REVIEW
published: 02 October 2020
doi: 10.3389/fpsyg.2020.575328

Phosphorylation Hypothesis of Sleep
Koji L. Ode 1 and Hiroki R. Ueda 1,2*
1
Department of Systems Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan, 2 Laboratory
for Synthetic Biology, RIKEN Center for Biosystems Dynamics, Osaka, Japan

Edited by:
Daniela D. Pollak,
Medical University of Vienna, Austria
Reviewed by:
Marcos G. Frank,
Washington State University Health
Sciences Spokane, United States
Seth G. N. Grant,
The University of Edinburgh,
United Kingdom*Correspondence:
Hiroki R. Ueda
uedah-tky@umin.ac.jp
Specialty section:
This article was submitted toComparative Psychology,
a section of the journal
Frontiers in Psychology
Received: 23 June 2020
Accepted: 15 September 2020
Published: 02 October 2020
Citation:
Ode KL and Ueda HR (2020)
Phosphorylation Hypothesis of Sleep.
Front. Psychol. 11:575328.
doi: 10.3389/fpsyg.2020.575328

Sleep is a fundamental property conserved across species. The homeostatic inductionof sleep indicates the presence of a mechanism that is progressively activated by
the awake state and that induces sleep. Several lines of evidence support that suchfunction, namely, sleep need, lies in the neuronal assemblies rather than specific brain
regions and circuits. However, the molecular mechanism underlying the dynamics of
sleep need is still unclear. This review aims to summarize recent studies mainly inrodents indicating that protein phosphorylation, especially at the synapses, could be themolecular entity associated with sleep need. Genetic studies in rodents have identified aset of kinases that promote sleep. The activity of sleep-promoting kinases appears to beelevated during the awake phase and in sleep deprivation. Furthermore, the proteomic
analysis demonstrated that the phosphorylation status of synaptic protein is controlled
by the sleep-wake cycle. Therefore, a plausible scenario may be that the awakedependent activation of kinases modifies the phosphorylation status of synaptic proteinsto promote sleep. We also discuss the possible importance of multisite phosphorylation
on macromolecular protein complexes to achieve the slow dynamics and physiological
functions of sleep in mammals.
Keywords: NREM sleep, phosphorylation, kinase, proteomics, CaMKIIINTRODUCTION
Sleep is a fundamental property of the central nervous system. A stereotypical sleep-wake transition
occurs from seconds to minutes in which nearly all cortical neural activity changes to thesleep-firing pattern. Some animal species to adopt different styles of the sleep-wake cycle called
unihemispheric sleep observed in migratory birds (Rattenborg et al., 2016) and fur seals (Lyamin
et al., 2017). Neural architectures for achieving sleep-wake transition also vary. Even jellyfish with adiffuse nervous system undergoes sleep-wake cycle (Nath et al., 2017), suggesting that sleep is not a
specific function of animals with a central nervous system but a function that originated in an earlystage of metazoan lineage.
Despite such anatomical differences in regulating sleep, the characteristics of sleep dynamics
are well conserved throughout the phyla. Animals have homeostatic sleep control to ensure thatthey can keep a baseline sleep amount: if an animal spends more time in an awake state in one
day, the reduced sleep amount is restored by taking an extra amount of sleep in the subsequentdays (recovery sleep), suggesting that the amount of sleep loss is monitored and measured. The
baseline daily duration of sleep varied depending on the animal species (Campbell and Tobler,
1984; Siegel, 2005) and even within inbred mice strains (Franken et al., 1999; Diessler et al., 2018):
such differences are at least in part genetically inherited, suggesting that there are genetic factorsthat determine the normal amount of sleep per day.

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Protein Phosphorylation and Sleepinessin each cell is lost, and the behavioral circadian rhythmicity
no longer persists without the external stimulus (e.g., lightdark cycle), although the transcription rhythmicity in individual
cells continues to some extent (Tahara et al., 2012). Therefore,
the mechanism to maintain the 24-h time period lies in themolecular activity within the cell, and the synchrony among thecells is achieved by the hierarchical connection of intercellularinteraction, including humoral factors (Silver et al., 1996).
The molecular mechanism of Process S is still unclear, but
Process S is a good factor to evaluate and simulate the level ofsleep need in human and rodents. The physiological metric wellmatched with the expected level of Process S is the slow-wave
activity (SWA) of electroencephalogram (EEG) signal (typicallyfrom 0.5 to 4 Hz) (Borbely, 1982). The predicted Process S basedon the two-process model well fits the daily dynamics of SWA
and its response to sleep deprivation (Daan et al., 1984; Dijk et al.,1990a,b). The model successfully characterizes the different sleep
phenotypes among inbred mice (Franken et al., 2001) and humanindividuals (Rusterholz et al., 2010). Furthermore, the model was
also applied to classify the dynamics of the gene expression profile(Hor et al., 2019).
Although the original two-process model assumes that Process
S monotonically decreases and increases in response to thesleep-wake state, the mechanism for the regulation of the sleepwake cycle independent from the circadian clock component
may include a non-linear feedback system that could work
as an oscillator. A classic example is a phenomenon calledinternal desynchronization in humans, in which the sleep-wakerhythm has a different period length than the physiologicalrhythms driven by the circadian clock (e.g., rhythms in core
body temperature) (Aschoff, 1965; Kronauer et al., 1982; Dijk
and Czeisler, 1995). Under certain conditions, the sleep-wakecycle in rodents also exhibits autonomous behavioral rhythms
that appear to be independent of the circadian clocks. An
example is sleep-wake rhythmicity induced by the chronic
application of methamphetamine (methamphetamine-sensitive
circadian oscillator) (Honma et al., 1987). Methamphetaminesensitive sleep-wake rhythmicity can be observed in the absenceof both environmental time cues and canonical circadianclocks (e.g., SCN lesion or genetic background where canonicalmolecular factors essential for driving the circadian TTFLs were
knocked out) (Honma et al., 1987, 2008; Masubuchi et al., 2001;
Mohawk et al., 2009). A dopaminergic oscillator was proposedas the underlying mechanism of ultradian (∼6 h) behavioral
rhythmicity observed in the absence of functional circadianclocks (Blum et al., 2014). Furthermore, the methamphetaminesensitive rhythmicity is proposed as the period-modulated
dopaminergic oscillator (Blum et al., 2014).
The molecular mechanism determining the basal sleep
duration is also still unknown. In the two-process model, theduration of daily sleep is determined by the intercrossing point ofProcess S and Process C. Thus, the duration of baseline daily sleep
should be altered by the manipulation of the increase/decrease
rate of Process S or manipulation of the upper/lower threshold of
Process C (Aeschbach et al., 1996). However, expected different
response to the sleep deprivation by such manipulation was notobserved between long sleepers and short sleepers in humanIn this review, we introduce recent progress in research,
mainly focusing on the posttranslational regulation of proteins
inside neurons, that reveal that phosphorylation plays animportant role in the homeostatic regulation of sleep inmammals. Beyond the fact that phosphorylation is one of themost important modalities in the regulation of protein activity(Cohen, 2000, 2002; Nishi et al., 2014) and it is used to control thebasic function of neurons such as long-term potentiation (LTP)(Soderling and Derkach, 2000), the global phosphorylation status
in neurons appear to be controlled by sleep-wake cycles (Wang Z.
et al., 2018; Bruning et al., 2019). Also, genetic evidence indicatesthat some classes of kinases have significant sleep-inducing effects(Funato et al., 2016; Tatsuki et al., 2016; Mikhail et al., 2017).
Thus, phosphorylation may actively regulate the dynamics of
sleep in mammals.
Sleep is composed of qualitatively different types of sleep
modes, NREM and REM. The following sections mainly focus
on NREM sleep because this mode of sleep constitutes mostof the total sleep episodes (e.g., ∼90% in C57BL/6 mice), andseveral genetic studies discussed below reported the changes in
the NREM sleep duration. To address how the phosphorylationoriented view of sleep regulation complements the previous
models of sleep control in mammals, we will begin with abrief overview of representative models proposed to explain the
homeostatic regulation of sleep. In the following sections, we will
discuss sleep in mammals, unless we explicitly mention the sleepof non-mammalian species.

HIERARCHICAL ORGANIZATION OF
CIRCADIAN CLOCKS AND SLEEPHOMEOSTASIS
Two-Process Model of Sleep-Wake Cycle
The two-process model, which has been the landmark for
explaining the behavioral rhythmicity of the daily sleep-wakecycle, describes the relationship between sleep homeostasis,characterized by “Process S,” and the circadian clock,characterized by “Process C” (Borbely, 1982; Borbely et al.,2016). Process S represents the amount of sleep need, such that
Process S increases in an awake state and decreases in a sleep
state. When the level of Process S increases to the certain upper
level (upper threshold), the animals go to the sleep phase, andwhen the level Process S decreases to the certain lower boundary(lower threshold), the animals turn to the awake phase. These
upper and lower threshold levels are modeled to show a daily
oscillation by the function of Process C. In the typical diurnal
animals’ Process C, the peak of the lower threshold oscillation isaround the morning and trough of it is around the night.
The mechanism driving Process C is a cellular circadian clock,in which the transcription-translation feedback loops (TTFLs)
generate a cell-autonomous oscillation of transcription activity(Takahashi, 2017; Patke et al., 2020). The nearly 24-h rhythmicity
of cellular activity is synchronized by a circadian clock in a
specific brain region called the suprachiasmatic nucleus (SCN);
if the SCN is disrupted, the synchrony of circadian clocks

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Protein Phosphorylation and Sleepinesspower of EEG (1–4 Hz) distributes heterogeneously and increases
within the cortical areas that showed higher activity during
the preceding awake period (Huber et al., 2006, 2007). Theseobservations support the hypothesis of local use-dependent
sleep, proposing that sleep need can be stored within a smaller
number of neuron/glial cells and distributed throughout thecortex heterogeneously (Krueger and Obal, 1993; Krueger et al.,2008, 2019). The minimum cellular set for sleep homeostasis
may be at the synapse level; the synaptic homeostasis hypothesis
employs the sum of synaptic strength as the hallmark of thecellular component of sleepiness (Tononi and Cirelli, 2003, 2006)
and is supported by the 3D electron microscopic quantification ofsynapses showing a decrease in the interface between axons andspines during sleep (de Vivo et al., 2017; Spano et al., 2019). In linewith this view, cultured neuronal/glial cells develop a sleep-likefiring pattern in a homeostatic manner upon the administrationof wake-inducing stimulation (Saberi-Moghadam et al., 2018). If
the sleep need does not rely on a specific set of brain circuits
but on a smaller number of neuronal/glial networks, it is worth
considering the possibility that molecular signaling inside theneurons, rather than the brain–circuit-level activity, is the directsource of the sleep need.

(Aeschbach et al., 1996). Also, mice lines that have similarparameters for the increase and decrease of Process S show
different daily sleep durations (Franken et al., 2001). Thesestudies suggest that daily sleep duration is determined by acomplex mechanism that is not covered by the simple twoprocess model.
In summary, although the two-process model is successful
in explaining and predicting the organism-level sleep-wakebehavior in mammals, the mechanisms underlying the behaviorof Process S and the interaction between Process S andProcess C are largely unknown. The understanding of molecularcomponents involved in the homeostatic regulation of sleep will
be a critical step to dissect, quantify and understand the feedback
mechanism controlling the behavioral sleep-wake rhythmicity.

Cortex Neuronal Activity During NREM
Sleep
At the neuronal-level dynamics associated with the sleep-wakecycle, synchronous activity among neurons is a typical featureof NREM sleep, in which the cortical neurons exhibit a uniquefiring pattern that alternates between a bursting (up state) andresting (down state) phase at 0.5–1 Hz (slow oscillation). The slow
oscillation occurs synchronously among the cortical neurons
such that the synchronized bursting/resting pattern is thought
to be the source of SWA observed in the EEG signal during
NREM sleep. The synchronized bursting/resting pattern ofcortical neurons during NREM sleep becomes less synchronized
in response to the decrease of sleep need (Vyazovskiy et al., 2009).
The excitability of cortical neurons is modulated by the levelof excitatory neurotransmitters (e.g., dopamine, norepinephrine,
acetylcholine, etc.). The level of neurotransmitters is controlled
through the inputs from several brain nuclei, including sleepactive (e.g., ventrolateral preoptic nucleus) and wake-active
nuclei (e.g., locus coeruleus, tuberomammillary nucleus, Raphe)that are supposed to be mutually regulated (flip-flop model)(Saper and Fuller, 2017). The inputs can be external stimuli, suchas wake-inducing caffeine, that are shown to affect the sleep-wakecycle through acting on the A2A receptor expressed at the nucleus
accumbens (Lazarus et al., 2011).
The synchronous slow oscillation appears to be the inherent
property of cortical neuron assemblies because the oscillation
can be found in ex vivo cortical slabs/slices (Sanchez-Vives andMcCormick, 2000; Timofeev et al., 2000). It was even shownthat the cultured neurons exhibit a synchronous firing patternsimilar to that of slow oscillation, and the synchronous firing
pattern can be converted to asynchronous one by applying
chemical(s) including excitatory neurotransmitters (Hinard et al.,2012; Kaufman et al., 2014; Saberi-Moghadam et al., 2018).
Overall, the synchronous slow oscillation of cortical neurons canbe modulated through multiple layers of neurotransmitters, brain
circuits, and neuronal networks. However, the layer on which
sleep need is encoded is still unclear.
Interestingly, several studies reported a phenomenon called
local sleep, where a part of the cortex exhibits slow oscillation
in a use-dependent manner even when the entire brain is in an
awake state (Krueger et al., 2019). It is also known that the delta

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PHOSPHORYLATION-DEPENDENTCONTROL OF THE SLEEP-WAKE CYCLE
To investigate candidate genes involved in the molecularsignaling pathway responsible for sleep homeostasis control in
mammals, researchers have been conducting various genetic
studies to find genetic manipulations that alter the animals’
baseline sleep duration, SWA and its response to sleep
deprivation (Jan et al., 2020). To validate whether the candidate
gene product controls the animals’ sleep in physiologicalcondition, it is also critical to confirm that the activity ofcandidate protein is changed in accordance with the sleepwake cycle or sleep-deprived situation. In the following
sections, we introduce recent genetic and biochemical studies
indicating the importance of protein kinases in inducing sleep(Figure 1). Because the activity of kinases is often regulated byphosphorylation modifications, changes in the phosphorylation
state of the kinases have also been investigated quantified alongwith the sleep-wake cycle.

CaMKIIα/CaMKIIβ
Camk2a/Camk2b were found as sleep-promoting kinases by areverse-genetic screening of sleep-regulating genes using the
CRISPR/Cas9 system in mice (Tatsuki et al., 2016). Embryonicknockout of Camk2a and Camk2b, but not Camk2d andCamk2g, leads to a significant decrease in sleep duration perday. Camk2a/Camk2b encodes calcium-/calmodulin-dependent
kinase II (CaMKII) α/β, expressed widely in the brain.
Because the knockout mice had shorter sleep duration,
CaMKIIα/CaMKIIβ appears to have a role in promoting sleep.
It should be noted that several studies suggest the wake/REM-associated roles of CaMKII in specific brain regions(Stack et al., 2010; Datta et al., 2011; Cui et al., 2016), and3

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FIGURE 1 | Genetic and phosphoproteomics approach to investigate the role of protein phosphorylation in the regulation of sleep. Genetic manipulation to either
increase or decrease the kinase activity tests the causal effect of kinase activity on the animals’ sleep control. It would be valuable to evaluate whether the up- anddownregulation of the same kinase activity affects the animals’ sleep phenotype bidirectionally. Although such kinase activity is not fully established, this criterion
would discriminate whether the kinase activity serves as an external input to the core homeostatic process or if the kinase is involved in the core component of sleep
homeostasis. Phosphoproteomics analysis provides complementary information to evaluate whether the kinases’ activity correlates with the expected sleep pressure
in vivo. Note that this figure depicts the case of sleep-promoting kinases; when the kinases serve as a wake-promoting factor, the relationship between enzyme
activity and sleep phenotype would be the opposite direction (i.e., loss of wake-promoting kinase would result in the increased sleep duration).

embryonic knockout study alone cannot identify the brain
area and cell type responsible for the phenotype. Even for theCamk2a/Camk2b, which are well understood as the enriched
expression in (excitatory) neuron cells, both CaMKII isoforms
are also expressed in astrocytes (Takeuchi et al., 2000). In
addition, embryonic knockout of Camk2a and Camk2b would beaccompanied by the effect on neuronal development (Wayman
et al., 2008) that may have a chronic effect on the sleep-wakecycle. Therefore, the exact mechanism of sleep induction by
CaMKIIα/CaMKIIβ needs to be clarified through the post-natal
brain-area-/cell-type-specific perturbation of these kinases.
CaMKII has a unique self-regulatory mechanism dependingon the auto-phosphorylation activity. CaMKII is typically
activated by the binding of calcium/calmodulin after synaptic
stimulation. The active CaMKII then auto-phosphorylates the
CaMKII itself. Autophosphorylation occurs on T286 (CaMKIIα)or T287 (CaMKIIβ). The T286/287 phosphorylation keeps thekinase in an active form, even in the absence of Ca2+ /calmodulin(Miller et al., 1988). Several studies in mice demonstrated that thephosphorylation of T286/287 is increased after sleep deprivationor during the awake/dark phase (Vyazovskiy et al., 2008; Diering
et al., 2017; Wang Z. et al., 2018; Bruning et al., 2019), suggestingthat the kinase activity of CaMKIIα/CaMKIIβ is upregulated inthese conditions where sleep need would be elevated.
CaMKII is one of the best-studied kinases for its criticalrole in regulating synaptic plasticity (Coultrap and Bayer, 2012;
Bayer and Schulman, 2019), and its regulatory mechanism
has been extensively studied. The autophosphorylation at
T286/287 is required for LTP (Giese et al., 1998), although
the Ca2+ /calmodulin-independent kinase activity is notneeded for maintaining long-term memory (Buard et al.,2010). After the activation CaMKII, autophosphorylation
continues to the other multiple residues. Autophosphorylation

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at T305/T306 (CaMKIIα) and T306/T307 (CaMKIIβ) are
well known downstream autophosphorylation targets. The
subsequent T305/T306 phosphorylation inhibits the bindingof Ca2+ /calmodulin to the kinase (Colbran and Soderling,1990), and disruption of the auto-inhibitory mechanism also
affects whether the activated CaMKII facilitates or depresses thesynaptic strength (Pi et al., 2010b). The roles of these multisite
autophosphorylation events in regulating sleep duration remaina mystery. Given the multi-step regulation encoded as aprotein function of CaMKII, it may be worth investigating
how CaMKIIα/CaMKIIβ with different phosphorylation states
control the sleep phenotype differently.

SIK3
The second discovery of sleep-promoting kinase in mammals wasderived from large-scale forward-genetic screening (Funato et al.,2016). The screening of >8,000 mice generated by ENU-based
mutagenesis identified the Sleepy mutant showing prolonged
NREM sleep. The extended NREM sleep is caused by a mutationthat leads to the exon skipping the salt-inducible kinase (SIK) 3.
The exon skipping is suggested to be the gain-of-function of SIK3;
thus, SIK3 is also a member of sleep-promoting kinases.
SIK3 activity is regulated by phosphorylation by other kinases(Sakamoto et al., 2018). The exon skipped in the Sleepy mutant
involves a phosphorylation site, S551, modified by protein
kinase A (PKA). SIK3 also has several phosphorylation sites,some of which have higher phosphorylation levels because of
sleep deprivation or the Sleepy mutation (Funato et al., 2016).
Among the multiple phosphorylation sites, S551 appears tobe critical for promoting NREM sleep, because the CRISPRmediated embryonic introduction of S551A point mutation
reproduced the effect of sleep promotion (Honda et al., 2018).
By using the similar CRISPR-mediated targeted mutagenesis, it

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was demonstrated that the PKA-targeted site plays a role in the
NREM sleep promotion in other SIK family proteins, SIK1 and
SIK2 (Park et al., 2020). Interestingly, the role of SIK family
in the sleep regulation appears to be conserved in invertebrate
animals because the temporal induction of S563A (corresponding
to the S551A in mice) SIK3 mutant also increase sleep duration
in Drosophila (Funato et al., 2016).
However, the exact role of phosphorylation at this residue
is still unclear. This is because not only S551D, mimicking
the phosphorylated status of Ser residue, but also S551A,
mimicking the unphosphorylated state of the residue, resulted
in a similar phenotype of Sleepy exon skipping (Honda et al.,2018). Although D/A mutations do not always mimic thephosphorylated/unphosphorylated status of Ser residue, theresult implies that the reversible phosphorylation that occurs
on this site may not account for the reversible regulation of
sleep duration. The role of SIK3 and the phosphorylationdependent regulation of SIK3 in regulating natural sleep will need
to be assessed through the sleep phenotype of SIK3 knockout
mammals, which has not been reported to date. Notably, Sik3null mutant of C. elegans and hypomorphic Sik3 mutant ofDrosophila have decreased amount of sleep-like quiescencebehavior, consistent with the sleep-promoting role of SIK3 inmammals (Funato et al., 2016).

Sleep Need, SWA, and Recovery Sleep
Does perturbation of the sleep-promoting kinases affectthe indicators of Process S? Significant reduction of daily
sleep amount observed in Camk2a/Camk2b knockout micesuggests that CaMKIIα/CaMKIIβ is involved in the mechanism
determining the basal sleep duration (Tatsuki et al., 2016). In the
study of Tatsuki et al., the sleep phenotype was recorded usinga respiratory-based system (Sunagawa et al., 2016), which can
non-invasively measure sleep and awake with high accuracy,
but, to date, cannot discriminate NREM and REM sleeps.
Accordingly, the SWA and its response to sleep deprivation
were not validated in Camk2a/Camk2b knockout mice, andthey ought to be investigated in a future study. As for SIK3,
Sleepy mice have an increased level of SWA, suggesting thatthe mice maintain a higher level of sleep need, resulting in
the longer sleep phenotype (Funato et al., 2016). Sleepy mice
were also shown to be susceptible to sleep deprivation. Taken
together, the Sleepy mice may have accelerated accumulation
and/or decelerated clearance of Process S. On the other
hand, knocking out ERK1 or ERK2 reduces NREM sleep
duration to the same extent in both normal and recovery sleep(Mikhail et al., 2017). Interestingly, inhibition of ERK1/ERK2
activity does not result in the changes in the delta powerof EEG. Thus, in contrast to the case of SIK3, ERK1/ERK2
may not be involved in the accumulation process of sleep
need but rather in the induction of NREM sleep downstreamof the sleep need.
The examples of SIK3 activation and ERK1/ERK2 inhibition
imply that these kinases are involved in the regulation of normal
sleep duration but do not significantly impair the inductionof recovery sleep. This is in stark contrast to the phenotype
caused by the inhibition of gliotransmission. Inhibition ofgliotransmission by the expression of the dominant-negative
form of SNARE leads to the suppression of rebound sleep after
sleep deprivation and the suppression of acute sleep inducedby the inflammatory response (Halassa et al., 2009; Nadjar
et al., 2013). Under this condition, however, the basal levelof sleep duration does not change. At this point, we shallrevisit the essence of the two-process model: the two-processmodel summarizes the dynamics of sleep need and recovery
sleep as a single scaler “Process S,” but in the molecular
view, a mechanism that maintains a homeostatic daily amountof sleep and other mechanisms that induce additional sleep
pressure upon an unusual situation, such as significant sleep
deprivation or inflammatory response, may be separated. Someof the chemicals secreted outside the cell called sleep-inducing
substances (SISs) appear to be involved in the latter mechanism.
For example, knockout of the adenosine A1 receptor impairs
the increase of SWA after sleep deprivation without affecting
total sleep (Bjorness et al., 2009). The knockout of adenosine
A2 also does not show a significant effect on normal sleep
but diminishes the wake-promoting effect of caffeine (Huang
et al., 2005). The knockout of receptors for SISs related to the
inflammatory responses (e.g., TNF-α) also results in normal
sleep duration, although the SWA during NREM sleep isparadoxically increased in TNF-α receptor knockout mice (Fanget al., 1997; Kaushal et al., 2012). Compared with these SISs,

ERK1/ERK2
Extracellular signal-regulated kinase (ERK) is also a sleeppromoting kinase. This was first suggested in Drosophila, where
pan-neuronal expression of the kinase-active form of ERK
increases sleep duration and application of ERK inhibitor reduces
sleep (Vanderheyden et al., 2013). However, the hypomorphic
ERK mutant flies show sleep duration similar to wild type flies(Vanderheyden et al., 2013), raising the question of how much of
the basal sleep duration is under ERK-specific control. The roleof mammalian ERK as a sleep-promoting kinase was then clearly
demonstrated in mice (Mikhail et al., 2017). Embryonic knockoutof ERK1 and conditional ERK2 knockout largely in corticalneurons both result in a decrease in NREM sleep duration. The
administration of ERK1-/ERK2-selective inhibitor (U0126) also
decreases NREM sleep, suggesting that the ERK1/ERK2 regulate
sleep independent of neurodevelopmental effect. Consistent withthis observation, SWA was decreased in the U0126-treated mice.
Conversely, the level of phosphorylated ERK1/ERK2 (kinaseactive ERK1/ERK2) is higher in the arousal state than thatin the NREM sleep.
The role of mammalian ERK as a sleep-promoting kinase
may lie in the control of gene expression associated withsleep-wake cycle. This notion was demonstrated throughunique studies using cultured cortical neurons, which show
electrophysiological activity similar to slow oscillation, together
with the homeostatic recovery response to the administrationof chemical/electrical stimulation (Hinard et al., 2012; SaberiMoghadam et al., 2018). The cortical neurons also show alteredgene expression by chemical stimulation mimicking the awake
state. Notably, inhibition of ERK signaling antagonized thechanges in gene expression caused by the wake-mimicking
stimulation (Mikhail et al., 2017).

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Protein Phosphorylation and Sleepinesstheir phosphorylation status along with the sleep-wake cycle(Figure 1). It is well understood that most of the transcriptome
profile is under the control of circadian clocks (Reppert andWeaver, 2002). Accordingly, researchers have attempted to findgenes in which the expression pattern shows daily rhythmicity
and changes upon the accumulation of sleep need (e.g., sleep
deprivation condition) (Elliott et al., 2014; O’Callaghan et al.,2019). However, advances in next-generation sequencing andmass spectrometer-based -omics technologies indicate that thegene expression pattern does not always reflect the expression
pattern of gene products (Figure 2). Deep sequencing allows
researchers to compare intron and exon cycling genes in theliver and has revealed that many of them are not overlapped,
indicating that the rhythmicity of a significant amount of mRNA
expression is controlled through posttranscriptional regulation(Koike et al., 2012). A similar conclusion has been obtained bycomparing nascent-RNA sequencing (Menet et al., 2012), RNApolymerase II occupancy (Le Martelot et al., 2012), and modelingstudies aiming to clarify the contribution of mRNA degradation
to the rhythmicity of mRNA abundances (Wang J. et al., 2018).
A quantitative proteomics study of liver samples further
revealed the uncoupling between the peak phase of cycling
proteins and their corresponding mRNAs. Moreover, ∼20% ofrhythmic protein may not be accompanied by rhythmic mRNA
expression, indicating the importance of posttranscriptional
mechanisms to generate the rhythmic protein expression (Robles
et al., 2014). At the posttranslational level, rhythmic protein
activities appear to be enhanced by protein phosphorylation.
Comparison of the proteomic and phosphoproteomic profiles
showed that the amplitude of the rhythmic component (protein
abundance or phosphopeptide abundance) was several times
higher on average for the phosphorylated peptide (Robles et al.,2017). Therefore, the oscillatory dynamics of protein function
seem to be generated through not only promoter-driven gene
expression but also posttranscriptional and posttranslational
control and enhanced at the layer of protein phosphorylation.

the sleep-promoting kinases appear to have a role in regulating
normal sleep duration.
The sleep-promoting kinases may be involved in the interplay
between the circadian clocks and sleep control. Although
circadian clocks and sleep homeostasis can run independently,
circadian genes affect the sleep-wake behavior and vice versa(Franken and Dijk, 2009). In the case of sleep-promoting kinases,
all of the abovementioned kinases are suggested to have a role
in regulating circadian clocks. CaMKII plays a critical rolein the robust oscillation of the circadian clock in the SCN
slice through the phosphorylation of a circadian transcription
factor called CLOCK (Kon et al., 2014). Notably, inhibitionof CaMKII also attenuated the circadian oscillation of nonneuronal cultured cells, indicating that CaMKII plays a role
independent of neural firing for circadian rhythmicity (Kon et al.,2015). SIK3 is shown to be involved in regulating circadianperiod. The knockout mice for Sik3 show several abnormalities
in circadian rhythmicity, including a longer circadian period in
the behavioral rhythmicity and impaired synchrony of individual
cells in the SCN (Hayasaka et al., 2017). A molecular link
between SIK3 and circadian clocks could be the SIK3-dependent
phosphorylation and degradation of PER2 (Hayasaka et al., 2017).
Abnormal circadian rhythmicity and PER protein dynamics arealso observed in Drosophila lacking Sik3 function (Fujii et al.,2017). ERK was reported to be involved in the resetting processof circadian clocks (Akashi and Nishida, 2000). In addition,
chemical screening studies have identified ERK inhibitors as
potent perturbator of cellular circadian oscillation (Isojima et al.,2009; Kon et al., 2015), although part of ERK inhibitors also
targets casein kinase 1 (CKI) δ/ε, which is a critical kinase forthe circadian period determination (Isojima et al., 2009). There
are several pathways connecting ERK activity and the circadianmolecular feedback loop, mainly through the transcription
control by ERK of the downstream signaling cascade (Goldsmithand Bell-Pedersen, 2013). One example is the transcription factor
Dpb, which composes the circadian TTFLs. The Dpb expression
is suppressed in sleep-deprived rats (Cirelli et al., 2004) as
well as cultured cortical neurons stimulated by wake-mimicking
chemicals (Mikhail et al., 2017).
In summary, sleep-promoting kinases markedly affect normal
sleep duration. The kinases may shape the molecular dissectionof the mechanism involved in Process S and mechanism
determining basal sleep duration, and reveal the difference
between sleep need to maintain daily sleep duration andthe induction of recovery/inflammatory sleep. The kinases
may also work as a hub to connect sleep homeostasis andcircadian regulation.

Impact of Sleep Need on Proteomic andPhosphoproteomic Profile
Modern phosphoproteomic analysis has also been applied todetermine factors responding to sleep deprivation. An analysisof postsynaptic density (PSD) fraction identified several proteinsand phosphopeptides showing different abundances between
light and dark phases (Diering et al., 2017). Among them, thelevel of CaMKIIα with activated autophosphorylation (T286) was
found to be approximately twofold higher in the dark phase atwhich the nocturnal mice spend much of its time in an awake
state. Thus, this kinase seems to have higher activity during theawake phase. Interestingly, one of the other phosphorylation
residues of CaMKIIα (T310) is lower in the dark phase,suggesting that not only the amount of phosphorylation but also
the pattern of multisite phosphorylation varies, depending on thesleep/awake state.
The rhythmic dynamics of each layer of -omics profiles canbe generated directly by circadian TTFLs or indirectly as aconsequence of clock-controlled changes in cellular activity, such

SLEEP-WAKE-DEPENDENT CONTROLOF PHOSPHOPROTEOME PROFILE
Uncoupling Between Transcriptome andProteome Profiles
A complementary approach for investigating the importance ofprotein phosphorylation is to quantify protein abundances andFrontiers in Psychology | www.frontiersin.org

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FIGURE 2 | Posttranscriptional, posttranslational, and protein phosphorylation shape the daily rhythmicity of protein action. Daily rhythmicity of mRNA and protein
abundance is created not only by the timing of transcription initiation but also through multiple layers of posttranscriptional and posttranslational regulation. The
continuous elevation of sleep pressure predominantly attenuates the rhythmicity observed in protein and phospho-protein abundance at the synapse.

the action of kinases (or phosphatases). This is because themajority of rhythmic phosphopeptides are derived fromproteins that do not exhibit daily oscillations. Even for minor
rhythmic phosphopeptides derived from rhythmic proteins, theamplitude of oscillations is much higher in phosphopeptides.
The computational prediction assigned CaMKII and PKC as
kinases that are activated at the transition phase from sleep
to awake, suggesting that the activities of these kinases are
regulated rhythmically. The Western blot against these kinases
with phosphorylation that marks the kinase-active forms showed
an interesting daily oscillation pattern: the active CaMKIIα(phosphorylated T286; autophosphorylation site) continuously
increases during the awake period and then decreases during thesleep period. This behavior mirrors the anticipated dynamics ofProcess S and is well matched with the expected role of CaMKII asa sleep-promoting kinase. The active PKC (phosphorylated S660;
autophosphorylation site) behaves symmetrically to CaMKII: the(auto)phosphorylated PKC increases during the sleep phase anddecreases during the awake phase. This reciprocal activation of
PKC and CaMKII is of particular interest in that both kinases are
involved in the induction of LTP (Malenka et al., 1989; Malinow
et al., 1989); the phosphorylation dynamics suggests that this
PKC has a mutually exclusive role with CaMKII in sleep-wakeregulation, although the conclusive role of PKC in the regulationof the sleep-wake cycle needs to be clarified.
Similar to the case of rhythmicity in protein abundances,
the rhythmic phosphopeptide dynamics are dominated by sleepwake transition rather than the circadian clocks (Bruning et al.,2019). The 4-h sleep deprivation applied prior to each sampling
point across the day eliminated the rhythmicity of ∼98%

as sleep-wake transitions. As for the case of proteomics andphosphoproteomics profiles in the brain, a recent study showsthat the latter profile is dominant. Time-course comparison
between synaptic transcriptome and proteome profiles found a
striking effect of sleep deprivation on the daily rhythmicity ofsynaptic proteins (Noya et al., 2019). This study revealed that
67% of synaptic transcripts are rhythmically expressed in thesynaptosome, but in the entire forebrain fraction, most of thetranscripts are not rhythmic. Therefore, the source of rhythmicity
for the synaptic transcripts lies in the posttranscriptional control
at synapses. In addition, 27% of such rhythmic transcripts still
remain rhythmic even when the mice are challenged to 4-h
sleep deprivation prior to each sampling time point. However,the sleep deprivation protocol diminishes the rhythmicity in theprotein abundances, which are otherwise observed in accordancewith the rhythmic transcripts. Therefore, translational and/or
posttranslational control at the synapse is dominated by sleepwake states, and 4-h sleep deprivation locks the proteomic
profile into a sleep-deprived (or awake) state. Assuming thatmost of the cellular functions are determined by the geneproducts rather than the expressed RNA itself, the effect of sleep
deprivation on protein abundance that is strong enough to mask
the rhythmic transcription indicates that the temporal synaptic
status is largely determined by the sleep-wake state rather thanthe circadian phase.
A phosphoproteomic profile of the synaptosome further
revealed circadian-phase-specific activation of kinase signaling(Bruning et al., 2019). In the synaptosome, ∼30% of quantified
phosphopeptides show daily rhythmicity. Most of thephosphorylation rhythm is supposed to be generated by

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Protein Phosphorylation and Sleepinessphosphatase 1 regulatory subunit 9B) results in the reduced
sleep duration per day analyzed by the piezoelectric-sensorbased activity recording1 (Jan et al., 2020). In the transcriptome
analysis of rodents, several catalytic and regulatory subunits ofprotein phosphatases were identified as genes whose expression
levels are affected by sleep deprivation (Cirelli et al., 2004;
Mackiewicz et al., 2007; Maret et al., 2007). Interestingly,
knockout of calcineurin, a Ca2+ /calmodulin-dependent protein
phosphatase, and its regulator sarah in Drosophila resulted in asignificant reduction of sleep duration (Nakai et al., 2011; Tomita
et al., 2011), suggesting a possible connection between Ca2+
signaling and protein phosphorylation status in the sleep control
mechanism. The expression of constitutive active calcineurinleads to controversial results in two studies (Nakai et al., 2011;
Tomita et al., 2011), potentially indicating the importance of theproper level of phosphatase activity.
The third possibility is about protein turnover control.
Because newly synthesized proteins are unphosphorylated form,
increased protein turnover rate (e.g., increased proteolysis
activity and increased transcription/translation activity) would
help to decrease the phosphorylation level. It has been shownthat the genes involved in the “macromolecular biosynthesis”(including protein synthesis and transport) are upregulated in
the sleep phase (Mackiewicz et al., 2007). If such biosynthesis
machinery contributes to refresh proteins, it can be assumed thatthe rate of protein turnover alters the global phosphorylation
status in sleep-wake cycle. Stable-isotope labeling technique
can be used to evaluate the omics-scale protein turnover rate
in neurons (Dorrbaum et al., 2018), and it would be worth
investigating the proteome-wide changes in the protein turnoverrate along with the sleep-wake cycle. In addition, the turnoverrate of specific proteins is often controlled through the ubiquitinproteasome pathway. There are some studies suggesting the
involvement of the proteasome system in the sleep-wake control.
Mouse with a knockout of Ube3a, an E3 ubiquitin ligase, atmaternal allele is a model animal for Angelman syndrome andcause a reduced SWA in NREM sleep and in response to sleep
deprivation (Colas et al., 2005; Ehlen et al., 2015). In Drosophila,
impaired activities of Cul3 and insomniac, an E3 ubiquitin ligase
and its adaptor, respectively, resulted in the reduced sleep amount(Stavropoulos and Young, 2011; Pfeiffenberger and Allada, 2012).
Overall, phosphoproteomics studies demonstrate that sleepwake transition dominates the phosphorylation status of synaptic
proteins. The clever strategy used to find the SNIPPs strengthens
the hypothesis that the phosphorylation status of protein
marks the level of sleep need. The proteomics measurements
alone reveal only the correlation between phosphorylation andthe expected level of sleepiness; however, given the fact that
phosphoproteomics analysis also suggests that CaMKII activity
increases with the transition from awake to asleep and withthe stimulation of sleepiness by two independent perturbations,
sleep-promoting kinases, which have been shown to have a causal
effect on sleep duration, can be reasonably regarded as moleculesthat promote sleep according to the accumulation of the awake
period in vivo.

otherwise rhythmic phosphopeptides. In agreement with this, thepeak points of rhythmic phosphopeptides were accumulated at
the transition of either the sleep-to-wake or wake-to-sleep phase.
The drastic changes in the phosphoproteomic profile imply thatthe phosphorylation status can be a molecular representationof sleep need. However, sleep deprivation increases not only
the level of sleep need but also the preceding awake duration.
Because some phosphorylation responds rapidly to the priorsleep/wake state (Mikhail et al., 2017), distinguishing whether
the changes in the phosphorylation status are caused by theincreased sleep need or signals associated with the increased
awake state (e.g., visual input) is difficult. Recently, Sleepy
mice were used for phosphoproteomic analysis to study thisproblem (Wang Z. et al., 2018). This study compared theproteomic and phosphoproteomic profiles caused by sleep
deprivation and the Sleepy mutation. Both perturbations aresupposed to increase the sleep need, but the behavioral
effects caused by these perturbations are opposite: Sleepy miceshould spend a longer time in the sleep state, while sleep
deprivation disturbs the sleep state. The results demonstrated
that both perturbations similarly increase phosphorylated
proteins without much increase in the amount of total (nonphosphorylated) protein; indeed, the researchers have a seriesof phosphorylated proteins, named as the sleep-need-indexphosphoproteins (SNIPPs), that increase in both sleep-deprived
and the Sleepy mice. Many SNIPPs are involved in thesynaptic regulation and, interestingly, include CaMKII. CaMKII
activation during sleep deprivation may simply be due toincreased synaptic input during the wake period, but similar
activation of CaMKII in the Sleepy mice with short awake
duration strongly suggests that CaMKII activity is associated withan increased need for sleep.
There could be several (not mutually exclusive) reasons for the
global changes in the phosphorylation states. The first possibility
is that altered neuronal excitability and synaptic inputs associatedwith different arousal state affect several types of kinases aspredicted in phosphoproteomic studies (Wang Z. et al., 2018;
Bruning et al., 2019). Because current bioinformatics analyses canonly predict kinase-substrate relationships for a limited numberof substrates (Needham et al., 2019), perturbations to multiple
kinases may result in changes in the phosphorylation state ofa larger number of residues than one would expect based on
current biochemical information.
The second possibility would be that the sleep-wake cycle
may affect protein phosphatase activity. Protein phosphorylationlevels in cells are reversibly controlled by the balance between
kinase and phosphatase activities. Phosphatases had been
generally considered as less-specific promiscuous enzymes
because they act on many substrate sequences. This view,
however, is being revised as phosphatase-substrate specificity
is controlled through various combinations of catalytic andregulatory subunits (Moorhead et al., 2007; Sacco et al.,2012). Several regulatory subunits appear to be involved in
the mammalian sleep-wake control. The phenotype catalog ofgene knockout mice developed by the International Mouse
Phenotyping Consortium (Brown and Moore, 2012a,b; Ring
et al., 2015) reported that knockout of Ppp1r9b (protein

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Protein Phosphorylation and Sleepinesscircadian clocks. In addition, given the fact that the sleep-wakecycle is strongly affected by external stimuli, the third aspect willbe that the specific phosphorylation events should also respondto the environmental and internal signals that promote either
sleep or wakefulness.
The most evident signal to regulate sleep is the neuronal
input, which can be directly transduced and integrated into
CaMKII activation by the intracellular Ca2+ signaling. This
would be straightforward: activation of sleep-promoting CaMKII
is mediated by the increased neuronal spikes presumably
associated with the awake state. Factors that may transduce thewake-associated information to the kinase are not limited tothe synaptic input. Several monoamines working as excitatoryneuromodulators (e.g., norepinephrine or dopamine) that
promote wakefulness (Fuller et al., 2011) may also alter theactivity of CaMKII by modulating the excitability of neurons.
The SIK family is controlled by the multisite phosphorylation
caused by elevated cAMP and subsequent activation of PKA. The
neuronal cAMP level is modulated by several G protein-coupled
receptors, including receptors of the excitatory monoamine
neuromodulators. Although PKA has been known to mediate anawake-promoting effect caused by octopamine neurotransmitterin Drosophila (Hendricks et al., 2001; Crocker et al., 2010), the
PKA-SIK pathway in mammals might be involved in the sleeppromoting effect of neurotransmitters which activates the cAMPin cortical neurons. On the other hand, ERK is activated bya wide range of cell signaling from cell differentiation to cell
death (Coulthard et al., 2009). A potential pathway related tosleep control is stress-induced activation of ERK, which may betriggered by sleep deprivation that is known to be associatedwith the elevation of inflammatory responses (Clinton et al.,2011). If a part of these signals activates the sleep-promoting
kinases, the kinases may bridge the cellular signaling to the
induction of sleep.
Alternatively, because sleep-wake transition and sleep
deprivation alter the phosphorylation status of sleep-promoting
kinases, the altered kinase activities would affect not only thesleep regulation, but also cellular functions associated witheach sleep-promoting kinase. In other words, sleep-promoting
kinases may serve as hubs for cellular signaling cascades linking
environmental/internal signals that regulate sleep and cellularfunctions that are regulated by sleep. For example, the wellunderstood function of CaMKII is its role in regulating synaptic
plasticity (Coultrap and Bayer, 2012; Bayer and Schulman,2019), and in the hippocampus, the altered phosphorylation
status of CaMKII affects the synapse size (Pi et al., 2010a).
The sleep-wake cycle would also affect synaptic strength.
However, whether NREM sleep contributes in the direction ofstrengthening or weakening synaptic strength, or both, is stilla matter of debate (Puentes-Mestril and Aton, 2017; Klinzing
et al., 2019). Several studies showed that synaptic strength isstrengthened (Chauvette et al., 2012) and spine formation at a
specific set of dendrite is promoted (Yang et al., 2014) during
NREM sleep. These synaptic enhancements during NREM
sleep would depend on the synaptic inputs during the awake
phase that precedes the sleep episode. By contrast, other lines
of evidence indicate that the size of the axon-spine interface

PHOSPHORYLATION HYPOTHESIS OF
MOLECULAR SLEEPINESS: A
PERSPECTIVE
Connecting Physiological Context to the
Sleep Regulation
We first propose a kinase-centric view of sleep need, namely,a phosphorylation hypothesis of molecular sleepiness, based on
the finding of the sleep-promoting kinase CaMKIIα/CaMKIIβ(Tatsuki et al., 2016). This hypothesis has been strengthenedby the subsequent finding of other sleep-promoting kinases aswell as phosphoproteomic investigations of molecular dynamics
according to the sleep-wake cycle and its disturbance. The
identified sleep-promoting kinases are involved in a numberof cellular functions rather than just a specific sleep-promoting
event; the example includes involvement of CaMKIIα/CaMKIIβin the regulation of learning and memory (Coultrap and Bayer,2012), ERK for stress responses (Coulthard et al., 2009), and
SIK for the regulation of metabolic processes (Sakamoto et al.,2018). Because of these multiple functions of the sleep-promoting
kinases, the kinase-centric view of sleep need opens an avenue
to reevaluate the physiological and biochemical aspects of sleep
control by integrating multiple inputs and outputs associatedwith sleep-wake control (Figure 3).
If protein activities modified by phosphorylation are the denovo molecular signature of sleep need, at least the following
aspects should be explained by critical phosphorylation events(Figure 4). First, the phosphorylation status should be directly
related to the homeostatic regulation of sleep. This will beachieved by demonstrating a causal sleep-promoting effect of a
specific phosphorylation event(s) that is, in turn, regulated by thesleep-wake cycle (feedback regulation). Second, given the strong
effect of circadian clocks on the behavioral sleep-wake cycle, thespecific phosphorylation events should be linked with circadianclock control. Such phosphorylation events will account for theunidentified molecular interface between sleep homeostasis and

FIGURE 3 | Phosphorylation hypothesis of molecular sleepiness.
Sleep-promoting kinases may play important roles in integrating several
signals that affect sleep-wake dynamics. The activities of sleep-promoting
kinases, in turn, may affect sleep-wake dynamics as well as physiological
functions associated with neural activity.

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Protein Phosphorylation and Sleepiness

FIGURE 4 | Three properties that the central factor responsible for sleep homeostasis should have. If sleep-promoting kinases compose a core mechanism of sleep
homeostasis, first, the dynamics of kinase activity would correspond to the level of sleep need. Second, the kinase activity would be directly or indirectly regulated bycircadian clocks as an interface of sleep homeostasis and circadian clocks. Third, the kinase activities may mediate various external signals to affect the arousal stateof animals.

types of neuronal plasticity and learning, including hippocampal
LTP and spatial learning (Mayford et al., 1996; Giese et al.,
1998; Elgersma et al., 2002; Yamagata et al., 2009). The
question is how the relatively short term event, such as the
autophosphorylation at CaMKII T286, which occurs within
few minutes after the simulation (Coultrap and Bayer, 2012),
can be converted to cellular outputs with slower dynamics.
Apparently, changes in the synaptic strength and memory
consolidation involve cellular events downstream of CaMKII
kinase action, including the translocation of CaMKII andsubsequent modification of synaptic architecture (Lisman et al.,2012). CaMKII itself can also encode information spanninga longer time duration because this kinase undergoes thecomplex nature of regulation, including dodecameric (andtetradecameric) oligomerization with variable structures (Myerset al., 2017; Bayer and Schulman, 2019) and phosphorylation at∼30 residues (Baucum et al., 2015).
Multisite phosphorylation is also the core mechanism for the
regulation of ERK and SIK3. A role for multisite phosphorylation
has been proposed to encode slower dynamics (Salazar et al.,2010). An increasing number of phosphorylation sites cancreate a time delay to reach a fully phosphorylated state.
Even if phosphorylation at a specific residue is critical for the
biological function, multisite phosphorylation can delay thetiming of the specific phosphorylation by serving as a decoy(Kim and Ferrell, 2007). The multisite phosphorylation often
occurs on the flexible region of a substrate protein, which may
form intrinsically disordered regions (IDRs), and altered local
electrostatic state produced by the multiple phosphorylation
may greatly affect IDR structure (Iakoucheva et al., 2004;

in several cortical areas and hippocampal CA1 region are,on average, decreased in the sleep phase compared with theawake phase (de Vivo et al., 2017; Spano et al., 2019). It is also
demonstrated that REM sleep but not NREM sleep is important
for the selective elimination of newly formed synapses dependingon the dendritic Ca2+ spikes (Li et al., 2017). To uncovercellular and sub-cellular responses upon the sleep induction,
local and molecular induction of sleep pressure would be a
critical experiment. Thus, the sleep-promoting kinases would
be key molecules and pertabator to uncover the molecularmechanism of reciprocal interaction between synaptic function
and sleep-wake regulation.

Connecting Different Timescales
The other perspective concerns the regulation of different
timescales. The phosphorylation reaction typically occurs in atimescale of seconds to minutes, while the accumulation of sleep
need should occur on a scale of minutes to hours (or evendays) (Franken et al., 2001; Van Dongen et al., 2003; Wisor andClegern, 2011). Is it possible that simple phosphorylation inside aneuron can count and integrate events that occur on such a slow
timescale?
Researchers have been tackling this problem for CaMKII interms of the regulation of synaptic plasticity and memory
function. Although the activity of CaMKII is elevatedcontinuously by repeptide synaptic inputs, the integrated
CaMKII activity seems not to be maintained on more than aminutes scale (Chang et al., 2017). Nonetheless, it has been
well understood that CaMKII kinase activity and proper
autophosphorylation at T286 and other sites are important for

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Protein Phosphorylation and Sleepinessthe simple transfer of the phosphoryl group governs the arousal
level of mammals.

Wright and Dyson, 2015). These processes may collectively
cause slow dynamics: excellent examples can be found in theregulation of the circadian 24-h timescale. A transcription
factor, PER, in mammals/flies and its functional homolog FRQ
in fungi undergo progressive multisite phosphorylation
within a 24-h period. Many biochemical and genetic
analyses have confirmed that progressive phosphorylation
is important for the 24-h period of circadian oscillation(Gallego and Virshup, 2007).
PER and FRQ are typical proteins with IDRs, and biochemical
studies indicate that the structures of PER and FRQ are
significantly changed according to phosphorylation status. FRQ
protein has a biased distribution of charged residues with an
isoelectric point (pI) of the N terminal domain around 9.7
but that of the C terminal domain around 4.3. With such an
uneven electrostatic distribution, FRQ protein is thought to be
in a closed state in its natural form. Multisite phosphorylationon the N terminal domain equilibrates the pI of the twoterminal domains and, through their repulsive interaction,
opens the protein conformation (Querfurth et al., 2011). Like
FRQ, it has been shown that PER in flies and mammals
undergoes phosphorylation-dependent structural change (Chiuet al., 2011; Zhou et al., 2015). In addition, PER/FRQ and their
responsible kinase CKI, as well as other binding partners, form amacromolecular complex (Aryal et al., 2017; Liu et al., 2019), thestatus of which (e.g., binding affinity of each component) may bechanged slowly by multisite phosphorylation within the complex.
In a sense, the slow dynamics produced by the collective andcooperative interaction of protein complexes driven by multisite
phosphorylation may act as a timer to count the length of a
24-h period in eukaryotes (Larrondo et al., 2015; Dunlap andLoros, 2018; Ode and Ueda, 2018; Diernfellner and Brunner,
2020; Loros, 2020; Partch, 2020).
Turning back to the sleep control, the essential next step is tofind the substrate protein and phosphorylation residues critical
for the sleep-inducing effect of the sleep-promoting kinases.
Because kinase is one of the best-studied protein class andthere are sophisticated methodologies to find target substratesof each kinase and to perturb specific kinase activity such asengineered analog-sensitive kinase (Bishop et al., 2000; Lopezet al., 2014) and photo-switchable kinase inhibitor (Murakoshi
et al., 2017). Do those kinases work in neurons in sleep control?
If so, which brain area and subcellular region would it be? The
substrates of sleep-promoting kinase should affect the neuronalfiring pattern to induce the synchronized cortical slow oscillation:
the direct substrates of sleep-promoting kinases might include
ion channels and pumps that expressed cortex to modulate theneuronal membrane potential and also are highlighted theirroles in the control of basal sleep duration and SWA by mice
genetic studies (Espinosa et al., 2004; Lee et al., 2004; Anderson
et al., 2005; Douglas et al., 2007; Sunagawa et al., 2016; Tatsuki
et al., 2016; Yoshida et al., 2018; Muheim et al., 2019). Also,
the kinases might indirectly affect the neuronal excitability bymodulating the other enzymatic and cellular transport systems
that control the level of neurotransmitters and neuromodulators.
Upon the identification of the part of sleep-promoting kinase andsubstrate, further studies will illuminate the mechanism of how

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CONCLUSION
The importance of kinases for the regulation of sleep has been
supported by genetic and phosphoproteomic studies. Genetic
studies indicated that CaMKII, SIK3, and ERK are potent
kinases that induce sleep. Phosphoproteomic studies indicatedthat the sleep-wake cycle dominates the phosphorylation statusof synaptic proteins. In particular, SIK3 appears to induce
a similar phosphoproteomic profile to that caused by sleep
deprivation, and the dynamics of CaMKII activation correlate
well with the expected accumulation of sleep need. These
results imply that kinases transduce and store cumulative
neuronal activity as the phosphorylation status of their substates,
including the phosphorylation status of the kinases themselves.
In this view, the molecular entity of sleepiness is, at leastin part, protein phosphorylation: we shall call this idea aphosphorylation hypothesis of molecular sleepiness–the kinasecentric understanding of sleep-wake transition may integrate themolecular mechanism of sleepiness and physiological functionsof sleep to control protein activity.
So far, experimental data supporting this hypothesis havecome primarily from studies of mouse models. Therefore,
this hypothesis would be applicable to NREM sleep inmammals. However, since protein regulation by phosphorylationis conserved across species, and the sleep-promoting enzymes areconserved from vertebrates to invertebrates, it is worthwhile toinvestigate the extent to which the kinases and their substrates
are involved in sleep regulation in other species as well. The
possibility that the need for sleep can be estimated as aprotein phosphorylation state suggests that the same index may
be used to assess sleep status in species such as primitive
invertebrates, where EEG cannot be used to measure sleep status.
In combination with a rigorous evaluation of this hypothesis
in mammalian sleep, it is important to investigate whether thishypothesis holds true in a variety of organisms. This should leadto a molecular exploration of the extent to which the regulatory
mechanisms and functions of sleep are conserved across species.

AUTHOR CONTRIBUTIONS
KO and HU wrote the manuscript, conducted the literature
survey, and approved the submitted version. Both authors
contributed to the article and approved the submitted version.

FUNDING
This work was supported by grants from the Japan Agency
for Medical Research and Development – Core Research
for Evolutional Science and Technology (AMED-CREST)(JP17gm0610006, HU), Brain Mapping by Integrated
Neurotechnologies for Disease Studies (Brain/MINDS)

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Ode and Ueda

Protein Phosphorylation and Sleepiness(JP17DM0207049, HU), Basic Science and Platform Technology
Program for Innovative Biological Medicine (JP17AM0301025,
HU), KAKENHI Grant-in-Aid from JSPS (Scientific Research S
18H05270, HU and Scientific Research C 20K06576, KO), HFSP
(Human Frontier Science Program) Research Grant Program(HFSP RGP0019/2018, HU), Exploratory Research for Advanced
Technology (ERATO) grant (JPMJER1904, HU) from the Japan
Science and Technology Agency (JST), and an intramural

Grant-in-Aid from the RIKEN Center for Biosystems Dynamics
Research to HU.

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ACKNOWLEDGMENTS
The authors would like to thank Enago (www.enago.jp) for the
English language review.

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