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Cyclin-dependent Kinase 5 (Cdk5) Regulates the Function of CLOCK Protein by Direct Phosphorylation*

Open AccessPublished:November 14, 2013DOI:https://doi.org/10.1074/jbc.M113.494856
      Circadian rhythm is a biological rhythm governing physiology and behavior with a period of ∼24 h. At the molecular level, circadian output is controlled by a molecular clock composed of positive and negative feedback loops in transcriptional and post-translational processes. CLOCK is a transcription factor known as a central component of the molecular clock feedback loops generating circadian oscillation. Although CLOCK is known to undergo multiple post-translational modifications, the knowledge of their entities remains limited. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is involved in various neuronal processes. Here, we report that Cdk5 is a novel regulator of CLOCK protein. Cdk5 phosphorylates CLOCK at the Thr-451 and Thr-461 residues in association with transcriptional activation of CLOCK. The Cdk5-dependent regulation of CLOCK function is mediated by alterations of its stability and subcellular distribution. These results suggest that Cdk5 is a novel regulatory component of the core molecular clock machinery.

      Introduction

      Circadian rhythm is an internally generated biological rhythm with a period of ∼24 h under control of day/night cycle. Circadian rhythm enables our body to adapt to the environmental changes by optimizing a wide variety of physiological processes such as the sleep/wake cycle, hormonal response, and feeding behaviors (
      • Reppert S.M.
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      Molecular analysis of mammalian circadian rhythms.
      ,
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      A web of circadian pacemakers.
      ,
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      ). This biological rhythm is found in most tissues, including brain (
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      ,
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      Molecular mechanisms of the biological clock in cultured fibroblasts.
      ). The rhythm is generated by transcriptional and post-translational feedback loops that are composed of networks of clock proteins at the cellular level (
      • Reppert S.M.
      • Weaver D.R.
      Molecular analysis of mammalian circadian rhythms.
      ,
      • Schibler U.
      • Sassone-Corsi P.
      A web of circadian pacemakers.
      ,
      • Lowrey P.L.
      • Takahashi J.S.
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      ,
      • Ko C.H.
      • Takahashi J.S.
      Molecular components of the mammalian circadian clock.
      ). This cell-autonomous event is initiated by the positive limb of feedback loops comprised of the CLOCK/BMAL1 heterodimer. The CLOCK/BMAL1 complex enters nucleus by BMAL1-dependent shuttling and binds to E-box enhancers to drive the rhythmic transcription of clock-controlled genes, including Per and Cry (
      • Tamaru T.
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      • Takei K.
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      • Takamatsu K.
      Nucleocytoplasmic shuttling and phosphorylation of BMAL1 are regulated by circadian clock in cultured fibroblasts.
      ,
      • Kwon I.
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      • Chang S.H.
      • Jung N.C.
      • Lee B.J.
      • Son G.H.
      • Kim K.
      • Lee K.H.
      BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer.
      ). Newly synthesized PER and CRY proteins heterodimerize, translocate into the nucleus, and repress the transcriptional activity of the CLOCK/BMAL1 complex, forming the core part of the negative feedback loop.
      Various clock components undergo post-translational modifications, such as phosphorylation (
      • Reppert S.M.
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      Molecular analysis of mammalian circadian rhythms.
      ,
      • Lowrey P.L.
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      Mammalian circadian biology: elucidating genome-wide levels of temporal organization.
      ,
      • Lowrey P.L.
      • Takahashi J.S.
      Genetics of the mammalian circadian system: Photic entrainment, circadian pacemaker mechanisms, and posttranslational regulation.
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      • Reppert S.M.
      Posttranslational mechanisms regulate the mammalian circadian clock.
      ,
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      • Saez L.
      Posttranscriptional and posttranslational regulation of clock genes.
      ) and acetylation (
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      The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control.
      ,
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      SIRT1 regulates circadian clock gene expression through PER2 deacetylation.
      ), which are critical for their stability, intracellular localization, and transcriptional activity. Interestingly, CLOCK has its own histone acetyltransferase activity; thus, it acetylates both histone and non-histone proteins, including BMAL1 and PER2 (
      • Nakahata Y.
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      • Alt F.W.
      • Schibler U.
      SIRT1 regulates circadian clock gene expression through PER2 deacetylation.
      ). In addition to its histone acetyltransferase enzymatic activity, a CLOCK-dependent phosphorylation of BMAL1 has also been reported (
      • Kwon I.
      • Lee J.
      • Chang S.H.
      • Jung N.C.
      • Lee B.J.
      • Son G.H.
      • Kim K.
      • Lee K.H.
      BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer.
      ,
      • Yoshitane H.
      • Takao T.
      • Satomi Y.
      • Du N.H.
      • Okano T.
      • Fukada Y.
      Roles of CLOCK phosphorylation in suppression of E-box-dependent transcription.
      ,
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      • Kondratova A.A.
      • Gorbacheva V.Y.
      • Gudkov A.V.
      • Antoch M.P.
      BMAL1-dependent circadian oscillation of nuclear CLOCK: posttranslational events induced by dimerization of transcriptional activators of the mammalian clock system.
      ). CLOCK itself is known to be regulated by cGMP-dependent protein kinase (PKG) and PKC phosphorylations that are important for temporal progression into the circadian daytime and resetting of the molecular clock (
      • Tischkau S.A.
      • Mitchell J.W.
      • Pace L.A.
      • Barnes J.W.
      • Barnes J.A.
      • Gillette M.U.
      Protein kinase G type II is required for night-to-day progression of the mammalian circadian clock.
      ,
      • Shim H.S.
      • Kim H.
      • Lee J.
      • Son G.H.
      • Cho S.
      • Oh T.H.
      • Kang S.H.
      • Seen D.S.
      • Lee K.H.
      • Kim K.
      Rapid activation of CLOCK by Ca2+-dependent protein kinase C mediates resetting of the mammalian circadian clock.
      ). Recently, glycogen synthase kinase 3β (GSK3β)
      The abbreviations used are: GSK3β
      glycogen synthase kinase 3β
      ANOVA
      analysis of variance
      IP
      immunoprecipitation.
      has also been reported as a kinase that phosphorylates CLOCK in a BMAL1-dependent manner, thereby regulating degradation and activation of CLOCK (
      • Spengler M.L.
      • Kuropatwinski K.K.
      • Schumer M.
      • Antoch M.P.
      A serine cluster mediates BMAL1-dependent CLOCK phosphorylation and degradation.
      ). Moreover, it has also been reported that dominant negative CLOCK (CLOCKΔ19) lacking the CLOCK-interacting protein, circadian (CIPC)-binding domain shows less phosphorylation and more stability than wild-type CLOCK does (
      • Yoshitane H.
      • Takao T.
      • Satomi Y.
      • Du N.H.
      • Okano T.
      • Fukada Y.
      Roles of CLOCK phosphorylation in suppression of E-box-dependent transcription.
      ,
      • Zhao W.N.
      • Malinin N.
      • Yang F.C.
      • Staknis D.
      • Gekakis N.
      • Maier B.
      • Reischl S.
      • Kramer A.
      • Weitz C.J.
      CIPC is a mammalian circadian clock protein without invertebrate homologues.
      ). Therefore, it appears that post-translational modifications widely occur in clock components and play crucial roles in maintaining the circadian feedback loop, but additional post-translational modification-mediated regulation of the molecular clock remains unidentified.
      Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine-threonine kinase that is controlled by the neural specific activators, p35 and p39. Cdk5 controls various neuronal processes such as neurogenesis, neuronal migration, and axon guidance (
      • Dhavan R.
      • Tsai L.H.
      A decade of CDK5.
      ,
      • Su S.C.
      • Tsai L.H.
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      • Veeranna
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      • Pant H.C.
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      • Kulkarni A.B.
      • Mikoshiba K.
      Synergistic contributions of cyclin-dependant kinase 5/p35 and Reelin/Dab1 to the positioning of cortical neurons in the developing mouse brain.
      ). It has also been proposed that Cdk5 acts as a modulator of the brain reward system, mediating the response to various drugs including psychostimulants (
      • Bibb J.A.
      • Chen J.
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      • Svenningsson P.
      • Nishi A.
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      • Sagawa Z.K.
      • Ouimet C.C.
      • Nairn A.C.
      • Nestler E.J.
      • Greengard P.
      Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5.
      ,
      • Benavides D.R.
      • Quinn J.J.
      • Zhong P.
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      • DiLeone R.J.
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      • Yan Z.
      • Taylor J.R.
      • Bibb J.A.
      Cdk5 modulates cocaine reward, motivation, and striatal neuron excitability.
      ). Some reports suggest that Cdk5 activities in the brain are linked to various psychiatric diseases related conditions (
      • Engmann O.
      • Hortobágyi T.
      • Pidsley R.
      • Troakes C.
      • Bernstein H.G.
      • Kreutz M.R.
      • Mill J.
      • Nikolic M.
      • Giese K.P.
      Schizophrenia is associated with dysregulation of a Cdk5 activator that regulates synaptic protein expression and cognition.
      ,
      • Zhu W.L.
      • Shi H.S.
      • Wang S.J.
      • Xu C.M.
      • Jiang W.G.
      • Wang X.
      • Wu P.
      • Li Q.Q.
      • Ding Z.B.
      • Lu L.
      Increased Cdk5/p35 activity in the dentate gyrus mediates depressive-like behaviour in rats.
      ), in which CLOCK also has been reportedly associated (
      • Benedetti F.
      • Dallaspezia S.
      • Fulgosi M.C.
      • Lorenzi C.
      • Serretti A.
      • Barbini B.
      • Colombo C.
      • Smeraldi E.
      Actimetric evidence that CLOCK 3111 T/C SNP influences sleep and activity patterns in patients affected by bipolar depression.
      ,
      • Roybal K.
      • Theobold D.
      • Graham A.
      • DiNieri J.A.
      • Russo S.J.
      • Krishnan V.
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      • Peevey J.
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      • McClung C.A.
      Mania-like behavior induced by disruption of CLOCK.
      ,
      • Mukherjee S.
      • Coque L.
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      • Kumar J.
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      • Graham A.
      • Gordon E.
      • Enwright 3rd, J.F.
      • DiLeone R.J.
      • Birnbaum S.G.
      • Cooper D.C.
      • McClung C.A.
      Knockdown of Clock in the ventral tegmental area through RNA interference results in a mixed state of mania and depression-like behavior.
      ).
      In this study, we demonstrated that Cdk5 can directly phosphorylate CLOCK, thereby modulating the robustness of the positive limb of the molecular clock. This finding refines the current model for the molecular basis of circadian rhythm by placing Cdk5 as a novel regulatory component in the molecular clock.

      DISCUSSION

      It is known that CLOCK undergoes rhythmical phosphorylation, which has been proposed as an important regulatory mechanism for the molecular clock (
      • Lee C.
      • Etchegaray J.P.
      • Cagampang F.R.
      • Loudon A.S.
      • Reppert S.M.
      Posttranslational mechanisms regulate the mammalian circadian clock.
      ,
      • Yoshitane H.
      • Takao T.
      • Satomi Y.
      • Du N.H.
      • Okano T.
      • Fukada Y.
      Roles of CLOCK phosphorylation in suppression of E-box-dependent transcription.
      ). Indeed, abnormal function of a mutant clock protein, CLOCKΔ19, has been associated with reduced phosphorylation (
      • Yoshitane H.
      • Takao T.
      • Satomi Y.
      • Du N.H.
      • Okano T.
      • Fukada Y.
      Roles of CLOCK phosphorylation in suppression of E-box-dependent transcription.
      ). Moreover, cGMP-dependent protein kinase and PKC phosphorylate CLOCK and activate the positive arm of the molecular clock feedback loop and thus promote the initiation of the subjective day phase (
      • Tischkau S.A.
      • Mitchell J.W.
      • Pace L.A.
      • Barnes J.W.
      • Barnes J.A.
      • Gillette M.U.
      Protein kinase G type II is required for night-to-day progression of the mammalian circadian clock.
      ,
      • Shim H.S.
      • Kim H.
      • Lee J.
      • Son G.H.
      • Cho S.
      • Oh T.H.
      • Kang S.H.
      • Seen D.S.
      • Lee K.H.
      • Kim K.
      Rapid activation of CLOCK by Ca2+-dependent protein kinase C mediates resetting of the mammalian circadian clock.
      ). GSK3 has also been identified as a kinase for phosphorylation of CLOCK at Ser-427 in a BMAL1-dependent manner (
      • Spengler M.L.
      • Kuropatwinski K.K.
      • Schumer M.
      • Antoch M.P.
      A serine cluster mediates BMAL1-dependent CLOCK phosphorylation and degradation.
      ). Ser-38 and Ser-42 residues of CLOCK have also been reported to be phosphorylated by unknown kinases, thereby affecting nuclear localization and DNA binding (
      • Yoshitane H.
      • Takao T.
      • Satomi Y.
      • Du N.H.
      • Okano T.
      • Fukada Y.
      Roles of CLOCK phosphorylation in suppression of E-box-dependent transcription.
      ). In the present study, we report that Cdk5 directly phosphorylates CLOCK and alters its transcriptional activity. Moreover, reduced Cdk5 activity significantly decreased oscillatory power in the mRNA level of CLOCK target genes. Therefore, phosphorylation of CLOCK by CdK5 can serve as a novel regulatory component for a robust activation of molecular clock. Notably, the Thr-451/461 residues are in close proximity to the phospho-degron regulated by GSK3 (
      • Spengler M.L.
      • Kuropatwinski K.K.
      • Schumer M.
      • Antoch M.P.
      A serine cluster mediates BMAL1-dependent CLOCK phosphorylation and degradation.
      ), which implies the possibility that multiple kinases and phosphorylation sites are functionally interlinked, for the regulation of CLOCK protein function. Collectively, we believe that current findings and further studies may provide important clues for understanding the roles of CLOCK modifications in the molecular clock and possibly linked cellular system.
      Although, roscovitine is widely used as an inhibitor of Cdk5, its specificity is not restricted to Cdk5. For example, roscovitine is known to inhibit other Cdks and MAP kinase in a concentration-dependent manner (
      • Meijer L.
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      • Mulner O.
      • Chong J.P.
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      • Inagaki N.
      • Inagaki M.
      • Delcros J.G.
      • Moulinoux J.P.
      Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5.
      ,
      • Wang D.
      • de la Fuente C.
      • Deng L.
      • Wang L.
      • Zilberman I.
      • Eadie C.
      • Healey M.
      • Stein D.
      • Denny T.
      • Harrison L.E.
      • Meijer L.
      • Kashanchi F.
      Inhibition of human immunodeficiency virus type 1 transcription by chemical cyclin-dependent kinase inhibitors.
      ,
      • Bach S.
      • Knockaert M.
      • Reinhardt J.
      • Lozach O.
      • Schmitt S.
      • Baratte B.
      • Koken M.
      • Coburn S.P.
      • Tang L.
      • Jiang T.
      • Liang D.C.
      • Galons H.
      • Dierick J.F.
      • Pinna L.A.
      • Meggio F.
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      • Schächtele C.
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      • Carnero A.
      • Wan Y.
      • Gray N.
      • Meijer L.
      Roscovitine targets, protein kinases and pyridoxal kinase.
      ). In our results, in NIH3T3 cells where p35 is not readily detectible, roscovitine treatment displayed a significant impact on functionalities of CLOCK especially when Cdk5/p35 was co-expressed. Moreover, unlike cell division-related Cdks, Cdk5 is known to be predominantly active in post-mitotic neurons (
      • Dhavan R.
      • Tsai L.H.
      A decade of CDK5.
      ,
      • Hellmich M.R.
      • Pant H.C.
      • Wada E.
      • Battey J.F.
      Neuronal cdc2-like kinase: a cdc2-related protein kinase with predominantly neuronal expression.
      ), where roscovitine showed significant effect. These indicate that the roscovitine effect in our experimental setting is likely to be mediated by Cdk5 inhibition. However, it is still possible that other Cdks may also affect the function of CLOCK, as the cell cycle-related Cdks and Cdk5 share consensus substrate motifs. Actually, the link between circadian clock and cell-cycle system has been proposed (
      • Fu L.
      • Lee C.C.
      The circadian clock: pacemaker and tumour suppressor.
      ). Obviously, more detailed mechanistic analyses on the interface between circadian clock and cell cycle are in demand.
      In mammals, resetting the circadian rhythm is mainly accomplished by the master clock located in the hypothalamic suprachiasmatic nucleus. However, robust circadian rhythmicity of the clock genes that are expressed in other brain regions has also been discovered (
      • Abe H.
      • Honma S.
      • Namihira M.
      • Masubuchi S.
      • Ikeda M.
      • Ebihara S.
      • Honma K.
      Clock gene expressions in the suprachiasmatic nucleus and other areas of the brain during rhythm splitting in CS mice.
      ,
      • Masubuchi S.
      • Honma S.
      • Abe H.
      • Ishizaki K.
      • Namihira M.
      • Ikeda M.
      • Honma K.
      Clock genes outside the suprachiasmatic nucleus involved in manifestation of locomotor activity rhythm in rats.
      ,
      • Sahar S.
      • Zocchi L.
      • Kinoshita C.
      • Borrelli E.
      • Sassone-Corsi P.
      Regulation of BMAL1 protein stability and circadian function by GSK3β-mediated phosphorylation.
      ). Notably, although the master clock is mainly governed by light stimulation, the peripheral clock, which is located outside of the suprachiasmatic nucleus, cannot only be affected by the suprachiasmatic nucleus-dependent resetting signal but also affected by non-photic stimulation, such as food intake or psycho-active drugs (
      • Wakamatsu H.
      • Yoshinobu Y.
      • Aida R.
      • Moriya T.
      • Akiyama M.
      • Shibata S.
      Restricted feeding-induced anticipatory activity rhythm is associated with a phase-shift of the expression of mPer1 and mPer2 mRNA in the cerebral cortex and hippocampus but not in the suprachiasmatic nucleus of mice.
      ,
      • Honma K.
      • Honma S.
      The SCN-independent clocks, methamphetamine and food restriction.
      ). Considering previous reports that demonstrated enhanced Cdk5 activity from a chronic psychostimulant administration and increased food reinforcement upon loss of Cdk5 function (
      • Benavides D.R.
      • Quinn J.J.
      • Zhong P.
      • Hawasli A.H.
      • DiLeone R.J.
      • Kansy J.W.
      • Olausson P.
      • Yan Z.
      • Taylor J.R.
      • Bibb J.A.
      Cdk5 modulates cocaine reward, motivation, and striatal neuron excitability.
      ,
      • Chen P.C.
      • Chen J.C.
      Enhanced Cdk5 activity and p35 translocation in the ventral striatum of acute and chronic methamphetamine-treated rats.
      ), Cdk5 activity may be involved with non-photic stimuli in the brain. Thus, it is intriguing to speculate that Cdk5 might be a link between the circadian system and non-photic inputs that are mostly related to the peripheral clock, demanding further studies at the organism level.
      Previously, it has been reported that transcriptional activation of CLOCK is tightly coupled with its nuclear translocation and degradation (
      • Kwon I.
      • Lee J.
      • Chang S.H.
      • Jung N.C.
      • Lee B.J.
      • Son G.H.
      • Kim K.
      • Lee K.H.
      BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer.
      ). For example, GSK3-mediated phosphorylation of CLOCK regulates transcriptional activity and degradation simultaneously (
      • Spengler M.L.
      • Kuropatwinski K.K.
      • Schumer M.
      • Antoch M.P.
      A serine cluster mediates BMAL1-dependent CLOCK phosphorylation and degradation.
      ). In addition CLOCKΔ19, a mutant CLOCK that displays reduced phosphorylation and transcriptional activity, is resistant to degradation compared with wild-type CLOCK (
      • Yoshitane H.
      • Takao T.
      • Satomi Y.
      • Du N.H.
      • Okano T.
      • Fukada Y.
      Roles of CLOCK phosphorylation in suppression of E-box-dependent transcription.
      ,
      • Jung H.
      • Choe Y.
      • Kim H.
      • Park N.
      • Son G.H.
      • Khang I.
      • Kim K.
      Involvement of CLOCK:BMAL1 heterodimer in serum-responsive mPer1 induction.
      ). These molecular events are consistent with the “black widow model”; the stability and transcriptional activity of certain transcription factors, including Jun, Fos, Myc, p53, and HIF1-α, exhibit a negative correlation that is critical for temporal fine tuning of gene expression (
      • Kwon I.
      • Lee J.
      • Chang S.H.
      • Jung N.C.
      • Lee B.J.
      • Son G.H.
      • Kim K.
      • Lee K.H.
      BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer.
      ,
      • Sahar S.
      • Zocchi L.
      • Kinoshita C.
      • Borrelli E.
      • Sassone-Corsi P.
      Regulation of BMAL1 protein stability and circadian function by GSK3β-mediated phosphorylation.
      ,
      • Muratani M.
      • Tansey W.P.
      How the ubiquitin-proteasome system controls transcription.
      ). In the present study, we showed that the stability of CLOCK was decreased by the expression of Cdk5. It was noteworthy that Cdk5 promoted the degradation of BMAL1 only under co-expression of CLOCK, indicating that the Cdk5-mediated destabilization of CLOCK induces the degradation of BMAL1. Our results also showed that Cdk5 elicited transcriptional activation and altered translocation of CLOCK. Interestingly, CLOCK-dependent degradation of BMAL1 is known to be coupled with translocation of CLOCK/BMAL1 complex in a good correlation with enhanced transcriptional activity (
      • Kwon I.
      • Lee J.
      • Chang S.H.
      • Jung N.C.
      • Lee B.J.
      • Son G.H.
      • Kim K.
      • Lee K.H.
      BMAL1 shuttling controls transactivation and degradation of the CLOCK/BMAL1 heterodimer.
      ,
      • Lee C.
      • Etchegaray J.P.
      • Cagampang F.R.
      • Loudon A.S.
      • Reppert S.M.
      Posttranslational mechanisms regulate the mammalian circadian clock.
      ,
      • Kondratov R.V.
      • Chernov M.V.
      • Kondratova A.A.
      • Gorbacheva V.Y.
      • Gudkov A.V.
      • Antoch M.P.
      BMAL1-dependent circadian oscillation of nuclear CLOCK: posttranslational events induced by dimerization of transcriptional activators of the mammalian clock system.
      ,
      • Sahar S.
      • Zocchi L.
      • Kinoshita C.
      • Borrelli E.
      • Sassone-Corsi P.
      Regulation of BMAL1 protein stability and circadian function by GSK3β-mediated phosphorylation.
      ). Thus, our results suggest that Cdk5-mediated phosphorylation of CLOCK affects molecular events following dimerization of CLOCK and BMAL1, thereby triggering translocation, transcriptional activation, and eventually degradation of CLOCK/BMAL1 complex, which appears to fit to the “black widow model.” Thus, the functional relationship between CLOCK and Cdk5 identified in this study may provide an additional example of this model.
      A potential link between the circadian system and mood disorders has been suggested. For example, an insufficient length of light phase has been linked to seasonal affective disorder by affecting entrainment of the molecular clock (
      • Rosenthal N.E.
      • Sack D.A.
      • Jacobsen F.M.
      • Skwerer R.G.
      • Wehr T.A.
      Seasonal affective disorder & light: past, present and future.
      ,
      • Dalgleish T.
      • Rosen K.
      • Marks M.
      Rhythm and blues: the theory and treatment of seasonal affective disorder.
      ). In addition, abnormalities of the sleep/wake cycle, hormonal function, and daily activity are prominent symptoms of mood disorders, and normalization of daily cycles is mostly associated with mood stabilization (
      • Souêtre E.
      • Salvati E.
      • Belugou J.L.
      • Pringuey D.
      • Candito M.
      • Krebs B.
      • Ardisson J.L.
      • Darcourt G.
      Circadian rhythms in depression and recovery: evidence for blunted amplitude as the main chronobiological abnormality.
      ,
      • Boivin D.B.
      Influence of sleep-wake and circadian rhythm disturbances in psychiatric disorders.
      ). Moreover, therapies controlling light stimulation effectively reverse the symptoms of mood disorders (
      • Barbini B.
      • Benedetti F.
      • Colombo C.
      • Dotoli D.
      • Bernasconi A.
      • Cigala-Fulgosi M.
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      Dark therapy for mania: a pilot study.
      ,
      • Sit D.
      • Wisner K.L.
      • Hanusa B.H.
      • Stull S.
      • Terman M.
      Light therapy for bipolar disorder: a case series in women.
      ,
      • Phelps J.
      Dark therapy for bipolar disorder using amber lenses for blue light blockade.
      ,
      • Gómez-Bernal G.
      Dark therapy for schizoaffective disorder. A case report.
      ). Recently, it has been reported that the CLOCKΔ19 mouse displays phenotypes strikingly similar to human mania in terms of their hyperactive ventral tegmental area dopaminergic neurons and manic-like behaviors (
      • Roybal K.
      • Theobold D.
      • Graham A.
      • DiNieri J.A.
      • Russo S.J.
      • Krishnan V.
      • Chakravarty S.
      • Peevey J.
      • Oehrlein N.
      • Birnbaum S.
      • Vitaterna M.H.
      • Orsulak P.
      • Takahashi J.S.
      • Nestler E.J.
      • Carlezon Jr., W.A.
      • McClung C.A.
      Mania-like behavior induced by disruption of CLOCK.
      ). Moreover, decreased CLOCK expression of CLOCK in the ventral tegmental area elicits a mixed state of mania and depression-like behaviors (
      • Mukherjee S.
      • Coque L.
      • Cao J.L.
      • Kumar J.
      • Chakravarty S.
      • Asaithamby A.
      • Graham A.
      • Gordon E.
      • Enwright 3rd, J.F.
      • DiLeone R.J.
      • Birnbaum S.G.
      • Cooper D.C.
      • McClung C.A.
      Knockdown of Clock in the ventral tegmental area through RNA interference results in a mixed state of mania and depression-like behavior.
      ). These reports indicate that abnormal CLOCK may be related to an abnormal dopaminergic system and mood conditions. Intriguingly, Cdk5 is well known to regulate dopamine signaling by regulating key players such as tyrosine hydroxylase and DARPP-32 in both the presynaptic and postsynaptic parts (
      • Dhavan R.
      • Tsai L.H.
      A decade of CDK5.
      ,
      • Su S.C.
      • Tsai L.H.
      Cyclin-dependent Kinases in Brain Development and Disease.
      ), and increased Cdk5/p35 activity was associated with depressive-like behaviors in rats (
      • Zhu W.L.
      • Shi H.S.
      • Wang S.J.
      • Xu C.M.
      • Jiang W.G.
      • Wang X.
      • Wu P.
      • Li Q.Q.
      • Ding Z.B.
      • Lu L.
      Increased Cdk5/p35 activity in the dentate gyrus mediates depressive-like behaviour in rats.
      ). To this end, our results may provide a mechanistic link between dopamine system-related mood conditions and the molecular clock, which has long been postulated.

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