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1 Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-0033, Japan
2
Radiation Biology Centre, Kyoto University, Yoshida-konoecho, Sakyo-Ku, Kyoto 606-8501, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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In the autoregulatory feedback loop, post-translational modifications such as phosphorylation of clock gene products are likely to contribute to maintenance of the 24-h rhythm by producing an appropriate time-lag (Dunlap 1999). In mammals, PER protein is phosphorylated and destabilized by casein kinase I
(Takano et al. 2000; Vielhaber et al. 2000; Lee et al. 2001), a homologue of Drosophila double-time whose missence alleles result in alteration of PER stability and the period length of the circadian rhythm (Kloss et al. 1998; Price et al. 1998). Also, a defect in hamster casein kinase I corresponds to the short period Tau mutation (Lowrey et al. 2000), and these observations indicate a significant contribution of protein kinase to the circadian time-keeping mechanism. Like PER protein, positive regulators, BMAL1 and CLOCK seem to be phosphorylated in clock structures (Lee et al. 2001), and one candidate kinase responsible for BMAL1 phosphorylation is mitogen-activated protein kinase (MAPK) which phosphorylates and negatively regulates BMAL1 (Sanada et al. 2002). Notably, MAPK (Sanada et al. 2000) and p38 stress-activated protein kinase (Hayashi et al. 2003) play important roles for the circadian time-keeping in the chick pineal clock system.
MAPK exhibits circadian activation in various clock structures such as the mouse SCN (Obrietan et al. 1998; Nakaya et al. 2003), chicken pineal gland (Sanada et al. 2000; Hayashi et al. 2001), bullfrog retina (Harada et al. 2000) and chicken retina (Ko et al. 2001), and a transient inhibition of MAPK delays the phase of the oscillator in chick pineal and bullfrog retinal culture (Harada et al. 2000; Sanada et al. 2000). Recent studies suggest that MAPK-mediated signals play more divergent roles in various aspects of the clockwork, such as photic (Butcher et al. 2002) and non-photic (Akashi & Nishida 2000) entrainment pathways, time-keeping of the oscillator (Harada et al. 2000; Sanada et al. 2000, 2002) and an output pathway (Ko et al. 2001; Williams et al. 2001). Within clock structures such as the SCN, functionally distinct populations of clock cells seem to exist in terms of mode of circadian and photic regulation of MAPK (Obrietan et al. 1998; Lee et al. 2003; Butcher et al. 2003; Nakaya et al. 2003). Consideration of these circumstances led us to examine a possibility that MAPK may phosphorylate diverse regulators of the clock system. In the present study, we found that MAPK binds to and phosphorylates mCRY1 and mCRY2 and that the phosphorylation alters their functional properties, suggesting temporal regulation of mCRYs’ function in the vertebrate clock structures.
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Results |
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Each mCRY has four potential sites for MAPK phosphorylation with a minimal consensus sequence Ser/Thr-Pro (Davis 1993), and three of them are found at positions conserved between mCRY1 and mCRY2. These sites are Ser247, Ser252 and Ser281 in mCRY1 and Ser265, Ser270 and Thr299 in mCRY2. To examine MAPK-catalysed phosphorylation of mCRY proteins, we employed in vitro kinase assay in which bacterially expressed glutathione-S-transferase (GST)-fused mCRYs (GST-mCRYs) was incubated with either phosphorylated or non-phosphorylated MAPK (P-myc-MAPK or myc-MAPK, respectively). As shown in Fig. 2A, both GST-mCRY1 and GST-mCRY2 were phosphorylated by P-myc-MAPK but not by myc-MAPK, whereas no phosphorylation of GST was observed. Quantitative analysis of the time course of the reaction revealed that mCRY1 and mCRY2 incorporated approx. 1.5 pmol of phosphate per pmol of protein (Fig. 2B), predicting at least two sites in each mCRY protein to be phosphorylated by activated MAPK.
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To identify the phosphorylation sites, GST-mCRY2 was phosphorylated by P-myc-MAPK in vitro and digested with trypsin. Then, the tryptic fragments of GST-mCRY2 were separated by reversed-phase HPLC (Fig. 3, middle trace), and the isolated fragments were subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). As a control sample, GST-mCRY2 was incubated with non-phosphorylated myc-MAPK that catalyses no measurable phosphorylation of mCRY2 (Fig. 2A), and its tryptic fragments were similarly isolated (Fig. 3, top trace) for MALDI-TOF/MS analysis. In this control experiment, three peak fractions (P1–P3) showed mass signals at m/z 914.8 (P1), 1906.4 (P2) and 1729.9 (P3), corresponding to the mCRY2 sequences, Ala550-Lys559, Met257-Arg274 and Asn297-Arg311, respectively (Table 1), in which four putative MAPK phosphorylation sites were found. MS-based sequence analysis corroborated the assignment of these fragments kept in non-phosphorylated forms. On the other hand, a careful comparison between the elution profiles of the two samples (phosphorylated and non-phosphorylated mCRY2) revealed two unique peaks (P4 and P6) in the phosphorylated sample (Fig. 3), suggesting a phosphorylation-dependent shift in elution position of peptides. A fragment in peak P4 exhibited a MALDI-TOF/MS signal at m/z 994.7, which agreed with the calculated mass (994.9) of Ala550-Lys559 (915.0) plus one phosphate group (79.9; see Table 1). A nearby peak P5 gave a mass signal at m/z 915.2 corresponding to the same sequence in the non-phosphorylated state, and its elution position coincided with that of peak P1 containing the same peptide (Fig. 3). Similarly, peak P6 and its nearby peak P7 gave mass signals at m/z 1986.0 and 1905.9, respectively, the former of which corresponded to the singly phosphorylated form of the latter peptide Met257-Arg274 (calculated mass: 1906.2; see Table 1). The fragment in peak P8 was assigned to Asn297-Arg311 (Table 1), and its phosphorylated form was not detected in the eluted peaks though the peptide sequence contains one potential site for MAPK phosphorylation.
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Mutations of Ser247 in mCRY1 and Ser265/Ser557 in mCRY2 diminish MAPK-mediated phosphorylation
In vitro phosphorylation of the Ser residues in mCRY proteins was examined by using mCRY mutants, in which individual phospho-acceptor Ser residues were replaced with Ala. As compared to wild-type mCRY2 (Fig. 4, lane 3), its mutants mCRY2(S265A) and mCRY2(S557A) showed significantly lower degrees of MAPK-catalysed phosphorylation (reduced to 
60%, lane 4 and to 26%, lane 5), and the double mutant mCRY2(S265A/S557A) almost completely lost the ability to be phosphorylated (lane 6), indicating that these two Ser residues are the principal phosphorylation sites. Similarly, mCRY1(S247A) showed a much lower degree of phosphorylation than wild-type mCRY1 (reduced to 55%, lanes 1 and 2), supporting Ser247 phosphorylation. Considering the maximal degree of mCRY1 phosphorylation (1.5 pmol phosphate per pmol of protein; Fig. 2B), we assume MAPK-catalysed mCRY1 phosphorylation at Ser247 and at least one additional site that was not identified here.
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A functional significance of the Ser phosphorylation of mCRYs was examined by transcription assay. In the absence of exogenous mCRYs, coexpression of mouse BMAL1 and CLOCK markedly stimulated E-box element-dependent transcription of a luciferase reporter gene in HEK293 cells (Fig. 5A, left panel). This transactivation was suppressed by coexpression of wild-type mCRY1 (middle) or mCRY2 (right) in a manner dependent on the amount of transfected plasmids expressing mCRYs (open bars). We then constructed mutants in which phospho-acceptor Ser residues were replaced with Ala or Asp, either to prevent phosphorylation (Ser to Ala mutants) or to mimic the negative charge due to phosphorylation (Ser to Asp mutants), and the resultant mCRY1 mutants (S247A and S247D) and mCRY2 mutants (S265A, S265D, S557A and S557D) were tested for their abilities to inhibit the transactivation. All the mutants transfected at high concentrations (100 ng or 250 ng plasmid) inhibited BMAL1:CLOCK-mediated transcription to a degree similar to that achieved by each cognate wild-type mCRY (Fig. 5A), indicating maintenance of CRY function in these mutants. At lower dosage of plasmid (2 ng or 5 ng), however, two Asp mutants mCRY1(S247D) and mCRY2(S265D) exhibited significantly reduced inhibitory effect on BMAL1:CLOCK-mediated transcription (solid bars) than respective wild-type mCRYs (P < 0.01, ANOVA). In parallel, we examined protein expression levels of wild-type and each mutant in transfected HEK293 cells, and higher doses of the plasmids (400 ng and 800 ng) were employed for the evaluation because of the detection limit in immunoblotting. Under the condition, protein expression levels of wild-type and the mutant mCRYs increased in a manner dependent on the amount of each plasmid (Fig. 5B), and at a fixed amount of the plasmid, the protein level of each mutant was comparable to or slightly higher than that of wild-type, with no significant negative effect of the mutations on the expression level (P > 0.01, ANOVA, Fig. 5B).
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We then asked whether Ser247 of mCRY1 and Ser265 of mCRY2 were phosphorylated by MAPK in vivo. Since both mCRYs share identical amino acid sequences encompassing the phospho-acceptor Ser (Fig. 7A), we raised antisera against a single phospho-peptide [CNSLLAS(PO4)SPTGLS] corresponding to the conserved region. To characterize the affinity-purified antibody (termed anti-pS247/265-mCRYs; see Experimental procedures), bacterially expressed GST-mCRYs were phosphorylated by P-myc-MAPK in vitro and subjected to immunoblotting. The anti-pS247/265-mCRYs antibody detected the phosphorylated form of both GST-mCRY1 and GST-mCRY2, and showed no detectable cross-reaction with the unphosphorylated form (Fig. 7B). These results clearly demonstrate that the antibody specifically recognizes Ser247-phosphorylated mCRY1 and Ser265-phosphorylated mCRY2. We finally determined the phosphorylation state of mCRYs expressed in COS7 cells. When myc-CRYs were transiently expressed together with FLAG-MAPK and DE-MEK, the phospho-specific antibody detected a band corresponding to myc-mCRYs (Fig. 7C, lanes 3 and 5). In contrast, when coexpressed with kinase-dead MAPK (FLAG-KR-MAPK), neither myc-mCRY1 nor myc-mCRY2 was recognized by the antibody (Fig. 7C, lanes 2 and 4). Collectively, Ser247 of mCRY1 and Ser265 of mCRY2, capable of being phosphorylated by MAPK in mammalian cells, would be functionally important sites for MAPK-mediated regulation of the clock machinery.
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Discussion |
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CRY is a member of the DNA photolyase/cryptochrome flavoprotein family, and each member contains reduced flavin-adenine dinucleotide (FADH-) as a cofactor. The FAD-binding sites are highly conserved among the members, and the phospho-acceptor residue, Ser247 of mCRY1 or Ser265 of mCRY2, is located in close proximity to FAD-contacting amino acid residues (e.g. Pro266-Ser270 of mCRY2) (Park et al. 1995). Functional interaction of FAD with nearby residues in CRY has been demonstrated by recent observations that the CRY functions are suppressed by mutations of amino acid residues that are implicated in FAD binding and in electron transport (Stanewsky et al. 1998; Zhu & Green 2001b; Froy et al. 2002). In the present study, introduction of a negative charge (Ser to Asp mutation) to the phospho-acceptor site near the FAD-binding pocket was shown to attenuate the mCRYs’ ability to inhibit BMAL1:CLOCK-mediated transcription (Fig. 5). In addition, the Ala mutation at this position had no effect on the mCRY activity, supporting a genuine importance of the negative charge in this region for negative regulation of mCRYs. The phosphorylation might induce electrostatic repulsion and/or attraction between phospho-Ser and its nearby amino acid residues and lead to change in mCRY structure (Froy et al. 2002), affecting FAD positioning and/or electron transport. It is also conceivable that the Asp mutation may reduce mCRY's affinity to BMAL1:CLOCK complex, because the effect of the Asp mutation became invisible when the mutant was expressed at relatively high level (Fig. 5A). If the Asp mutation may cause lowering of mCRY's affinity for BMAL1:CLOCK complex, then the mutant would exhibit a reduced activity only at a lower dose, and the higher dose of the mutant should mask the effect of the decreased affinity by mass action.
In contrast to the FAD-binding domain highly conserved among the members in the DNA photolyase/cryptochrome flavoprotein family, unique C-terminal extension is found in plant and animal CRYs, and another phospho-acceptor residue Ser557 lies in this region of mCRY2. Arabidopsis CRY protein associates with phytochrome A through the C-terminal extension, which is phosphorylated by phytochrome A-associated kinase activity and plays an important role in the maintenance of the circadian rhythms (Ahmad et al. 1998). Likewise the C-terminal extension of vertebrate CRYs is implicated in protein-protein interaction (Todo 1999), to which Ser557 of mCRY2 might contribute as a phosphorylation-dependent regulator. Knockout studies in mice have revealed that mCRY1 and mCRY2 contribute differently to the clockwork (van der Horst et al. 1999), so it is interesting to characterize the role of mCRY2-specific phosphorylation to uncover a functional difference of mCRY1 and mCRY2. In contrast to the phosphorylation near the FAD-binding site, phosphorylation of Ser557 in mCRY2 is unlikely to affect its molecular activity directly (Fig. 5). Ser557 phosphorylation may rather have an indirect regulatory role of facilitating cooperative phosphorylation at Ser265, as predicted from the robust inhibitory effect of S557A mutation on total mCRY2 phosphorylation (Fig. 4).
In the mouse SCN, protein levels of both mCRY1 and mCRY2 rise at late subjective day peaking at CT12 and decrease during the subjective night, showing robust circadian rhythms (Kume et al. 1999). Noticeably, MAPK activity increases during the subjective night in the central region of the mouse SCN (Obrietan et al. 1998; Lee et al. 2003; Nakaya et al. 2003), suggesting that mCRY activity would be progressively reduced during the nighttime by cooperative effects of both decreasing protein level and the MAPK-mediated attenuation of the activity. It is speculated that mCRYs are timely phosphorylated in vivo when the phosphorylation significantly affects their activities toward effective depletion of the residual mCRY activity at the later declining phase of mCRYs protein level. Such well-ordered degradation and inactivation of negative regulators could be very important for their cyclic expression because this process eliminating feedback inhibition of the transcription allows their RNA levels to rise again at an appropriate time of the day. Recently we have found MAPK-mediated negative regulation of BMAL1 transcriptional activity (Sanada et al. 2002). In the molecular feedback loop, BMAL1:CLOCK-mediated transcription of mCry/mPer is kept suppressed until protein levels of mCRY/mPER reach significantly low. This builds an appropriate time-lag from the phase of mCRY/mPER protein rhythm to that of mCry/mPer mRNA rhythm, and this lag should be important for the stable 24-h rhythm of the molecular oscillation. We speculate that MAPK may contribute to generation of the time-lag by regulating both positive and negative elements; i.e. MAPK promotes decrease in mCRYs’ activity on one hand, and at the same time delays a rise of mCry/mPer mRNA levels by suppressing BMAL1-mediated transactivation. Consequently, the increase of mCry/mPer mRNA is postponed even in the declining phase of the protein/activity level of mCRY during the subjective night.
All the phosphorylation sites of mCRYs identified in the present study are supposed to be the targets of other proline-directed kinases such as p38 stress-activated protein kinase. It is also reported that casein kinase I phosphorylates mCRYs, though its functional significance remains unclear (Eide et al. 2002). Thus mCRYs are likely subject to regulation by multiple kinases in the clock structures, and further evaluation of post-translational regulation of mCRY function in clock structures would provide an insight into the mechanism underlying maintenance of a 24-h oscillation and photic entrainment of the circadian clock.
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Experimental procedures |
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For bacterial expression of mCRY1 and mCRY2 as GST-fusion proteins, open reading frames (ORFs) of mCRY1 and mCRY2 were ligated into pGEX-4T expression vector (Amersham Biosciences, Inc.). Mammalian expression vectors encoding myc epitope-tagged mCRY proteins were generated by inserting ORFs of mCRY1 and mCRY2 into EcoRI/XhoI sites and BamHI/XhoI sites of pCMV-Tag3B (Stratagene), respectively. Mutations (Ser to Ala or Asp) were introduced into mCrys by using a site-directed mutagenesis kit (Stratagene). The mammalian expression vector encoding FLAG epitope-tagged human K54R-ERK2 (kinase-dead mutant) was a kind gift of Masato Ogata (Osaka Medical School). The expression vectors for myc-epitope tagged chicken MAPK (myc-MAPK), constitutive active form of chicken MEK2 (DE-MEK) and GST-fused DE-MEK (GST-DE-MEK) were generated as described (Sanada et al. 2002). For transcription assay, mouse Bmal1 (a kind gift of Yoshiaki Fujii-Kuriyama at Tohoku University) were subcloned into pcDNA3.1 vector (Invitrogen). Mouse Clock subcloned into pcDNA3.1 is a kind gift of Charles J. Weitz at Harvard Medical School.
Preparation of recombinant proteins
GST-mCRY1, GST-mCRY2 and their mutants were expressed in E. coli strain BL21(DE3) cultured with 30 µM isopropyl-ß-D-thiogalactopyranoside at 25 °C for 5 h. GST-fusion proteins expressed were purified by a glutathione-Sepharose column (Amersham Biosciences, Inc.) as described (Sanada et al. 2002). After concentration by Centricon YM-30 concentrator (Amicon), the protein content was estimated by densitometry of SDS-PAGE gel bands in comparison with those of serial dilutions of BSA standards in the same gel. Phosphorylated form of bacterially expressed myc-MAPK was prepared as described (Sanada et al. 2002).
Protein kinase assay
Recombinant myc-MAPK or P-myc-MAPK (0.3 µg) was incubated with 1.8 µg of GST-mCRY, its mutant or GST alone in 40 µL of a kinase buffer (50 mM Tris-HCl, 20 mM MgCl2, 50 µM Na3VO4, 0.003% (w/v) Brij 35; pH 7.8) at 30 °C for 20 min in the presence of 400 µM[
-32P]ATP (200 Bq/pmol). The reaction mixture was then subjected to SDS-PAGE, and the phosphorylation levels of GST-mCRYs and their mutants were estimated by autoradiography of the gel.
Cell culture, transfection, immunoprecipitation and immunofluorescence
COS7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal calf serum in wells of 6-well plates. Cells were transiently transfected using LipofectAMINE PLUS (Invitrogen) with a total of 1.4 µg of various protein expression plasmids. Cells were then cultured in DMEM supplemented with 10% foetal calf serum for 36 h, washed with ice-cold phosphate-buffered saline, and solubilized for 1 h on ice in 300 µL of a lysis buffer (20 mM HEPES-NaOH, 2 mM MgCl2, 2 mM EGTA, 1 mM benzamidine, 4 µg/mL leupeptin, 4 µg/mL aprotinin, 50 mM NaF, 1% (w/v) Triton X-100, and 1 mM Na3VO4; pH 7.4 at 4 °C). Cell extracts were then centrifuged for 30 min at 15 000 x g. The supernatant (220 µg proteins) was precleared by incubation with 15 µL of Protein G-Sepharose (Amersham Biosciences, Inc.) for 30 min and then incubated with 4 µg of anti-FLAG antibody (clone M2, Sigma) at 4 °C for 3 h, followed by incubation with 15 µL of Protein G-Sepharose for 1 h. The beads were washed seven times with the lysis buffer containing 1% (w/v) sodium deoxycholate, mixed with SDS-PAGE sample solution, boiled, and centrifuged. The supernatant was then subjected to immunoblotting with anti-FLAG antibody (0.2 µg/mL), anti-myc antibody (clone 9E10, 0.2 µg/mL, SantaCruz Biotechnology), or anti-phospho-MAPK antibody (0.1 µg/mL, New England Biolab) as described (Sanada et al. 2000).
For immunofluorescence, COS7 cells were fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with 0.2% Triton X-100 in PBS for 10 min, and then blocked in 3% bovine serum albumin and 0.2% Triton X-100 in PBS (blocking solution) for 30 min. After that, the cells were incubated with anti-myc antibody (clone 9E10, 1 : 200, SantaCruz Biotechnology) in the blocking solution for 1 h. After three washes with PBS, the cells were incubated with Alexa 488-conjugated anti-mouse IgG (1 : 1000; Molecular probe) in the blocking solution, followed by nuclear staining with DAPI.
Phosphopeptide analysis by reversed-phase HPLC and MALDI-TOF/MS
GST-mCRY1 or GST-mCRY2 (18 µg each) was incubated at 30 °C for 2 h with P-myc-MAPK (1.8 µg) in 40 µL of the kinase buffer containing 1 mM ATP. GST-mCRY proteins were then separated by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. The blot containing GST-mCRY protein band was excised, washed five times with water, and incubated for blocking with 100 mM acetic acid containing 0.5% (w/v) polyvinylpyrrolidone-40 at 37 °C for 1 h. After washing five times with water, the blot was incubated with 2.0 µg of TPCK-treated trypsin at 37 °C for 18 h in 200 µL of 50 mM Tris buffer (pH 8.0) containing 10 mM CaCl2 and 10% (w/v) acetonitrile. The mixture was further supplemented with 2.0 µg TPCK-treated trypsin, incubated for additional 8 h, and subjected to reversed-phase HPLC (Sanada et al. 1994). All the fragments eluted from the column were analysed by MALDI-TOF/MS. Voyager MALDI-TOF mass spectrometer (PerSeptive Biosystems) was equipped with a delayed extraction ion source and operated in the linear mode. For sequence analysis of phosphopeptides, the HPLC-purified peptide was lyophilized, dissolved in 10 µL of 10% (w/v) HCl, incubated at 50 °C for 6 h and subjected to MALDI-TOF/MS analysis (Sanada et al. 2002).
Transcription assay
HEK293EBNA cells cultured in wells of 6-well plates were transiently transfected using LipofectAMINE PLUS (Invitrogen) with various combinations of plasmids expressing mouse BMAL1, CLOCK and CRY, together with 50 ng of firefly luciferase reporter plasmid and 0.5 ng of pRL-CMV (Renilla) luciferase plasmid (Promega) as an internal control. The firefly luciferase reporter plasmid was constructed as described (Okano et al. 2001). In brief, oligonucleotides, CACGTG E-box element and its flanking sequences within the promoter/enhancer region of mouse vasopressin gene were synthesized, and the three copies linked in tandem were inserted into pGL3-Promoter plasmid (Promega). Total amount of DNA applied per well was adjusted to 1.4 µg by adding pCMV-Tag3B empty plasmid. The cell extract was prepared 24 h after the transfection, and subjected to a dual-luciferase assay by luminometry (Promega) according to the manufacturer's protocol. The values were normalized to transfection efficiency with the aid of the internal control value in each cell culture.
Generation of antibody specific to phospho-Ser247 of mCRY1 and phospho-Ser265 of mCRY2
To generate polyclonal antibody (anti-pS247/265-mCRYs) which recognizes both Ser247-phosphorylated mCRY1 and Ser265-phosphorylated mCRY2, a synthetic phospho-peptide [CNSLLAS(PO4)SPTGLS] corresponding to the amino acids 241–253 of mCRY1 and 259–271 of mCRY2 was conjugated to keyhole limpet haemocyanin. After three cycles of immunization with the conjugate, the New Zealand white rabbits were bled, and the serum was subjected to an affinity-purification column of the phospho-peptide-conjugated EAH Sepharose (Amersham Biosciences, Inc.). The antibody was further purified by passing through a non-phospho-peptide[CNSLLASSPTGLS]-coupled EAH Sepharose column. The antibody thus purified was termed anti-pS247/265-mCRYs, and it was used at 1 : 200 dilution for immunoblotting.
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Acknowledgements |
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Footnotes |
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Present address: aDepartment of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA;
bDivision of Neuroscience, Children's Hospital, Harvard Medical School, 300 Longwood Avenue, Boston, MA 02115, USA
These authors contributed equally to this work.
* Correspondence: E-mail: sfukada{at}mail.ecc.u-tokyo.ac.jp |
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Received: 23 March 2004
Accepted: 17 May 2004
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