A novel protein interacts with a clock-related protein, rPer1

Brain Research 916 (2001) 1–10 www.elsevier.com / locate / bres

Research report

A novel protein interacts with a clock-related protein, rPer1 Tohru Matsuki 1 , Atsuko Kiyama 1 , Masahiro Kawabuchi, Masato Okada, Katsuya Nagai* Division of Protein Metabolism, Institute for Protein Research, Osaka University, 3 -2 Yamada-Oka, Suita, Osaka 565 -0871, Japan Accepted 10 July 2001

Abstract Mammalian Per proteins are thought to be important in the mechanism of circadian rhythm. We identified a novel protein PIPS (Per1 interacting protein of the suprachiasmatic nucleus) with the yeast two-hybrid system using PAS domain of rat Per1 (rPer1) as a bait. PIPS is about a 180-kDa protein and expressed mainly in the brain, especially in the hypothalamus including the suprachiasmatic nuclei (SCN). PIPS interacts with mouse Per1 (mPer1) in vitro and in cultured cells transfected with both molecules. Furthermore, it was found that mPer1 translocated PIPS into the nuclei in the cultured cells. Thus, these findings suggest a possibility that PIPS is involved in the feedback loop or output mechanism of circadian rhythm through interacting with Per1 in the SCN.  2001 Elsevier Science B.V. All rights reserved. Theme: Neural basis of behavior Topic: Biological rhythms and sleep Keywords: Circadian rhythm; Yeast two-hybrid system; Suprachiasmatic nucleus; mPer1; Protein–protein interaction

1. Introduction Almost all living organisms have a self-oscillatory clock mechanism with a period of about 24 h, and show circadian rhythms in their physiological phenomena driven by the mechanism. In Drosophila, it has been shown that the interaction of Period (Per) and Timeless (Tim) proteins make them translocated into the nucleus [11] and that they suppress the transcription of their genes by inhibiting the transcription activity of dClock–Cycle heterodimer [4]. This negative and positive transcriptional feedback loop is thought to be essential for sustaining circadian oscillation. In mammals, it has been established that a master circadian oscillator exists in the hypothalamic suprachiasmatic nucleus (SCN) [8]. The molecular mechanism of mammalian circadian rhythm is seemed to be similar to Drosophila, but it has not been fully clarified yet [10]. Three Per orthologues were identified in mice. mPer1 is proposed to be necessary for phase resetting mechanism [1] and mPer2 were thought to be essential for sustaining circadian

oscillation [23]. Although the role of mPer3 has remained unclear, three Per proteins seem to be very important for circadian clock functions. mCry1, mCry2 and casein kinase 1e (CK1e) are known to interact with mPer proteins [5,16,21]. mCry proteins are strong inhibitor for Clock– BMAL1 heterodimer that activate the transcription of mper1 and some other clock regulated genes [9]. mCry proteins are reported to be necessary for sustaining circadian oscillation [20]. But the role of the interactions between mCry and mPer are not clear. CK1e phosphorylate mPer proteins and relate with degradation or translocation of Per proteins [16,21]. We thought that there would exist another interacting proteins with Per and it would be important to find new molecules to understand the role of mPer proteins. In this study, we identified and characterized a novel molecule interact with Per1 screened from rat brain cDNA library with a yeast two-hybrid screening system.

2. Materials and methods *Corresponding author. Tel.: 181-6-6879-8632; fax: 181-6-68798633. E-mail address: k [email protected] (K. Nagai). ] 1 These authors equally contributed.

2.1. Materials The cDNA library of rat hippothalamus was purchased

0006-8993 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 01 )02857-8

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T. Matsuki et al. / Brain Research 916 (2001) 1 – 10

from Takara Biomedicals (Tokyo, Japan). pBluescript vectors were from Stratagene (La Jolla, CA). Northern blot membrane of mouse tissues were from Clontech (Tokyo, Japan). pGEX vector, proteinG sepharose, glutathione sepharose, HiTrap NHS-activated column, hybridization buffer for Northern blot, Cy3-labeled goat anti-mouse IgG, [a- 32 P]dCTP and [ 35 S]methionine were from Amersham Pharmacia Biotech (Sweden). pMAL vector and amylose resin were from New England Biolabs (Beverly, MA). TNT coupled reticulocyte lysate transcription / translation systems were from Promega (Tokyo, Japan). BigDye DNA sequencing kit was from Applied Biosystems (Tokyo, Japan). Anti-neuron specific enolase (NSE) antibody and horseradish peroxidase (HRP) conjugated goat anti rabbit IgG antibody were from Zymed (San Francisco, CA). PVDF membrane for immunoblot (Immobilon) was from Millipore (Bedford, MA). Luminol-based chemiluminescence detection kit (Renaissance) was from NEN Life Technologies Inc. (Boston, MA). X-ray film was from Kodak (Tokyo, Japan). Biotinylated anti-rabbit IgG antibody and Vectastain Elite ABC kit were form Vector Laboratories (Burlingame, CA). Propium iodide, Alexa Fluor 488 anti-rabbit IgG antibody and SlowFade were from Molecular Probe (Eugene, OR). LipofectAmine 2000 were from Gibco BRL (Gothenburg, MD). FuGene6 were from Rosh Diagnostic (Indianapolis, IA). Anti-hemagglutinin (HA) monoclonal antibody was from Boehringer Mannheim (Germany). pCMV-Tag1 expression vector were from Invitrogen (San Diego, CA).

The cDNA library of rat hypothalamus was screened with [a- 32 P]dCTP-labelled cDNA fragment of PIPS obtained from the yeast two-hybrid method. DNA sequencing was carried out as described above.

2.2. Isolation of the partial DNA fragment of rat Per1 (rPer1)

2.5. Northern blot

The helix-loop-helix (HLH)-PAS domain of rPer1 was cloned from rat cDNA library of rat hypothalamus by PCR using mper1 gene specific primers (sense: 59TCTCAGCGGAATTCTCATAGTTCCT-39, antisense: 59GCTCCTGGATATCAGAGTCCAGGGA-39). The amplified DNA was subcloned into pBluescript vectors and sequenced by dideoxynucleotide chain-reaction method using BigDye sequencing kit and ABI Prism model 310 genetic analyzer (Perkin-Elmer–Applied Biosystems, Tokyo, Japan). The amino acid sequence of HLH-PAS domain of rPer1 is almost identical with that of mPer1 (amino acid residues 66–488), the only exception is Ala475 replaced with valine.

2.3. Screening with yeast two-hybrid system The HLH-PAS domain of rPer1 cDNA was cloned in-frame in the yeast expression vector pGBT9, which contains the Gal4 DNA binding domain. Then it was introduced into the yeast strain CG-1945 by lithium acetate transformation. As pGBT9 contains the tryptophan gene, transformed yeast were selected on tryptophan-deficient medium. Rat brain library fused to the Gal4 transactivation

domain was introduced into Gal4-rPer1 expressing CG1945. Since the library vector pGAD10 carries the leucine gene, it conferred the capacity to grow in leucine-deficient medium. In addition, binding of library-encoded products to the rPer1 resulted in expression of his and lacZ genes. These two genes allowed growth on histidine-deficient medium and green staining in the presence of 5-bromo-4chloro-3-indolyl-b-D-galactopyranoside (X-gal), respectively. Since his gene was weakly expressed in CG-1945, 1 mM 3-amino-1,2,4-triazole (3-AT) was added to the selection medium. A total of 8.5310 5 library transformants were screened in this manner. A total of 74 of these colonies were positive for his; while 25 of 74 colonies were positive for lacZ. Library plasmids were recovered from these 25 colonies by DNA extraction and transferred to E. coli in order to obtain larger amounts of DNA. We concluded 19 of 25 colonies were false positive, because they have known sequences out of frame or unknown sequences with many stop codons. Six colonies coded three identical unknown clones. We analyzed one of them in this study.

2.4. Isolation and nucleotide sequence analysis of the cDNA clones

A Northern blot membrane was hybridized with specific probes mentioned above in hybridization buffer and washed following manufacture’s manuals. Radioactivities were detected by BAS 3000 image analyzer (Fuji Film, Tokyo, Japan).

2.6. Expression of fusion proteins and antibody production The C-terminal fragment of PIPS (encoding amino acid residue 1235–1319) were proliferated by PCR using specific primers and subcloned into pGEX vector or pMAL vector. The constructs were transformed into E. coli strain JM109. Expressions of GST-PIPS (1235–1319) fusion protein or MBP-PIPS (1235–1319) were induced by a final concentration of 0.5 mM isopropyl-thiogalactoside for 1 h at 308C. GST-PIPS (1235–1319) was purified with glutathione-sepharose from the bacterial lysate and MBP-PIPS (1235–1319) was purified with amylose-resin. Anti-PIPS antiserum was raised against GST-PIPS (1235–1319) with rabbits. The antiserum against PIPS was purified with the HiTrap NHS-activated column, to which MBP-PIPS (1235–1319) were coupled.

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2.7. Pull down assay of PIPS and mPer1 The in vitro translation products of full-length PIPS and mPer1 were prepared using TNT Coupled reticulocyte lysate systems and [ 35 S]methionine according to the manufacturer’s protocol. GST fused PIPS fragments and GST fused mPer1 fragments were prepared with the similar manner as written above. The in vitro translation products of PIPS or mPer1 were incubated with glutathione sepharose beads bound to each GST-PIPS fragment or GST-mPer1 fragment for 1 h at 48C in the TNE buffer (20 mM Tris–HCl, pH 7.4, 1 mM EDTA, 1% NP-40, 5% glycerol, 0.15 M NaCl and 5 mM 2-mercaptoethanol). The beads were washed three times with the TNE buffer, and bound proteins were solubilized in the SDS sample buffer and separated by the SDS–PAGE. After the electrophoresis, the gel was dried up on the gel dryer (Bio-Rad, Hercules, CA) and the radioactivities were detected with BAS 3000. To qualify the radioactivities, the images were transferred to a Macintosh computer and analyzed with NIH-Image software.

2.8. Immunoblot of PIPS Male Wistar rats (Nippon Dobutsu, Japan) aged 3–4 weeks old were maintained at 24618C under a 12-h light / dark (LD) cycle for at least 2 weeks before the experiments. The experiments under constant dim red light (less than 0.2 lux) condition (DD) were performed 2 days after transition. Animals were sacrificed by decapitation at various times under LD or DD conditions. The brains were quickly removed and frozen in dry ice. Coronal sections with a thickness of 700 mm including the SCN were made using a cryomicrotome (Moriyasu Koki, Japan), and bilateral SCN and other regions were punched out from the brain sections using a 20-gauge needle as described previously [14]. The punched out samples were homogenized in RIPA buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 10 mg / ml aprotinin, 10 mg / ml leupeptin, 1 mM PMSF, 5 mM 2-mercaptoethanol), and the supernatants obtained by 15 0003g centrifugation for 10 min were separated with 8% polyacrylamide gel electrophoresis and transferred to PVDF membrane. Membranes were incubated with purified anti-PIPS antiserum or anti-NSE antibody and then with HRP-goat anti-rabbit IgG antibody. Immunoreactivities (IR) of PIPS or NSE were visualized with a Renaissance chemiluminescence detection kit and exposed to X-ray films.

2.9. Immunohistochemical staining Under pentobarbital anesthesia, rats were intracardially perfused with 200 ml of saline with 1 mg / ml NaNO 2 , then with 200 ml of 4% paraformaldehyde in 0.05 M phosphate-buffered saline (PBS) (pH 7.4), and finally with 200

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ml of 1% glutaraldehyde in citrate buffer (pH 2.5). After perfusion, brains were removed and dehydrated in 30% sucrose in PBS for cryoprotection. The brains were cut into 25-mm thick coronal sections using a cryomicrotome (Leica, Germany). The sections were incubated with purified anti-PIPS antiserum and visualized with ABC elite kit in conjunction with diaminobenzidine chromogen. For fluorescent staining, the 15-mm thick sections were incubated with 10 mg / ml RNase for 20 min at 378C and then incubated with purified anti-PIPS antiserum. PIPS-IR were visualized with Alexa 488 anti-rabbit IgG antibody and the nuclei were counterstained with 0.3 mg / ml propium iodide. The sections were mounted on slide glasses, enclosed under coverslips with SlowFade and viewed on a confocal laser scanning microscope (LSM510, Zeiss, Germany).

2.10. Transient expression and coimmunoprecipitation assays in COS-7 cells PIPS cDNA was subcloned into the pCMV-Tag1 expression vector. mPer1 cDNA was subcloned into the pHM6 plasmid to connect the HA-tag to N-terminal of mPer1 (HA-mPer1). COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum. Cells were plated at 90–95% confluence and were transiently transfected with expression vectors carrying PIPS and HA-mPer1 using LipofectAmine 2000 according to the manufacturer’s protocol. After a 24-h incubation, the cells were solubilized with RIPA buffer. The supernatants of cell lysates obtained by a centrifugation (16 0003g) were immunoprecipitated with the purified anti-PIPS antiserum or anti-HA monoclonal antibody coupled with proteinG sepharose. After washing the resin with homogenizing buffer at least four times, bound proteins were analyzed by the immunoblot method as described above using the purified anti-PIPS antiserum and anti-HA monoclonal antibody.

2.11. Cell staining COS-7 cells were plated onto coverslips in 35-mm culture dishes at a density of 10 4 cells / cm 2 and cultured overnight. PIPS and HA-mPer1 were transfected with FuGENE 6 transfection reagent was used according to the manufacturer’s protocol. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed twice with phosphate-buffered saline (PBS) (pH 7.4), and permeabilized by incubation with 0.2% Triton X-100 in PBS for 10 min at room temperature. The fixed cells were incubated with purified anti-PIPS antiserum and anti-HA monoclonal antibody for 2 h at room temperature. PIPS and HA-mPer1 were visualized with Alexa Fluor 488labeled goat anti-rabbit IgG and Cy3-labeled goat antimouse IgG, respectively. Observations were done as described above.

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3. Results

3.1. Identification and full-length cDNA cloning of rPer1 PAS-domain interacting protein As described in Section 2, we identified three unknown clones that putatively interact with the PAS domain of rPer1 with the yeast two-hybrid method. The two unknown molecules that we did not analyze in this paper are currently being studied. We cloned a full-length cDNA from adult rat brain cDNA library using the identified sequence by yeast twohybrid method as a probe. The length of cDNA was 6429 bp and the open reading frame was suggested to be 4041 bp (1346 amino acids) (Fig. 1A). A homology search for the DNA sequence showed little homology with any DNA sequence except KIAA0443 (Genebank accession no.: NM]014710) [7], whose function is unknown. Since this gene was markedly expressed in the SCN as described later, we termed this protein product PIPS (Per1 interacting protein of the SCN). Fig. 1A shows deduced amino acid sequences of PIPS. The start methionine was decided by comparing with amino acid sequences of KIAA0443 and molecular size of the in vitro translation products of pips cDNA (Fig. 1C). The homology of primary structures between PIPS and KIAA0443 was 74% calculated using CLUSTALW (data not shown). Then it was suggested that KIAA0443 was human homolog of PIPS. There was no functional domain detected in the primary structure of PIPS except putative nuclear localization signal (NLS) at amino acid residues 211–215 by information obtained from the PROSITE and PSORT database. We termed a domain of PIPS protein composed of amino acid residues 412–893 as interacting domain (ID), because this sequence was identified as an interacting protein by the two-hybrid system (Fig. 1A, broken underlined). A Northern blot analysis of rat tissues showed that the size of pips transcript was about 6.5 kb. Expressions of pips gene were observed markedly in the brain, faintly in the heart, but not in the spleen, lung, liver, skeletal muscle, kidney and testis (Fig. 1B). In order to analyze PIPS in protein level, we raised an antiserum against C-terminal fragment of PIPS (Fig. 1A, underlined). The molecular size of PIPS-IR in the brain lysate was same with that of the in vitro translation product of pips cDNA (Fig. 1C, middle). Moreover, PIPS-IR was absorbed by preincubation of the antiserum with the antigen (Fig. 1C, right). These findings suggest that the purified anti-PIPS antiserum specifically recognizes PIPS.

3.2. Identifications of interacting domains of PIPS and mPer1 In order to identify the interacting domains of PIPS and Per1, we made [L- 35 S]methionine labeled full-length

mPer1 protein by in vitro translation method and GSTPIPS fragment proteins, and examined their interaction with pull-down assay (Fig. 2A,B). GST-domain1 and domain4 efficiently pulled down radiolabeled mPer1. GSTID and domain2 bound about half amount of mPer1 compared with domain1 or 4. The other fragments pulled down less than one-third amount of mPer1. The radioactivity pulled down by GST alone was less than 2% compared with that by GST-domain4. Next, we performed pull-down assays using full-length [ 35 S]methionine-labeled PIPS proteins produced by the in vitro translation method and GST-fused partial mPer1 proteins containing N-terminal (amino acid residue 1– 127), HLH-PAS (66–485), Bam-Bgl (352–548), and BglBgl (548–799) region (Fig. 2C). GST fused C-terminal domain of Per1 (800–1291) degraded too much to use for pull-down assay. As seen in Fig. 2C, full-length PIPS was pulled down by any fragments of mPer1 except N-terminal region (N end in Fig. 2C). However, the HLH-PAS domain of mPer1 had relatively high affinity to PIPS compared to other fragments (relative amounts of bound PIPS to GSTHLH-PAS were more than five times compared with other fragments). The radioactivity pulled down by GST alone was less than 2% compared with that by GST-HLH-PAS. These data indicate that mPer1 and PIPS can interact with each other in vitro, and that the N-terminal area (domain1 and 2) and domain4 in ID of PIPS and HLHPAS domain of mPer1 are important for interaction.

3.3. Interaction of PIPS and Per1 in vivo It was examined whether PIPS interact with mPer1 in vivo when these two proteins were co-expressed in COS-7 cells. COS-7 cells do not express PIPS with immunoblot or immunocytochemistry (data not shown). In this experiment, full-length PIPS and HA-mPer1 were co-transfected in COS-7 cells. PIPS-IR was co-immunoprecipitated from the cell lysate with anti-HA antibody (Fig. 3A) On the other hand, HA-mPer1 was co-precipitated with purified anti-PIPS antiserum (Fig. 3B). These results suggest that full-length PIPS and full-length mPer1 interact with each other in vivo.

3.4. Expression pattern of PIPS First, the rat brain sections were immunohistochemically stained using the purified anti-PIPS antiserum. In this study, it was found that PIPS-IR was detected in almost every part of the brain including cortex, hippocampus and cerebellum (data not shown) but most markedly in the SCN and supraoptic nucleus (SON) in the hypothalamus (Fig. 4A, left). The PIPS-IR was mainly observed in the dorsomedial and ventral parts of the SCN (Fig. 4A, right). When the purified anti-PIPS antiserum was preabsorbed with the antigen, the immunoreactivity disappeared (data not shown). To analyze the subcellular localization of PIPS

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Fig. 1. Deduced amino acid sequences and distributions of PIPS. (A) Deduced amino acid sequence of PIPS was presented. Broken underline indicates interactive domain (ID), which was identified as an interactive sequence to rPer1 with yeast two-hybrid method. Underline shows the fragment used for antigen. (B) Tissue distribution of pips mRNA by Northern blot analysis is shown. The molecular size of pips mRNA was about 6.5 kbp. Note that pips transcript was mainly expressed in the brain. The lower panel shows the expression of actin mRNA as a control. (C) The left lane shows the in vitro translation products of pips gene. The molecular size was approximately 180 kDa. The middle and right lanes show the results of immunoblot analysis of the SCN lysate using purified anti-PIPS antiserum with (right) and without (middle) absorbance by the antigen. Note the PIPS-IR was observed in the same molecular size as that of in vitro translation product (arrowhead).

6 T. Matsuki et al. / Brain Research 916 (2001) 1 – 10 Fig. 2. Putative interaction domains of PIPS and mPer1. (A) Pull-down assay using the radiolabeled in vitro translation product of full-length mper1 gene and GST-fused fragments of PIPS protein was done. (B) Schematic representation of GST-fusion proteins of PIPS fragments. The number indicated the amino acid residues of each fragment. (C) Pull-down assay using the radiolabeled in vitro translation products of full-length pips gene and GST-fused fragments of mPer1 protein was done. (D) Schematic representation of GST-fused fragments of mPer1. The number of amino acid residues indicated the areas of each fragment.

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Fig. 3. PIPS interacts with mPer1 in vivo. (A) Lysates of COS-7 cells co-transfected with PIPS and HA-mPer1 were immunoprecipitated with 0 mg (lane 2), 1 mg (lane 3), and 5 mg (lane 4) of anti-HA antibody or purified anti-PIPS antiserum (lane 1). These immunoprecipitates were subjected to SDS–PAGE and the immunoblot analysis with purified anti-PIPS antiserum. (B) Lysate of COS-7 cells co-transfected with PIPS and HA-mPer1 was immunoprecipitated with anti-HA (lane1) antibody or purified anti-PIPS antiserum (lane 2). Lysate of untransfected COS-7 cells are also immunoprecipitated with anti-PIPS antibody (lane 3). These immunoprecipitates were subjected to SDS–PAGE and the immunoblot analysis with the anti-HA antibody.

in the SCN, we visualized PIP-IR with Alexa Fluor 488 (green in Fig. 4B) and counterstained nuclei with propium iodide (red in Fig. 4B). The immunoreactivities were observed both in the cytoplasms (arrowhead in Fig. 4B) and in the nuclei (arrow in Fig. 4B). To clarify whether PIPS shows a circadian rhythm in the SCN, expression levels of PIPS in the tissues examined under a light and dark cycle (L:D512 / 12 h) and constant dim light (dark) (DD) conditions with the immunoblot method (Fig. 5C). The expression of PIPS-IR was higher in the SCN than in the cortex as seen in immunohistochemistry. PIPS-IR in the SCN showed no significant daily fluctuations under LD or DD condition. PIPS-IR in the cortex also did not show the circadian oscillations.

3.5. PIPS–Per1 interaction affects their subcellular localization To analyze the function of PIPS–Per1 interaction, both genes were expressed transiently in COS-7 cells. When PIPS or HA-mPer1 was expressed individually, they located in the cytoplasm (Fig. 5A). Interestingly, when PIPS and HA-mper1 genes were co-transfected in COS-7

cells, PIPS-IR were observed in nuclei as well as cytoplasm in almost all cases (96.1%, Fig. 5B). On the contrary, mPer1 remained mainly in the cytoplasm. In few cases (2.3%), mPer1 as well as PIPS moved to nuclei (Fig. 5C). In the other case (1.6%), both PIPS and mPer1 remained in cytoplasm, but the expression level of both proteins are very low (data not shown). These data suggest that PIPS–Per1 interaction affects subcellular localizations of them.

4. Discussion Up to now, three mouse period genes (mper1, mper2 and mper3 ) were cloned as a mammalian ortholog of Drosophila period gene [13,15,17–19]. Furthermore, these mper transcripts were found to have robust circadian oscillations in the SCN, and the mper1 and mper2 transcripts were induced also after light-exposure in the subjective dark period [2,17,24]. Recent reports about mutant mice of mper genes suggested that mper2 was necessary for circadian transcriptional feedback loop and

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Fig. 4. Expression of PIPS in the SCN. (A) Photomicrographs of coronal brain sections immunohistochemically stained with purified anti-PIPS antiserum. The left panel shows at a lower magnification. Note the high PIPS-IR in the SCN (arrowhead) and SON (arrows). The right panel shows the SCN at a higher magnification. PIPS-IR were detected mainly in dorsomedial and ventral part of the SCN. (B) Fluorescent imaging of PIPS-IR (green) in the SCN. The nuclei were counterstained with propium iodide (red). The right panel indicates the combined image of PIPS-IR and propium iodide. Note PIPS-IR were seen mainly in cytoplasm in some neurons (arrowhead in right panel) and both cytoplasm and nucleus in other group of cells (yellow, arrow in right panel). (C) The SCN and the cortex was collected at 6-h intervals under LD cycle (ZT; left) or under DD condition (CT; right). ZT1 means the time 1 h after the light onset, and CT1 indicates 1 h after the onset of the subjective day. Tissue lysates (10 mg protein / lane) were subjected to immunoblot analysis with the antibodies against PIPS or NSE as a control.

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Fig. 5. mPer1 promotes nuclear entry of PIPS. COS-7 cells were transfected with HA-mPer1 (A, left panel), PIPS (A, right panel) or both HA-mPer1 and PIPS (B, C). mPer1 were visualized with Cy3 (red) and PIPS-IR with Alexa Fluor 488 (green). Co-localizations of HA-mPer1 and PIPS are pictured in yellow (B, C). The number of cells we checked were 1963. In 96.1% case, PIPS partially moved into the nuclei but mPer1 remained in cytoplasm (B). In rare case (2.3%), both PIPS and mPer1 localized in the nucleus (C). In the other case (1.6%), both molecules remained in cytoplasm but both PIPS-IR and mPer1-IR were very faint (data not shown).

that mper1 seemed to be related with output signaling pathway of the clock [3,22,23]. In this study, using the yeast two-hybrid system we found three identical clones that can interact with rPer1 from the rat brain cDNA library. Thus, we cloned a full sequence of one of the clones and examined whether this molecule (PIPS) could really interact with mPer1. The other two clones are now under analyzing. Using the database searches, we found that there exists a human expression sequence tag with 74% identical amino acid sequence to PIPS. This putative molecule, KIAA0443, may be a human homolog of PIPS. The possible existence of PIPS homologs in other species suggests that PIPS have important functions. The pull-down assays and co-immunoprecipitation experiment showed that PIPS and mPer1 could interact with each other in vivo as well as in vitro. We also found that HLH-PAS domain of mPer1 were most important for the interaction with PIPS (Fig. 2C). This result suggests two possibilities: (1) since PAS domain of mPer1 is important for interaction with other molecules, the interaction of mPer1 and PIPS can affects the interaction of mPer1 and other molecules via PAS domain; (2) PIPS can interact with other clock-related proteins that have PAS domain (Per2, Per3, BMAL1, BMAL2 and Clock). Because domain3 in the N terminal fragment of PIPS could not bind to mPer1 (Fig. 2A), it is able to suppose that mPer1 interact with PIPS with separated two areas (domain1–2 and domain4 in ID). This estimation raises a possibility that PIPS interact with two different proteins with HLHPAS domain and form functional complex. Northern blot and immunoblot analysis showed that

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PIPS is mainly expressed in the brain, especially in the hypothalamus containing the SCN (Figs. 1B and 4A). This data also suggests the relation between PIPS and circadian clock functions. Although PIPS-IR did not show circadian fluctuations in the SCN, Clock also does not show circadian rhythm in its expression [12]. Fluorescent staining of PIPS counterstained with propium iodide showed that PIPS localized in the cytoplasms as well as in the nuclei (Fig. 4C). This subcellular localization of PIPS in the SCN is quite unique, because the other clock-related proteins localize only in the nuclei [6,9]. The data of co-transfection experiments were also very interesting. A part of PIPS in the cytoplasm moved into nuclei under the existence of mPer1 (Fig. 5B). The subcellular localization of PIPS in the co-transfection experiments agreed with that in the SCN (Fig. 4B). Because mPer1 remained mainly in the cytoplasm, it is not logical to suppose that mPer1 itself work as a transporter of PIPS into the nucleus. It is possible that unknown modifications such as phosphorylation were induced by PIPS–mPer1 interaction, and PIPS entered into the nucleus. The other hypothesis is that PIPS–mPer1 interaction affects the interaction of PIPS with another molecule that keeps PIPS in cytoplasm. Because PIPS has putative two binding domains as described above and the one of the domains contained putative NLS, it can be suggested that the interaction with mPer1 via domain4 affect the binding with another molecule at domain1–2 including putative NLS. Although further analysis must be necessary, these data about the subcellular localization of PIPS in vitro and in vivo suggested the hypothesis that cytoplasmic PIPS or nuclear PIPS transfer some signals related with the circadian clock function in the SCN and that subcellular localization of PIPS were controlled by mPer1. Although the actual functions of PIPS remain unclear, it can be supposed that PIPS is related with the output signaling of circadian clock because of the following three reasons: (1) mPer1 is thought to be involved in output signaling [3,22]; (2) PIPS mainly localizes in the cytoplasm; and (3) the subcellular localization of PIPS are controlled by mPer1. Whether this is the case must be examined in future.

Acknowledgements We especially thank Dr Yasushi Isojima for his advice and discussions about this research. This work was partly supported by Grant-in-Aid for Science Research (B) 10044283 and Priority Areas (C) 12206053 from the Ministry of Education, Science, Sports and Culture (Japan).

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