25

0

NIH3T3(A.U.)

SB202190

96

SP600125
Bioluminescence

Roscovitine

2

10
0

(A.U.)

Luc

mPer2

U2OS

50

Concentration (µM)

C

NIH3T3

U2OS

18

20

0 5 10 15 20 25

DRB

NIH3T3

Bioluminescence28

24SU5416

Period Length (h, real)32

Period Length (h, CT)

TG003
NIH3T3

U2OS

20

0

40
36
32
28
24
0 10 20 30 40 50

Period Length (h, CT)

Roscovitine
NIH3T3(A.U.)

Period Length (h, CT)

NIH3T3-mPer2-Luc / U2OS-hPer2-Luc
Roscovitine(A.U.)

A

0

48

96 1445.6 µM
16.7 µM50.0 µM

0
0

48

96

0

48

96 144

Time (hr)

E
TG003
MEF

SCN
80000

0

0
0

60

120

30000

0
0 48 96 144 192

060

SB202190
MEF

MEF

80000

4000

0

60

120

SCN
120000

7000

10000

0

30000

0
0 48 96 144 192

0

60

(cpm)

Bioluminescence

MEF

DRB
MEF

SCN
80000

0

60

20000

0
0 48 96 144 192

120

0

60

MEF
(cpm)

Bioluminescence

SP600125
MEF

80000

0
60

20000

0
0 48 96 144 192

120

0

60

MEF
(cpm)

Bioluminescence

PPT
4000

MEF

SCN

6.25 µM
12.5 µM
25.0 µM50.0 µM
100.0 µM

60

120

SCN

4000

30000

0

70000
0

0 48 96 144 192

120

17-OHP

250000

0

SCN
90000

6000

20000
0

0 48 96 144 192

120

CGS-15943
SCN

8000

SCN
110000

4000

10000

0

0 48 96 144 192

120

SU54164000

0 48 96 144 192

120

PD169316
SCN

(cpm)

Bioluminescence

SCN
110000

5000

(cpm)

Bioluminescence

Roscovitine
MEF
6000

0 48 96 144 192

15000
0

60

120

0 48 96 144 192

Time (hr)Fig. S1. Dose-response of period length in the NIH 3T3-mPer2-Luc and U2OS-hPer2-Luc cells (A), primary cultures of MEFs (B), slice cultures of SCN (C) frommPer2Luc mice, and raw bioluminescence data (D and E). The period length were represented both in real time (right axis in B and C) and in circadian time (i.e.,
the period length of control samples was adjusted to 24 h, left axis). Blue circles in (A) represented average of NIH 3T3 (n ⫽ 2) and red squares U2OS (n ⫽ 3). Eachline represented independent experiments. Each value in (B and C) represents the mean ⫾ SEM. At the concentrations without data points, the cells behaved
arrhythmically. (D) Time course of the reporter activity in NIH 3T3-mPer2-Luc cells (left panels) and U2OS-hPer2-Luc cells (right panels) after forskolin stimulationand the addition of compound. The gray lines represent the average of control samples (n ⫽ 768 for NIH 3T3, and n ⫽ 96 for U2OS) and colored lines represent
the averages of samples containing compounds at the concentrations indicated at the base of the figure (n ⫽ 2 for NIH 3T3, and n ⫽ 3 for U2OS). The compound
name is displayed at the top of each set of four panels. The upper and lower panels within each group indicate separate experiments. (E) Time course of thereporter activity in MEFs (left panels) and SCN slices (right panels) after stimulation with compound with (MEFs) or without (SCN) forskolin. The gray lines
represent the average of control samples (n ⫽ 5 for MEF, and n ⫽ 4 for SCN) and colored lines represent the average of samples containing compound at theconcentrations indicated at the bottom of the figure (n ⫽ 3 for MEF, and n ⫽ 4 for SCN). The compound represented in the graphs is written above each pairof panels.

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Fig. S2. Effects of gene knockdowns on the period length in U2OS-hPer2-Luc cells. (A) Relative expression levels of CKI␧, CKI␦ and genes related to the 10 potent
chemical compounds that lengthened the period length (see Table S3), examined by the quantitative real-time PCR method. The graph indicates the average⫹ SEM of the gene expressions relative to GAPDH (n ⫽ 6). The expression of the FLT1 gene was undetectable. (B–G) Relationship between gene knockdown effects
and period length in U2OS-hPer2-Luc cells. Two sets of U2OS-hPer2-Luc cells were transfected with predesigned Silencer Select siRNA: One was cultured on the
PMT-Tron system for measurement of the period length, and the other was analyzed for gene expression levels 24 h after the transfection. The x-axis indicatesthe expression level of genes relative to the samples transfected with control siRNA. The y-axis indicates the period length, described in circadian time (CT), withthe control samples assigned as 24 h. Each symbol represents the mean ⫾ SEM. of independent experiments (n ⱖ 3). (B) Gene knockdowns of kinases inhibitedby SP600125. MAPK8, mitogen-activated protein kinase 8 [MAPK8, also c-Jun N-terminal kinase 1 (JNK1)]; MAPK9, MAPK9 (JNK2). (C) Gene knockdowns of kinases
inhibited by TG003. CLK1, CDC-like kinase 1 (CLK1); CLK2, CLK2; CLK4, CLK4. (D) Gene knockdowns of kinases inhibited by Roscovitine. CDK1, Cyclin-dependent
kinase 1 (CDK1); CDK2, CDK2; CDK4, CDK4. (E) Gene knockdowns of kinases inhibited by SB202190 and PD169316. MAPK14, MAPK14 [also p38 MAPK ␣ (p38␣)];
MAPK11, MAPK11 (p38␤); MAPK12, MAPK12 (p38␥). (F) Gene knockdowns of kinases inhibited by DRB. CSNK2A1, CK2␣; CSNK2A2, CK2␣⬘. (G) Gene knockdownsof adenosine receptors inhibited by CGS-15943. ADORA1, Adenosine A1 receptor; ADORA2A, Adenosine A2a receptor; ADORA2B, Adenosine A2b receptor

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Fig. S3. Effect of the potent effective compounds on the stability of mPER2 proteins in cellulo. (A) mPER2 stabilization in 293T cells. Bioluminescence from 293T
cells cotransfected with a reporter (LUC::mPER2 or LUC) and CKI␧(tau) was measured, and the ratio of the bioluminescence of LUC::mPER2 to LUC was plotted.
Each value represents the mean ⫾ SEM. (n ⫽ 16 for 0 ␮M, n ⫽ 4 for chemically treated samples). All of the compounds except 17-OHP exhibited a significant
dose-dependent effect (P ⬍ 0.01; one-way ANOVA). (B) mPER2 degradation rate in 293T cells. The averages (n ⫽ 3; closed circles) of the normalizedbioluminescence of 293T cells cotransfected with the LUC::mPER2 reporter and CKI␧(tau) after treatment with CHX in the presence (green) or absence (gray) ofa chemical compound were plotted. The fitted curve is also indicated in each panel. The time course of all of the samples except 17-OHP was significantly different
from that of the control samples (P ⬍ 0.001; two-way ANOVA). (C) The potent inhibitors dominantly affected the CKI-dependent degradation of LUC::mPER2
but not the CKI-independent degradation. The average ⫾ SEM. (n ⫽ 3) of the normalized bioluminescent activities of 293T cells cotransfected with the
LUC::mPER2 reporter and CKI␧(tau) (right panel) or control vector (left panel) with 100 ␮M SP600125 (red squares), TG003 (blue diamonds), or solvent only (0.25%
DMSO; gray circles) after treatment with CHX was plotted. The fitted curve is also indicated in each panel.

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Fig. S4. Effects of SP600125 and TG003 on mPER2 stabilization in 293T cells. (A) 293T cells cotransfected with Myc-tagged mPER2 (Myc-mPER2) and CKI␧(tau)were incubated with or without 100 ␮M SP600125 (Left) or TG003 (Right) for the indicated times, and the lysates were collected and analyzed by Western blotting.
Equal portions of three samples were mixed and analyzed in each lane. Myc-mPER2 was detected with an anti-Myc antibody (upper panels) and Tubulin was also
blotted as an internal control (lower panels). Note that the two compounds inhibited the mobility shift of mPER2 caused by the coexpression with CKI␧(tau). (B)Quantification of the results of (A). The densities of immunoreactive bands were measured, and the density of Myc-mPER2 was normalized to that of Tubulin
in the same sample (Myc-mPER2/tubulin). The graph represents the relative value of Myc-mPER2/Tubulin at the indicated incubation times with 100 ␮M SP600125(Left) or TG003 (Right) compared with the control (DMSO) samples.

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Fig. S5.
CKI␧(tau) destabilizes the LUC::mPER2 and LUC::mPER1 proteins but not LUC or LUC::mBMAL1. (A) Effects of CKI␧(tau) on the stability ofLuciferase-fused clock proteins in 293T cells. The Luciferase activity in 293T cells transfected with the expression vector for LUC, LUC::mPER2, LUC::mPER1, or
LUC::mBMAL1, and CKI␧(tau) was measured 48 h after transfection. Note that the co-expression with CKI␧(tau) attenuated the bioluminescent activity of
LUC::mPER2 and LUC::mPER1, but not of LUC or LUC::mBMAL1. Each value represents the mean ⫹ SEM (n ⫽ 4). (B) Effects of CKI␧(tau) on the degradation ratesof Luciferase-fused clock proteins in 293T cells. The average ⫾ SEM. (n ⫽ 3) of the normalized bioluminescent activities of 293T cells cotransfected with LUC-fusedclock protein reporters (LUC::mPER2, LUC::mPER1, or LUC::mBMAL1) and CKI␧(tau) (circles) or control vector (squares) after treatment with CHX was plotted. The
fitted curve is also indicated in each panel.

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Fig. S6. Temperature insensitivity of the CKI␧ phosphorylation activity. (A) Schematic diagram of PERIOD2 (PER2) and the sequence alignment around the
␤TrCP-binding (SSGYGS) and FASPS motif. Locations of the PAS domains (light blue), the ␤TrCP-binding motif (orange), the CKI-binding domain (red), and the
CRYPTOCHROME-binding domain (yellow) are indicated. Location corresponding to the amino acid sequence of the synthetic peptide substrate is indicated bybars above the diagram. Sequence corresponding to the ␤TrCP-peptide (amino acids 473–502, a serine-rich region around the ␤TrCP-binding motif in PER2) and
FASPS-peptide (amino acids 642– 666, a region around FASPS-related phosphorylation sites) are underlined in the aligned sequence. (B) The catalytic domains
of CKI␧ (wt) and CKI␧ (tau) [⌬CKI␧(tau) and ⌬CKI␧(wt)] and full-length CKI␧ were purified as described in the Materials and Methods, separated by electrophoresis,
and stained with Coomassie Brilliant Blue. (C) Autophosphorylation of CKI␧ before performing in vitro kinase assays. Dephosphorylated CKI␧ was incubated withor without ATP for 60 min. Note that CKI␧ was phosphorylated by the incubation with ATP (right-most lane). Lanes ‘‘M’’ in both (B) and (C) were standard
molecular weight markers. (D) Phosphorylation of FASPS-peptide by ⌬CKI␧(wt) (circles) and ⌬CKI␧(tau) (squares) was assayed at 25 °C (blue) and 35 °C (red). (Inset)
Expanded plot of the ⌬CKI␧(tau) phosphorylation activity. Assay conditions are described in the experimental procedures.

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Table S1
Table S2
Table S3
Table S4

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