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Design Principles of PhosphorylationDependent Timekeeping in Eukaryotic
Circadian Clocks
Koji L. Ode1,2 and Hiroki R. Ueda1,2
1

Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1,
Bunkyo-ku, Tokyo 113-0033, Japan

2

Laboratory for Synthetic Biology, RIKEN Quantitative Biology Center, 1-3 Yamadaoka, Suita, Osaka
565-0871, Japan

Correspondence: uedah-tky@umin.ac.jp

The circadian clock in cyanobacteria employs a posttranslational oscillator composed of asequential phosphorylation–dephosphorylation cycle of KaiC protein, in which the dynamics
of protein structural changes driven by temperature-compensated KaiC’s ATPase activity are
critical for determining the period. On the other hand, circadian clocks in eukaryotes employ
transcriptional feedback loops as a core mechanism. In this system, the dynamics of protein
accumulation and degradation affect the circadian period. However, recent studies of eukaryotic circadian clocks reveal that the mechanism controlling the circadian period can be
independent of the regulation of protein abundance. Instead, the circadian substrate is often
phosphorylated at multiple sites at flexible protein regions to induce structural changes. The
phosphorylation is catalyzed by kinases that induce sequential multisite phosphorylationsuch as casein kinase 1 (CK1) with temperature-compensated activity. We propose that thedesign principles of phosphorylation-dependent circadian-period determination in eukaryotes may share characteristics with the posttranslational oscillator in cyanobacteria.

T

he circadian clock produces near-24-hrhythms in physiological activities. From
multicellular animals to plants, circadian clocks
regulate cellular metabolic pathways and otherphysiological responses of the organism, such as
sleep–wake cycles and immune responses (Leviand Schibler 2007; Bass and Takahashi 2010;
Mohawk et al. 2012; Albrecht 2013; Greenhamand McClung 2015; Lu et al. 2017; Sanchez and
Kay 2017). The evolutionary advantage of a circadian clock was demonstrated by coculture experiments with prokaryotic cyanobacteria, inwhich strains with a circadian period matching
the light–dark cycle of their environment became dominant in the population (Woelﬂe
et al. 2004). This advantage should apply tomany species; circadian rhythmicity of physiological activities is observed in evolutionarily
distant species. Conserved features of the molecular mechanisms that drive circadian clocks
in different phyla have been revealed throughstudies in a variety of organisms, including conventional models such as rodents in mammals,
Drosophila melanogaster in insects, Arabidopsis

Editors: Paolo Sassone-Corsi, Michael W. Young, and Akhilesh B. Reddy
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K.L. Ode and H.R. Ueda

thaliana in higher plants, Neurospora crassa infungi, and Synechococcus elongatus in cyanobacteria. In all organisms, circadian rhythmicity is cell-autonomous. Even in mammals,various cell types show robust circadian rhythmicity in gene expression and physiological activity, even when cultured in vitro (Welsh et al.
1995; Yagita et al. 2001; Nagoshi et al. 2004;
Yoo et al. 2004). Cell-autonomous circadianoscillation is produced by the system-level orchestration of transcription–translation feedback and/or a posttranslational interaction network (Gallego and Virshup 2007; Ukai andUeda 2010; Millius and Ueda 2017), but themolecular components of the oscillators especially for transcriptional or translational regulators differ between species (Rosbash 2009;
Brown et al. 2012; van Ooijen and Millar
2012; Takahashi 2017). However, the question
arises whether a common design principle underlies the molecular components of circadianclocks in different organisms.
In most organisms, circadian molecular oscillators seem to be based on transcription–translation feedback loops (TTFLs). However,
how circadian clocks maintain a robust 24-hoscillation is a mystery. The suprachiasmatic
nucleus, a region of the brain central to circadian rhythm in mammals, can sustain oscillating
gene expression for years when cultured in vitro(Yamazaki and Takahashi 2005). However, theperiodic gene expression in artiﬁcial biologicaloscillators encoded as TTFLs is less stable whensubjected to cell-intrinsic or cell-extrinsic
changes (Elowitz and Leibler 2000; Danino
et al. 2010). This observation suggests that anadditional layer exists in the period-keeping
mechanism of circadian cellular oscillators.
Here, we review the circadian-clock features invarious organisms from the perspective of
TTFL-based design principles and posttranslational modiﬁcations of oscillator components.
Through the comparison between a phosphorylation-based oscillator in cyanobacteria andmultisite phosphorylation of clock componentsthat can regulate the circadian period in eukaryotes, we propose that shared design principles
can be found in the circadian period-determination mechanisms.
2

TRANSCRIPTION NEGATIVE FEEDBACK IN
EUKARYOTIC CIRCADIAN OSCILLATORS

Figure 1 summarizes the major molecular components of circadian oscillators in different phyla. Eukaryote oscillators have conserved negative
feedback loops in the transcription–translation
network (Fig. 1A). The PERIOD protein (PER)
is a key transcriptional repressor in mammals
and Drosophila. PER binds and inhibits a basic
helix–loop–helix transcription-activator complex consisting of CLOCK and BMAL1 in mammals (Fig. 1B). In Drosophila, PER inhibits acomplex of CLOCK (CLK) and CYCLE (CYC),a BMAL1 ortholog (Fig. 1C). The CLOCK–BMAL1 (CLK-CYC in Drosophila) complex activates Per transcription; thus, the overall transcription network creates a negative feedback
loop. PER binds other partners in various organisms to act on transcription activators. In Drosophila, PER represses its own transcription bybinding TIMELESS (TIM), the vertebrate homolog of which maintains chromosomal integrity (Chou and Elledge 2006; Tanaka et al. 2009).
In contrast, mammalian PER forms a complex
with CRYPTOCHROME (CRY). The cryptochrome superfamily is widely conserved frombacteria to plants, but nonmammalian cryptochrome functions as a blue-light receptor rather
than as a circadian transcription repressor (Linand Todo 2005; Ozturk et al. 2007). Phylogenetically, the closest homolog of mammalian cryptochrome is 6–4 photolyase, which catalyzes therepair of damaged DNA residues using energy
from blue light. These functional divergence of
TIM and CRY in maintaining DNA integrity
and circadian rhythmicity may be related tothe evolutionarily close relationship of DNAdamage responses and circadian clocks in anenvironment with daily oscillations in sunlight.
The molecular components of circadiannegative feedback loops differ between fungiand plants. White Collar complex (WCC) andFREQUENCY (FRQ) are the primary drivers of
Neurospora circadian clocks (Fig. 1D). WCC activates FRQ transcription, which in turn inhibits
WCC and creates the negative feedback loop.
The canonical core feedback architecture of
the circadian clock in plants is more complex

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Phosphorylation-Dependent Timekeeping in Eukaryotes

A Canonical design principle

P
X

B Mammals

C Fruit fly

P

P

P

X genePer gene
Cry gene

BMAL1
CLOCK

D Fungi

CRY
P
PER P

TIM
P
PER P

per gene
tim gene

CYC
CLK

E Higher plants

P
FRQ

LHY
P
CCA1 P

P
EC
PRRs
TOC1

WC1
WC2

frq gene

CCA1 gene
LHY gene

Figure 1. Shared structure of transcription–translation feedback loop (TTFL)-based eukaryotic circadian clocks.
(A) Diagram showing the basic structure of a circadian negative-feedback transcription oscillator. The transcription repressor is regulated by multisite phosphorylation and proteasome-mediated proteolysis. (B–E) Major
components of the circadian TTFL-based oscillator in each species. Note that the indicated components andpathways were selected based on the context of this review; the actual circadian systems are far more complex
reaction networks involving numerous gene products. EC (evening complex) composed of ELF4, LUX, and ELF3.

(Fig. 1E). In Arabidopsis, the canonical model
involves transcription feedback loops around
two transcription repressors, CCA1 and LHY,
which suppress the transcription of both activators and inhibitors of CCA1/LHY expression,
creating an architecture combining positive
and negative feedback.
Theoretical studies emphasize the importance of a negative feedback loop encoded as a
TTFL in autonomous oscillation (Novak andTyson 2008; Kim 2016). In a general sense, theperiod of TTFL-based oscillators is determinedthrough the dynamics of protein quantity: therepression phase is initiated by the accumulation of transcription repressor(s) and ceases
when the repressors have disappeared. Indeed,
the circadian period is reported to be regulated
through the delayed timing ( phase) of the accumulation of mammalian transcriptional repressor Cry messenger RNA (mRNA) (Ukai-Tadenuma et al. 2011) or Per mRNA in mammals
and Drosophila (Kadener et al. 2008; Fustin et al.

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K.L. Ode and H.R. Ueda

2013). These changes are regulated by the timing of transcription and the mRNA maturation
process, independent of the biochemical characteristics of the CRY/PER proteins themselves.
However, evidence suggests that the dynamics ofmRNA abundance are less important in eliciting
rhythmic transcription; noncircadian expression of the per gene in Drosophila (Ewer et al.
1988; Frisch et al. 1994; Vosshall and Young
1995; Yang and Sehgal 2001) and a constitutive
supply of CRY protein in mammals can support
circadian oscillation (Fan et al. 2007; Ukai-Tadenuma et al. 2011), indicating the importanceof protein-level dynamics in the regulation of
circadian clocks. Consistent with this view,
rhythmic changes in mammalian PER levels
were observed when PER was expressed undera constitutive promoter (Yamamoto et al. 2005;
Fujimoto et al. 2006; Nishii et al. 2006).
In protein-level regulation of the circadianperiod, several theoretical models predict theimportance of transcriptional repressor degradation rates (Gerard et al. 2009). In a simpliﬁed
negative feedback TTFL model such as theGoodwin model, accelerating the degradation
rate shortens the circadian period (Forger2011), and this relationship is observed in model organisms. The degradation rate of corecircadian components is ﬁne-tuned by theirphosphorylation and subsequent proteasomedependent degradation. In mammals and Drosophila, the circadian period is shortened or
lengthened by mutations that destabilize or stabilize PER, respectively (Vanselow et al. 2006;
Chiu et al. 2008; Meng et al. 2008; Syed et al.
2011). Mammalian CRY also regulates the circadian period; genetic and pharmacologicalperturbations that stabilize CRY lengthen the
period (Siepka et al. 2007; Hirota et al. 2012;
Gao et al. 2013; Hirano et al. 2017), while destabilizing CRY shortens it (Hirano et al. 2013,2016; Yoo et al. 2013). The causal relationshipbetween CRY degradation and a shorter period
was demonstrated by tuning the period throughCRY-speciﬁc artiﬁcial proteolysis (Ode et al.
2017). In Neurospora, FRQ stability is also
well correlated with the circadian period (Liuet al. 2000; Gorl et al. 2001; Baker et al. 2009;
Tang et al. 2009).
4

Recent ﬁndings, however, indicate that period-determination processes can be independentof transcriptional-repressor clearance rates. Deleting a ubiquitin-ligase component leading to
the proteolysis of FRQ stabilized FRQ but, surprisingly, had little effect on rhythmic FRQ expression (Larrondo et al. 2015). Several mutant
alleles of mammalian CRY1 altered the circadian period but had little effect on CRY1’s stability(Ode et al. 2017). One chemical biology study
designed a compound that stabilizes CRY1 andshortens the circadian period (Oshima et al.
2015). These studies suggest that the regulationof protein quantity (the rates of protein production and degradation) does not tell the entire
story of circadian-period determination, and aquestion remains about the general applicabilityof the TTFL model in determining circadianperiods (Gallego and Virshup 2007; Blau 2008).
POSTTRANSLATIONAL CIRCADIANOSCILLATOR DESIGN PRINCIPLESIN CYANOBACTERIA

The prokaryotic cyanobacterial circadian clock
is an example of circadian-period determination
independent of protein-quantity dynamics. This
clock is driven by the proteins KaiA, KaiB, andKaiC (Ishiura et al. 1998). KaiC represses transcription, and its targets include KaiC itself.
Thus, it was proposed that KaiC forms a TTFL
(Fig. 2A). However, a subsequent study showedthat the circadian clock in cyanobacteria can operate in the absence of global transcription andtranslational activities (Tomita et al. 2005). Indeed, autonomous circadian rhythmicity in thephosphorylation status of puriﬁed KaiC can bereconstituted by incubating a mixture of KaiC,
KaiA, and KaiB with ATP in vitro (Nakajima
et al. 2005). Thus, KaiC provides a posttranslational oscillator (PTO) as well as a TTFL-based
negative-feedback architecture. The coupling of
PTO and TTFL-based oscillators appears to beimportant for cyanobacteria circadian clocks,
depending on growth conditions (Kitayama
et al. 2008; Teng et al. 2013). PTO rhythmicity
requires ﬁne-tuning the expression of KaiA,KaiB, and KaiC at speciﬁc ratios for oscillationin vitro (Nakajima et al. 2010). Their expression

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Phosphorylation-Dependent Timekeeping in Eukaryotes

A

P
KaiC P

B
P
KaiC
Autophosphorylation

Autophosphorylation
P
KaiC P

KaiC

Auto-dephosphorylation
KaiC gene

Auto-dephosphorylation
KaiC PCATP
ATPaseSequential (de-)phosphorylation
P P
P

ADP
ATP hydrolysis

Local structural change(temperature compensated)

Changes in protein–protein interactions andenzymatic activities

Figure 2. The KaiA/B/C-dependent posttranslational circadian oscillator in cyanobacteria. (A) KaiC represses its
own transcription, and thus forms a transcriptional negative-feedback loop. Although every reaction should bereversible, biased reaction rates between antagonizing kinase/phosphatase reactions are required to create aposttranslational oscillator (PTO). (B) A mixture of KaiA/B/C with ATP can reconstitute a PTO in vitro. (C)The period-determining step of the KaiC oscillator is temperature-insensitive ATP hydrolysis, which induces astructural change in KaiC.

is also regulated by translational efﬁciency: nonoptimal codon usage in kaiBC genes tunes theamplitude of the circadian clock in response to
growth conditions (Xu et al. 2013). However,detailed analysis of a reconstituted KaiC oscillator revealed a mechanism in which protein function itself creates the circadian oscillation anddetermines 24-h rhythmicity.
KaiC is biochemically unique. It functions asa homohexamer and has autophosphorylation,
autodephosphorylation, and ATPase activities.
KaiC has two sites that are autophosphorylated
and autodephosphorylated in a deﬁned order,
such that KaiC undergoes four distinct phosphorylation states over the course of the circadian cycle (Fig. 2B) (Nishiwaki et al. 2007; Rustet al. 2007). Each phosphorylation state has adistinct afﬁnity to KaiA and KaiB. In turn,
KaiA and KaiB regulate KaiC’s autophosphorylation and autodephosphorylation to autono-

mously switch between a “daytime” KaiC complex with autophosphorylation activity and a“nighttime” complex with autodephosphorylation activity. These switches include dynamic
structural alterations in a ﬂexible loop domain
in KaiC (Chang et al. 2011; Tseng et al. 2014)
and a fold switch in the KaiB structure (Changet al. 2015).
The force that drives KaiC status switching isprovided through KaiC-intrinsic ATP hydrolysis and ADP/ATP exchange. The ATP-hydrolysis rate is relatively slow (∼10 ATP molecules perday), is well correlated with the circadian period
in vivo for several period-modulating mutants(Terauchi et al. 2007), and is unaffected bychanges in incubation temperature (Terauchi
et al. 2007; Murakami et al. 2008). Thus, in cyanobacteria, temperature-insensitive ATPase
activity is proposed to support temperaturecompensation of the circadian period, a mech-

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K.L. Ode and H.R. Ueda

anism for maintaining a constant circadian period irrespective of environmental temperature.
The crystal structure of KaiC reveals that the
ATP-hydrolysis rate is determined by the position of ATP’s phosphorus atom and a watermolecule (Abe et al. 2015). The unfavored position of the water molecule slows down ATP hydrolysis, which is coupled to an alternation of
KaiC’s structure at ATP-binding surfaces, further slowing ATP hydrolysis (Abe et al. 2015)
and driving a structural change in KaiC thatinduces it to interact with KaiA/B (Snijder
et al. 2017; Tseng et al. 2017).
Based on the KaiC oscillator mechanism, we
can deduce an abstractive design principle of theposttranslational circadian oscillator (Fig. 2C):
energy from temperature-compensated ATPhydrolysis induces local structural alterations
that induce biochemical changes in protein–protein interactions, enzymatic activities, andmodiﬁcation patterns. Transitions between protein states have a signiﬁcant energy barrier that
determines the length of the circadian period. In
this scenario, the circadian period is not determined by the dynamics of protein quantity, butrather by the dynamics of status change or thequality of each protein molecule.
MULTISITE PHOSPHORYLATION
DETERMINES THE PERIOD OF EUKARYOTICCIRCADIAN CLOCKS

Although transcription-independent circadianoscillation marked by cellular redox states has
been found in eukaryotic cells (O’Neill andReddy 2011; O’Neill et al. 2011; Edgar et al.
2012; Reddy and Rey 2014), it is not known
whether components in TTFL-based eukaryotic
circadian oscillators can elicit posttranslational
oscillation. If you recall, however, that the cyanobacteria circadian clock adopts a combinationof PTO and TTFL-based oscillators, it may be
useful to consider that some part of the designprinciples observed in the cyanobacteria circadian PTO might be also applicable to the regulation of eukaryotic circadian oscillators.
A PTO can theoretically be constructed using
a generic kinase, phosphatase, and substrate (Jolley et al. 2012) with a similar oscillation mecha6

nism to that proposed through the modeling of
the KaiA/B/C oscillator (Fig. 3A) (Clodong et al.
2007; Rust et al. 2007; van Zon et al. 2007). A PTO
constructed with a kinase, phosphatase, and substrate protein (assuming that processes are catalyzed in the Michaelis–Menten scheme) mustuse a substrate that is phosphorylated at multiple
sites, because reversible phosphorylation at onesite alone cannot produce autonomous oscillation in the substrate’s phosphorylation states(Angeli and Sontag 2008; Conradi and Shiu2015). Interestingly, various lines of evidenceshow that several components of the eukaryotic
circadian oscillators are also phosphorylated at
multiple sites (Fig. 1), and that such multisite
phosphorylation is critical for determining theoscillation period. Indeed, as discussed in thefollowing sections, several features of designprinciples found in cyanobacterial PTO can bealso found in the eukaryotic circadian substratesof the multisite phosphorylation and kinases catalyzing the phosphorylation.
CK1, CK2, and Glycogen Synthase Kinase(GSK)3βCasein kinase 1 (CK1) is a conserved component that markedly regulates the circadian-clock
period in mammals and Drosophila. CK1ε was
ﬁrst characterized as a period-controlling kinase
coded by the Drosophila gene doubletime, mutations of which shorten or lengthen the circadian period (Kloss et al. 1998; Price et al. 1998).
CK1ε was subsequently identiﬁed as the molecule responsible for the shorter circadian period
found in tau mutant hamsters (Lowrey et al.
2000). Another CK1 isoform, CK1δ, was identiﬁed as a mutation associated with a familial
advanced sleep-phase syndrome (FASPS) pedigree (Xu et al. 2005). The genetic evidence for
CK1’s contribution to period determination was
further supported by high-throughput screening of small molecules or short interfering
RNAs (siRNAs), which identiﬁed chemicals/siRNAs that lengthen the circadian period byinhibiting CK1δ/ε (Hirota et al. 2008, 2010; Isojima et al. 2009; Zhang et al. 2009; Chen et al.
2012). Screening revealed that GSK3β, casein
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Phosphorylation-Dependent Timekeeping in Eukaryotes

CA

CK1 + nonphosphorylated substrateP
Substrate

Lower temperature

KinaseKinaseSubstrateP
Substrate P

Phosphatase

Higher temperature

CK1
ATP
Substrate

CK1
ATP
Substrate

Increase thekinase-substrate affinity

Phosphatase

D

Substrate P

Decrease thekinase-substrate affinity

CK1 + product with multisite phosphorylation
Lower temperature
CK1
ADP

B
CK1

ADP

ATP

CK1
ADP
P P
Substrate

P P
Substrate

P
S

P

Higher temperature

Amino terminal

Carboxy terminal

Decrease thekinase-product affinity

Increase thekinase-product affinity

E
CK1

GSK3β

ADP

ATP
P
S

Lower temperature

Higher temperature

P P

P P

P

Amino terminal

Carboxy terminal

P

CK2

ADP

P
PER

P P

PER

ATP
P
S
Amino terminal

P

P

Carboxy terminal

Destabilizing PER

Stabilizing PER(accelerate the clock)(decelerate the clock)Figure 3. Multisite phosphorylation is the core mechanism of period determination in eukaryotic circadianclocks. (A) Multisite phosphorylation is a prerequisite for forming a posttranslational oscillator (PTO) from ageneric kinase, a phosphatase, and a substrate. (B) The kinases responsible for period determination preferentially promote sequential phosphorylation. “S” denotes the target residues for phosphorylation. (C–D) Ueda andhis colleagues found that raising the temperature decreases casein kinase 1 (CK1)’s afﬁnity for the substrate(Shinohara et al. 2017). Raising the temperature also prompts CK1 to bind the phosphorylated product. These
effects decrease the turnover number and thus decelerate the enzyme’s reaction rate. (E) Virshup, Forger, andtheir colleagues (2015) found that raising the temperature promotes PERIOD protein (PER) phosphorylation by
CK1, which stabilizes PER and thereby lengthens the period (Zhou et al. 2015).

cadian period (Hirota et al. 2008, 2010; Maieret al. 2009; Zhang et al. 2009). Although otherkinases such as AMP-activated protein kinase(AMPK), calmodulin-dependent protein kinase
II (CaMKII), and mitogen-activated protein kinase (MAPK) regulate circadian clocks by in-corporating various cellular and environmental
signals (Gallego and Virshup 2007; Reischl andKramer 2011), the importance of CK1, CK2,
and GSK3β in period determination is highlighted both by screening studies and in the conservation of their roles across species. Among

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K.L. Ode and H.R. Ueda

these kinases, CK1 may have one of the mostprominent effects on the circadian period because the inhibition of CK1δ/ε can double theperiod up to 48 h (Isojima et al. 2009) and because CK1δ/ε-dependent phosphorylation invitro and CK1δ/ε-dependent degradation in cellulo are both temperature-compensated (Isojima et al. 2009).
Kinases can phosphorylate multiple sites ona substrate in a random manner, phosphorylating each site with a random access, or in a sequential manner in which the kinase phosphorylates the target sites in a speciﬁc order (Yamada
and Forger 2010). Intriguingly, CK1, GSK3β,
and CK2 undergo sequential phosphorylation(Fig. 3B). CK1’s substrate consensus sequence
is pS/T-X-X-S/T ( pS/T denotes phosphorylated
serine or threonine, X denotes any amino acids,
and S/T denotes the target residue). The CK1
kinase preferentially phosphorylates residues
downstream of prephosphorylated residues(Flotow et al. 1990). The consensus sequenceof GSK3β is S/T-X-X-X-pS/T (Frame and Cohen 2001), and that of CK2 is S/T-X-X-D/E,
where substituting pS into the +1 and +3 positions optimize the phosphorylation efﬁciency(St-Denis et al. 2015). Thus, all three of theseimportant period-controlling kinases preferentially phosphorylate residues near prephosphorylated residues. When a kinase with thisproperty acts on multiple and clustered target
residues, phosphorylation events preferentiallyfollow a deﬁned order. For instance, CK1 preferentially phosphorylates sites sequentially,
from the peptide amino terminus toward thecarboxy terminus. This sequential phosphorylation also makes it easy to enrich local protein
regions with phosphorylated residues.
PER

Transcription repressors in the eukaryotic circadian oscillator are regulated through multisite
phosphorylation, catalyzed at least partly by theabove-mentioned kinases. PER in ﬂy and mammals is among the best-understood substrates of
CK1δ/ε (DBT in Drosophila). PER is progressively phosphorylated as the circadian cycle progresses (Edery et al. 1994; Lee et al. 2001). The
8mammalian PER sequence contains a repeat of
the CK1 motif. Once the amino-terminal side of
the S-X-X-S repeat is phosphorylated (i.e., priming phosphorylation), CK1 can efﬁciently phosphorylate the downstream serine cluster (Vanselow et al. 2006). A mutation on the priming
phosphorylation site, found in a FASPS pedigree(Toh et al. 2001), destabilizes the PER protein(Vanselow et al. 2006), indicating that phosphorylation at this site helps stabilize PER and regulate
the circadian period. The kinase responsible forthe priming site is unclear, but CK1δ/ε may beable to act on the nonphosphorylated PER-derived sequence (Isojima et al. 2009). Another
PER phosphorylation domain is important forproteasome-dependent PER degradation (Eideet al. 2005; Shirogane et al. 2005; Shanware
et al. 2011; Zhou et al. 2015). CK2 also controls
the period of the mammalian circadian clock byphosphorylating PER (Tsuchiya et al. 2009). In
Drosophila, DBT phosphorylates PER at multiple sites (Chiu et al. 2008). DBT-dependent multisite phosphorylation is primed by NEMO kinase (Chiu et al. 2011; Yu et al. 2011). In addition,
CK2 (Lin et al. 2002; Akten et al. 2003; Nawathean and Rosbash 2004) and GSK3β (Martinek
et al. 2001; Fang et al. 2007; Ko et al. 2010; Haraet al. 2011) regulate the action of Drosophila PER,
although the effect is probably mediated partly
through the phosphorylation of TIM bound withPER (Top et al. 2016).
CRY

Mammalian CRY also has multiple phosphorylation sites (Lamia et al. 2009; Gao et al. 2013;
Ode et al. 2017) targeted by various kinases,
including CK1δ/ε (Eide et al. 2002; Qin et al.
2015; Ode et al. 2017) and GSK3β (Kurabayashi
et al. 2010). The multisite phosphorylation of
CRY plays a critical role in period determination
since combinatorial mutations in the phosphorylation sites results in the twofold change of theperiod length from 19 to 38 h (Ode et al. 2017).
CRY phosphorylation sites appear to be concentrated in the P-loop and carboxy terminus, andmutations in several phosphorylation sites inthese regions signiﬁcantly affect the circadianperiod (Gao et al. 2013; Liu and Zhang 2016;

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Phosphorylation-Dependent Timekeeping in Eukaryotes

Ode et al. 2017). Furthermore, a recent study
identiﬁed a FASPS pedigree CRY mutation inwhich an alanine in the P-loop region is replacedby threonine (Hirano et al. 2016). This substitution might create a de novo phosphorylation sitearound the P-loop that modulates the circadianperiod. Indeed, the threonine-substitution phenotype can be recapitulated by aspartic-acidsubstitution, which mimics the phosphorylated
residue (Hirano et al. 2016).
FRQ

Like PER, FRQ is progressively phosphorylated
during the circadian cycle (Garceau et al. 1997)
at multiple sites, many of which are presumably
phosphorylated by CK1 and CK2 (Baker et al.
2009; Tang et al. 2009). CK1 stably binds FRQ
and is responsible for its clustered phosphorylation (He et al. 2006; Baker et al. 2009; Querfurth
et al. 2011). CK2 also directly phosphorylates
FRQ (Yang et al. 2003; Mehra et al. 2009).
CCA1

In Arabidopsis, CCA1 is phosphorylated by CK2
to obtain the proper circadian period (Sugano
et al. 1998, 1999; Daniel et al. 2004). Depleting
CK2 or increasing its activity lengthens or shortens the circadian period, respectively (Sugano
et al. 1999; Lu et al. 2011). Although the kinases
responsible for phosphorylating CCA1 are not aswell understood as those in other conventionalmodel organisms, several circadian proteins exhibit circadian variations in their phosphorylation state (Fujiwara et al. 2008), suggesting thatmultisite phosphorylation is critical in regulating the circadian period in plants (Seo and Mas
2014). A study using the unicellular plant Ostreococcus tauri revealed that pharmacological inhibition of CK1 markedly lengthens the period
over 8 h, suggesting that the central role of CK1
in determining the circadian period is conservedin the plant kingdom (van Ooijen et al. 2013).
Temperature Compensation

CK1 and CK2 and their substrates are important
for the temperature compensation that pre-serves the circadian period. Mammalian CK1
is linked to temperature compensation (Tosiniand Menaker 1998; Isojima et al. 2009; Zhou
et al. 2015), and CK2 is at least partly responsible
for the temperature compensation in Neurospora and higher plants (Mehra et al. 2009; Portoles
and Mas 2010).
In general, increased temperature accelerates biochemical reactions such as phosphorylation and protein degradation, thus raising thetemperature should accelerate clock speed.
However, a previous study indicated that CK1δ/ε kinase activity markedly inﬂuences the speedof mammalian circadian clocks and is largely
insensitive to temperature changes in vitro (Isojima et al. 2009). A subsequent study revealed
that temperature-insensitive kinase activity isachieved through CK1δ/ε’s ability to bind substrate and phosphorylated products in a temperature-dependent manner (Shinohara et al.
2017). When CK1 acts on a substrate with asingle phosphorylation site, which may be similar to the reaction step for catalyzing the priming phosphorylation site, raising the temperature decreases the afﬁnity between the kinase
and the substrate to be phosphorylated (Fig.
3C). In addition, when CK1 acts on a substratewith multiple phosphorylation sites, raising thetemperature increases the afﬁnity between the
kinase and the phosphorylated product at multiple sites in the presence of ADP, reducing theturnover number of CK1 action (Fig. 3D). Thesetemperature-sensitive afﬁnity alterations reduce
substrate binding and product release at higher
temperatures. Therefore, the net kinase-reaction
rate stays nearly constant despite temperaturechanges. Ueda and his colleagues successfully
conferred temperature insensitivity on anothertemperature-sensitive kinase protein by introducing an evolutionarily conserved CK1-specific domain that seems to bind to substrates andphosphorylated product.
The presence of a temperature-insensitive
kinase such as CK1 can provide another layerof regulation to achieve the circadian temperature compensation. Detailed analysis of the degradation kinetics of mammalian PER showedthat raising the temperature stabilizes PER,
which were proposed by the combination ofCite this article as Cold Spring Harb Perspect Biol 2018;10:a028357

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K.L. Ode and H.R. Ueda

temperature-insensitive and temperature-sensitive kinases (Zhou et al. 2015). At higher temperatures, CK1δ/ε preferentially phosphorylates
PER on the PER-stabilizing domain (which contains the site of the mutation found in the FASPS
pedigree), while at lower temperatures, theCK1δ/ε preferentially phosphorylates PER on
the PER-destabilizing domain (Fig. 3E). Thus,
increasing the temperature decelerates the PER
degradation, slowing down the pace of the circadian clock. The similar temperature-dependent stability mechanism was also proposed in
Neurospora FRQ. A study proposed that increasing the temperature accelerates FRQ stabilization and counteracts any temperature-dependent acceleration of the circadian clock(Mehra et al. 2009). This balance can be modulated by mutations of either CK2 or the CK2phophorylation site on FRQ. These studies indicated that temperature-dependent kinase activity counteracts the canonical acceleration ofclock speed in response to a rise in temperatureby modulating the substrate degradation rate.
To summarize, the CK1δ/ε kinase reaction
itself can be insensitive to temperature changes,
providing the mechanism of temperature compensation at the systems’ component level. Thistemperature-insensitive kinase together withother temperature-sensitive kinases (or probably phosphatases) may provide the system-leveltuning of FRQ/PER proteolysis, which further
compensates the effect of temperature changes.
These mechanisms are not mutually exclusive incircadian clocks (Hogenesch and Ueda 2011);
however, the former model suggests that temperature-insensitive reactions are not exclusive
to the circadian substrate. Rather, if the afﬁnity
of CK1δ/ε for phosphorylated products is important for temperature-insensitive kinase reactions, such temperature-compensated reactions
may also function in other biological processes.
In a study of temperature-compensated budding
yeast metabolic oscillation, the oscillation period was sensitive to CK1 and GSK3β (Causton
et al. 2015). Indeed, a budding yeast homologue
of CK1 also preserves temperature-compensated kinase activity (Shinohara et al. 2017). Therefore, the CK1 enzyme might confer robust biochemical reaction rates on circadian clock and10other systems to protect against environmental
temperature ﬂuctuations.
MULTISITE PHOSPHORYLATION AT
FLEXIBLE PROTEIN REGIONS

The multisite phosphorylation process itself
may contribute to period determination independent of the regulation of protein degradation. In a recent study in Neurospora, mutationsin FRQ phosphorylation sites strongly affected
the circadian period even in a genetic background in which FRQ’s stability was unchanged(Larrondo et al. 2015). In another study, a seriesof phosphorylation-site mutants of mammalian
CRY severely affected the circadian period, eventhough CRY’s stability was only marginally affected (Ode et al. 2017). Furthermore, a deletion-mutant CRY found in delayed sleep-phase
disorder extends the circadian period without adetectable change in the stability of the CRY
protein (Patke et al. 2017). In plants, CK2 phosphorylation regulates CCA1 transcription without affecting the amount of CCA1 protein (Portoles and Mas 2010).
KaiC studies strongly suggest that circadianclock speed is encoded as the time necessary forchanges in protein structure. On the other hand,a common feature of other circadian-period regulatory processes is that the period is largely
determined by multisite phosphorylation by kinases that elicit clustered and sequential phosphorylation, although the events downstream ofthe phosphorylation may vary among circadiansystems. Are there any links connecting the design principles of the KaiC oscillator and multisite phosphorylation-based oscillation? Serial
enzymatic reactions such as multisite phosphorylation can produce cooperative responses and a
sufﬁcient delay to produce fully modiﬁed substrates, and can potentially determine period
length (Salazar and Hofer 2009; Salazar et al.
2010; Ferrell and Ha 2014). Studies suggestthat even apart from its time-related characteristics, multisite and clustered phosphorylation
might be able to strongly modulate protein
structure.
Multisite phosphorylation often affects protein structure and function by acting on intrin-

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Phosphorylation-Dependent Timekeeping in Eukaryotes

sically disordered regions (IDRs) of substrate
proteins (Valk et al. 2014; Wright and Dyson
2015). In IDRs, the polypeptide structure ishighly ﬂexible, not rigid as in an α-helical conformation. IDRs tend to be located at the protein
surface where they are accessible to kinases. Indeed, statistical analyses of proteomics data indicated that phosphorylation sites and that IDRs
are located close together (Iakoucheva et al.
2004; Xie et al. 2007), and IDRs are often regulated by phosphorylation (Holt et al. 2009; Tyanova et al. 2013; Sharma et al. 2014). Multiple
phosphorylations on IDRs dramatically alter thebulk electrostatic potential of the region andmay induce dynamic structural changes (Fig.
4A) (Theillet et al. 2014). When the ﬂexibilityof an IDR strongly affects intramolecular interactions, multisite phosphorylation can change
the overall structure of the proteins themselves.
If the IDR is located at the interface of protein–protein interactions, such structural changes
could affect binding afﬁnity.
Multisite phosphorylations on IDRs may
have a critical role for circadian-period control.
It has been suggested that FRQ is an intrinsicallydisordered protein (IDP) (Hurley et al. 2013).
Multisite phosphorylation changes the structureof FRQ (Querfurth et al. 2011), and the nonphosphorylated FRQ is proposed to have a compact form through an interaction between thebasic amino-terminal domain and the acidic
carboxy-terminal domain. Multisite phosphorylation of the amino-terminal domain loosens
the interaction, and the FRQ becomes moresensitive to limited protease digestion. The multisite phosphorylation on PER appears to be
similar to FRQ. Although PER contains functionally important structured domains, such as
Par-Arnt-Sim (PAS) domains and the CRYbinding domain (Hennig et al. 2009; Kucera

A

P
P

P

P
P

Cooperative protein–protein interaction

B

P

P

P
Modulating local protein structure

Switching global protein structure

Kinase
P

ATP
S

2- 2P
P

KinaseADP
P

Kinase

22P P
P P

P
P

KinaseP
S

Sequential phosphorylation

Multisite phosphorylation changes(temperature compensated?)

the local electrostatic state

Changes in protein–protein interactions andenzymatic activities

Figure 4. Multisite phosphorylation affects protein structure and function. (A) Multisite phosphorylation,
particularly around the ﬂexible protein regions, affects protein–protein interactions and local/global protein
structure. (B) The multisite phosphorylation process could be a period-determining step in eukaryotic circadianclocks. Reaction rates of the kinases responsible for these events can be temperature-insensitive. This abstractive
principle may be compatible with the design principle of cyanobacterial circadian-period determination (seeFig. 2C).

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K.L. Ode and H.R. Ueda

et al. 2012; Nangle et al. 2014), most of the otherregions are predicted to be disordered (Gustafson and Partch 2015). In both mammalian andDrosophila PER, phosphorylation states control
the PER global structure, as indicated by an altered susceptibility to protease treatment (Chiuet al. 2011; Zhou et al. 2015). In contrast to FRQ
and PER, the structure of the photolyase homology domain of cryptochrome superfamily proteins is well conserved, indicating that CRY is
not an IDP. Nonetheless, clustered phosphorylation sites that are critical for controlling thecircadian period are located near the ﬂexible
P-loop (Hirano et al. 2016; Liu and Zhang
2016; Ode et al. 2017). Multisite phosphorylation appears to additively regulate the circadianperiod, and the number of phosphorylation sitesaround this loop domain may serve as a cumulative timer for the circadian period (Ode et al.
2017). These lines of evidence suggest that structural changes in ﬂexible regions, elicited by multisite phosphorylation, are a critical step in determining the circadian period.
Although the detailed mechanisms of period-determination remain unknown, the outcome of phosphorylation-dependent protein
structural change could be coupled to TTFLbased architecture via proteolysis (Chiu et al.
2011; Zhou et al. 2015). Changes in the multiple
phosphorylation state would also affect the protein–protein interaction. We further point out
that the substrate with multiple phosphorylationsites can reciprocally regulate the action of kinases such as CK1. For example, PER protein
alters the CK1’s substrate preference (Qin et al.
2015). Anionic small molecules and phosphorylated peptides can bind to the allosteric site of
CK1 and thereby modulate the CK1’s enzymatic
activity (Shinohara et al. 2017). These substrate–
CK1 interactions may imply that phosphorylation events in a stable protein complex,
including CK1, PER, and CRY, should not beinterpreted as a simple mixture of kinases andsubstrates. Instead, the components in a kinase–substrate complex cooperatively regulate theprocess of multiple phosphorylation, providing
the temperature-compensated and accurate period-determining information for the eukaryotic circadian clocks.
12

CONCLUDING REMARKSThe importance of multisite phosphorylationand structural changes in eukaryotic circadianperiod control remind us of the unsolved questions: What is the source of the 24-h period?
What are the rate-limiting steps? Why is thecircadian period is so slow (a scale of minutes)
compared with biochemical events in general (ascale of seconds), but still accurate (Akiyama2012). Studies of the KaiC oscillator revealed astructural basis for slow kinetics (Abe et al.
2015). In contrast, a structural basis for its accuracy remains unknown. Although transcription–translation dynamics are at the core ofnoncyanobacterial circadian clocks, we may
well ﬁnd phosphorylation-dependent structuralchanges at the core of timekeeping mechanisms
in these organisms. In addition, a recent study
proposed that the slow cis/trans isomerization of
speciﬁc peptide bonding in BMAL1 is a timekeeping mechanism in mammalian circadianclocks (Gustafson et al. 2017). Such rate-limiting
events, encoded as protein structures, could be
induced directly (via control of the local electrostatic state) or indirectly (via protein–protein
interactions) through multisite phosphorylation. Furthermore, the temperature-compensation property of reaction speed could be encoded via multisite phosphorylation by CK1(Isojima et al. 2009; Shinohara et al. 2017).
From this perspective, the circadian timekeeping mechanism in the KaiC protein oscillator and eukaryotic circadian oscillator may converge as the progressive regulation of protein
structure powered by temperature-compensatedenzymatic activities—KaiC’s ATPase activity
and CK1’s phosphorylation activity (Fig. 4B),
thus encoding circadian-period determination
as a quality rather than quantity of protein dynamics (Larrondo et al. 2015; Ode et al. 2017).
The rate-limiting step should be encoded as aprotein structural property to keep the period
length constant regardless of ﬂuctuations in
expression (Dibner et al. 2009), especially for
low-abundance circadian-transcription factors(Forger and Peskin 2005; Narumi et al. 2016).
To evaluate this perspective, we must execute a
structure-based analysis of the clock proteins,

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Phosphorylation-Dependent Timekeeping in Eukaryotes

and reconstruct and design circadian timekeeping steps in eukaryotic circadian systems.
ACKNOWLEDGMENTS

Studies from our laboratory introduced in thismanuscript were supported in part by Japan
Agency for Medical Research and Development(AMED)-Core Research for Evolutional Scienceand Technology (CREST), CREST, Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS), the Basic Scienceand Platform Technology Program for Innovative Biological Medicine, Japan Society for
the Promotion of Science (JSPS) (25221004,
23115006, 25830146, 16K14653), Rikagaku
Kenkyusho (Institute of Physical and Chemical
Research, Japan—RIKEN), and the Takeda Science Foundation.
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Design Principles of Phosphorylation-Dependent Timekeeping inEukaryotic Circadian Clocks
Koji L. Ode and Hiroki R. Ueda
Cold Spring Harb Perspect Biol 2018; doi: 10.1101/cshperspect.a028357 originally published online
October 16, 2017
Subject Collection

Circadian Rhythms

Introduction to Chronobiology
Sandra J. Kuhlman, L. Michon Craig and Jeanne F.
Duffy
Design Principles of Phosphorylation-Dependent
Timekeeping in Eukaryotic Circadian Clocks
Koji L. Ode and Hiroki R. Ueda
Circadian Posttranscriptional Regulatory
Mechanisms in Mammals
Carla B. GreenA 50-Year Personal Journey: Location, Gene
Expression, and Circadian Rhythms
Michael Rosbash
Regulating the Suprachiasmatic Nucleus (SCN)
Circadian Clockwork: Interplay between
Cell-Autonomous and Circuit-Level Mechanisms
Erik D. Herzog, Tracey Hermanstyne, Nicola J.
Smyllie, et al.
Systems Chronobiology: Global Analysis of Gene
Regulation in a 24-Hour Periodic World
Jérôme Mermet, Jake Yeung and Felix Naef

Interplay between Microbes and the Circadian
Clock
Paola Tognini, Mari Murakami and Paolo
Sassone-Corsi
Coordination between Differentially Regulated
Circadian Clocks Generates Rhythmic Behavior
Deniz Top and Michael W. Young
Cellular Timekeeping: It's Redox o'Clock
Nikolay B. Milev, Sue-Goo Rhee and Akhilesh B.
Reddy
Molecular Mechanisms of Sleep Homeostasis in
Flies and Mammals
Ravi Allada, Chiara Cirelli and Amita Sehgal
Membrane Currents, Gene Expression, andCircadian Clocks
Charles N. Allen, Michael N. Nitabach andChristopher S. Colwell
The Plant Circadian Clock: From a Simple
Timekeeper to a Complex Developmental Manager
Sabrina E. Sanchez and Steve A. Kay

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