Nuclear import of mPER3 in Xenopus oocytes and HeLa cells requires complex formation with mPER1 Susanne Loop and Tomas Pieler Abteilung Entwicklungsbiochemie, Zentrum fu ¨ r Biochemie und Molekulare Zellbiologie, Georg-August Universita ¨ t, Go ¨ ttingen, Germany The genetic control of circadian rhythmicity was first analysed in Drosophila. A central autoregulatory feed- back loop that involves different transcriptional regula- tors was uncovered. The bHLH transcription factors CLOCK (CLK) and CYCLE (CYC) drive expression of the period (per) and timeless (tim) genes. Conversely, Period and Timeless proteins (PER and TIM) inhibit CLK ⁄ CYC-mediated transcription of their own genes, resulting in a gradual loss of PER and TIM proteins. At a critically reduced level of PER and TIM protein activity, CLK ⁄ CYC repression is relieved and per ⁄ tim gene expression returns [1–5]. A similar mechanism seems to operate in verte- brates. In mammals, CLOCK–BMAL1 heterodimers activate transcription of Period (mPer) and Crypto- chrome ( mCry) genes. mPER and mCRY proteins act as negative regulators of their own expression by directly interacting with and thereby inhibiting CLOCK–BMAL1 [6,7]. Gene duplications have gener- ated three different mPER proteins (mPER1, mPER2 and mPER3) and two different mCRY proteins (mCRY1 and mCRY2). Functional diversity among the individual members of each of these clock protein subfamilies has been reported [8–15]. Post-translational control constitutes a further important level of regulation in both vertebrate and invertebrate systems. In Drosophila, phoshorylation of both PER and TIM affects stability and ⁄ or nuclear transport [16–21]. Phosphorylated forms of the two proteins are targeted for degradation by the ubiquitin– proteasome pathway [22–24]. It has also been pro- posed that PER⁄ TIM phosphorylation may promote nuclear transfer, but more recent studies argue in favour of phosphorylation positively regulating their transcriptional repressor activity [25]. Conversely, the regulated rhythmic dephosphorylation of PER by pro- tein phosphatase 2A stabilizes PER, thereby contribu- ting to the rhythmicity of PER protein concentrations [26]. Mammalian PER proteins have also been found to become phosphorylated; mPER1, mPER2 and mPER3 are subjected to rhythmical phosphorylation mediated Keywords circadian rhythm; mCRY, mPER; nuclear import; Xenopus oocytes Correspondence T. Pieler, Abteilung Entwicklungsbiochemie, Zentrum fu ¨ r Biochemie und Molekulare Zellbiologie, Georg-August Universita ¨ t, Justus von Liebig Weg 11, D-37077 Go ¨ ttingen, Germany Fax: +49 551 3914614 Tel: +49 551 395683 E-mail: tpieler@gwdg.de (Received 14 March 2005, revised 27 May 2005, accepted 31 May 2005) doi:10.1111/j.1742-4658.2005.04798.x Several transcription factors with the function of setting the biological clock in vertebrates have been described. A detailed understanding of their nucleocytolasmic transport properties may uncover novel aspects of the regulation of the circadian rhythm. This assumption led us to perform a systematic analysis of the nuclear import characteristics of the different murine PER and CRY proteins, using Xenopus oocytes and HeLa cells as experimental systems. Our major finding is that nuclear import of mPER3 requires complex formation with mPER1. We further show that the nuclear localization signal (NLS) function of mPER1 and not activation of a masked NLS in mPER3 is critical for the import of the mPER1–mPER3 complex. Finally, and as previously described in other cell systems, nuclear import of mPER proteins in Xenopus oocytes correlates positively with their phosphorylation. Abbreviations CK, casein kinase; NLS, nuclear localization signal. 3714 FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS by casein kinases (CKIe and CKId) [27–30]. The phos- phorylation status of murine PER proteins, similar to that reported for Drosophila PER, influences stability and nuclear transport; phosphorylated forms of mPER1 and mPER3 are rapidly degraded [31,32]. Furthermore, mPER1 mutant mice have been used to demonstrate that mPER1 is required for phosphoryla- tion and nuclear transfer of mPER3 [33]. Phosphoryla- tion of mPER1 itself correlates with nuclear transport [34]. Earlier studies had already indicated that nuclear translocation of mPER3 is promoted by mPER1 in NIH3T3 cells [14]. Other studies, using different cell systems, had come to additional and sometimes apparently contradictory conclusions. Yagita et al. [35], using COS7 cells, repor- ted that mPER3 by itself is predominantly cytoplas- mic, and nuclear accumulation is obtained by serum shock-induced formation of mPER1 ⁄ 3 or mPER2 ⁄ 3 heterodimers. Furthermore, Vielhaber et al. [36] had observed that mPER1 is predominantly nuclear, whereas mPER2 is predominantly cytoplasmic in HEK293 cells; CKIe-mediated phosphorylation of mPER1 was reported to lead to masking of the nuclear localization signal (NLS) and coexpression of mPER1 with mPER2 and cytoplasmic localization of the heterodimer. Finally, Miyazaki et al. [37], using COS1 cells, had observed that mammalian PER2 has a posit- ive regulatory function with respect to the nuclear import of mCRY1. What all these studies have in common is the idea that dimerization of the different mammalian PER and CRY proteins modulates their nucleocytoplasmic dis- tribution and thereby probably also their function as transcriptional repressors. In the work presented here, we systematically analyzed nuclear import of the dif- ferent murine PER and CRY proteins, either individu- ally or in all possible heterodimeric combinations, primarily using Xenopus oocytes as an experimental system. We found that interaction with mPER1 is required for the nuclear import of mPER3, and we observed a positive correlation between nuclear import of mPER proteins and their phosphorylation. Results Positive correlation between phosphorylation and nuclear import of mPER proteins in Xenopus oocytes The different individual murine PER and CRY pro- teins were generated by in vitro translation and injected into the cytoplasm of Xenopus oocytes. The kinetics of nuclear import were analysed after manual separation of cytoplasmic and nuclear fractions by gel electro- phoresis (Fig. 1A). The data obtained reveal that, whereas the mCRY proteins, as well as mPER1 and mPER2, are readily imported into the nucleus of Xeno- pus oocytes, mPER3 is not. We also observed reduced A B Fig. 1. Nuclear import of murine PER and CRY proteins in Xenopus oocytes. mPER1 and mPER2, but not mPER3, are phosphorylated and imported into the nucleus of Xenopus oocytes. (A) 35 S-labelled mPER1, mPER2, mPER3 and derived protein fragments fused to six copies of the myc tag in tandem repeat were translated in vitro and injected into the cytoplasm of Xenopus oocytes. The nucleus and cytoplasm were separated manually at the time points indicated. Proteins were immunoprecipitated from 10 pooled nuclear and cytoplasmic fractions using the myc antibody and analysed by SDS ⁄ PAGE and phosphorimaging. To test for phosphorylation, immunoprecipitated nuclear or cyto- plasmic fractions were incubated with lambda protein phosphatase (k PPase) before gel electrophoresis. (B) mCRY1 and mCRY2 are impor- ted into the nucleus of Xenopus oocytes. S. Loop and T. Pieler Nuclear import of circadian clock proteins FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS 3715 electrophoretic mobility of mPER1 and mPER2, but not of mPER3, which increases with the time of incu- bation after microinjection. It also seems that the relative amount of the phosphorylated forms of the proteins is higher in the nucleus than in the cytoplasm. Reduced electrophoretic mobility suggests chemical modification events, such as phosphorylation. Phos- phatase treatment of cytoplasmic and nuclear protein fractions isolated from microinjected oocytes equalizes the electrophoretic mobility of all samples tested, revealing that mPER1 and mPER2 are indeed phos- phorylated after injection into Xenopus oocytes. Thus, we found a positive correlation between phosphoryla- tion and nuclear import for mPER1 and mPER2, whereas mPER3, which is not imported into the nuc- leus, is also not phosphorylated. On the other hand, there is no evidence for phosphorylation of mCRY1 and mCRY2, which are readily imported into the nuc- leus of injected oocytes (Fig. 1B). The absence of nuclear import of mPER3 injected into Xenopus oocytes suggests that the protein is devoid of a nuclear import signal that is functional in this experimental system. To address this question, all three murine PER proteins were broken down into four fragments, and each one tested for nuclear import activity in Xenopus oocytes (Fig. 2). In agree- ment with earlier NLS-mapping experiments in other experimental systems [31,35–37], the corresponding region (fragment 3) of all three PER proteins har- bours a functional NLS. Mutation of the putative NLS in mPER3 abrogates import activity (data not shown). In extension of previous studies, we further detected a novel, additional NLS located in the C-ter- minal portion (fragment 4) of mPER1 within the 186 C-terminal amino acids (Fig. 3, fragment 4b). We also noted faint nuclear signals for the corresponding C-terminal fragments derived from mPER2 and mPER3 (Fig. 2A). However, nuclear import rates A B Fig. 2. mPER1 contains an additional NLS in its C-terminal domain. (A) Mapping of NLS function in murine Per proteins. Fragments corres- ponding to different portions of mPER1, mPER2 and mPER3 (as indicated) were assayed for nuclear import in Xenopus oocytes. MPER2 Frag2 was rapidly degraded in Xenopus oocytes. mPER1: myc Frag 1, aa 1–323; myc Frag 2, aa 324–645; myc Frag 3, aa 646–972; myc Frag 4, aa 973–1291. mPER2: myc Frag 1, 1–314; myc Frag 2, aa 314–628; myc Frag 3, 629–942; myc Frag 4, aa 943–1257. mPER3: myc Frag 1, aa 1–280; myc Frag 2, 281–559; myc Frag 3, 560–835; myc Frag 4, 836–1113. (B) Schematic representation of the fragments used for map- ping experiments and percentage of nuclear import in multiple independent experiments. The grey boxes define the location of the NLSs in the Per proteins (in bold the newly identified NLS2 in mPER1). Nuclear import of circadian clock proteins S. Loop and T. Pieler 3716 FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS below 10% (Fig. 2B) are considered nonsignificant. A primary sequence comparison of the three murine PER proteins revealed a high degree of structural diversity in the C-terminal domain (data not shown), correlating with functional diversity with respect to NLS activities. Mutation or deletion of one of the two NLSs in mPER1 led to reduced nuclear import. A complete block occurred only after mutation ⁄ dele- tion of both NLSs (Fig. 3, myc- mPER1mutNLSDC); phosphorylation was not affected in these mutants (data not shown). Alternative explanations exist for the observed absence of mPER3 nuclear import when injected by itself; either it is rapidly degraded in the nucleus or rapid export prevents its nuclear accumulation. How- ever, in a separate study on the nuclear export of clock proteins in Xenopus oocytes [38], we observed that, after nuclear injection of mPER3, the protein is only slowly exported and there is no indication of protein degradation in the nucleus. Thus, in summary, microinjection of individual iso- lated murine PER proteins reveals that mPER1 and mPER2 become phosphorylated and are imported into the nucleus of Xenopus oocytes. In contrast, mPER3 is not phosphorylated and not transferred to the nucleus, even though it contains an NLS that is functional in this system. Furthermore, deletion analysis uncovered a novel NLS (NLS2) that is specific to the C-terminal region of mPER1. Complex formation with mPER1 promotes nuclear import of mPER3 in Xenopus oocytes As heterodimerization of clock proteins is known to modulate nuclear import activity, we tested whether complex formation with either mPER1 or mPER2 would enable transfer of mPER3 into the nucleus of Xenopus oocytes. For this purpose, mPER dimers were formed in vitro (Fig. 4A); we found that cotranslation of different combinations of mPER proteins allowed heterodimerization, whereas coincubation after in vitro translation did not. In good agreement with earlier studies [35,39], we also found that the entire PAS domain in mPER1 was required for complex forma- tion with mPER3 (data not shown), and the NLS-defi- cient mPER1 mutant was not impaired with respect to its ability to interact with mPER3 (Fig. 4A). Microinjection of complexes formed with different combinations of mPER proteins into the cytoplasm of Xenopus oocytes revealed that, whereas mPER3 by itself (Fig. 1A) or in complex with mPER2 was not imported, it was readily transferred to the nucleus in complex with mPER1 (Fig. 4B). As expected, a com- plex of mPER1 and mPER2 was also imported. Thus, A B Fig. 3. Mutation of the NLS function in mPER1 blocks nuclear import activity. (A) Different mutants of mPER1 (as indicated) were assayed for nuclear import activity in Xenopus oocytes. To mutate mPER1-NLS1, three of the basic amino acids were chan- ged to alanine (RRHHCRSKAKRSR). In mPER1DC and mPER1mutNLS1DC, the 186 C-terminal amino-acid sequence containing NLS2 was deleted. myc mPER1 Frag 4a, aa 973–1104; myc mPER1 Frag 4b, 1105–1291; myc mPER1mutNLS1, aa 1–1291; myc mPER1DC, aa 1–1104; myc mPER1mutNLS1DC, aa 1–1291. (B) Percent- age of nuclear import of multiple independ- ent experiments. S. Loop and T. Pieler Nuclear import of circadian clock proteins FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS 3717 mPER1 seems to serve as an adaptor for the nuclear import of mPER3 in Xenopus oocytes. As both mPER1 and mPER3 contain functional NLSs (as described above), we tested whether complex formation with mPER1 would unmask the NLS activity in full-length mPER3. We constructed a mutant version of mPER1 that had lost both of its two NLSs but retained its ability to form a heterodimer with mPER3 (mPER1mutNLS1DC; Figs 3 and 4A). In complex with this mutant mPER1 variant, mPER3 was no longer transferred to the nucleus (Fig. 4C). Conversely, upon mutation of the NLS in mPER3, the mPER1 ⁄ mPER3mutNLS heterodimer was still imported into the nucleus of Xenopus oocytes (Fig. 4C), suggesting that it A BC Fig. 4. mPER3 is imported into the nucleus of Xenopus oocytes in complex with mPER1. (A) Homodimer and heterodimer formation of mPER proteins. Flag-tagged mPER3 was cotranslated in vitro with myc-tagged versions of full-length mPER1, mPER2, mPER3 and mPER1mutNLS1DC. Complex formation was detected by coimmunoprecipitation using a flag antibody (bottom panel). As a negative control, myc tagged period proteins were translated without flag mPER3 and immunprecipitated by using the flag antibody (left hand panel). 50% of the input was loaded on the SDS ⁄ polyacrylamide gel. (B) Complexes formed by cotranslation of different combinations of myc-tagged mPER1, mPER2 and mPER3 (as indicated) were injected into the cytoplasm of Xenopus oocytes and assayed for nuclear import after 3 and 6 h incubation at 18 °C as described in Fig. 1. (C) The NLS function in mPER1 is required for mPER3 import. The heterodimer of myc mPER3 and flag mPER1mutNLS1DC was injected into the cytoplasm of Xenopus oocytes; nuclear and cytoplasmic fractions were immuno- precipitated by using the myc and flag antibodies at the time points indicated. Myc-tagged, cotranslated mPER1 and mPER3mutNLS were analysed for nuclear import. All proteins were treated with lambda protein phosphatase before electrophoresis. Fig. 5. The NLS functions of mPER proteins are also active in HeLa cells. (A) Schematic representation of mPER proteins and derived fragments used for transient transfection into HeLa cells and their nucleocytoplasmic distribution. (B) HeLa cells were transfected with the indicated myc-tagged mPER proteins. The intracellular localization of these proteins was detected by immunofluorescence staining using Cy3-coupled myc antibodies (red). The nuclei were visualized by DAPI DNA staining (blue). (C) Quantitative analysis. The subcellular localiza- tion of the different protein constructs was categorized as nuclear (N), nuclear and cytoplasmic (N ⁄ C), or cytoplasmic (C). For each construct, 50–100 transfected cells were analysed. Nuclear import of circadian clock proteins S. Loop and T. Pieler 3718 FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS A B C S. Loop and T. Pieler Nuclear import of circadian clock proteins FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS 3719 is the NLS activity in mPER1, and not unmasking of the NLS in mPER3, that is responsible for the nuclear transfer of the mPER1–mPER3 complex. Nuclear import of mPER3 in HeLa cells also requires complex formation with mPER1 We further investigated whether the above import characteristics of murine PER proteins reflect specific features of nucleocytoplasmic transport in Xenopus oocytes. HeLa cells were transiently transfected with the same set of mPER protein fragments as used in the oocyte microinjection experiments. We found that the main effects, i.e. the lack of nuclear import of mPER3 and the presence of an additional NLS at the C-terminus of mPER1, can be reproduced in these cells (Fig. 5). In addition, we also observed weak nuclear import activity for the C-terminal fragment of mPER2 (Fig. 5, mPER2 Frag 4). Next, we analysed whether, similar to the situation with Xenopus oocytes, complex formation with mPER1 is sufficient for nuclear import of mPER3 in HeLa cells. mPER proteins alone, or specific combinations of mPER3 with mPER1 or mPER1mutNLS1DC, were used in the transient transfection assay (Fig. 6). Indeed, in combination with mPER1, but not with mPER2, mPER3 was mostly nuclear; analysis of a combination of mPER3 with mPER1mutNLS1DC revealed that, again as in the oocyte system, it is the NLS function of mPER1 that is required for the nuc- lear import of mPER3 in the heterodimeric complex with mPER1. Thus, the requirement of complex for- mation with mPER1 for the nuclear import of mPER3 appears to be a general phenomenon that is not restricted to the Xenopus oocyte system. Discussion Analysis of the nucleocytoplasmic transport activities of murine PER and CRY proteins in Xenopus oocytes and HeLa cells reveals that mPER1 serves as a nuclear import adaptor for mPER3, even though mPER3 con- tains a functional NLS that appears to be masked in the full-length protein. We also mapped a novel NLS to the C-terminus of mPER1. Nuclear import of the mPER1–mPER3 complex requires a functional NLS in mPER1, and the silent NLS in mPER3 is not necessary. Finally, nuclear import of mPER1 and mPER2 corre- lates with their phosphorylation in Xenopus oocytes. A systematic fragmentation analysis of the three dif- ferent murine PER proteins produced two main obser- vations. First, mPER1 contains a second NLS at its extreme C-terminus in addition to the one that had been described previously [36], which is functional in both Xenopus oocytes and HeLa cells. Secondly, mPER3 contains a silent NLS that is repressed in the context of the full-length protein. The corresponding protein fragment contains a basic stretch of amino acids that is conserved in all three murine PER proteins. Previous studies with COS7 cells also found cytoplasmic retention of mPER3 which could be relieved by cotransfection of CKIe [31]. The molecular mechanism responsible for the masking of the NLS in mPER3 remains to be elucidated. The NLS in mPER3 may be masked by intramolecular protein folding or by interaction with an unknown inhibitory factor. With respect to the elucidation of the mechanism that eventually relieves the cytoplasmic sequestration of mPER3, previous studies used different cell lines and produced partially contradictory observations. Our finding that the nuclear import of mPER3 is strongly enhanced in Xenopus oocytes and in HeLa cells by the presence of mPER1 is consistent with results obtained in COS7 and NIH3T3 cells [14,35]. In further support of such a scenario, mPER3 has been reported to always be cytoplasmic in the livers of mPER1-deficient mice [33]. However, Vielhaber et al. [36] reported that coexpression of mPER1 with mPER2 results in cytoplasmic localization of the het- erodimer in HEK293 cells. This result is inconsistent with our observations in microinjected oocytes and transiently transfected HeLa cells. We cannot exclude the possibility that this apparent contradiction is a result of the use of different experimental systems. Several independent studies also describe a positive correlation between mPER3 phosphorylation and nuclear accumulation [29,31,33]. In Xenopus oocytes, cytoplasmic mPER3 was not found to be phosphoryl- ated, whereas nuclear import of mPER1 and mPER2 correlated with protein phosphorylation. As mPER3 was also shown to require mPER1 for stable inter- action with CKIe and phosphorylation [33], there may be a direct functional link between phoshorylation and activation of the ‘silent’ NLS in mPER3. However, Vielhaber et al. [36] proposed that CKIe-mediated phosphorylation of mPER1 leads to NLS masking in HEK293 cells. Again, this apparent contradiction may be due to the differences in the experimental systems used. Experimental procedures Plasmids For in vitro translation, mPer and mCry cDNAs were sub- cloned into the pCSMT vector containing six myc epitopes Nuclear import of circadian clock proteins S. Loop and T. Pieler 3720 FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS [40], or into the pCSflag vector, in which the myc tag was replaced by a double-stranded oligonucleotide sequence containing a kozak element and the flag epitope (5¢-GATC GCCGCCATGGACTACAAGGACGAGGATGACAA-3¢). The mPER2 cDNA was subcloned into the NcoI restriction site of pCSMT; the resulting construct possesses five copies A B Fig. 6. Nuclear import of clock proteins in HeLa cells. (A) The cells were transiently transfected with myc-tagged and flag- tagged proteins as indicated. The intracellu- lar localization of these proteins was detec- ted by immunofluorescence staining using myc-Cy3 (red) or flag-fluorescein isothio- cyanate (FITC) (green) antibodies. The nuclei were visualized by DAPI DNA staining (blue). (B) Quantitative analysis of the nucleocytoplasmic distribution of mPER3 cotransfected with with other mPER variants, as indicated (see also the legend to Fig. 5C). S. Loop and T. Pieler Nuclear import of circadian clock proteins FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS 3721 of the myc epitope. All mPER1 fragments were amplified by PCR with 5¢ primers containing the EcoRI restriction site and 3¢ primers containing the StuI restriction site. All mPER2 fragments were amplified by PCR with 5¢ primers containing the NcoI restriction site and 3¢ primers contain- ing the XhoI restriction site. MPER3 Frag1 was amplified by PCR with 5¢ primers containing the StuI restriction site and 3¢ primers containing the XbaI restriction site. mPER3 Frag2 and mPER3 Frag4 were amplified by PCR with 5¢ primers containing the EcoRI restriction site and 3¢ primers containing the StuI restriction site. MPER3 Frag3 was amplified by PCR with 5¢ primers containing the EcoRI restriction site and 3¢ primers containing the XbaI restric- tion site. The mPER1 mutants used were constructed by using the Quick Change site-directed mutagenesis kit (Strat- agene, La Jolla, CA, USA) using the user’s protocol provi- ded by the manufacturer. Protein expression Radiolabelled proteins were expressed as fusions with the myc or flag epitope in a coupled transcription ⁄ translation (TNT) system (Promega, Madison, WI, USA) in the pres- ence of 20 lCi [ 35 S]methionine (Amersham, Little Chalfont, Bucks, UK). The in vitro translated proteins products were analysed by SDS ⁄ PAGE and phosphorimaging (Molecular Dynamics, Sunnyvale, CA, USA). Coimmunoprecipitation experiments For coimmunoprecipitation experiments, cDNAs were mixed and in vitro cotranslated in the coupled TNT sys- tem (Promega). The samples were incubated for 120 min at 30 °C, and 2 lL each sample added to protein G–Seph- arose–myc–antibody pellets. The coimmuoprecipitation was performed in a final volume of NET-2 [50 mm Tris ⁄ HCl, pH 7.4, 150 mm NaCl, 0.05% (v ⁄ v) Nonidet P40] for 1 h at 4 °C. After being washed three times with NET-2, proteins were analysed by SDS ⁄ PAGE and phos- phorimaging. Microinjection into Xenopus laevis oocytes Oocytes were prepared for microinjection as described previously [41]. All measures were taken to minimise pain and discomfort of the frogs in accord with the German regulations on experimental use of animals. About 15 nL protein injection mix was microinjected into the cytoplasm of oocytes. To determine the nucleocytoplasmic distribu- tion, the nucleus and cytoplasm were manually separated after different time intervals. Proteins fused to the myc epitope were purified from pooled nuclear and cytoplas- mic fractions by immunoprecipitation as described by Rudt & Pieler [42]. The following antibodies were used: mouse anti-myc (9E10; Sigma, St Louis, MO, USA) and mouse anti-flagM2 (Sigma). Phosphatase treatment After immunoprecipitation, immunopellets were resuspended in phosphatase buffer supplemented with 2 mm MnCl 2 and incubated with 200 U lambda protein phosphatase (New England Biolabs, Beverly, MA, USA) for 30 min at 30 °C. The addition of SDS ⁄ PAGE sample buffer stopped the reac- tion. Cell culture and transfection Hela cells were cultured in Eagle’s minimal essential med- ium supplemented with 10% (v ⁄ v) fetal bovine serum (Biochrom, Cambridge, UK). Approximately 3 · 10 5 cells per well were plated in a six-well dish one day before trans- fection. Plasmid (4 lg) was transfected with Lipofectamine 2000 (Invitrogen, San Diego, CA, USA) using the user’s protocol provided by the manufacturer. Immunocytochemistry The cells were grown on coverslips and fixed with 3% (v ⁄ v) paraformaldehyde in NaCl ⁄ P i at room temperature for 15 min. After treatment with 0.5% (v ⁄ v) Triton X-100 in NaCl ⁄ P i , nonspecific staining was blocked with 3% (w ⁄ v) BSA in NaCl ⁄ P i . The immunostaining was performed with the myc-Cy3 or flag-FITC (Sigma). The cells were embed- ded with Vectashield containing 4’,6-diamidino-2-phenyl- indole (DAPI; Linaris, Bettingen, Germany). Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 523) to T.P. We thank Dr Gregor Eichele and Dr Pablo Szendro for the mPer and mCry encoding plasmids, Dr Katja Koebernick for pCSflag, and Andreas Nolte for DNA sequencing. References 1 Rutila JE, Suri V, Le M, So WV, Rosbash M & Hall JC (1998) CYCLE is a second bHLH-PAS clock protein essential for circadian rhythmicity and transcription of Drosophila period and timeless. Cell 93, 805–814. 2 Lee C, Bae K & Edery I (1998) The Drosophila CLOCK protein undergoes daily rhythms in abundance, phos- phorylation, and interactions with the PER-TIM com- plex. Neuron 21, 857–867. 3 Lee C, Bae K & Edery I (1999) PER and TIM inhibit the DNA binding activity of a Drosophila CLOCK-CYC ⁄ Nuclear import of circadian clock proteins S. Loop and T. Pieler 3722 FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS dBMAL1 heterodimer without disrupting formation of the heterodimer: a basis for circadian transcription. Mol Cell Biol 19, 5316–5325. 4 Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, Weitz CJ, Takahashi JS & Kay SA (1998) Closing the circadian loop: CLOCK- induced transcription of its own inhibitors per and tim. Science 280, 1599–1603. 5 Allada R, White NE, So WV, Hall JC & Rosbash M (1998) A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless. Cell 93, 791–804. 6 Bunger MK, Wilsbacher LD, Moran SM, Clendenin C, Radcliffe LA, Hogenesch JB, Simon MC, Takahashi JS & Bradfield CA (2000) Mop3 is an essential component of the master circadian pacemaker in mammals. Cell 103, 1009–1017. 7 Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS & Weitz CJ (1998) Role of the CLOCK protein in the mammalian circadian mechanism. Science 280, 1564–1569. 8 Tei H, Okamura H, Shigeyoshi Y, Fukuhara C, Ozawa R, Hirose M & Sakaki Y (1997) Circadian oscillation of a mammalian homologue of the Drosophila period gene. Nature 389, 512–516. 9 Zylka MJ, Shearman LP, Weaver DR & Reppert SM (1998) Three period homologs in mammals: differential light responses in the suprachiasmatic circadian clock and oscillating transcripts outside of brain. Neuron 20, 1103–1110. 10 van der Horst GT, Muijtjens M, Kobayashi K, Takano R, Kanno S, Takao M, de Wit J, Verkerk A, Eker AP, van Leenen D, et al. (1999) Mammalian Cry1 and Cry2 are essential for maintenance of circadian rhythms. Nature 398, 627–630. 11 Sun ZS, Albrecht U, Zhuchenko O, Bailey J, Eichele G & Lee CC (1997) RIGUI, a putative mammalian ortho- log of the Drosophila period gene. Cell 90, 1003–1011. 12 Shearman LP, Sriram S, Weaver DR, Maywood ES, Chaves I, Zheng B, Kume K, Lee CC, van der Horst GT, Hastings MH & Reppert SM (2000) Interacting molecular loops in the mammalian circadian clock. Science 288, 1013–1019. 13 Shearman LP, Zylka MJ, Weaver DR, Kolakowski LF Jr & Reppert SM (1997) Two period homologs: circa- dian expression and photic regulation in the suprachias- matic nuclei. Neuron 19, 1261–1269. 14 Kume K, Zylka MJ, Sriram S, Shearman LP, Weaver DR, Jin X, Maywood ES, Hastings MH & Reppert SM (1999) mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop. Cell 98, 193–205. 15 Albrecht U, Sun ZS, Eichele G & Lee CC (1997) A differ- ential response of two putative mammalian circadian reg- ulators, mper1 and mper2, to light. Cell 91, 1055–1064. 16 Akten B, Jauch E, Genova GK, Kim EY, Edery I, Raabe T & Jackson FR (2003) A role for CK2 in the Drosophila circadian oscillator. Nat Neurosci 6, 251–257. 17 Bao S, Rihel J, Bjes E, Fan JY & Price JL (2001) The Drosophila double-timeS mutation delays the nuclear accumulation of period protein and affects the feedback regulation of period mRNA. J Neurosci 21, 7117–7126. 18 Lin JM, Kilman VL, Keegan K, Paddock B, Emery-Le M, Rosbash M & Allada R (2002) A role for casein kinase 2alpha in the Drosophila circadian clock. Nature 420, 816–820. 19 Martinek S, Inonog S, Manoukian AS & Young MW (2001) A role for the segment polarity gene shaggy ⁄ GSK- 3 in the Drosophila circadian clock. Cell 105, 769–779. 20 Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B & Young MW (1998) double-time is a novel Drosophila clock gene that regulates PERIOD protein accumula- tion. Cell 94, 83–95. 21 Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS & Young MW (1998) The Drosophila clock gene double-time encodes a protein closely related to human casein kinase Iepsilon. Cell 94, 97–107. 22 Naidoo N, Song W, Hunter-Ensor M & Sehgal A (1999) A role for the proteasome in the light response of the timeless clock protein. Science 285, 1737–1741. 23 Grima B, Lamouroux A, Chelot E, Papin C, Limbourg- Bouchon B & Rouyer F (2002) The F-box protein slimb controls the levels of clock proteins period and timeless. Nature 420, 178–182. 24 Ko HW, Jiang J & Edery I (2002) Role for Slimb in the degradation of Drosophila Period protein phosphory- lated by Doubletime. Nature 420, 673–678. 25 Nawathean P & Rosbash M (2004) The doubletime and CKII kinases collaborate to potentiate Drosophila PER transcriptional repressor activity. Mol Cell 13, 213–223. 26 Sathyanarayanan S, Zheng X, Xiao R & Sehgal A (2004) Posttranslational regulation of Drosophila PER- IOD protein by protein phosphatase 2A. Cell 116, 603– 615. 27 Lowrey PL, Shimomura K, Antoch MP, Yamazaki S, Zemenides PD, Ralph MR, Menaker M & Takahashi JS (2000) Positional syntenic cloning and functional characterization of the mammalian circadian mutation tau. Science 288, 483–492. 28 Keesler GA, Camacho F, Guo Y, Virshup D, Mondadori C & Yao Z (2000) Phosphorylation and destabilization of human period I clock protein by human casein kinase I epsilon. Neuroreport 11, 951–955. 29 Takano A, Shimizu K, Kani S, Buijs RM, Okada M & Nagai K (2000) Cloning and characterization of rat casein kinase 1epsilon. FEBS Lett 477, 106–112. 30 Toh KL, Jones CR, He Y, Eide EJ, Hinz WA, Virshup DM, Ptacek LJ & Fu YH (2001) An hPer2 phosphory- lation site mutation in familial advanced sleep phase syndrome. Science 291, 1040–1043. S. Loop and T. Pieler Nuclear import of circadian clock proteins FEBS Journal 272 (2005) 3714–3724 ª 2005 FEBS 3723 [...]... RA, Snider L & Weintraub H (1994) Xenopus embryos regulate the nuclear localization of XMyoD Genes Dev 8, 1311–1323 Claussen M, Rudt F & Pieler T (1999) Functional modules in ribosomal protein L5 for ribonucleoprotein complex formation and nucleocytoplasmic transport J Biol Chem 274, 33951–33958 Rudt F & Pieler T (1996) Cytoplasmic retention and nuclear import of 5S ribosomal RNA containing RNPs EMBO.. .Nuclear import of circadian clock proteins 31 Akashi M, Tsuchiya Y, Yoshino T & Nishida E (2002) Control of intracellular dynamics of mammalian period proteins by casein kinase I epsilon (CKIepsilon) and CKIdelta in cultured cells Mol Cell Biol 22, 1693– 1703 32 Eide EJ, Woolf MF, Kang H, Woolf P, Hurst W, Camacho F, Vielhaber EL, Giovanni A & Virshup DM (2005) Control of mammalian circadian... proteins in mammalian cells Genes Dev 14, 1353–1363 36 Vielhaber E, Eide E, Rivers A, Gao ZH & Virshup DM (2000) Nuclear entry of the circadian regulator mPER1 3724 S Loop and T Pieler 37 38 39 40 41 42 is controlled by mammalian casein kinase I epsilon Mol Cell Biol 20, 4888–4899 Miyazaki K, Mesaki M & Ishida N (2001) Nuclear entry mechanism of rat PER2 (rPER2): role of rPER2 in nuclear localization of. .. localization of CRY protein Mol Cell Biol 21, 6651–6659 Loop S, Katzer M & Pieler T (2005) mPER1- mediated nuclear export of mCRY1 ⁄ 2 is an important element in establishing circadian rhythm EMBO Rep 6, 341–347 Gekakis N, Saez L, Delahaye-Brown AM, Myers MP, Sehgal A, Young MW & Weitz CJ (1995) Isolation of timeless by PER protein interaction: defective interaction between timeless protein and long-period mutant... PERIOD and CKIepsilon is critical for a functioning circadian clock Mol Cell Biol 24, 584–594 34 Takano A, Isojima Y & Nagai K (2004) Identification of mPer1 phosphorylation sites responsible for the nuclear entry J Biol Chem 279, 32578–32585 35 Yagita K, Yamaguchi S, Tamanini F, van Der Horst GT, Hoeijmakers JH, Yasui A, Loros JJ, Dunlap JC & Okamura H (2000) Dimerization and nuclear entry of mPER proteins . Nuclear import of murine PER and CRY proteins in Xenopus oocytes. mPER1 and mPER2, but not mPER3, are phosphorylated and imported into the nucleus of Xenopus oocytes. (A) 35 S-labelled mPER1, . the C-terminal region of mPER1. Complex formation with mPER1 promotes nuclear import of mPER3 in Xenopus oocytes As heterodimerization of clock proteins is known to modulate nuclear import activity,. function of mPER1 and not activation of a masked NLS in mPER3 is critical for the import of the mPER1 mPER3 complex. Finally, and as previously described in other cell systems, nuclear import of mPER