PER2 Degradation Research Articles

Hierarchical and scaffolded phosphorylation of two degrons controls PER2 stability

The duration of the transcription-repression cycles that give rise to mammalian circadian rhythms is largely determined by the stability of the PERIOD protein, the rate-limiting components of the molecular clock. The degradation of PERs is tightly regulated by multisite phosphorylation by Casein Kinase 1 (CK1δ/ε). In this phosphoswitch, phosphorylation of a PER2 degron (Degron 2, D2) causes degradation, while phosphorylation of the PER2 Familial Advanced Sleep Phase (FASP) domain blocks CK1 activity on the degron, stabilizing PER2. However, this model and many other studies of PER2 degradation do not include the second degron of PER2 that is conserved in PER1, termed Degron 1, D1. We examined how these two degrons contribute to PER2 stability, affect the balance of the phosphoswitch, and how they are differentiated by CK1. Using PER2-luciferase fusions and real-time luminometry, we investigated the contribution of both D2 and of CK1-PER2 binding. We find that D1, like D2, is a substrate of CK1 but that D1 plays only a ‘backup’ role in PER2 degradation. Notably, CK1 bound to a PER1:PER2 dimer protein can phosphorylate PER1 D1 in trans. This scaffolded phosphorylation provides additional levels of control to PER stability and circadian rhythms.

Lithium affects the circadian clock in the choroid plexus – A new role for an old mechanism

Lithium is an effective mood stabilizer, but the mechanism of its therapeutic action is not well understood. We investigated the effect of lithium on the circadian clock located in the ventricle barrier complex containing the choroid plexus (CP), a part of the glymphatic system that influences gross brain function via the production of cerebrospinal fluid. The mPer2Luc mice were injected with lithium chloride (LiCl) or vehicle, and their effects on the clock gene Nr1d1 in CP were detected by RT qPCR. CP organotypic explants were prepared to monitor bioluminescence rhythms in real time and examine the responses of the CP clock to LiCl and inhibitors of glycogen synthase kinase-3 (CHIR-99021) and protein kinase C (chelerythrine). LiCl affected Nr1d1 expression levels in CP in vivo and dose-dependently delayed the phase and prolonged the period of the CP clock in vitro. LiCl and CHIR-99021 had different effects on 1] CP clock parameters (amplitude, period, phase), 2] dexamethasone-induced phase shifts of the CP clock, and 3] dynamics of PER2 degradation and de novo accumulation. LiCl-induced phase delays were significantly reduced by chelerythrine, suggesting the involvement of PKC activity. The effects on the CP clock may be involved in the therapeutic effects of lithium and hypothetically improve brain function in psychiatric patients by aligning the function of the CP clock-related glymphatic system with the sleep-wake cycle. Importantly, our data argue for personalized timing of lithium treatment in BD patients.

The role of cell-autonomous circadian oscillation of Cry transcription in circadian rhythm generation.

The current model of the mammalian circadian clock describes cell-autonomous and negative feedback-driven circadian oscillation of Cry and Per transcription as the core circadian rhythm generator. However, the actual contribution of this oscillation to circadian rhythm generation remains undefined. Here we perform targeted disruption of cis elements indispensable for cell-autonomous Cry oscillation. Mice lacking overt cell-autonomous Cry oscillation show robust circadian rhythms in locomotor activity. In addition, tissue-autonomous circadian rhythms are robust in the absence of overt Cry oscillation. Unexpectedly, although the absence of overt Cry oscillation leads to severe attenuation of Per oscillation at the cell-autonomous level, circadian rhythms in Per2 accumulation remain robust. As a mechanism to explain this counterintuitive result, Per2 half-life shows cell-autonomous circadian rhythms independent of Cry and Per oscillation. The cell-autonomous circadian clock may therefore remain partially functional even in the absence of overt Cry and Per oscillation because of circadian oscillation in Per2 degradation.

Mutation of a PER2 phosphodegron perturbs the circadian phosphoswitch

Casein kinase 1 (CK1) plays a central role in regulating the period of the circadian clock. In mammals, PER2 protein abundance is regulated by CK1-mediated phosphorylation and proteasomal degradation. On the other hand, recent studies have questioned whether the degradation of the core circadian machinery is a critical step in clock regulation. Prior cell-based studies found that CK1 phosphorylation of PER2 at Ser478 recruits the ubiquitin E3 ligase β-TrCP, leading to PER2 degradation. Creation of this phosphodegron is regulated by a phosphoswitch that is also implicated in temperature compensation. However, in vivo evidence that this phosphodegron influences circadian period is lacking. Here, we generated and analyzed PER2-Ser478Ala knock-in mice. The mice showed longer circadian period in behavioral analysis. Molecularly, mutant PER2 protein accumulated in both the nucleus and cytoplasm of the mouse liver, while Per2 messenger RNA (mRNA) levels were minimally affected. Nuclear PER1, CRY1, and CRY2 proteins also increased, probably due to stabilization of PER2-containing complexes. In mouse embryonic fibroblasts derived from PER2-Ser478Ala::LUC mice, three-phase decay and temperature compensation of the circadian period was perturbed. These data provide direct in vivo evidence for the importance of phosphorylation-regulated PER2 stability in the circadian clock and validate the phosphoswitch in a mouse model.

Site-specific phosphorylation of casein kinase 1 δ (CK1δ) regulates its activity towards the circadian regulator PER2.

Circadian rhythms are intrinsic ~24 hour cycles that regulate diverse aspects of physiology, and in turn are regulated by interactions with the external environment. Casein kinase 1 delta (CK1δ, CSNK1D) is a key regulator of the clock, phosphorylating both stabilizing and destabilizing sites on the PER2 protein, in a mechanism known as the phosphoswitch. CK1δ can itself be regulated by phosphorylation on its regulatory domain, but the specific sites involved, and the role this plays in control of circadian rhythms as well as other CK1-dependent processes is not well understood. Using a sensitized PER2::LUC reporter assay, we identified a specific phosphorylation site, T347, on CK1δ, that regulates CK1δ activity towards PER2. A mutant CK1δ T347A was more active in promoting PER2 degradation. This CK1δ regulatory site is phosphorylated in cells in trans by dinaciclib- and staurosporine-sensitive kinases, consistent with their potential regulation by cyclin dependent and other proline-directed kinases. The regulation of CK1δ by site-specific phosphorylation via the cell cycle and other signaling pathways provides a mechanism to couple external stimuli to regulation of CK1δ-dependent pathways including the circadian clock.

A Period2 Phosphoswitch Regulates and Temperature Compensates Circadian Period

Period (PER) protein phosphorylation is a critical regulator of circadian period, yet an integrated understanding of the role and interaction between phosphorylation sites that can both increase and decrease PER2 stability remains elusive. Here, wepropose a phosphoswitch model, where two competing phosphorylation sites determine whether PER2 has a fast or slow degradation rate. This mathematical model accurately reproduces the three-stage degradation kinetics of endogenous PER2. We predict and demonstrate that the phosphoswitch is intrinsically temperature sensitive, slowing down PER2 degradation as a result of faster reactions at higher temperatures. The phosphoswitch provides a biochemical mechanism for circadian temperature compensation of circadian period. This phosphoswitch additionally explains the phenotype of Familial Advanced Sleep Phase (FASP) and CK1ε(tau) genetic circadian rhythm disorders, metabolic control of PER2 stability, and how drugs that inhibit CK1 alter period. The phosphoswitch provides a general mechanism to integrate diverse stimuli to regulate circadian period.

Abstract 53: Interplay among E3 ubiquitin ligases regulate timely degradation of the circadian factor Period 2

Abstract Circadian rhythms are mechanisms that measure time on a scale of about 24 h and that adjusts our body to external environmental signals. Core circadian clock genes are defined as genes whose protein products are necessary components for the generation and regulation of circadian rhythms. Oscillatory rhythms are generated as result of the activation of positive and negative transcriptional feedback loops in which post-translational modifications, shuttling, and degradation are common themes. In mammals, the positive feedback loop involves regulation of bmal1 transcription. BMAL1/CLOCK selectively binds to E-box enhancers and drives the expression of per, cry and rev-erbα genes. REV-ERBa protein then represses bmal1 transcription through Rev-Erba/ROR response elements in its promoter. Then, the level of bmal1 RNA falls, as per and cry RNA levels peak. Period proteins (i.e., PER1, 2, and 3) accumulate in the cytoplasm, becomes phosphorylated by CK1e/d, ubiquitylated and degraded. Later in the day, cryptochrome accumulates (i.e., CRY2), associates with PER2/CK1e/d and this complex translocates to the nucleus where CRY disrupts the CLOCK/BMAL1-associated transcriptional complex, resulting in the inhibition of cry, per and rev-erba and de-repression of bmal1 transcription. In addition to the transcriptional loops, ubiquitin E3 ligases-mediated protein degradation is critical to sustain physiological rhythms. The F-box proteins b-TrCP1/2 target PER1 and PER2 for degradation via ubiquitination. Whereas knocking down β-trcp results in PER stabilization and period lengthening in synchronized culture cells; knock-out b-trcp1 mice do not display an abnormal circadian rhythmicity. Our findings indicate that PER2 ubiquitination engages two different types of E3 ligases, a process that directly influences the timely control of PER2 accumulation in the nucleus. Our data show that the mouse double-minute 2 homolog (Mdm2) RING finger E3 ligase binds to PER2 in multiple regions including an overlapping site with b-TrCP. We determined that PER2 is a specific substrate for Mdm2 and that PER2's polyubiquitination occurs both in vitro and in cells. Accordingly, overexpression Mdm2 results in the decrease of PER2 half-life, whereas, down-regulation of Mdm2 by siRNA increases PER2 stability. Furthermore, we found Mdm2-dependent post-translational modification of PER2 directly influences the amplitude of the circadian oscillatory response in synchronized cells and that both, b-TrCP and Mdm2 target PER2 at different circadian times. Overall, our findings support a model in which, the rate of nuclear degradation of PER2 leads to slow accumulation of the protein in the nucleus and, later in the day, to a temporal switch in which b-TrCP takes control over PER2 degradation. Thus, the interplaying between these two E3 ligases is indispensible for sustaining robust circadian oscillation. Citation Format: Carla V. Finkielstein, jingjing liu. Interplay among E3 ubiquitin ligases regulate timely degradation of the circadian factor Period 2. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 53. doi:10.1158/1538-7445.AM2015-53

Involvement of Stress Kinase Mitogen-activated Protein Kinase Kinase 7 in Regulation of Mammalian Circadian Clock

The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes, such as cell proliferation, apoptosis, differentiation, and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of an MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1ε. Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.

Temperature-insensitive reaction in the mammalian circadian clock

The striking feature of the circadian clock is its flexible yet robust response to various environmental conditions. A chemical-biological approach with 1260 pharmacologically active compounds identified “potent” compounds that markedly (>10 SD) lengthened the period. Most inhibited casein kinase Ie (CKIe) phosphorylation of the PER2 protein. By examining period extending effect and PER2 degradation inhibition of the 10 potent compounds, we verified the CKIe-dependent phosphorylation as a period-determining process. Moreover, the CKIe-dependent phosphorylation was temperature-insensitive in living clock cells. This temperature-insensitivity was preserved in the CKIe-dependent phosphorylation of a synthetic peptide in vitro. In addition, overexpression of CKIe restored temperature-insensitivity of degradation of LUC::PER2 protein in NIH3T3 cells. These results indicated that CKIe-dependent phosphorylation is a temperature-insensitive period-determining process in the mammalian circadian clock.

A Phylogenetically Conserved DNA Damage Response Resets the Circadian Clock

The mammalian circadian clock influences the timing of many biological processes such as the sleep/wake cycle, metabolism, and cell division. Environmental cues such as light exposure can influence the timing of this system through the posttranslational modification of key components of the core molecular oscillator. We have previously shown that DNA damage can reset the circadian clock in a time-of-day-dependent manner in the filamentous fungus Neurospora crassa through the modulation of negative regulator FREQUENCY levels by PRD-4 (homologue of mammalian Chk2). We show that DNA damage, generated with either the radiomimetic drug methyl methane sulfonate or UV irradiation, in mouse embryonic fibroblasts isolated from PER2::LUC transgenic mice or in the NIH3T3 cell line, elicits similar responses. In addition to induction of phase advances, DNA damage caused a decrease in luciferase signal in PER2::LUC mouse embryonic fibroblast cells that is indicative of PER2 degradation. Finally, we show that the activity of the BMAL1 promoter is enhanced during DNA damage. These findings provide further evidence that the DNA damage-mediated response of the clock is conserved from lower eukaryotes to mammals.

Selective Inhibition of Casein Kinase 1ϵ Minimally Alters Circadian Clock Period

The circadian clock links our daily cycles of sleep and activity to the external environment. Deregulation of the clock is implicated in a number of human disorders, including depression, seasonal affective disorder, and metabolic disorders. Casein kinase 1 epsilon (CK1epsilon) and casein kinase 1 delta (CK1delta) are closely related Ser-Thr protein kinases that serve as key clock regulators as demonstrated by mammalian mutations in each that dramatically alter the circadian period. Therefore, inhibitors of CK1delta/epsilon may have utility in treating circadian disorders. Although we previously demonstrated that a pan-CK1delta/epsilon inhibitor, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462), causes a significant phase delay in animal models of circadian rhythm, it remains unclear whether one of the kinases has a predominant role in regulating the circadian clock. To test this, we have characterized 3-(3-chloro-phenoxymethyl)-1-(tetrahydro-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine (PF-4800567), a novel and potent inhibitor of CK1epsilon (IC(50) = 32 nM) with greater than 20-fold selectivity over CK1delta. PF-4800567 completely blocks CK1epsilon-mediated PER3 nuclear localization and PER2 degradation. In cycling Rat1 fibroblasts and a mouse model of circadian rhythm, however, PF-4800567 has only a minimal effect on the circadian clock at concentrations substantially over its CK1epsilon IC(50). This is in contrast to the pan-CK1delta/epsilon inhibitor PF-670462 that robustly alters the circadian clock under similar conditions. These data indicate that CK1epsilon is not the predominant mediator of circadian timing relative to CK1delta. PF-4800567 should prove useful in probing unique roles between these two kinases in multiple signaling pathways.

SIRT1 Regulates Circadian Clock Gene Expression through PER2 Deacetylation

The mammalian circadian timing system is composed of a central pacemaker in the suprachiasmatic nucleus of the brain that synchronizes countless subsidiary oscillators in peripheral tissues. The rhythm-generating mechanism is thought to rely on a feedback loop involving positively and negatively acting transcription factors. BMAL1 and CLOCK activate the expression of Period (Per) and Cryptochrome (Cry) genes, and once PER and CRY proteins accumulate to a critical level they form complexes with BMAL1-CLOCK heterodimers and thereby repress the transcription of their own genes. Here, we show that SIRT1, an NAD(+)-dependent protein deacetylase, is required for high-magnitude circadian transcription of several core clock genes, including Bmal1, Rorgamma, Per2, and Cry1. SIRT1 binds CLOCK-BMAL1 in a circadian manner and promotes the deacetylation and degradation of PER2. Given the NAD(+) dependence of SIRT1 deacetylase activity, it is likely that SIRT1 connects cellular metabolism to the circadian core clockwork circuitry.

β-TrCP1-Mediated Degradation of PERIOD2 Is Essential for Circadian Dynamics

Regulated degradation of circadian clock proteins is a crucial step for rhythm generation per se but also for establishing a normal circadian period. Here, the authors show that the F-box protein beta-transducin repeat containing protein 1 (beta-TrCP1) as part of the E3 ubiquitin ligase complex is an essential component of the mammalian circadian oscillator. Down-regulation of endogenous beta-TrCP1 as well as expression of a dominant-negative form both result in lengthening of the circadian period in oscillating fibroblasts. These phenotypes are due to an impaired degradation of PERIOD (PER) proteins, since expression of beta-TrCP interaction-deficient PER2 variants--but not wild-type PER2--results in a dramatic stabilization of PER2 protein as well as in the disruption of circadian rhythmicity. Mathematical modeling conceptualizes the authors' findings and suggests that loss of sustained rhythmicity in cells with eliminated beta-TrCP-mediated PER2 degradation is due to excessive nuclear repression, a prediction they verified experimentally.

Modeling of a Human Circadian Mutation Yields Insights into Clock Regulation by PER2

Circadian rhythms are endogenous oscillations of physiological and behavioral phenomena with period length of approximately 24 hr. A mutation in human Period 2 (hPER2), a gene crucial for resetting the central clock in response to light, is associated with familial advanced sleep phase syndrome (FASPS), an autosomal dominant condition with early morning awakening and early sleep times. The FASPS hPER2 S662G mutation resulted in PER2 being hypophosphorylated by casein kinase I (CKI) in vitro. We generated transgenic mice carrying the FASPS hPER2 S662G mutation and faithfully recapitulate the human phenotype. We show that phosphorylation at S662 leads to increased PER2 transcription and suggest that phosphorylation at another site leads to PER2 degradation. Altering CKIdelta dosage modulates the S662 phenotype demonstrating that CKIdelta can regulate period through PER2 in vivo. Modeling a naturally occurring human variant in mice has yielded novel insights into PER2 regulation.

Control of Mammalian Circadian Rhythm by CKIε-Regulated Proteasome-Mediated PER2 Degradation

The mammalian circadian regulatory proteins PER1 and PER2 undergo a daily cycle of accumulation followed by phosphorylation and degradation. Although phosphorylation-regulated proteolysis of these inhibitors is postulated to be essential for the function of the clock, inhibition of this process has not yet been shown to alter mammalian circadian rhythm. We have developed a cell-based model of PER2 degradation. Murine PER2 (mPER2) hyperphosphorylation induced by the cell-permeable protein phosphatase inhibitor calyculin A is rapidly followed by ubiquitination and degradation by the 26S proteasome. Proteasome-mediated degradation is critically important in the circadian clock, as proteasome inhibitors cause a significant lengthening of the circadian period in Rat-1 cells. CKIepsilon (casein kinase Iepsilon) has been postulated to prime PER2 for degradation. Supporting this idea, CKIepsilon inhibition also causes a significant lengthening of circadian period in synchronized Rat-1 cells. CKIepsilon inhibition also slows the degradation of PER2 in cells. CKIepsilon-mediated phosphorylation of PER2 recruits the ubiquitin ligase adapter protein beta-TrCP to a specific site, and dominant negative beta-TrCP blocks phosphorylation-dependent degradation of mPER2. These results provide a biochemical mechanism and functional relevance for the observed phosphorylation-degradation cycle of mammalian PER2. Cell culture-based biochemical assays combined with measurement of cell-based rhythm complement genetic studies to elucidate basic mechanisms controlling the mammalian clock.