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

  • Yoshimi Uchida
    Footnotes
    Affiliations
    Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
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  • Tomomi Osaki
    Footnotes
    Affiliations
    Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
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  • Tokiwa Yamasaki
    Affiliations
    Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
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  • Tadanori Shimomura
    Affiliations
    Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
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  • Shoji Hata
    Affiliations
    Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
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  • Kazumasa Horikawa
    Affiliations
    Department of Physiology and Pharmacology, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan
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  • Shigenobu Shibata
    Affiliations
    Department of Physiology and Pharmacology, School of Advanced Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 162-8480, Japan
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  • Takeshi Todo
    Affiliations
    Department of Radiation Biology and Medical Genetics, Graduate School of Medicine, Osaka University, B4, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan
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  • Jun Hirayama
    Correspondence
    To whom correspondence should be addressed. Tel.: 81-3-5803-4658; Fax: 81-3-5803-5829
    Affiliations
    Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
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  • Hiroshi Nishina
    Affiliations
    Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
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  • Author Footnotes
    * This work was supported by a Grant-in-Aid for Scientific Research on a Priority Area from the Ministry of Education, Culture, Sport, Science, and Technology of Japan and the Ministry of Health, Labor, and Welfare of Japan.
    This article contains supplemental Figs. 1–4.
    1 Both authors contributed equally to this work.
      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.

      Introduction

      c-Jun N-terminal kinase (JNK) is a member of the family of mitogen-activated protein kinases (MAPKs), which are ubiquitously expressed and evolutionarily conserved (
      • Davis R.J.
      Signal transduction by the JNK group of MAP kinases.
      ,
      • Chang L.
      • Karin M.
      Mammalian MAP kinase signaling cascades.
      ). JNK is activated by many types of external stresses, including changes in osmolarity, heat shock, and UV irradiation, and this activity is regulated via the phosphorylation of particular tyrosine and threonine residues located in the kinase domain. JNK phosphorylation is catalyzed by two dual-specificity kinases, MKK4
      The abbreviations used are: MKK
      mitogen-activated protein kinase kinase
      CHX
      cycloheximide
      CRE
      Cre recombinase
      Dex
      dexamethasone
      DKO
      double knockout
      ES
      embryonic stem
      KN
      kinase negative
      MEF
      mouse embryonic fibroblast
      NLS
      nuclear localization signal
      Ab
      antibody
      WB
      Western blot
      pTP
      phosphorylated threonine-proline.
      and MKK7, that act in a synergistic manner (
      • Lawler S.
      • Fleming Y.
      • Goedert M.
      • Cohen P.
      Synergistic activation of SAPK1/JNK1 by two MAP kinase kinases in vitro.
      ,
      • Kishimoto H.
      • Nakagawa K.
      • Watanabe T.
      • Kitagawa D.
      • Momose H.
      • Seo J.
      • Nishitai G.
      • Shimizu N.
      • Ohata S.
      • Tanemura S.
      • Asaka S.
      • Goto T.
      • Fukushi H.
      • Yoshida H.
      • Suzuki A.
      • Sasaki T.
      • Wada T.
      • Penninger J.M.
      • Nishina H.
      • Katada T.
      Different properties of SEK1 and MKK7 in dual phosphorylation of stress-induced activated protein kinase SAPK/JNK in embryonic stem cells.
      ). Although most often activated in response to stress, phosphorylated JNK has been detected in unstressed cultured cells and in isolated mouse tissues, such as the brain (
      • Pizzio G.A.
      • Hainich E.C.
      • Ferreyra G.A.
      • Coso O.A.
      • Golombek D.A.
      Circadian and photic regulation of ERK, JNK, and p38 in the hamster SCN.
      ,
      • Chansard M.
      • Molyneux P.
      • Nomura K.
      • Harrington M.E.
      • Fukuhara C.
      c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms.
      ), indicating the importance of JNK signaling in physiological processes other than cellular stress responses. We previously showed that genetic inactivation of the JNK activator MKK7 in mice resulted in defective hepatocyte proliferation and embryonic lethality (
      • Wada T.
      • Joza N.
      • Cheng H.Y.
      • Sasaki T.
      • Kozieradzki I.
      • Bachmaier K.
      • Katada T.
      • Schreiber M.
      • Wagner E.F.
      • Nishina H.
      • Penninger J.M.
      MKK7 couples stress signaling to G2/M cell cycle progression and cellular senescence.
      ). In addition, loss of MKK7 in mouse embryonic fibroblasts (MEFs) led to their impaired proliferation, premature senescence, and G2/M cell cycle arrest (
      • Wada T.
      • Joza N.
      • Cheng H.Y.
      • Sasaki T.
      • Kozieradzki I.
      • Bachmaier K.
      • Katada T.
      • Schreiber M.
      • Wagner E.F.
      • Nishina H.
      • Penninger J.M.
      MKK7 couples stress signaling to G2/M cell cycle progression and cellular senescence.
      ). These findings indicated that MKK7 is crucial for cell proliferation even in the absence of stress and raised the possibility that the MKK7-JNK pathway might contribute to additional normal biological events.
      Circadian clocks are endogenous oscillators that drive the daily rhythms of organisms ranging from bacteria to humans (
      • Dunlap J.C.
      Molecular bases for circadian clocks.
      ,
      • King D.P.
      • Takahashi J.S.
      Molecular genetics of circadian rhythms in mammals.
      ). These clocks regulate various biochemical, physiological, and behavioral processes with a periodicity of ∼24 h (
      • King D.P.
      • Takahashi J.S.
      Molecular genetics of circadian rhythms in mammals.
      ). Under natural conditions, circadian rhythms are entrained to this 24-h day by environmental time cues, with light level being the most important (
      • Schibler U.
      • Sassone-Corsi P.
      A web of circadian pacemakers.
      ). The core of the clock mechanism in almost all organisms studied to date is a transcription/translation-based negative feedback loop that relies on positive and negative oscillators (
      • King D.P.
      • Takahashi J.S.
      Molecular genetics of circadian rhythms in mammals.
      ,
      • Reppert S.M.
      • Weaver D.R.
      Coordination of circadian timing in mammals.
      ). In mammals, three basic helix-loop-helix PAS (PER-ARNT-SIM) domain-containing transcription factors, called CLOCK, NPAS2, and BMAL1, constitute the positive elements. CLOCK or NPAS2 heterodimerizes with BMAL1 to form a transcriptionally active complex that binds to E-box elements (CACGTG) present in the promoters of members of the Period (Per) and Cryptochrome (Cry) gene families (Per1, -2, and -3 and Cry1 and -2). Once the PER and CRY proteins have been translated, they form heterodimers that can then translocate to the nucleus to repress CLOCK (NPAS2)-BMAL1-mediated transcription through direct protein-protein interaction. These interactions then set up the rhythmic oscillations of gene expression that drive the circadian clock.
      The functions of various clock proteins, including CLOCK, BMAL1, PER1, PER2, PER3, CRY1, and CRY2, are regulated via phosphorylation by various enzymes, including casein kinase-1 ϵ (CK1ϵ), CK1δ, glycogen synthase kinase-3β (GSK3β), and casein kinase-2 (CK2) (
      • Hirayama J.
      • Sassone-Corsi P.
      Structural and functional features of transcription factors controlling the circadian clock.
      ,
      • Gallego M.
      • Virshup D.M.
      Post-translational modifications regulate the ticking of the circadian clock.
      ,
      • Uchida Y.
      • Hirayama J.
      • Nishina H.
      A common origin. Signaling similarities in the regulation of the circadian clock and DNA damage responses.
      ). Genetic studies have revealed the important role this phosphorylation plays in mammalian clock function (
      • Hirayama J.
      • Sassone-Corsi P.
      Structural and functional features of transcription factors controlling the circadian clock.
      ,
      • Gallego M.
      • Virshup D.M.
      Post-translational modifications regulate the ticking of the circadian clock.
      ). For example, in the Syrian hamster, the tau mutation causing a short period phenotype affects the gene encoding CK1ϵ. CK1ϵ was subsequently demonstrated to phosphorylate PER2 (
      • Lowrey P.L.
      • Shimomura K.
      • Antoch M.P.
      • Yamazaki S.
      • Zemenides P.D.
      • Ralph M.R.
      • Menaker M.
      • Takahashi J.S.
      Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau.
      ), and the short period phenotype of tau hamsters was directly linked to their lower rate of CK1ϵ-dependent PER2 phosphorylation. Intriguingly, a defect in CK1ϵ-mediated PER2 phosphorylation has also been implicated in human sleep disorders. For example, familial advanced sleep phase syndrome is associated with a missense mutation in the human PER2 gene, and the corresponding mutated PER2 protein is less effectively phosphorylated by CK1ϵ in vitro than is wild type (WT) PER2 (
      • Toh K.L.
      • Jones C.R.
      • He Y.
      • Eide E.J.
      • Hinz W.A.
      • Virshup D.M.
      • Ptácek L.J.
      • Fu Y.H.
      An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome.
      ). At the molecular level, CK1ϵ-mediated PER2 phosphorylation has been shown to decrease the stability of PER2 protein by promoting its ubiquitination (
      • Akashi M.
      • Tsuchiya Y.
      • Yoshino T.
      • Nishida E.
      Control of intracellular dynamics of mammalian period proteins by casein kinase I ϵ (CKIϵ) and CKIδ in cultured cells.
      ,
      • Eide E.J.
      • Woolf M.F.
      • Kang H.
      • Woolf P.
      • Hurst W.
      • Camacho F.
      • Vielhaber E.L.
      • Giovanni A.
      • Virshup D.M.
      Control of mammalian circadian rhythm by CKIϵ-regulated proteasome-mediated PER2 degradation.
      ). Notably, changes in PER2 stability have been linked to changes in the period length of circadian rhythms (
      • Vanselow K.
      • Vanselow J.T.
      • Westermark P.O.
      • Reischl S.
      • Maier B.
      • Korte T.
      • Herrmann A.
      • Herzel H.
      • Schlosser A.
      • Kramer A.
      Differential effects of PER2 phosphorylation. Molecular basis for the human familial advanced sleep phase syndrome (FASPS).
      ,
      • Xu Y.
      • Toh K.L.
      • Jones C.R.
      • Shin J.Y.
      • Fu Y.H.
      • Ptácek L.J.
      Modeling of a human circadian mutation yields insights into clock regulation by PER2.
      ).
      In this study, we present evidence that PER2 may also be regulated by MKK7-JNK-mediated phosphorylation, establishing a role for the stress kinase MKK7 in controlling the mammalian circadian clock. Importantly, we demonstrate that the MKK7-JNK signaling pathway has an effect opposite to that of CK1ϵ-induced PER2 destabilization. Thus, MKK7-JNK signaling may provide a balancing influence on clock protein functions that helps to maintain the normal periodicity of the circadian clock machinery.

      EXPERIMENTAL PROCEDURES

       Plasmids, Reagents, Cells, and Transfection

      An EcoRI fragment of full-length nuclear localization signal-fused CRE recombinase (NLS-CRE) (
      • Kishimoto H.
      • Nakagawa K.
      • Watanabe T.
      • Kitagawa D.
      • Momose H.
      • Seo J.
      • Nishitai G.
      • Shimizu N.
      • Ohata S.
      • Tanemura S.
      • Asaka S.
      • Goto T.
      • Fukushi H.
      • Yoshida H.
      • Suzuki A.
      • Sasaki T.
      • Wada T.
      • Penninger J.M.
      • Nishina H.
      • Katada T.
      Different properties of SEK1 and MKK7 in dual phosphorylation of stress-induced activated protein kinase SAPK/JNK in embryonic stem cells.
      ) was inserted in the corresponding site of pCLNCX (IMGENEX, San Diego, CA) retrovirus vector, generating NLS-CRE-pCLNCX. A HindIII-EcoRI fragment of Myc epitope was inserted in the corresponding sites of pEGFPN2 (Clontech), generating a Myc-GFP-expressing vector. A mutation was introduced into Myc-PER2/pCS2 using PCR-based site-directed mutagenesis (Per2S, 5′-gcaaggccgaggctgtggtgtccctcac-3′; Per2A, 5′-gtgagggacaccacagcctcggccttgc-3′). Expression vectors for CK1ϵ and HA-ubiquitin were the kind gifts of Dr. A. Takano-Hayata (Osaka University) and Dr. K. Nakayama (Tokyo Medical and Dental University), respectively. Other plasmids used in this study have been described elsewhere (
      • Rennefahrt U.E.
      • Illert B.
      • Kerkhoff E.
      • Troppmair J.
      • Rapp U.R.
      Constitutive JNK activation in NIH 3T3 fibroblasts induces a partially transformed phenotype.
      ,
      • Hirayama J.
      • Sahar S.
      • Grimaldi B.
      • Tamaru T.
      • Takamatsu K.
      • Nakahata Y.
      • Sassone-Corsi P.
      CLOCK-mediated acetylation of BMAL1 controls circadian function.
      ).
      MEFs, HeLa cells, and 293T cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS). ES cells were cultured in DMEM (Invitrogen) containing 15% FBS, 0.1% 2-mercaptoethanol (Sigma), and leukemia inhibitory factor (propagation medium). Cultured cells were transfected with Fugene (Roche Applied Science) according to the manufacturer's protocol. SP600125 and calyculin A were purchased from Calbiochem and Wako Pure Chemical Industries, respectively.

       Generation of Stable Reporter Cell Lines and Monitoring of Real-time Luciferase Activity

      To obtain lines of Mkk7flox/flox MEFs stably expressing firefly luciferase from a 1.8-kb Per2 promoter fragment, Mkk7flox/flox MEFs were co-transfected with linearized mouse Per2 promoter-pGL3basic (7 μg) and pcDNA3.1 (1 μg) vectors. After 1 day in standard culture, 0.5 mg/ml G418 was added, and cultures were selected for 2 weeks. Colonies were picked, and their real-time luciferase activities were determined using a Kronos system (ATTO). Two cell lines that showed robust circadian patterns of luciferase activity were selected for further experimentation.

       Antibodies

      Antibodies (Abs) recognizing the following proteins were used in this study: Myc (9E10), JNK (sc-571), JNK (sc-137018), CLOCK, and actin (all from Santa Cruz Biotechnology, Inc.); ERK, c-Jun, phosphorylated c-Jun, and phosphorylated JNK (Cell Signaling); CK1ϵ (Abcam); mouse PER2 (Alpha Diagnostic International Inc.); HA (Immunology Consultants Laboratory); and FLAG (Sigma). All other Abs have been described elsewhere (
      • Hirayama J.
      • Sahar S.
      • Grimaldi B.
      • Tamaru T.
      • Takamatsu K.
      • Nakahata Y.
      • Sassone-Corsi P.
      CLOCK-mediated acetylation of BMAL1 controls circadian function.
      ,
      • Yagita K.
      • Tamanini F.
      • Yasuda M.
      • Hoeijmakers J.H.
      • van der Horst G.T.
      • Okamura H.
      Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein.
      ).

       Co-immunoprecipitation

      Co-immunoprecipitation assays were performed as described previously with some modifications (
      • Hirayama J.
      • Sahar S.
      • Grimaldi B.
      • Tamaru T.
      • Takamatsu K.
      • Nakahata Y.
      • Sassone-Corsi P.
      CLOCK-mediated acetylation of BMAL1 controls circadian function.
      ). 293T cells or MEFs were washed twice with phosphate-buffered saline (PBS) and homogenized in binding buffer (150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 1 mm EGTA, 5% glycerol, and 20 mm Tris-HCl, pH 7.4) containing protease inhibitor mixture tablets. Extracts were clarified by centrifugation for 10 min at 15,000 × g, and supernatants were incubated with 15 μl of protein G-agarose beads (GE Healthcare) for 1 h at 4 °C. The bead mixture was centrifuged, and the supernatant was incubated for 12 h at 4 °C with the Abs described in each figure legend plus 20 μl of protein G-agarose beads. The beads were washed three times with binding buffer, boiled in SDS sample buffer, and centrifuged. The supernatant was fractionated by SDS-PAGE and analyzed by Western blotting, as described below.

       Western Blotting

      Immunoprecipitated materials and total cell extracts obtained as described above were fractionated by SDS-PAGE and transferred electrophoretically onto polyvinylidene difluoride membranes. Membranes were blocked with 2 or 5% nonfat milk and incubated for 10 h at 4 °C with the Abs indicated in each figure legend. The blots were incubated with the appropriate secondary Ab plus peroxidase-conjugated anti-mouse or anti-rabbit IgG Ab (Santa Cruz Biotechnology, Inc.) and developed with the ECL Western blotting detection system (Amersham Biosciences).

       Ubiquitination Assay

      Myc-PER2 was co-expressed with HA-tagged ubiquitin in 293T cells. The cells were lysed in 1% SDS buffer (1% SDS in TBS), boiled for 10 min, and diluted 10-fold in incubation buffer (1% Triton X-100 in TBS) prior to immunoprecipitation with anti-Myc.

       Retroviral Transduction

      Retroviruses used in this study were produced using the RetroMax expression system (IMGENEX) according to the manufacturer's instructions. NLS-CRE was expressed in Mkk7flox/flox MEFs using the pCLNCX-NLS-CRE vector, with pMD.G/vsv-g as the enveloping vector. The high infection efficiency (95–100%) of this system was confirmed by infecting MEFs with pCLNCX vector expressing GFP (data not shown).

      RESULTS

       Genetic Inactivation of Mkk7 Gene Alters Circadian Gene Expression

      To investigate whether MKK7 was involved in circadian regulation, we examined cultured cells under conditions that provide a good model of the cell-autonomous circadian oscillation that occurs in mammalian peripheral tissues (
      • Tsuchiya Y.
      • Nishida E.
      Mammalian cultured cells as a model system of peripheral circadian clocks.
      ). When cultured human and mouse cells are treated with dexamethasone (Dex), a rhythm of circadian gene expression is entrained (
      • Tsuchiya Y.
      • Nishida E.
      Mammalian cultured cells as a model system of peripheral circadian clocks.
      ). To visualize circadian rhythms in cultured MEFs, we used previously generated mice carrying a conditional Mkk7 allele (Mkk7flox/flox mice) (
      • Schramek D.
      • Kotsinas A.
      • Meixner A.
      • Wada T.
      • Elling U.
      • Pospisilik J.A.
      • Neely G.G.
      • Zwick R.H.
      • Sigl V.
      • Forni G.
      • Serrano M.
      • Gorgoulis V.G.
      • Penninger J.M.
      The stress kinase MKK7 couples oncogenic stress to p53 stability and tumor suppression.
      ,
      • Yamasaki T.
      • Kawasaki H.
      • Arakawa S.
      • Shimizu K.
      • Shimizu S.
      • Reiner O.
      • Okano H.
      • Nishina S.
      • Azuma N.
      • Penninger J.M.
      • Katada T.
      • Nishina H.
      Stress-activated protein kinase MKK7 regulates axon elongation in the developing cerebral cortex.
      ) to establish cultures of mutant MEFs (Mkk7flox/flox MEFs) in which MKK7 could be genetically inactivated by expression of NLS-CRE (Fig. 1A, left). We then generated two independent lines of Mkk7flox/flox MEFs that stably expressed firefly luciferase from a 1.8-kb DNA fragment containing the mouse Per2 promoter (Per2-Luc/Mkk7flox/flox line 1, Per2-Luc/Mkk7flox/flox line 2). Retroviral transduction of NLS-CRE resulted in genetic inactivation of MKK7 in both cell lines (Fig. 1A, middle and right). When we treated these cells with Dex and monitored real-time Per2 promoter-driven luciferase bioluminescence, we found that NLS-CRE-mediated inactivation of MKK7 significantly lengthened the circadian period of bioluminescence in both Per2-Luc/Mkk7flox/flox line 1 and Per2-Luc/Mkk7flox/flox line 2 MEFs (Fig. 1, B and C, and supplemental Fig. 1, A and B). Thus, MKK7 influences circadian gene expression in unstressed cells.
      Figure thumbnail gr1
      FIGURE 1MKK7 controls circadian gene expression in cultured cells. A, confirmation of loss of MKK7 expression. Mkk7flox/flox MEFs and two lines of Mkk7flox/flox MEFs expressing luciferase under the control of the Per2 promoter (Per2-Luc/Mkk7flox/flox line 1 and Per2-Luc/Mkk7flox/flox line 2), were infected with NLS-CRE-expressing (+) or control (−) retroviral vectors. MKK7 levels were determined by WB with anti-MKK7 Ab. ACTIN, loading control. B and C, loss of MKK7 alters the circadian period. Per2-Luc/Mkk7flox/flox line 1 (B) and Per2-Luc/Mkk7flox/flox line 2 (C) MEFs were infected with retrovirus expressing NLS-CRE or control vector and synchronized by Dex treatment. Per2 reporter bioluminescence was monitored over the indicated time course. Detrended data representative of three independent experiments are shown in the upper panels. Blue trace, control bioluminescence rhythm; red trace, bioluminescence rhythm in the absence of MKK7. Tables in the lower panels show the calculated periods of bioluminescence rhythms expressed as the mean ± S.D. (n = 3). p values as compared with the control are indicated. D, effects of calyculin A treatment on JNK phosphorylation. Mkk7flox/flox MEFs infected with NSL-CRE-expressing retrovirus (Mkk7−/−) or empty vector (Mkk7flox/flox) were treated with 5 nm calyculin A. At the indicated time points, lysates were analyzed by WB using anti-phospho-JNK (p-JNK), anti-JNK (JNK), and anti-actin (ACTIN) Abs. Black arrowheads, phosphorylated JNK. *, nonspecific band. For all experiments, results shown are representative of at least three independent trials.
      Because MKK7 is a specific activator of JNK (
      • Davis R.J.
      Signal transduction by the JNK group of MAP kinases.
      ,
      • Chang L.
      • Karin M.
      Mammalian MAP kinase signaling cascades.
      ), we speculated that the altered circadian periodicity we observed following MKK7 inactivation might be associated with impaired JNK function. We therefore examined the effect of MKK7 inactivation on JNK phosphorylation. Previous reports have established that, even in the absence of external stress, cultured cells exhibit a low level of JNK phosphorylation (
      • Pizzio G.A.
      • Hainich E.C.
      • Ferreyra G.A.
      • Coso O.A.
      • Golombek D.A.
      Circadian and photic regulation of ERK, JNK, and p38 in the hamster SCN.
      ,
      • Chansard M.
      • Molyneux P.
      • Nomura K.
      • Harrington M.E.
      • Fukuhara C.
      c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms.
      ), a result we confirmed in our Mkk7flox/flox MEFs (Fig. 1D, left). When we treated Mkk7flox/flox MEFs with the phosphatase inhibitor calyculin A, JNK phosphorylation was markedly increased. However, when MKK7 was genetically deleted, JNK phosphorylation was significantly reduced both in untreated and calyculin A-treated MEFs (Fig. 1D, right), showing that the stress-independent JNK activation observed in cultured cells depends on the kinase activity of MKK7. We then examined the effect of the kinase inhibitor SP600125 (which blocks JNK activity) on circadian gene expression and found that SP600125 treatment lengthened the circadian period of bioluminescence in both Per2-Luc/Mkk7flox/flox line 1 and 2 MEFs (supplemental Fig. 2). Importantly, the effects of SP600125 on circadian gene expression were strikingly similar to those of MKK7 genetic inactivation (Fig. 1, B and C). In addition, our results are consistent with previous studies showing that SP600125 extends the period of circadian transcription in cultured cells and in ex vivo organ culture systems (
      • Chansard M.
      • Molyneux P.
      • Nomura K.
      • Harrington M.E.
      • Fukuhara C.
      c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms.
      ,
      • Yagita K.
      • Yamanaka I.
      • Koinuma S.
      • Shigeyoshi Y.
      • Uchiyama Y.
      Mini screening of kinase inhibitors affecting period-length of mammalian cellular circadian clock.
      ). Thus, our findings provide evidence that the MKK7-JNK signaling pathway is involved in the circadian regulation of gene expression.

       MKK7-JNK Fusion Protein Induces PER2 Phosphorylation

      To identify the MKK7-JNK target(s) involved in regulating the mammalian circadian clock, we overexpressed an MKK7-JNK (WT) fusion protein in 293T or HeLa cells and performed Western blotting (WB). It has been previously demonstrated that expression of this MKK7-JNK (WT) fusion protein, but not of a kinase-negative (KN) version (MKK7-JNK (KN)), results in trans-phosphorylation of JNK by the fused MKK7 and a subsequent marked increase in JNK catalytic activity (
      • Rennefahrt U.E.
      • Illert B.
      • Kerkhoff E.
      • Troppmair J.
      • Rapp U.R.
      Constitutive JNK activation in NIH 3T3 fibroblasts induces a partially transformed phenotype.
      ). Consistent with these results, we found that phosphorylation of endogenous c-Jun, a well known JNK target (
      • Davis R.J.
      Signal transduction by the JNK group of MAP kinases.
      ), was strongly induced by overexpression of MKK7-JNK (WT) but not by MKK7-JNK (KN) (Fig. 2A). We then examined the effect of MKK-JNK (WT) on the co-expression of a set of Myc-tagged clock proteins. Interestingly, MKK7-JNK (WT) caused a significant shift in the electrophoretic mobility of Myc-PER2 and Myc-BMAL1 (Fig. 2B) that could be reversed by phosphatase treatment (Fig. 2, C and D). These data indicate that JNK-mediated phosphorylation can induce a mobility shift in clock proteins, implying a concrete interaction between MKK7-JNK signaling and the clock machinery.
      Figure thumbnail gr2
      FIGURE 2Co-expression of MKK7-JNK fusion protein induces the phosphorylation of exogenous PER2 and BMAL1. A, confirmation of kinase properties. 293T cells were transiently transfected with vectors expressing MKK7-JNK (WT) or MKK7-JNK (KN), and lysates were analyzed by WB with anti-phospho-c-Jun (p-c-Jun), anti-c-Jun, anti-JNK, and anti-ERK Abs to detect phospho-c-Jun, c-Jun, MKK7-JNK, and ERK proteins, respectively. Only MKK7-JNK (WT) expression resulted in phosphorylation of the JNK target c-Jun (p-c-Jun). B, mobility shift of clock proteins. MKK7-JNK (WT) or empty control vector (−) was co-expressed in HeLa cells with vectors expressing Myc-CLOCK, Myc-CRY1, Myc-PER2, or Myc-BMAL1. Lysates were analyzed by WB with anti-Myc Ab to detect the indicated Myc-tagged clock proteins or with anti-JNK Ab to detect MKK7-JNK protein. Brackets indicate the shift in the PER2 and BMAL1 bands induced by co-expression of MKK7-JNK (WT). C and D, sensitivity to phosphatase. HeLa cells were co-transfected with vectors expressing Myc-PER2 (C) or Myc-BMAL1 (D), plus either empty vector (−) or vector expressing MKK7-JNK (WT) (+). Lysates were treated (+) or not (−) with alkaline phosphatase (PPA) and analyzed by WB with anti-Myc Ab to detect Myc-PER2 or Myc-BMAL1 and with anti-JNK Ab to detect MKK7-JNK (WT). E, MKK7-JNK-mediated increase in PER2 pTP motifs. 293T cells transiently expressing Myc-PER2 were co-transfected with empty vector (−) or MKK7-JNK (WT) (+). Lysates were immunoprecipitated (IP) using anti-Myc Ab to isolate PER2. This immunoprecipitate was subjected to WB analysis with anti-pTP Ab to detect phosphorylated PER2 (p-PER2) (top) and with anti-Myc Ab to detect Myc-PER2 (middle). Bottom, WB analysis of total cell lysate using anti-JNK Ab to detect MKK7-JNK (WT).
      A particular threonine-proline motif is a consensus site for JNK phosphorylation (
      • Gdalyahu A.
      • Ghosh I.
      • Levy T.
      • Sapir T.
      • Sapoznik S.
      • Fishler Y.
      • Azoulai D.
      • Reiner O.
      DCX, a new mediator of the JNK pathway.
      ). We overexpressed Myc-PER2 or Myc-BMAL1 in the presence or absence of MKK7-JNK (WT) in cultured cells and examined whether these proteins were phosphorylated on this motif. We immunoprecipitated Myc-tagged clock proteins with anti-Myc Ab followed by WB analysis using an Ab specifically recognizing proteins bearing the phosphorylated threonine-proline (pTP) motif. We found that PER2 was modestly phosphorylated on its threonine-proline motif(s) in control cells but that this PER2 phosphorylation was greatly enhanced by MKK7-JNK (WT) (Fig. 2E). However, no band corresponding to BMAL1 phosphorylation was detected in either control or MKK7-JNK (WT)-expressing cells (data not shown). We therefore speculated that PER2 is the more likely circadian target of MKK7-JNK signaling and thus focused our subsequent analyses mainly on PER2.

       JNK Physically Interacts with PER2

      We tested whether JNK could physically interact with clock proteins using co-immunoprecipitation assays. First, we co-expressed FLAG-JNK with Myc-PER2, Myc-BMAL1, or Myc-LacZ (negative control) in 293T cells and subjected lysates to immunoprecipitation with anti-FLAG Ab. We found that these exogenous forms of PER2 and BMAL1 both co-immunoprecipitated with FLAG-JNK (Fig. 3A). To confirm an interaction between PER2 and JNK at the endogenous level, we used anti-PER2 Ab to immunoprecipitate endogenous PER2 from Dex-synchronized WT MEFs at various time points and performed WB to detect endogenous JNK. Indeed, endogenous JNK co-immunoprecipitated with endogenous PER2 in a manner that appeared to depend on the abundance of PER2 protein (Fig. 3B).
      Figure thumbnail gr3
      FIGURE 3PER2 and BMAL1 can physically interact with JNK. A, exogenous level. FLAG-JNK was co-expressed with Myc-BMAL1, Myc-PER2, or Myc-LacZ in 293T cells. Lysates were immunoprecipitated (IP) with anti-FLAG Ab and analyzed by WB with anti-Myc Ab to detect clock proteins or with anti-JNK (sc-571) Ab to detect JNK. Input, total lysates subjected to WB with anti-Myc Ab. B, endogenous level. WT MEFs were synchronized with Dex, and protein extracts were prepared at the indicated times (h). Top, extracts were immunoprecipitated with anti-PER2 Ab, and the immunoprecipitate was analyzed by WB with anti-JNK or anti-PER2 Abs. CNT, Immunoprecipitation using rabbit IgG (negative control). Input, total lysates subjected to WB with anti-JNK or anti-ERK Abs.

       MKK7-JNK Signaling Stabilizes PER2 Protein

      During the course of the above experiments, we realized that MKK7-JNK (WT) expression increased the abundance of PER2 protein, suggesting that MKK7-JNK signaling might affect PER2 stability. To test this possibility, we co-expressed increasing amounts of MKK7-JNK (WT) with Myc-PER2 and Myc-GFP in 293T cells and detected a dose-dependent increase in PER2 protein (Fig. 4A). This PER2 up-regulation was almost entirely dependent on the enzymatic activity of JNK because it was not observed when Myc-PER2 was co-expressed with MKK7-JNK (KN). A more modest increase in Myc-PER2 protein was observed in HeLa cells expressing MKK7-JNK (WT) (data not shown), indicating that this phenomenon varies by cell type. We believe that the MKK7-JNK-induced increase in PER2 protein is not due to an elevation in Per2 mRNA because MKK7-JNK co-expression did not affect levels of Myc-GFP protein (Fig. 4A), whose transcription was controlled by the same CMV promoter as that controlling Per2.
      Figure thumbnail gr4
      FIGURE 4MKK7-mediated JNK activation stabilizes PER2 protein. A, increased PER2 protein. 293T cells were transfected with Myc-PER2 and Myc-GFP and either empty vector (−); 0.01, 0.04, 0.16, or 0.64 μg of MKK7-JNK (WT); or 0.64 μg of MKK7-JNK (KN). Lysates were analyzed by WB with anti-Myc (Myc-PER2 and Myc-GFP), anti-JNK (MKK7-JNK), anti-phospho-c-Jun (p-c-Jun), and anti-ERK (ERK) Abs. B, enhanced PER2 stability. 293T cells were transfected with vectors expressing Myc-PER2 and Myc-GFP and either empty vector (−), MKK7-JNK1 (WT), or MKK7-JNK1 (KN). Transfected cells were treated with 20 μg/ml CHX at time 0. At the indicated times after CHX treatment, protein extracts were analyzed by WB with anti-Myc Ab to detect Myc-PER2 and Myc-GFP. C, quantitation of B. Relative intensities of the Myc-PER2 and Myc-GFP bands in the blot in B were determined by densitometry. Myc-PER2 values were normalized to the Myc-GFP value for a given time point, and values at time 0 were set to 1. The graph shown is for a single experiment representative of three trials. D, decreased PER2 protein in MKK-DKO ES cells. Protein extracts from WT and MKK-DKO ES cells were subjected to WB using anti-phospho-JNK (p-JNK), anti-JNK (JNK), anti-PER2 (PER2), anti-BMAL1 (BMAL1), anti-CLOCK (CLOCK), anti-CRY1 (CRY1), and anti-ERK (ERK) Abs. E, JNK activity decreases PER2 ubiquitination. 293T cells were transfected with Myc-PER2 and HA-ubiquitin (HA-Ub) and either empty vector (−), MKK7-JNK (WT), or MKK7-JNK (KN). Top, lysates of transfected cells were immunoprecipitated with anti-Myc Ab, and the immunoprecipitate was subjected to WB analysis with anti-HA Ab to detect ubiquitinated PER2 (PER2-(HA-Ub)n). Middle, WB analysis of immunoprecipitate using anti-Myc Ab to detect Myc-PER2. Bottom, WB analysis of total cell lysate with anti-JNK Ab to detect MKK7-JNK (WT) and MKK7-JNK (KN).
      We next investigated the effect of MKK7-JNK (WT) on the half-life of PER2 protein. To this end, we co-transfected cultured cells expressing Myc-PER2 plus Myc-GFP with empty vector, MKK7-JNK (WT), or MKK7-JNK (KN) and treated the cells with cycloheximide (CHX) to prevent new protein synthesis. Intriguingly, MKK7-JNK (WT), but not MKK7-JNK (KN), markedly extended the half-life of Myc-PER2 protein (Fig. 4, B and C). Thus, the PER2 protein stabilization attributable to MKK7-JNK (WT) depends on the kinase activity of JNK.
      We previously established mouse ES cells bearing disruptions of both the Mkk4 and Mkk7 genes (MKK-DKO ES cells) (
      • Nishitai G.
      • Shimizu N.
      • Negishi T.
      • Kishimoto H.
      • Nakagawa K.
      • Kitagawa D.
      • Watanabe T.
      • Momose H.
      • Ohata S.
      • Tanemura S.
      • Asaka S.
      • Kubota J.
      • Saito R.
      • Yoshida H.
      • Mak T.W.
      • Wada T.
      • Penninger J.M.
      • Azuma N.
      • Nishina H.
      • Katada T.
      Stress induces mitochondria-mediated apoptosis independent of SAPK/JNK activation in embryonic stem cells.
      ). Because the circadian machinery is not functional in ES cells (
      • Yagita K.
      • Horie K.
      • Koinuma S.
      • Nakamura W.
      • Yamanaka I.
      • Urasaki A.
      • Shigeyoshi Y.
      • Kawakami K.
      • Shimada S.
      • Takeda J.
      • Uchiyama Y.
      Development of the circadian oscillator during differentiation of mouse embryonic stem cells in vitro.
      ), we can study this cell type to avoid the effects of time-dependent regulation of clock protein expression and degradation. We confirmed that JNK phosphorylation, and therefore activity, was markedly diminished in MKK-DKO ES cells (Fig. 4D), consistent with our previous report (
      • Nishitai G.
      • Shimizu N.
      • Negishi T.
      • Kishimoto H.
      • Nakagawa K.
      • Kitagawa D.
      • Watanabe T.
      • Momose H.
      • Ohata S.
      • Tanemura S.
      • Asaka S.
      • Kubota J.
      • Saito R.
      • Yoshida H.
      • Mak T.W.
      • Wada T.
      • Penninger J.M.
      • Azuma N.
      • Nishina H.
      • Katada T.
      Stress induces mitochondria-mediated apoptosis independent of SAPK/JNK activation in embryonic stem cells.
      ). Interestingly, PER2 protein was also dramatically reduced in MKK-DKO ES cells. In contrast, the expression of other clock proteins, including CLOCK, BMAL1, and CRY1, was comparable in WT and MKK-DKO ES cells. These data support our results obtained using the MKK7-JNK fusion protein (Fig. 4, A and B) and indicate that JNK activity has a specific effect on PER2 protein stability.
      Ubiquitination is an important step in the targeting of PER2 protein for proteasomal degradation (
      • Akashi M.
      • Tsuchiya Y.
      • Yoshino T.
      • Nishida E.
      Control of intracellular dynamics of mammalian period proteins by casein kinase I ϵ (CKIϵ) and CKIδ in cultured cells.
      ,
      • Eide E.J.
      • Woolf M.F.
      • Kang H.
      • Woolf P.
      • Hurst W.
      • Camacho F.
      • Vielhaber E.L.
      • Giovanni A.
      • Virshup D.M.
      Control of mammalian circadian rhythm by CKIϵ-regulated proteasome-mediated PER2 degradation.
      ,
      • Yagita K.
      • Tamanini F.
      • Yasuda M.
      • Hoeijmakers J.H.
      • van der Horst G.T.
      • Okamura H.
      Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein.
      ). We therefore tested the effect of MKK7-JNK (WT) or MKK7-JNK (KN) expression on PER2 ubiquitination. Myc-PER2 was co-expressed in cultured cells with HA-ubiquitin plus empty vector, MKK7-JNK (WT), or MKK7-JNK (KN). Immunoprecipitation of extracts followed by WB showed that Myc-PER2 underwent prominent ubiquitination in control cultures (Fig. 4E), consistent with previous reports (
      • Akashi M.
      • Tsuchiya Y.
      • Yoshino T.
      • Nishida E.
      Control of intracellular dynamics of mammalian period proteins by casein kinase I ϵ (CKIϵ) and CKIδ in cultured cells.
      ,
      • Eide E.J.
      • Woolf M.F.
      • Kang H.
      • Woolf P.
      • Hurst W.
      • Camacho F.
      • Vielhaber E.L.
      • Giovanni A.
      • Virshup D.M.
      Control of mammalian circadian rhythm by CKIϵ-regulated proteasome-mediated PER2 degradation.
      ,
      • Yagita K.
      • Tamanini F.
      • Yasuda M.
      • Hoeijmakers J.H.
      • van der Horst G.T.
      • Okamura H.
      Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein.
      ). Significantly, this ubiquitination of Myc-PER2 was efficiently inhibited by co-expression of MKK7-JNK (WT) but not by co-expression of MKK7-JNK (KN) (Fig. 4E). These results imply that the enzymatic activity of MKK7-activated JNK is required to block PER2 ubiquitination.

       MKK7-JNK Inhibits CK1ϵ-mediated PER2 Degradation

      It has been reported that CK1ϵ phosphorylates PER2 and induces subsequent PER2 degradation via ubiquitination (
      • Akashi M.
      • Tsuchiya Y.
      • Yoshino T.
      • Nishida E.
      Control of intracellular dynamics of mammalian period proteins by casein kinase I ϵ (CKIϵ) and CKIδ in cultured cells.
      ,
      • Eide E.J.
      • Woolf M.F.
      • Kang H.
      • Woolf P.
      • Hurst W.
      • Camacho F.
      • Vielhaber E.L.
      • Giovanni A.
      • Virshup D.M.
      Control of mammalian circadian rhythm by CKIϵ-regulated proteasome-mediated PER2 degradation.
      ). We found that overexpression of CK1ϵ caused a significant shift in the mobility of PER2 protein that was consistent with its destabilization (Fig. 5, A (compare lanes 1–3 with lanes 4–6) and B). This electrophoretic retardation resulted from phosphorylation because it could be reversed by phosphatase treatment (supplemental Fig. 3), a finding consistent with previous reports (
      • Akashi M.
      • Tsuchiya Y.
      • Yoshino T.
      • Nishida E.
      Control of intracellular dynamics of mammalian period proteins by casein kinase I ϵ (CKIϵ) and CKIδ in cultured cells.
      ,
      • Eide E.J.
      • Woolf M.F.
      • Kang H.
      • Woolf P.
      • Hurst W.
      • Camacho F.
      • Vielhaber E.L.
      • Giovanni A.
      • Virshup D.M.
      Control of mammalian circadian rhythm by CKIϵ-regulated proteasome-mediated PER2 degradation.
      ). Notably, co-expression of MKK7-JNK (WT) did not inhibit the electrophoretic retardation of the PER2 protein band induced by CK1ϵ. Instead, MKK7-JNK (WT) caused an additional shift in the PER2 band (Fig. 5C) that was also reversed by phosphatase treatment (supplemental Fig. 3). These data suggest that CK1ϵ and JNK do not compete in phosphorylating PER2. To confirm this hypothesis, we mutated the Ser-659 residue of mouse PER2 protein to alanine (PER2 (S659A)), thereby altering the residue that corresponds to the best-characterized CKIϵ phosphorylation site of the human PER2 protein (
      • Toh K.L.
      • Jones C.R.
      • He Y.
      • Eide E.J.
      • Hinz W.A.
      • Virshup D.M.
      • Ptácek L.J.
      • Fu Y.H.
      An hPer2 phosphorylation site mutation in familial advanced sleep phase syndrome.
      ). We found that MKK7-JNK (WT) was still able to efficiently phosphorylate the PER2 (S659A) protein (Fig. 5D), indicating that MKK7-JNK phosphorylates PER2 at amino acid residue(s) other than Ser-659. When we assessed the effect of MKK7-JNK (WT) expression on CK1ϵ control over PER2 stability, we found that the enzymatic activity of JNK reduced the destabilization of PER2 induced by CK1ϵ (Fig. 5, A (compare lanes 4–6 with lanes 10–12) and B). Furthermore, MKK7-JNK (WT) co-expression suppressed CK1ϵ-induced PER2 ubiquitination (Fig. 5E). Thus, our results clearly show that the MKK7-JNK signaling pathway and CK1ϵ activity oppose each other in regulating PER2 protein stability.
      Figure thumbnail gr5
      FIGURE 5MKK7-activated JNK inhibits CK1ϵ-mediated PER2 degradation. A, effect of CHX. 293T cells were transfected with vectors expressing Myc-PER2 and Myc-GFP, plus empty vector (−), CK1ϵ, or MKK7-JNK (WT) alone or in combination, as indicated. Transfected cells were treated with 20 μg/ml CHX at time 0. At the indicated times after CHX treatment, protein extracts were analyzed by WB with anti-Myc, anti-CK1, or anti-JNK Ab to detect Myc-PER2 and Myc-GFP, CK1ϵ, and MKK7-JNK (WT) proteins, respectively. B, quantitation of A. Relative band intensities in the blot in A were determined and analyzed as for C. C, mobility shift of PER2. 293T cells were transfected with vectors expressing Myc-PER2 and Myc-GFP plus either empty vector, MKK7-JNK (WT), MKK7-JNK (KN), or CK1ϵ, as indicated. Protein extracts were analyzed by WB with anti-Myc, anti-CK1, and anti-JNK. The black arrowhead indicates the band shift induced by co-expression of CK1ϵ; the white arrowhead indicates the band supershift induced by co-expression of CK1ϵ and MKK7-JNK (WT). D, sensitivity to phosphatase. Top, HeLa cells were co-transfected with vector expressing Myc-PER2 (S659A) plus either empty vector (−) or vector expressing MKK7-JNK (WT) (+). Myc-PER2 (S659A) was immunoprecipitated with anti-Myc Ab, treated (+) or not (−) with alkaline phosphatase (PPA), and analyzed by WB with anti-Myc Ab. Bottom, MKK7-JNK (WT) was detected by WB with anti-JNK Ab. E, MKK7-JNK suppresses CK1ϵ-induced PER2 ubiquitination. 293T cells were transfected with Myc-PER2 and HA-Ub plus empty vector (−), MKK7-JNK (WT), or CK1ϵ alone or in combination, as indicated. Top, lysates were immunoprecipitated (IP) with anti-Myc Ab and subjected to WB analysis with anti-HA Ab to detect ubiquitinated PER2 (PER2-(HA-Ub)n). Middle, lysates were immunoprecipitated with anti-Myc Ab and analyzed by WB using anti-Myc Ab to detect Myc-PER2. Bottom, total cell lysates were analyzed by WB using anti-JNK (MKK7-JNK) or anti-CK1ϵ (CK1ϵ) Abs.

      DISCUSSION

      Previous studies have shown that phosphorylation levels of JNK, and thus its kinase activity, fluctuate in a circadian manner in both the suprachiasmatic nucleus, the site of the master clock, and in cultured mammalian cells (
      • Pizzio G.A.
      • Hainich E.C.
      • Ferreyra G.A.
      • Coso O.A.
      • Golombek D.A.
      Circadian and photic regulation of ERK, JNK, and p38 in the hamster SCN.
      ,
      • Chansard M.
      • Molyneux P.
      • Nomura K.
      • Harrington M.E.
      • Fukuhara C.
      c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms.
      ). In addition, the use of the JNK inhibitor SP600125 and siRNA-mediated suppression of Jnk have both been reported to lengthen the period of circadian transcription in cultured cells and in ex vivo organ culture systems (
      • Chansard M.
      • Molyneux P.
      • Nomura K.
      • Harrington M.E.
      • Fukuhara C.
      c-Jun N-terminal kinase inhibitor SP600125 modulates the period of mammalian circadian rhythms.
      ,
      • Yagita K.
      • Yamanaka I.
      • Koinuma S.
      • Shigeyoshi Y.
      • Uchiyama Y.
      Mini screening of kinase inhibitors affecting period-length of mammalian cellular circadian clock.
      ,
      • Zhang E.E.
      • Liu A.C.
      • Hirota T.
      • Miraglia L.J.
      • Welch G.
      • Pongsawakul P.Y.
      • Liu X.
      • Atwood A.
      • Huss 3rd, J.W.
      • Janes J.
      • Su A.I.
      • Hogenesch J.B.
      • Kay S.A.
      A genome-wide RNAi screen for modifiers of the circadian clock in human cells.
      ). These findings have provided evidence of the involvement of the JNK signaling pathway in circadian regulation. However, several laboratories, including ours, have demonstrated that SP600125 suppresses the activities of kinases other than JNK, such as phosphatidylinositol 3-kinase and Cdk1 (
      • Tanemura S.
      • Momose H.
      • Shimizu N.
      • Kitagawa D.
      • Seo J.
      • Yamasaki T.
      • Nakagawa K.
      • Kajiho H.
      • Penninger J.M.
      • Katada T.
      • Nishina H.
      Blockage by SP600125 of Fcϵ receptor-induced degranulation and cytokine gene expression in mast cells is mediated through inhibition of phosphatidylinositol 3-kinase signaling pathway.
      ,
      • Kim J.A.
      • Lee J.
      • Margolis R.L.
      • Fotedar R.
      SP600125 suppresses Cdk1 and induces endoreplication directly from G2 phase, independent of JNK inhibition.
      ). Thus, to examine circadian gene expression in the specific absence of JNK activity, we used genetic inactivation of MKK7, which activates JNK alone. We have shown that abrogation of MKK7 function in unstressed cultured cells lengthens the period of circadian gene expression (Fig. 1), identifying for the first time a function for MKK7 as a circadian clock regulator.
      Several studies have demonstrated that external stimuli, such as ionizing radiation, UV light, and hydrogen peroxide affect circadian gene expression by cultured cells and tissues (
      • Fu L.
      • Pelicano H.
      • Liu J.
      • Huang P.
      • Lee C.
      The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo.
      ,
      • Oklejewicz M.
      • Destici E.
      • Tamanini F.
      • Hut R.A.
      • Janssens R.
      • van der Horst G.T.
      Phase resetting of the mammalian circadian clock by DNA damage.
      ). We have also confirmed that hydrogen peroxide altered circadian gene expression and the accumulation of PER2 protein (supplemental Fig. 4). Because MKK7-JNK signaling is activated by these physical and chemical stresses (
      • Davis R.J.
      Signal transduction by the JNK group of MAP kinases.
      ,
      • Chang L.
      • Karin M.
      Mammalian MAP kinase signaling cascades.
      ), this pathway may mediate stress-induced molecular clock regulation, an intriguing possibility to examine in the future. Our present study has focused on the role of MKK7 in unstressed cells, and we have shown that MKK7 contributes to the regulation of the molecular clock in cells at steady state. These findings provide insight into the roles of stress-responsive molecules in the maintenance of normal cellular homeostasis.
      In our hands, the effects of SP600125 on the periodicity of circadian gene expression were more severe than those of MKK7 inactivation (Fig. 1, B and C, and supplemental Fig. 2, C and D). The more drastic phenotype achieved with SP600125 may be due to its known effects on several other kinases (
      • Tanemura S.
      • Momose H.
      • Shimizu N.
      • Kitagawa D.
      • Seo J.
      • Yamasaki T.
      • Nakagawa K.
      • Kajiho H.
      • Penninger J.M.
      • Katada T.
      • Nishina H.
      Blockage by SP600125 of Fcϵ receptor-induced degranulation and cytokine gene expression in mast cells is mediated through inhibition of phosphatidylinositol 3-kinase signaling pathway.
      ,
      • Kim J.A.
      • Lee J.
      • Margolis R.L.
      • Fotedar R.
      SP600125 suppresses Cdk1 and induces endoreplication directly from G2 phase, independent of JNK inhibition.
      ). Indeed, it has been reported that LY294002 can lengthen the period of circadian gene expression in cultured cells by inhibiting phosphatidylinositol 3-kinase (
      • Yagita K.
      • Yamanaka I.
      • Koinuma S.
      • Shigeyoshi Y.
      • Uchiyama Y.
      Mini screening of kinase inhibitors affecting period-length of mammalian cellular circadian clock.
      ). On the other hand, our data may indicate that JNK phosphorylation mediated by MKK4, the other key JNK activator (
      • Davis R.J.
      Signal transduction by the JNK group of MAP kinases.
      ,
      • Chang L.
      • Karin M.
      Mammalian MAP kinase signaling cascades.
      ), can also contribute to circadian clock regulation. This possibility is under investigation.
      At the molecular level, our study has provided several lines of evidence that the MKK7-JNK pathway regulates the circadian feedback loop by interacting with and phosphorylating PER2. First, the MKK7-JNK (WT) fusion protein, which is a constitutively active form of JNK, can phosphorylate PER2 (Fig. 2). Importantly, the threonines in the consensus JNK phosphorylation motifs present in PER2 were phosphorylated in cultured cells, and co-expression of MKK7-JNK (WT) enhanced this phosphorylation (Fig. 2E). Second, we showed that JNK interacts with PER2 at both the exogenous and endogenous levels (Fig. 3). Third, overexpression of MKK7-JNK (WT) inhibited PER2 ubiquitination and stabilized PER2 protein (Fig. 4). These results, together with our observation that the genetic inactivation of MKK7 has an influence on circadian gene expression (Fig. 1), suggest that the MKK7-JNK pathway contributes to circadian rhythm regulation by controlling PER2 protein levels and thus PER2 functions.
      Many kinases, including CK1ϵ, CK1δ, CK2, and GSK3β, have been shown to regulate PER2 functions (
      • Gallego M.
      • Virshup D.M.
      Post-translational modifications regulate the ticking of the circadian clock.
      ,
      • Uchida Y.
      • Hirayama J.
      • Nishina H.
      A common origin. Signaling similarities in the regulation of the circadian clock and DNA damage responses.
      ,
      • Akashi M.
      • Tsuchiya Y.
      • Yoshino T.
      • Nishida E.
      Control of intracellular dynamics of mammalian period proteins by casein kinase I ϵ (CKIϵ) and CKIδ in cultured cells.
      ,
      • Tsuchiya Y.
      • Akashi M.
      • Matsuda M.
      • Goto K.
      • Miyata Y.
      • Node K.
      • Nishida E.
      Involvement of the protein kinase CK2 in the regulation of mammalian circadian rhythms.
      ,
      • Maier B.
      • Wendt S.
      • Vanselow J.T.
      • Wallach T.
      • Reischl S.
      • Oehmke S.
      • Schlosser A.
      • Kramer A.
      A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock.
      ). Moreover, these kinases can cooperate in this regard because CK2 reportedly decreases PER2 protein stability by enhancing CK1ϵ-mediated PER2 degradation (
      • Tsuchiya Y.
      • Akashi M.
      • Matsuda M.
      • Goto K.
      • Miyata Y.
      • Node K.
      • Nishida E.
      Involvement of the protein kinase CK2 in the regulation of mammalian circadian rhythms.
      ). In our study, we found that co-expression of MKK7-JNK (WT) decreased CK1ϵ-mediated PER2 degradation (Fig. 5). However, our data show that MKK7-JNK signaling does not directly inhibit CK1ϵ-mediated PER2 phosphorylation and may instead phosphorylate PER2 residue(s) different from those targeted by CK1ϵ-dependent phosphorylation. In short, MKK7-JNK signaling and CK1ϵ appear to have opposing regulatory effects on PER2 protein stability that are mediated via differential phosphorylation. Future studies should establish how the MKK7-JNK cascade combines with other kinases either to control the oscillatory mechanism itself or to modulate the signaling pathway that drives circadian entrainment in response to external signals.
      The control of PER2 stability via phosphorylation is associated with the period length of circadian gene expression (
      • Vanselow K.
      • Vanselow J.T.
      • Westermark P.O.
      • Reischl S.
      • Maier B.
      • Korte T.
      • Herrmann A.
      • Herzel H.
      • Schlosser A.
      • Kramer A.
      Differential effects of PER2 phosphorylation. Molecular basis for the human familial advanced sleep phase syndrome (FASPS).
      ,
      • Xu Y.
      • Toh K.L.
      • Jones C.R.
      • Shin J.Y.
      • Fu Y.H.
      • Ptácek L.J.
      Modeling of a human circadian mutation yields insights into clock regulation by PER2.
      ). Notably, the phosphorylation of PER2 protein regulates its stability in multiple ways, and this phosphorylation depends both on which phosphorylation sites are used and on which kinases are performing the phosphorylation (
      • Hirayama J.
      • Sassone-Corsi P.
      Structural and functional features of transcription factors controlling the circadian clock.
      ,
      • Gallego M.
      • Virshup D.M.
      Post-translational modifications regulate the ticking of the circadian clock.
      ). In addition, interference with phosphorylation-dependent PER2 stability controls can lead to either short or long circadian periods, making it difficult to predict how a given level of PER2 stability will affect the period length of circadian gene expression (
      • Eide E.J.
      • Woolf M.F.
      • Kang H.
      • Woolf P.
      • Hurst W.
      • Camacho F.
      • Vielhaber E.L.
      • Giovanni A.
      • Virshup D.M.
      Control of mammalian circadian rhythm by CKIϵ-regulated proteasome-mediated PER2 degradation.
      ,
      • Vanselow K.
      • Vanselow J.T.
      • Westermark P.O.
      • Reischl S.
      • Maier B.
      • Korte T.
      • Herrmann A.
      • Herzel H.
      • Schlosser A.
      • Kramer A.
      Differential effects of PER2 phosphorylation. Molecular basis for the human familial advanced sleep phase syndrome (FASPS).
      ,
      • Xu Y.
      • Toh K.L.
      • Jones C.R.
      • Shin J.Y.
      • Fu Y.H.
      • Ptácek L.J.
      Modeling of a human circadian mutation yields insights into clock regulation by PER2.
      ,
      • Maier B.
      • Wendt S.
      • Vanselow J.T.
      • Wallach T.
      • Reischl S.
      • Oehmke S.
      • Schlosser A.
      • Kramer A.
      A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock.
      ,
      • Reischl S.
      • Vanselow K.
      • Westermark P.O.
      • Thierfelder N.
      • Maier B.
      • Herzel H.
      • Kramer A.
      β-TrCP1-mediated degradation of PERIOD2 is essential for circadian dynamics.
      ). For example, both mutation of PER2 Ser-662, which leads to a lower rate of CK1ϵ/δ-dependent phosphorylation, and mutation of PER2 N-terminal phosphoacceptor sites for CK2 lead to PER2 instability. However, these mutations have the opposite effect on the circadian period; Ser-662 mutation shortens the period, whereas N-terminal mutation extends it (
      • Vanselow K.
      • Vanselow J.T.
      • Westermark P.O.
      • Reischl S.
      • Maier B.
      • Korte T.
      • Herrmann A.
      • Herzel H.
      • Schlosser A.
      • Kramer A.
      Differential effects of PER2 phosphorylation. Molecular basis for the human familial advanced sleep phase syndrome (FASPS).
      ,
      • Xu Y.
      • Toh K.L.
      • Jones C.R.
      • Shin J.Y.
      • Fu Y.H.
      • Ptácek L.J.
      Modeling of a human circadian mutation yields insights into clock regulation by PER2.
      ,
      • Maier B.
      • Wendt S.
      • Vanselow J.T.
      • Wallach T.
      • Reischl S.
      • Oehmke S.
      • Schlosser A.
      • Kramer A.
      A large-scale functional RNAi screen reveals a role for CK2 in the mammalian circadian clock.
      ). Thus, further elucidation of the processes that affect PER2 stability are required to unravel the complex and largely mysterious post-translational controls governing circadian timing in mammals.
      Previous work has established that a single kinase is capable of phosphorylating multiple clock components (
      • Gallego M.
      • Virshup D.M.
      Post-translational modifications regulate the ticking of the circadian clock.
      ,
      • Uchida Y.
      • Hirayama J.
      • Nishina H.
      A common origin. Signaling similarities in the regulation of the circadian clock and DNA damage responses.
      ). For example, CK1ϵ can phosphorylate PER1, PER2, PER3, BMAL1, and CRY1, altering their subcellular localization, protein stability, and transcriptional activity and thus contributing to regulation of their functions (
      • Akashi M.
      • Tsuchiya Y.
      • Yoshino T.
      • Nishida E.
      Control of intracellular dynamics of mammalian period proteins by casein kinase I ϵ (CKIϵ) and CKIδ in cultured cells.
      ,
      • Takano A.
      • Shimizu K.
      • Kani S.
      • Buijs R.M.
      • Okada M.
      • Nagai K.
      Cloning and characterization of rat casein kinase 1ϵ.
      ,
      • Vielhaber E.
      • Eide E.
      • Rivers A.
      • Gao Z.H.
      • Virshup D.M.
      Nuclear entry of the circadian regulator mPER1 is controlled by mammalian casein kinase I ϵ.
      ,
      • Eide E.J.
      • Vielhaber E.L.
      • Hinz W.A.
      • Virshup D.M.
      The circadian regulatory proteins BMAL1 and cryptochromes are substrates of casein kinase Iϵ.
      ). Our results indicate that, in addition to PER2, BMAL1 is also a target for MKK7-JNK-mediated phosphorylation. We have not yet obtained enough data to prove that BMAL1 phosphorylation is required for circadian clock function, and the physiological importance of this phosphorylation remains under investigation.
      In addition to their circadian clock functions, PER2 and BMAL1 participate in non-circadian physiological processes, such as tumor suppression (
      • Fu L.
      • Pelicano H.
      • Liu J.
      • Huang P.
      • Lee C.
      The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo.
      ), aging (
      • Kondratov R.V.
      • Kondratova A.A.
      • Gorbacheva V.Y.
      • Vykhovanets O.V.
      • Antoch M.P.
      Early aging and age-related pathologies in mice deficient in BMAL1, the core componentof the circadian clock.
      ), immune responses (
      • Nakamura Y.
      • Harama D.
      • Shimokawa N.
      • Hara M.
      • Suzuki R.
      • Tahara Y.
      • Ishimaru K.
      • Katoh R.
      • Okumura K.
      • Ogawa H.
      • Shibata S.
      • Nakao A.
      Circadian clock gene Period2 regulates a time-of-day-dependent variation in cutaneous anaphylactic reaction.
      ), and metabolic control (
      • Shimba S.
      • Ishii N.
      • Ohta Y.
      • Ohno T.
      • Watabe Y.
      • Hayashi M.
      • Wada T.
      • Aoyagi T.
      • Tezuka M.
      Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis.
      ,
      • Grimaldi B.
      • Bellet M.M.
      • Katada S.
      • Astarita G.
      • Hirayama J.
      • Amin R.H.
      • Granneman J.G.
      • Piomelli D.
      • Leff T.
      • Sassone-Corsi P.
      PER2 controls lipid metabolism by direct regulation of PPARγ.
      ,
      • Marcheva B.
      • Ramsey K.M.
      • Buhr E.D.
      • Kobayashi Y.
      • Su H.
      • Ko C.H.
      • Ivanova G.
      • Omura C.
      • Mo S.
      • Vitaterna M.H.
      • Lopez J.P.
      • Philipson L.H.
      • Bradfield C.A.
      • Crosby S.D.
      • JeBailey L.
      • Wang X.
      • Takahashi J.S.
      • Bass J.
      Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinemia and diabetes.
      ). MKK7-JNK-dependent phosphorylation of circadian regulators, such as PER2 and BMAL1, may therefore be a key integrating event that translates internal and external signals into a variety of physiological cellular responses, including circadian rhythm adjustment.

      Acknowledgments

      We are grateful to all members of the Nishina and Katada laboratories (Tokyo University) for insightful comments.

      Supplementary Material

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