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Identification of mPer1 Phosphorylation Sites Responsible for the Nuclear Entry

2004; Elsevier BV; Volume: 279; Issue: 31 Linguagem: Inglês

10.1074/jbc.m403433200

1083-351X

Atsuko Takano, Yasushi Isojima, Katsuya Nagai,

Light effects on plants

Casein kinase 1 epsilon (CK1ϵ) is an essential component of the circadian clock in mammals and Drosophila. The phosphorylation of Period (Per) proteins by CK1ϵ is believed to be implicated in their subcellular localization and degradation, but the precise mechanism by which CK1ϵ affects Per proteins has not been determined. In this study, three putative CK1ϵ phosphorylation motif clusters in mouse Per1 (mPer1) were identified, and the phosphorylation status of serine and threonine residues in these clusters was examined. Phosphorylation of residues within a region defined by amino acids 653–663 and in particular of Ser-661 and Ser-663, was identified as responsible for the nuclear translocation of mPer1. Furthermore, phosphorylation of these residues may influence the nuclear translocation of a clock protein complex containing mPer1. These findings indicate that mPer1 phosphorylation is a critical aspect of the circadian clock mechanism. Casein kinase 1 epsilon (CK1ϵ) is an essential component of the circadian clock in mammals and Drosophila. The phosphorylation of Period (Per) proteins by CK1ϵ is believed to be implicated in their subcellular localization and degradation, but the precise mechanism by which CK1ϵ affects Per proteins has not been determined. In this study, three putative CK1ϵ phosphorylation motif clusters in mouse Per1 (mPer1) were identified, and the phosphorylation status of serine and threonine residues in these clusters was examined. Phosphorylation of residues within a region defined by amino acids 653–663 and in particular of Ser-661 and Ser-663, was identified as responsible for the nuclear translocation of mPer1. Furthermore, phosphorylation of these residues may influence the nuclear translocation of a clock protein complex containing mPer1. These findings indicate that mPer1 phosphorylation is a critical aspect of the circadian clock mechanism. The metabolism and behavior of organisms are influenced by endogenous circadian rhythms. Recently it has been revealed that a positive-negative transcriptional feedback mechanism may be responsible for the generation of the circadian clock signal in various species (1Danlap J.C. Cell. 1999; 96: 271-290Google Scholar, 2Young M.W. Kay S.A. Nat. Rev. Genet. 2001; 2: 702-715Google Scholar, 3Harmer S.L. Panda S. Kay S.A. Annu. Rev. Cell Dev. Biol. 2001; 17: 215-253Google Scholar, 4Reppert S.M. Weaver D.R. Nature. 2002; 418: 935-941Google Scholar). Experiments with cultured cells and knockout and transgenic mice have shown that the Clock, Bmal1, Per2, and Cryptochrome (Cry) 1The abbreviations used are: Cry, Cryptochrome protein; dPer, Drosophila Period protein; Dbt, Drosophila Double-time protein; aa, amino acid(s); FASPS, familial advanced sleep-phase syndrome; NLS, nuclear localization signal; GST, glutathione S-transferase; HA, hemagglutinin; CFP, cyan fluorescent protein; LMB, Leptomycin B; NES, nuclear export signal. 1The abbreviations used are: Cry, Cryptochrome protein; dPer, Drosophila Period protein; Dbt, Drosophila Double-time protein; aa, amino acid(s); FASPS, familial advanced sleep-phase syndrome; NLS, nuclear localization signal; GST, glutathione S-transferase; HA, hemagglutinin; CFP, cyan fluorescent protein; LMB, Leptomycin B; NES, nuclear export signal. 1 and 2 proteins are essential for generating circadian rhythms in mammals (5Vitaterna M.H. King D.P. Chang A.M. Kornhauser J.M. Lowrey P.L. McDonald J.D. Dove W.F. Pinto L.H. Turek F.W. Takahashi J.S. Science. 1994; 264: 719-725Google Scholar, 6Zheng B. Larkin D.W. Albrecht U. Sun Z.S. Sage M. Eichele G. Lee C.C. Bradley A. Nature. 1999; 400: 169-173Google Scholar, 7van der Horst G.T. Muijtjens M. Kobayashi K. Takano R. Kanno S. Takao M. deWit J. Verkerk A. Eker A.P. van Leenen D. Buijs R. Bootsma D. Hoeijmakers J.H. Yasui A. Nature. 1999; 398: 627-630Google Scholar, 8Vitaterna M.H. Selby C.P. Todo T. Niwa H. Thompson C. Fruechte E.M. Hitomi K. Thresher R.J. Ishikawa T. Miyazaki J. Takahashi J.S. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3859-3863Google Scholar).In this context, it has been suggested that the phosphorylation of clock-related proteins such as Per1 has an important role in regulating the circadian clock. The Drosophila Period protein (dPer) and mammalian Per proteins (Per1–3) have been reported to undergo temporal shifts in electrophoretic mobility due to phosphorylation (9Edery I. Zwiebel L.J. Dembinska M.E. Rosbash M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2260-2264Google Scholar, 10Lee C. Etchegaray J.P. Cagampang F.R.A. Loudon A.S.I. Reppert S.M. Cell. 2001; 107: 855-867Google Scholar). Drosophila Double-time (Dbt) protein, a homologue of mammalian CK1ϵ, was suggested to be a putative kinase for the dPer protein (11Kloss B. Price J.L. Saez L. Blau J. Rothenfluh A. Wesley C.S. Young M.W. Cell. 1998; 94: 97-107Google Scholar). Mutant alleles of dbt that confer shortened or lengthened circadian behavioral rhythms, or that abolish circadian rhythms altogether, have been identified (12Price J.L. Blau J. Rothenfluh A. Abodeely M. Kloss B. Young M.W. Cell. 1998; 94: 83-95Google Scholar). These alterations are correlated with changes in the phosphorylation and stability of dPer, indicating that Dbt is a necessary component of the circadian clock mechanism (12Price J.L. Blau J. Rothenfluh A. Abodeely M. Kloss B. Young M.W. Cell. 1998; 94: 83-95Google Scholar). Similar roles for mammalian Dbt have also been suggested by recent findings. The Syrian hamster Tau mutation confers free running periods of 22 h for heterozygotes and 20 h for homozygotes. The tau locus was cloned and found to encode the hamster homologue of CK1ϵ. The Tau mutation is a single nucleotide change that causes an arginine-to-cysteine substitution at amino acid (aa) residue 178, which is highly conserved in other members of the CK1 family. The mutant protein can still associate with mPer proteins, but its kinase activity is much lower than that of wild-type CK1ϵ (13Lowrey P.L. Shimomura K. Antoch M.P. Yamazaki S. Zemenides P.D. Ralph M.R. Menaker M. Takahashi J.S. Science. 2000; 288: 483-492Google Scholar). Furthermore, hPer2 was shown to be implicated in a human disorder, familial advanced sleep-phase syndrome (FASPS), an autosomal dominant circadian rhythm disorder characterized by a 4-h phase advance in the daily sleep-wake cycle (14Toh K.L. Jones C.R. He Y. Eide E.J. Hinz W.A. Virshup D.M. Ptacek L.J. Fu Y.H. Science. 2001; 291: 1040-1043Google Scholar). FASPS is caused by a serine-to-glycine substitution affecting aa 662 of hPer2, which is located within a domain thought to be bound and phosphorylated by CK1ϵ. The hPer2 mutant was shown to be less phosphorylated by CK1ϵ than the wild type hPer2 in vitro. In addition, CK1ϵ-1 phosphorylates not only Per proteins but also Cry1 and BMAL1 (15Eide E.J. Vielhaber E.L. Hinz W.A. Virshup D.M. J. Biol. Chem. 2002; 277: 17248-17254Google Scholar). Collectively, these data suggest that CK1ϵ is a component of the mechanism that regulates circadian rhythms.In experiments with COS-7 cells, we previously found evidence that CK1ϵ interacts with the mouse Per proteins mPer1, mPer2, and mPer3 and that it causes mPer1 and mPer3, but not mPer2, to enter the nucleus in a phosphorylation-dependent manner (16Takano A. Shimizu K. Kani S Buijs R.M. Okada M. Nagai K. FEBS Lett. 2000; 477: 106-112Google Scholar). In contrast, it was reported that coexpression of mPer1 and CK1ϵ in HEK293 cells causes mPer1 to be transferred to the cytoplasm from the nucleus, which was explained as being due to the masking of the mPer1 nuclear localization signal (NLS) by phosphorylation of the protein (17Vielhaber E. Eide E. Rivers A. Gao Z.H. Virshup D.M. Mol. Cell. Biol. 2000; 20: 4888-4899Google Scholar). Although these data suggested that CK1ϵ is important in generating circadian rhythms in mammals, the precise molecular effect of CK1ϵ on mPer proteins remained unclear.In this work, we identify putative CK1ϵ-dependent phosphorylation sites in mPer1 that are responsible for its entry into the nucleus. Our results suggest that one of three clusters of these sites is involved in controlling the subcellular localization of mPer1 and thus is implicated in the circadian clock mechanism.EXPERIMENTAL PROCEDURESIn Vitro Kinase Assay—Recombinant rat CK1ϵ (rCK1ϵ) was used for in vitro kinase assays. cDNA encoding the full-length protein or the catalytic domain (aa 1–297) of rCK1ϵ was subcloned into the vectors pTrcHisb (Invitrogen) or pGEX4T-3 (Qiagen) to produce hexahistidine-tagged rCK1ϵ (His-rCK1ϵ) or GST-fused rCK1ϵ (GST-rCK1ϵ), respectively. These expression vectors were transformed into the BL21(DE3) Escherichia coli expression system (Stratagene). His-rCK1ϵ was purified with nickel-nitrilotriacetic acid resin (Qiagen) and GST-rCK1ϵ with glutathione-Sepharose 4B (Amersham Biosciences), according to the manufacturers' protocol. Prior to use in kinase assays, recombinant His-rCK1ϵ was subjected to limited digestion with trypsin (15 min at 30 °C) to eliminate the COOH terminus and to reduce autophosphorylation, which hampers its activity toward exogenous substrate. Fragments of mPer1 fused to GST (GST-mPer) and a hemagglutinin-tagged full-length mPer1 (HA-mPer1) protein were used as substrates for the kinase assay. The GST-mPer fragment was produced in a manner similar to that described for GST-rCK1ϵ. The mPer1 fragment was mutated as described below. Overexpressed HA-mPer1 was purified by immunoprecipitation using an anti-HA monoclonal antibody (Invitrogen) from COS-7 cell lysates. Kinase reactions (Fig. 1) were performed for 10 min at 37 °C in a buffer containing 45 mm Tris-HCl, pH 7.4, 9 mm MgCl2, 0.9 mm β-mercaptoethanol, 40 μm ATP, and 92.5 Bq/pmol [γ-32P]ATP. For quantitative analysis (Fig. 5), recombinant GST-rCK1ϵ (8–10 pmol) was incubated with GST-mPer1 or mutant derivatives (3–5 pmol) in 20 μl of kinase buffer at 30 °C in the presence of 500 μm ATP and 7.4 Bq/pmol [γ-32P]ATP according to the method of Sanada K. et al. (18Sanada K. Okano T. Fukada Y. J. Biol. Chem. 2002; 277: 267-271Google Scholar). The reactions were stopped by the addition of SDS-PAGE sample buffer and electrophoresed in 10 or 6% polyacrylamide gels. The incorporation of radioactive phosphate into mPer1 fragments was detected with a BAS 3000 image analyzer (Fujifilm). These samples were also subjected to immunoblot analysis using the anti-HA antibody or stained with Coomassie Brilliant Blue to confirm the molecular size of the substrates.Fig. 5Effects of the nuclear export inhibitor Leptomycin B on the subcellular localizations of mPer1 and mutant derivatives in COS-7 cells.A, representative examples of the subcellular distribution patterns of HA-tagged wild type mPer1 (panels a and b), S[661,663]A (panels c and d), wild type mPer1 coexpressed with rCK1ϵ (panels e and f), and S[661,663]A coexpressed with rCK1ϵ (panels g and h) in COS-7 cells. Transfected cells were untreated (left panels) or treated with the nuclear export inhibitor LMB (right panels) for 6 h. The distribution of HA-mPer1 or mutant proteins is represented in red and that of rCK1ϵ in green. The colocalization of rCK1ϵ and HA-mPer1 and mutant proteins is indicated in yellow (merged). B, quantitative results of the subcellular localization of HA-mPer1 or S[661,663]A mutants. At least three independent trials, in which 30–100 cells were counted, were performed for each experiment.View Large Image Figure ViewerDownload (PPT)Cell Culture and Transfection—cDNAs encoding rCK1ϵ, mPer1, and mutant proteins were transfected into COS-7 cells as described previously (16Takano A. Shimizu K. Kani S Buijs R.M. Okada M. Nagai K. FEBS Lett. 2000; 477: 106-112Google Scholar). Briefly, site-directed mutagenesis was performed in a single step by PCR to change specific mPer1 serine and threonine residues to alanine or glycine. Mutated DNA was identified by DpnI selection for hemimethylated DNA, and the sequences were verified by the dideoxynucleotide chain reaction method using the BigDye sequencing kit and an ABI Prism model 310 genetic analyzer (PerkinElmer Life Sciences-Applied Biosystems). For expression in mammalian cells, cDNA encoding rCk1ϵ or a kinase-inactive mutant (rCk1ϵK38R) was subcloned into the mammalian expression vector pcDNA3 (Invitrogen). cDNAs encoding wild type and mutant versions of mPer1 were subcloned into the HA tag vector pHM6 (Invitrogen). In cotransfection experiments with three proteins (Fig. 7), mCry1 cDNA was subcloned into the vector pHM6 and mPer1 cDNA was subcloned into the vector pcDNA3. For identification of rCK1ϵ by CFP-specific fluorescence, rCk1ϵ cDNA was subcloned into the vector pCFP (kindly provided by Dr. A. Miyawaki, Brain Research Institute, RIKEN (The Institute of Physical and Chemical Research)). COS-7 cell cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Cells were transfected with expression vectors carrying mPer1, rCk1ϵ, and mCry1 sequences with LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocol.Fig. 7Subcellular localization of the clock-related protein complex depends on Ser-661 and Ser-663 of mPer1.A, representative examples of the subcellular distribution patterns of wild type mPer1 (panel b), S[661,663]A (panels e and h) in COS-7 cells coexpressed with CFP-rCK1ϵ (panels a, d, and g) and HA-mCry1 (panels c, f, and i). The distribution of mPer1 or mutant derivatives is represented in green (middle panels), HA-tagged protein in red (lower panels), and CFP-fused protein in blue (upper panels). Transfected cells were untreated (left and middle panels) or treated with the nuclear export inhibitor LMB (right panels) for 6 h. B, quantitative analysis of the subcellular localization of mPer1 or mutant derivatives. At least three independent trials, in which 30–100 cells were counted, were performed for each experiment.View Large Image Figure ViewerDownload (PPT)Immunoblot Analysis—Transfected COS-7 cells were lysed in TNE buffer containing 20 mm Tris-HCl (pH 7.4), 1 mm EDTA, 0.15 m NaCl, 1% Nonidet P-40, 5% glycerol, 5 mm β-mercaptoethanol, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride. For immunoprecipitation, the lysates were incubated for 2 h at 4 °C with the anti-HA antibody or an anti-rCK1ϵ antibody (16Takano A. Shimizu K. Kani S Buijs R.M. Okada M. Nagai K. FEBS Lett. 2000; 477: 106-112Google Scholar) bound to protein A-Sepharose beads (Amersham Biosciences). The precipitates were then washed three times with TNE buffer and resuspended in SDS sample buffer. The samples were separated by SDS-PAGE on 7.5% polyacrylamide gels and transferred electrophoretically to nitrocellulose membranes. After incubation with the anti-rCK1ϵ antibody (1: 5000) or anti-HA antibody (1:2000), the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5000) or mouse IgG (1:2000) antiserum (Zymed Laboratories Inc.) as a secondary antibody. Immunoreactive bands were visualized with a chemiluminescence detection system (Western Lightning kit, PerkinElmer Life Sciences) and exposed to x-ray films (Fujifilm).Antibody Production—A rabbit polyclonal anti-mPer1 antibody was raised against the peptide Cys-(ALPAEENSTS) corresponding to aa 1282–1291 of mPer1 (kindly synthesized by K. Kawakami and Dr. S. Aimoto, Institute for Protein Research, Osaka University) and purified by affinity chromatography using the antigen peptide coupled with SulfoLink (Pierce) according to the manufacturer's protocol.Cell Staining—For expression studies, the TransFast transfection reagent (Promega) was used according to the manufacturer's protocol. The transfected cells were fixed and performed staining as described previously (16Takano A. Shimizu K. Kani S Buijs R.M. Okada M. Nagai K. FEBS Lett. 2000; 477: 106-112Google Scholar).Leptomycin B Treatment—Twenty-four hours after transfection, COS-7 cells were treated with 10 ng/ml Leptomycin B (LMB) (Sigma) for 2 h. In all experiments, 50 μg/ml cycloheximide (Sigma) was added 6 h before LMB treatment to block de novo protein synthesis.RESULTSIdentification of CK1ϵ-dependent Phosphorylation Sites in mPer1—Previous reports by others and from our laboratory indicated that CK1ϵ phosphorylates mPer proteins and affects their subcellular localization and turnover (16Takano A. Shimizu K. Kani S Buijs R.M. Okada M. Nagai K. FEBS Lett. 2000; 477: 106-112Google Scholar, 17Vielhaber E. Eide E. Rivers A. Gao Z.H. Virshup D.M. Mol. Cell. Biol. 2000; 20: 4888-4899Google Scholar, 19Vielhaber E. Duricka D. Ullman K.S. Virshup D.M. J. Biol. Chem. 2001; 276: 45921-45927Google Scholar, 20Akashi M. Tsuchiya Y. Yoshino T. Nishida E. Mol. Cell. Biol. 2002; 22: 1693-1703Google Scholar). To clarify the function of CK1ϵ in the circadian clock mechanism, we identified mPer sites phosphorylated by CK1ϵ by performing in vitro kinase assays using a series of GST-mPer1 fragments as substrates for rCK1ϵ (Fig. 1A). We first found that rCK1ϵ phosphorylated a GST-mPer1 fragment containing aa 547–799 (Fig. 1A, lane 6). To further define the phosphorylated region of mPer1, four smaller GST-tagged fragments covering aa 549–799 were constructed (Fig. 1A, lanes 8–11). Three of these fragments (aa 627–667, aa 667–742, and aa 742–799) were phosphorylated by rCK1ϵ (Fig. 1A, lanes 9–11), but the fourth (aa 547–628) was not (Fig. 1A, lane 8). This result suggests that mPer1 contains at least three CK1ϵ phosphorylation sites, within the region from aa 628–799.The sites of CK1ϵ-mediated phosphorylation in mPer2 and mPer3 were also examined. rCK1ϵ was found to phosphorylate only a GST-tagged mPer2 fragment containing aa 486–793 and a GST-tagged mPer3 fragment containing aa 367–880 (data not shown).Next, we searched for the CK1ϵ-phosphorylation consensus motif (Ser/Thr-X-X-Ser/Thr or Asp-Asp-Asp-Asp-X-X-Ser/Thr) (21Flotow H. Graves P.R. Wang A.Q. Fiol C.J. Roeske R.W. Roach P.J. J. Biol. Chem. 1990; 265: 14264-14269Google Scholar) within the CK1ϵ-targeted regions of the mPer proteins. For mPer1, two consensus motif clusters were found, from aa 653 to 663 (ST653–663) and aa 714 to 726 (S714–726), as well as an isolated single motif from aa 784 to 787 (ST784–787) (Fig. 1B). The S714–726 region, which is conserved among all mPer proteins, has been suggested to be relevant for their ubiquitination (20Akashi M. Tsuchiya Y. Yoshino T. Nishida E. Mol. Cell. Biol. 2002; 22: 1693-1703Google Scholar). Moreover, a mutation in the corresponding region of hPer2 was reported to cause familial advanced sleep-phase syndrome (FASPS) (14Toh K.L. Jones C.R. He Y. Eide E.J. Hinz W.A. Virshup D.M. Ptacek L.J. Fu Y.H. Science. 2001; 291: 1040-1043Google Scholar). ST784–787 is conserved in mPer3 but not mPer2, and ST653–663 is not found in mPer2 or mPer3. To clarify whether these putative motifs are indeed phosphorylated and, if so, whether they are responsible for the phosphorylation-dependent nuclear entry of mPer proteins, we constructed several HA-mPer1 mutants. In three of these, the serine and threonine residues in a single consensus motif cluster were replaced by alanine (S/T[653–663]A, S[714–726]A, and S[784]A). In a fourth derivative all three consensus motif clusters were mutated (S/T[653–784]A). Another replacement mutant with alterations of the motif cluster from aa 902 to 916 (ST902–916), which has been reported to be a region phosphorylated by CK1ϵ (17Vielhaber E. Eide E. Rivers A. Gao Z.H. Virshup D.M. Mol. Cell. Biol. 2000; 20: 4888-4899Google Scholar), was also constructed (S/T[902–916]A) (Fig. 1B).Identification of Phosphorylation Sites Necessary for the Nuclear Entry of mPer1—To determine how many CK1ϵ-dependent phosphorylation sites are present in mPer1, the phosphorylation status of each mPer1 mutant in the presence of rCK1ϵ was examined. COS-7 cells were transfected with vectors expressing HA-tagged full-length or mutant mPer1 derivatives, along with wild type rCK1ϵ, a kinase-inactive rCK1ϵ mutant (rCK1ϵ-KN) in which the lysine residue at aa 38 was replaced by arginine or an empty control vector. The levels of phosphorylation of the mPer1 proteins were initially assessed by a shift in electrophoretic mobility on SDS-PAGE gels, as detected by immunoblot analysis. Wild type mPer1 and the mPer1 mutant S[784]A underwent an obvious shift in mobility in the presence of intact rCK1ϵ (Fig. 2A, lanes 1–3 and 10–12). However, the S/T[653–663]A mutant showed a lower mobility shift than did the wild type protein (Fig. 2A, lanes 4–6), and the S[714–726]A mutant exhibited a striking electrophoretic mobility even in the absence of rCK1ϵ protein (Fig. 2A, lane 7–9). The S/T[653–784]S mutant, in which all serine and threonine residues of the three consensus motif clusters were replaced, was still phosphorylated (Fig. 2A, lanes 13–15). Each mPer1 mutant could be coimmunoprecipitated with rCK1ϵ. These results suggest that there are multiple CK1ϵ-dependent phosphorylation sites in mPer1 and that each phosphorylation site differentially influences the phosphorylation status of mPer1.Fig. 2Effects of mutation of putative rCK1ϵ phosphorylation motif clusters in mPer1 on its subcellular localization in COS-7 cells coexpressing rCK1ϵ.A, immunoblot analyses of the interaction and phosphorylation of mPer1 and mutant derivatives by rCK1ϵ. HA-tagged mPer1 and mutant derivatives were coexpressed with the empty vector pcDNA3, rCK1ϵ, or rCK1ϵ-KN (K38R) in COS-7 cells, and the phosphorylation status of HA-mPer1 proteins was assessed by SDS-PAGE as a shift in electrophoretic mobility. All mPer1 mutants were coimmunoprecipitated with rCK1ϵ and exhibited mobility shifts. B, representative examples of the subcellular distribution patterns of HA-tagged wild type mPer1 (panels a and b), and the S/T[653–663]A (panels c and d), S[714–726]A (panels e and f), S[784]A (panels g and h), and S/T[914–926]A proteins (panels i and j) in COS-7 cells transfected with the empty vector pcDNA3 (upper panels) or rCK1ϵ (lower panels). The distribution of HA-mPer1 and mutant proteins is represented in red, and that of rCK1ϵ in green. Colocalization of rCK1ϵ and HA-mPer1 and mutant proteins is indicated in yellow (merged image). C, quantitative results of the subcellular localization of HA-mPer1 and mutant derivatives. At least three independent trials, in which 30–100 cells were counted, were performed for each experiment.View Large Image Figure ViewerDownload (PPT)Our previous study showed that the coexpression of rCK1ϵ causes mPer1 and mPer3, but not mPer2, to enter the nucleus in COS-7 cells (16Takano A. Shimizu K. Kani S Buijs R.M. Okada M. Nagai K. FEBS Lett. 2000; 477: 106-112Google Scholar). To identify the Ser/Thr residues responsible for mediating the phosphorylation-dependent nuclear entry of mPer1, HA-tagged wild type or mutant mPer1 proteins were coexpressed with rCK1ϵ in COS-7 cells, and their subcellular localizations were determined by immunocytochemistry (Fig. 2, B and C). When HA-mPer1 or mutant proteins were overexpressed alone, they localized mainly in the cytoplasm and were observed in the nucleus in less than one-third of the cells (Fig. 2B, panels a, c, e, g, and i). When wild type HA-mPer1 was coexpressed with rCK1ϵ, most of the protein was found in the nucleus (Fig. 2B, panel b). This difference in the subcellular localization of HA-mPer1 is likely due to its phosphorylation by rCK1ϵ, as indicated by its cytoplasmic localization when it was coexpressed with the kinase-inactive version of rCK1ϵ (Fig. 2C). Interestingly, coexpression of HA-mPer1 S/T[653–663]A or S[714–726]A with rCK1ϵ did not result in the nuclear localization of these proteins (Fig. 2B, panels d and f). HA-mPer1 S/T[653–663]A coexpressed with the empty vector or with kinase-inactive rCK1ϵ-KN did not appear in the nucleus, but approximately one-third of the HA-mPer1 S[714–726]A protein was observed in the nucleus when it was coexpressed with the vector or rCK1ϵ-KN. These data indicate that the rCK1ϵ-dependent phosphorylation motif clusters ST653–663 and S714–726 of mPer1 are important for its nuclear entry and that the ST653–663 region is of special importance, because the HA-mPer1 S/T[653–663]A mutant did not localize to the nucleus even when it was coexpressed with empty vector or the kinase-inactive rCK1ϵ-KN. In contrast, HA-mPer1 S[784]A and S/T[902–916]A entered the nucleus in a manner dependent on phosphorylation by rCK1ϵ, but the proportion of these mutant proteins in the nucleus was significantly lower compared with wild type mPer1 (Fig. 2B). For HEK 293 cells, which express considerable amounts of endogenous CK1ϵ, it was previously reported that overexpressed mPer1 was found in the nucleus but that in the presence of exogenous CK1ϵ it was exported to the cytoplasm (17Vielhaber E. Eide E. Rivers A. Gao Z.H. Virshup D.M. Mol. Cell. Biol. 2000; 20: 4888-4899Google Scholar). Therefore, we examined the subcellular localization of mPer1 and mutant derivatives in HEK 293 cells by methods similar to those used for COS-7 cells. Surprisingly, the HA-mPer1 S/T[653–663]A protein was observed only in the cytoplasm irrespective of the presence of rCK1ϵ (data not shown). Another mutant, HA-mPer1 S[714–726]A, was distributed in a pattern similar to that of wild type mPer1. These observations strongly suggest that the mPer1 ST653–663 motif cluster is essential for the nuclear entry of mPer1 mediated by CK1ϵ-dependent phosphorylation, because the mPer1 S/T[653–663]A protein is found only in the cytoplasm, irrespective of host cell type or the presence of rCK1ϵ.A Chimeric mPer2 Protein Is Transferred into the Nucleus by CK1ϵ—In our previous study (16Takano A. Shimizu K. Kani S Buijs R.M. Okada M. Nagai K. FEBS Lett. 2000; 477: 106-112Google Scholar), we showed that coexpression with CK1ϵ caused mPer1 and mPer3, but not mPer2, to enter the nucleus in COS-7 cells. Moreover, the mPer1 ST653–663 cluster motif is not conserved in mPer2 (Fig. 1C), and the absence of this region may therefore explain this difference. Thus, to confirm the nuclear import function of the ST653–663 motif cluster, we constructed a chimeric mPer2 mutant in which aa 611–620 were replaced by aa 652–667 of mPer1 (mPer2 [mPer1 652–667]). Furthermore, we constructed another HA-mPer2 mutant by replacing the alanine residue at position 723 with serine (mPer2 A[723]S), because this site corresponds to Ser-784 of mPer1, which is conserved in mPer3 but not in mPer2 (Fig. 1C). As expected, the HA-mPer2 [mPer1 652–667] protein entered the nucleus when coexpressed with rCK1ϵ in COS-7 cells, but the HA-mPer2 A[723]S protein did not (Fig. 3A, panels c–f). These data indicate that the ST653–663 motif cluster of mPer1 is essential for its phosphorylation-dependent entry into the nucleus. Both of these mPer2 mutants exhibited a shift in electrophoretic mobility similar to that of wild type mPer2 when coexpressed with rCK1ϵ (data not shown). Either the potential change in phosphorylation was beyond the sensitivity of the assay, or residues 653–663 may have an NLS function. These two possibilities should be addressed.Fig. 3The chimeric mPer2 protein with the S/T (653–663) region of mPer1 is transferred into the nucleus by CK1ϵ in a phosphorylation-dependent manner.A, representative examples of the subcellular distribution patterns of HA-tagged wild type mPer2 (panels a and b), mPer2[mPer1 652–667] (panels c and d), and mPer2 A[723]S (panels e and f) in COS-7 cells transfected with the empty vector (upper panels) or rCK1ϵ (lower panels). The distribution of HA-mPer1 or mutant proteins is represented in red and that of rCK1ϵ in green. Colocalization of rCK1ϵ and HA-mPer1 and mutant proteins is indicated in yellow (merged). B, quantitative results of the subcellular localization of HA-mPer1 and mutant derivatives. At least three independent trials, in which 30–100 cells were counted, were performed for each experiment.View Large Image Figure ViewerDownload (PPT)Ser-661 and Ser-663 May Be Essential for the CK1ϵ-dependent Nuclear Translocation of mPer1—To identify essential phosphorylation sites in the ST653–663 motif cluster that mediate the nuclear entry of mPer1, mutant proteins in which each serine or threonine residue of the cluster was replaced by alanine were constructed and expressed in COS-7 cells (Fig. 4, A and B). Only two mPer1 mutants, S[661]A and S[663]A, showed marked and significant decreases in the frequency of nuclear localization when coexpressed with rCK1ϵ (37.8% for S[661]A and 14.0% for S[663]A (Fig. 4A, panels i and j)), and the other mPer1 mutants were observed mainly in the nucleus. A mPer1 mutant in which both Ser-661 and Ser-663 were replaced by alanine (S[661,663]A) localized only in the cytoplasm, like mPer1 S/T[653–663]A (Fig. 4A, panels b and k).Fig. 4The mPer1 serine 661 and 663 residues are responsible for CK1ϵ-dependant nuclear translocation in COS-7 cells.A, representative examples of the subcellular distribution patterns of HA-tagged wild type mPer1 (a), S/T[653–663]A (b), S[653]A (c), S[655]A (d), S[656]A (e), T[658]A (f), S[660]A (g), S[661]A (h), S[663]A (i), and S[661,663]A (j) in COS-7 cells transfected with rCK1ϵ. The distribution of the merged image is represented in yellow. B, quantitative analysis of the subcellular localization of HA-mPer1 and mutant derivatives. At least three independent trials, in which 30–100 cells were counted, were performed for each experiment. C, representative examples of the subcellular distribution patterns of HA-tagged wild type mPer1 (a), and the S[661–663]A (b), an