Sequences that direct subcellular traffic of the Drosophila methoprene-tolerant protein (MET) are located predominantly in the PAS domains

Molecular and Cellular Endocrinology 345 (2011) 16–26

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Sequences that direct subcellular traffic of the Drosophila methoprene-tolerant protein (MET) are located predominantly in the PAS domains a _ Beata Greb-Markiewicz a,⇑, Marek Orłowski a, Jerzy Dobrucki b, Andrzej Ozyhar a b

´ skiego 27, 50-370 Wrocław, Poland Department of Biochemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzez_ e Wyspian Department of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland

a r t i c l e

i n f o

Article history: Received 24 March 2011 Received in revised form 2 June 2011 Accepted 28 June 2011 Available online 2 July 2011 Keywords: Methoprene-tolerant bHLH-PAS transcription factor NLS NES Juvenile hormone 20-Hydroxyecdysone

a b s t r a c t Methoprene-tolerant protein (MET) is a key mediator of antimetamorphic signaling in insects. MET belongs to the family of bHLH-PAS transcription factors which regulate gene expression and determine essential physiological and developmental processes. The ability of many bHLH-PAS proteins to carry out their functions is related to the patterns of intracellular trafficking, which are determined by specific sequences and indicate that a nuclear localization signal (NLS) or a nuclear export signal (NES) is present and active. Therefore, the identification of NLS and NES signals is fundamental in order to understand the intracellular signaling role of MET. Nevertheless, data on the intracellular trafficking of MET are inconsistent, and until now there hasn’t been any data on potential NLS and NES sequences. To analyze the trafficking of MET we designed a number of expression vectors encoding full-length MET, as well as various derivatives, that were fused to yellow fluorescent protein (YFP). Confocal microscopy analysis of the subcellular distribution of YFP–MET indicated that while this protein was localized mainly in the nucleus, it was also observed in the cytoplasm. This suggested the presence of both an NLS and NES in MET. Our work has shown that each of the two PAS domains of MET (PAS-A and PAS-B, respectively) contain one NLS and one NES sequence. Additional NES activity was present in the C-terminal fragment. The NLS activity located in PAS-B was dependent on the presence of juvenile hormone (JH), the potential ligand for MET. In contrast to this, JH didn’t seem to be required for the NLS in PAS-A to be active. However, on the basis of current knowledge about the function of PAS-A in other bHLH-PAS proteins, we suggest there might be other proteins that control the activity of the NLS and possibly the NES located in the PAS-A of MET. Thus, the intracellular trafficking of MET seems to be regulated by a rather complicated network of different factors. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction In contrast to the complexity of vertebrate hormone signaling pathways, Drosophila melanogaster has only two known physiologically active lipophilic hormones, the steroid hormone 20-hydroxyecdysone (20E) and the sesquiterpenoid juvenile hormone (JH). They regulate insect development, reproduction, and other important biological processes (Dubrovsky, 2005; Gruntenko and

Abbreviations: 20E, 20-hydroxyecdysone; CFP, cyan fluorescent protein; EcR, ecdysteroid receptor; GFP, green fluorescent protein; GCE, germ cells expressed; NES, nuclear export signal; NLS, nuclear localization signal; Usp, ultraspiracle; YFP, yellow fluorescent protein; JH, juvenile hormone; MET, methoprene-tolerant; bHLH, basic helix-loop-helix; PAS, Per-Arnt-Sim (period-aryl hydrocarbon receptor nuclear translocator-single-minded); FKBP39, 39 kDa FK506-binding nuclear protein; Chd64, calponin-like protein Chd64; SRC, steroid receptor co-activator. ⇑ Corresponding author. Tel.: +48 713206226; fax: +48 713206337. E-mail address: [email protected] (B. Greb-Markiewicz). 0303-7207/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2011.06.035

Rauschenbach, 2008). Furthermore, the fly genome contains much fewer nuclear receptors genes than the human genome. Nevertheless, all the main nuclear receptor subfamilies are represented in D. melanogaster nuclear receptors (King-Jones and Thummel, 2005). This together with the well-established genetic and genomic tools for studying the biology of D. melanogaster makes this insect an ideal model system for characterizing nuclear receptor function and regulation. The molecular mechanisms of 20E action have been extensively studied (Beckstead et al., 2007). In contrast, the mechanisms of JH signaling are poorly understood and many basic questions remain unanswered (Davey, 2000; Dubrovsky, 2005; Flatt et al., 2005; Restifo and Wilson, 1998; Wilson et al., 2006a,b). At the molecular level JH is known to modify or suppress the expression of genes involved in 20E signal transduction (Dubrovsky et al., 2000; Restifo and Wilson, 1998). Recently, the JH response element has been identified (Li et al., 2007) as well as genes whose expression is directly influenced by JH (Zhou et al., 2002). Additionally, it has been shown that the response for JH regulation can be

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modified by 20E. Thus, the interplay between JH and 20E is more complex than previously thought (Beckstead et al., 2007). Many attempts have been made to identify JH receptors. One of the candidates for this function is methoprene-tolerant protein (MET) (Ashok et al., 1998; Miura et al., 2005; Shemshedini et al., 1990). The Met gene was identified by screening mutagenized D. melanogaster larvae for both the toxic and morphogenetic effects of methoprene (Shemshedini and Wilson, 1990; Wilson and Ashok, 1998; Wilson and Fabian, 1986). It has been shown that MET binds JH in physiological concentrations (Miura et al., 2005; Shemshedini et al., 1990). Initially, it was difficult to believe that MET was a JH receptor because of the fact that D. melanogaster MET null mutants were viable (Wilson, 1996; Wilson et al., 2006a,b). However, Konopova and Jindra (2007) have documented that MET is an essential mediator of antimetamorphic JH signaling in Tribolium castaneum, which supports its putative role as a JH receptor (Parthasarathy and Palli, 2009; Parthasarathy et al., 2008). It has been shown that MET is able to interact with its homolog, germ cell expressed (GCE) protein (Godlewski et al., 2006). Additionally, MET interacts with proteins involved in the signal transduction of ecdysteroids, i.e., ecdysteroid receptor (EcR), ultraspiracle (USP), 39 kDa FK506-binding nuclear protein (FKBP39), calponin-like protein Chd64 (Chd64) (Bitra and Palli, 2009; Li et al., 2007) and steroid receptor co-activator (SRC) (Li et al., 2011; Zhang et al., 2011). As revealed from a comparison of the MET sequence to the sequences of proteins deposited in databases, MET has been classified as a member of the family of bHLH-PAS transcription factors (Ashok et al., 1998). The domains of both bHLH (basic helix-loophelix) and PAS (named for Per-Arnt-Sim, the first three proteins from this family) play important roles in the transcriptional activity of these kinds of transcription factors (Gu et al., 2000; Kewley et al., 2004). Interestingly, the MET sequence exhibits higher homology to vertebrates than other D. melanogaster bHLH-PAS proteins (Ashok et al., 1998). The bHLH-PAS proteins are critical regulators of the gene expression networks that are responsible for many essential physiological and developmental processes in invertebrates (Furness et al., 2007). For example, the aryl hydrocarbon receptor (AhR) regulates transcriptional responses to environmental pollutants (Hahn, 1998) and has been shown to be a modulator of anti-viral immunity (Head and Lawrence, 2009). The hypoxia inducible factor (HIF) is a bHLH-PAS receptor for low oxygen tension (Bracken et al., 2003; Déry et al., 2005), whereas single-minded (Sim) is responsible for neuronal development (Chrast et al., 1997; Nambu et al., 1991). Many aspects of the functional control of bHLH-PAS proteins are analogous to those defined for nuclear receptor signaling (Kewley et al., 2004). The bHLH-PAS proteins usually dimerise to form functional DNA binding complexes. The PAS domain shows the specificity of the complex formation and the distinct recognition of target genes (Chapman-Smith and Whitelaw, 2006; Zelzer et al., 1997). The ability to localize and translocate proteins to specific cellular compartments is fundamental to the organization and functioning of all living cells. For a number of transcription factors that mediate inducible gene regulation in response to extracellular signals, translocation from the cytoplasm to the nucleus is an important event, enabling the transcription factors to recruit coactivators (Kallio et al., 1998; Lee and Hannink, 2003). For example, it has been shown that steroid/nuclear receptors continuously shuttle between the cytoplasm and the nucleus and that their localization at any given point in time is a consequence of the fine balance between the operational strength of the sequences for the nuclear localization signal (NLS) and the nuclear export signal (NES) (Kumar et al., 2006). In the case of AhR, which belongs to the bHLH-PAS family, its subcellular localization is differentially regulated by one NLS and two NESs and depends on the interaction

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of other proteins (Berg and Pongratz, 2001; Ikuta et al., 1998; Kawajiri and Fujii-Kuriyama, 2007). Current knowledge about the subcellular traffic of MET has been very limited. The first study on the localization of the MET protein was done by analyzing JH binding in larval fat bodies, and localization was determined to be in the fat body cytosol (Shemshedini et al., 1990). The photoaffinity labeling technique made it possible to localize MET both in cytoplasmic (Shemshedini and Wilson, 1990) and nuclear (Shemshedini and Wilson, 1993) fractions of D. melanogaster cells. In contrast, immunolocalization studies of MET in a variety of D. melanogaster tissues showed this protein to be exclusively nuclear (Pursley et al., 2000). Miura et al. (2005) confirmed this using Drosophila Schneider cells and GFP (green fluorescent protein)-labeled MET. In order to better determine the presence of NLS and NES signals in the MET protein we decided to use mammalian cells, which do not produce juvenile hormone and 20-hydroxyecdysone. These cells are also devoid of some insect cell-specific endogenous proteins like GCE and the EcR/USP complex, which could influence results obtained in insect cells. Recently, mammalian cells have been successfully used to analyze the subcellular trafficking of EcR and Usp, nuclear receptors from D. melanogaster (Dutko-Gwóz´dz´ et al., 2008; Gwóz´dz´ et al., 2007; Nieva et al., 2005, 2007, 2008). Similarly, as was described for EcR and Usp, we decided to investigate the subcellular distribution of MET in living cells using yellow and cyan fluorescent proteins (YFP, CFP), which have been shown to be useful tags for monitoring the subcellular distribution of various proteins in living cells (Chalfie et al., 1994). This study is the first detailed characterization of the subcellular traffic of D. melanogaster methoprene-tolerant protein. We have shown that MET contains both NLS and NES activities which are localized in the PAS-A and PAS-B regions of MET. For the first time, we have documented that MET can translocate from the cytoplasm to the nucleus, and this process seems to be mediated through a mosaic of elements in a JH-dependent and/or JH-independent manner. 2. Materials and methods 2.1. Plasmid construction MET cDNA from D. melanogaster was a kind gift from Prof. Thomas G. Wilson (Department of Entomology, Ohio State University, USA). Full-length cDNA, encoding amino acid residues 1–716, was amplified by PCR and cloned into the EcoRI and SmaI restriction sites of the MCS of the pEYFP-C1, pEYFP-N1, pECFP-C1 and pECFP-N1 vectors. Deletion mutants of MET were cloned analogically in the pEYFP-C1 vector. Primers used for PCR are listed in Table 1 (Supplementary data). DNA constructs: YFP–MET/R98A/K102A, YFP–MET34–190/R98A/K102A and YFP–MET98–508/R98A/K102A, were obtained analogically to the full-length and deletion mutants, and for the PCR reaction, the mutated template MET/R98A/K102A was used thanks to a gift from Jakub Godlewski (Department of Entomology, Ohio State University, USA). The point mutants listed in Table 2 (Supplementary data) were obtained by the PCR of point mutation insertions according to Ko and Ma (2005) and cloned with LguI, EcoRI and SmaI restriction enzymes. All constructs were verified by DNA sequencing. 2.2. Cell culture and DNA transfection Chinese hamster ovary cells (CHO-K1), (ATCC CCL-61) were maintained in Ham’s F12 medium. African green monkey kidney fibroblasts COS-7 (ATCC CRL-1651) and human cervix adenocarci-

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noma HeLa cells (ATCC CCL-2) were maintained in Dulbecco’s modified Eagle medium (DMEM) supplemented with 1% nonessential amino acids (Gibco/Invitrogen), 1 mM sodium pyruvate and 2% glutamine (Gibco/Invitrogen). Both media were supplemented with fetal calf serum (FCS), 5% for CHO-K1 cells and 10% for COS-7 and HeLa cells. Cells were grown at 37 °C in a 95% air/ 5% CO2 atmosphere. Cells were transfected with 6 lg DNA/ 500,000 cells using jetPEI (Polyplus-transfection SA) according to the manufacturer’s instructions. Juvenile hormone III (JHIII) (Serva) was dissolved in DMSO (Sigma) to a concentration of 10 3 M and added during transfection to the medium to a final concentration of 10 6 M. 2.3. Confocal fluorescence microscopy Before conducting the imaging experiments cells were plated on 0.17-mm-thick round glass coverslips (Mentzel) submerged in a culture medium in 4-cm-diameter petri dishes. Prior to the microscopy experiment (20–24 h after transfection), coverslips with cell cultures were transferred into a steel holder and mounted in a microscope stage microincubator (Life Science Resources). The standard culture medium was replaced by DMEM/F12 buffered with 15 mM HEPES without phenol red (Sigma). During microscopy studies, the temperature of the cell cultures was maintained at 37 °C. Images of fluorescently labeled proteins were acquired using an MRC1024 confocal system (Bio-Rad), built on a Nikon Diaphot 300 inverted microscope (Nikon), and equipped with a 100-mW argon ion laser (ILT). A 60 PlanApo oil-immersion NA 1.4 objective lens was used. YFP fluorescence was excited using light at 514 nm and the emitted fluorescence was observed. 2.4. Electrophoresis and Western blot In tandem with the microscopy experiments SDS–PAGE and Western blot were performed to proof the expression of MET and MET mutants in cultured cells. For protein analysis, cells were solubilized 24 h after transfection in a lysis buffer (20 mM HEPES, pH 7.9, 20% Glycerin, 1% Nonidet P-40, 1 mM MgCl2, 0.5 mM EDTA, 0.1 M DTT) with the addition of 0.1 mM PMSF and the proteinase inhibitor cocktail Complete Mini (Roche), incubated for 30 min

on ice and then centrifuged 20 min at 4 °C 20,000g. The supernatant was put in new Eppendorf tubes and stored at 20 °C. The lysate was mixed with the SDS 4 gel loading buffer (Laemmli, 1970), boiled for 5 min and centrifuged for 5 min (13,000g). Proteins were separated by a 10% SDS–PAGE and transferred to a Protran Nitrocellulose Transfer Membrane (Schleicher and Schuell GmbH) with a mini Trans-Blot apparatus (Bio-Rad). The membrane was incubated overnight at 4 °C with the anti-GFP polyclonal antibody (Clontech) diluted 1:400 with a milk buffer which cross-reacts with CFP and YFP. The secondary goat anti-rabbit antibody coupled to a horseradish peroxidase (Vector Laboratories) was added (1:10,000) and incubated for 1 h at room temperature. Blots were developed using the ECL-plus chemiluminescence kit (Amersham Biosciences). 2.5. In silico analysis of the MET sequence To predict the secondary structure of MET we used PSIPRED (Protein Structure Prediction Server) (Jones, 1999), http:// www.psipred.net/psiform.html. To predict the domain architecture of MET we used SMART (simple modular architecture tool) (Schultz et al., 1998), http://smart.embl-heidelberg.de and PROSITE (a database of protein families and domains), http://expasy.org/ tools/scanprosite/. The sequence alignments were obtained by CLUSTAL_X (Thompson et al., 1997) http://www.clustal.org/. Predictions of the potential NLS signals were performed by NucPred (Brameier et al., 2007), http://www.sbc.su.se/~maccallr/nucpred/ and PSORTII (Nakai and Horton, 1999), http://www.psort.org/. Predictions of the potential NES signals were performed by the NetNes 1.1 server (La Cour et al., 2004), http://www.cbs.dtu.dk/services/ NetNES/. 3. Results 3.1. MET contains both NLS and NES sequences The expression of full-length MET tagged with the YFP and/or CFP in COS-7 cells was analyzed by Western blot using the antiGFP antibody. After the fusion of the YFP or CFP to the C-terminus

Fig. 1. Analysis of the subcellular distribution of full-length MET tagged with YFP in COS-7, CHO-K1 and HeLa cells. (A) Typical confocal images of cells expressing fusion proteins 20 h after transfection. (B) Confocal images of cells presenting an atypical cytoplasmic localization of expressed fusion proteins. Images were obtained using a confocal microscope 20 h after cells transfection. A 60 PlanApo oil-immersion NA 1.4 objective lens was used. YFP fluorescence was excited by a 514 nm wavelength light, and the emitted fluorescence was detected. Bar, 10 lm.

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of MET there was no specific band observed corresponding to the respective fusion protein, probably due to the process of degradation (data not shown). In contrast, the fusion proteins consisting of MET and CFP and/or YFP attached to the N-terminus of MET appeared to be more stable. Since the MET derivative containing YFP appeared principally as a single band corresponding to the full-length YFP–MET, the N-terminal tagged YFP derivatives were further used in all the experiments described below (data not shown). The stability of the MET derivatives was also checked in CHO-K1 and HeLa cells, and the results were essentially the same (data not shown). To test the subcellular localization of YFP–MET, COS-7 cells were transfected with an appropriate plasmid, and then, the distribution of YFP–MET was analyzed by confocal fluorescence microscopy. Results (presented in Fig. 1A) obtained 24 h after transfection indicate that YFP–MET was found in the nucleus or predominantly in the nucleus for more than 90% of the analyzed cells. In some cells YFP–MET was localized predominantly in the cytoplasm, and sometimes it was in the form of clusters (Fig. 1B).

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The pattern observed for COS-7 cells remained constant for at least 8 h after the first visible signs of expression. Similar experiments were also carried out for CHO-K1 and HeLa cells with essentially the same results (Fig. 1A and B). Based on the above results, we presume that there is a high probability that MET contains a sequence (or several sequences) that is responsible for its translocation to the nucleus (NLS) and for being active in all cell lines that were investigated. The appearance of cells where localization was predominantly in the cytoplasm and the lack of cells with a homogeneous distribution (Fig. 1 and data not shown) suggest that this protein may also contain an export signal(s) (NES). 3.2. Nuclear import and export signals are localized in different parts of MET The presence of NLS and NES signals has not been determined for MET until now. In order to identify these structural motifs we carefully prepared a series of deletion mutants (schematically pre-

Fig. 2. Analysis of the subcellular distribution of various MET deletion mutants tagged with YFP in COS-7 cells. (A) Schematic representation of full-length MET and its derivatives tagged with YFP. Individual regions of deletion were defined according to Ashok et al. (1998) and PSIPRED (http://www.psipred.net/psiform.html). The length of each domain in the diagram is arbitrary. (B) Confocal images of the subcellular distribution of the MET derivatives depicted in (A). Bar, 10 lm. For more details see text.

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sented in Fig. 2A). Regions of truncations were planned out according to the putative secondary structure motifs of MET in such a way that the fragments encompassed whole domains of the MET protein (Ashok et al., 1998). Some additional amino acid residues were included based on some of the predictions in the PROSITE and SMART databases (not shown). Consequently, we divided the MET sequence into six fragments corresponding to structural and/or functional motifs (Fig. 2A a). The N-terminal fragment encompassing amino acid residues 1–33 and containing many S and G residues does not have a known function. The bHLH domain occupies the area between residues 34 and 97. The 98–190 fragment is composed not only of the PAS-A domain but also of amino acid residues that connect bHLH with PAS-A. However, for simplicity’s sake the 98–190 fragment is referred to as PAS-A. The amino acid residues from 191 to 402 correspond to an area which has neither a defined structure nor a known function. The MET fragment composed of amino acid residues 403–508 corresponds to PAS-B. In other proteins belonging to the bHLH-PAS family this fragment is often responsible for ligand binding (Soshilov and Denison, 2008). The last fragment is the C-terminal part of MET encompassing amino acid residues 509–716, which has neither a defined structure nor a known function. All fragments were then N-terminally tagged with YFP (Fig. 2A c–h), and their subcellular localization was analyzed using confocal microscopy. In contrast to the full-length protein (see Figs. 2B a and 1A, B) MET fragments tagged with YFP revealed clear patterns of localiza-

tion in the COS-7 cells (Fig. 2B). The N-terminal fragment of MET alone (Fig. 2A c) and the N-terminal fragment connected with the bHLH region (Fig. 2A i) were observed both in the nucleus and in the cytoplasm (Fig. 2B c and i) analogically to the YFP protein expressed as a control (Fig. 2B b). However, bHLH alone (Fig. 2A d) resulted in fluorescence which was slightly more intensive in the nucleus than in the cytoplasm (Fig. 2B d). Clearly, nuclear localization was obtained for the MET fragment encompassing the PAS-A domain (Fig. 2A e and B e). The extension of PAS-A with the bHLH domain (Fig. 2A j) did not change this localization (Fig. 2B j). The region between the PAS-A and PAS-B domains (Fig. 2A f) doesn’t seem to have any localization signals (Fig. 2B f), and when connected only to PAS-A (Fig. 2A k) the resulting protein appeared in the nuclei of analyzed cells similarly as was observed for PAS-A alone (Fig. 2B k and compare with Fig. 2B e). The fragment encompassing PAS-B (Fig. 2A g) and the fragment encompassing the Cterminal part of MET (Fig. 2A h) was localized exclusively in the cytoplasm (Fig. 2B g and B h), and this was also the case when they were connected together (Fig. 2B m). The same was also true for PAS-B expressed together with the MET fragment encompassing the sequence that separates the PAS-B and PAS-A domains. (Fig. 2A l and B l). The expression of these fragments in CHO-K1 and HeLa cells analogically showed the same results (data not shown). Collectively, these results indicate that MET might contain both NLS and NES signals, and the activity of these signals may influence the distribution of MET in cells.

Fig. 3. Analysis of the subcellular distribution of bHLH-PAS-A area deletion and point mutants. (A) Schematic representation of full-length MET and its derivatives tagged with YFP. Regions of MET are depicted using different patterns. Individual regions of deletion were defined according to Ashok et al. (1998) and PSIPRED (http:// www.psipred.net/psiform.html). The length of each domain in the diagram is arbitrary. (B) Confocal images of the deletion fragments of YFP–MET/34–190 and fragments with point mutations R98A and K102A. Distribution of fusion proteins was determined in COS-7 cells. Bar, 10 lm. (C and D) A comparison of the MET sequence with the NLS from SV40 T large antigen and consensus Leu-rich NES using CLUSTAL_X (Brameier, 2007; Thompson et al., 1997). Asterisks indicate identical residues, and colons indicate similar residues. Residue numbering corresponding to the sequence of MET is given at the bottom of the alignment. Red asterisks indicate the mutated residues. For more details see text. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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3.3. The bHLH-PAS-A fragment possesses both NLS and NES signals To continue screening for NLS activity we paid close attention to the fragment encompassing the PAS-A domain which displayed clear nuclear localization (see above). To determine the position of the potential nuclear localization signal in this domain we decided to divide it into four fragments according to the PSIPRED (Jones, 1999) secondary structure prediction (not shown). Fragments were then N-terminally tagged with YFP, and their subcellular localization was analyzed using confocal microscopy. A schematic representation of the respective mutants is shown in Fig. 3A a–d. The localization of fragments encompassing amino acids 98–158 (Fig. 3B a) and 98–126 (Fig. 3B b) was strictly nuclear, whereas fragments 127–158 and 159–190 were observed both in the nucleus and cytoplasm (Fig. 3B c and d), which led us to the hypothesis that the nuclear localization of the PAS-A fragment is dependent on the presence of amino acid residues located between residues 98 and 126. Analysis of the primary structure of the 98– 126 sequence identified a cluster of basic amino acids (98–102) presenting high homology to the monopartite NLS from SV40 T large antigen (Kalderon et al., 1984) (Fig. 3C). Although the PSORTII predictor was not able to detect this sequence as an NLS (Nakai and Horton, 1999), the NucPred program predicted this sequence as promoting nuclear localization with a probability of 0.82 (Brameier et al., 2007; Heddad et al., 2004). Experimental proof for this hypothesis was obtained with a construct including a 34–190 fragment where R98 and K102 residues were substituted with A (Fig. 3A f). Contrary to the wild type fragment (Fig. 3A e) that accumulated in the nucleus (Fig. 3B e), the expressed mutant was located predominantly in the cytoplasm, which indicated that there was disruption of the NLS activity (Fig. 3B f) and suggested the presence of NES activity. We did not expect any NES activity

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in this area, based on the results we had obtained earlier (see above Fig. 3AB a–c). Program NetNes1.1 predicted the putative leucinerich NES signal (Bogerd et al., 1996) starting from L130 (see Fig. 3D). However, the fusion protein containing the MET fragment from amino acid residues 127–158 (Fig. 3A c) was distributed equally in both compartments of transfected cells (Fig. 3B c). A detailed sequence analysis of this area indicates that the presence of the L126 residue might be crucial for NES signal activity (Fig. 3D), and that in previous experiments (Fig. 3A c and B c) we had interrupted the putative NES signal by cutting off L126. Thus, for experimental proof of the presence of the NES we substituted L130 and 133 with A in the YFP–MET34–190/R98/K102A construct, which also included the substitution of L126 with A (Fig. 3A g). As shown in Fig. 3B g this construct was distributed equally. Thus, we have identified the sequence 98-RRRKK-102 as having NLS activity and the sequence 126-LTDTLMQLLDCCFL-139 as having NES activity. 3.4. PAS-B possesses NES and NLS signals As shown above (Fig. 2B g) the fusion protein YFP–MET/403– 508 encompassing the whole PAS-B region accumulated predominantly in the cytoplasm. To identify the putative NES in PAS-B, we decided to create deletion mutants by dividing PAS-B into three fragments according to the PSIPRED secondary structure prediction (not shown). Again, the fragments were N-terminally tagged with YFP, and their subcellular localization was analyzed using confocal microscopy. The schematic presentation of the respective mutants and the expression of the chimeric fragment proteins is presented in Fig. 4A and B, respectively. For fragment 403–441 (Fig. 4A a) we observed homogenous distribution in all analyzed cells (Fig. 4B a). Because of this and the fact that it didn’t seem to possess an NLS or NES, we decided to exclude it from further experiments. The frag-

Fig. 4. Analysis of the subcellular distribution of the PAS B area deletion and point mutants. (A) Schematic representation of full-length MET and PAS-B derivatives tagged with YFP. Individual regions of deletion were defined according to PSIPRED (http://www.psipred.net/psiform.html). The length of each domain in the diagram is arbitrary. (B) Confocal images of the MET derivatives depicted in (A). The distribution of fusion proteins was determined in COS-7 cells. Bar, 10 lm. (C and D) A comparison of the MET sequence with the bipartite NLS from Nucleoplasmin and consensus Leu-rich NES using CLUSTAL_X (Thompson et al., 1997), respectively. Asterisks indicate identical residues, and colons indicate similar residues. Residue numbering corresponding to the sequence of MET is given at the bottom of the alignment. Red asterisks indicate the mutated residues. For more details see text. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

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Fig. 5. Analysis of the subcellular distribution of the MET C-terminal area deletion and point mutants. The length of each domain in the diagram is arbitrary. Individual regions of deletion were defined according to PSIPRED (http://www.psipred.net/psiform.html). (B) Confocal images of MET mutants depicted in (A). Bar, 10 lm. (C) A comparison of the MET sequence with the RXR (Retinoid X Receptor) type NES using CLUSTAL_X (Thompson et al., 1997). Asterisks indicate identical residues, and colons indicate similar residues. Residue numbering corresponding to the sequence of MET is given at the bottom of the alignment. Red asterisks indicate the mutated residues. For more details see text. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

ment encompassing amino acids 442–465 (Fig. 4A b) was localized predominantly in the cytoplasm (Fig. 4B b), indicating the presence of NES activity. The NetNes1.1 server prediction performed for this fragment showed a positive result for the sequence 446–456 (VRWVIVALRQM). A comparison of this sequence with consensus leucine-rich NES (Bogerd et al., 1996) in CLUSTAL_X is presented in Fig. 4C. The MET fragment encompassing residues 466–508 (Fig. 4A c) exhibited predominantly nuclear distribution (Fig. 4B c) and indicated the presence of NLS activity, which was in agreement with our hypothesis that the putative NLS is present in this region of PAS-B. For final proof of the activity of localization signals in PAS-B, mutants of the MET fragment 403–508 were constructed. When V446 and I450 of the putative NES were substituted with residue A (Fig. 4A e), the mutant no longer localized in the cytoplasm. Contrary to the wild type localized in the cytoplasm (Fig. 4B d), fluorescence was observed mainly in the nucleus (Fig. 4B e). Thus, creating this mutant not only proved the existence of an active NES in PAS-B, but also allowed us an opportunity to observe the activity of a potential NLS. Predictors for an NLS in MET were not able to find any similarity with other known NLS’s in databases (not shown). However, the alignment of the fragment of the MET sequence 466–508 with bipartite Nucleoplasmin NLS (Dingwall et al., 1987) indicated some partial similarity (Fig. 4D). To validate our hypothesis, a mutant (YFP–MET403–508/V446A/I450A/G491A/ K494A/H496A) was prepared (Fig. 4A f) with YFP–MET403–508/ V446A/I450A as a background, where both the NES and putative NLS signals were switched off. The mutant protein displayed equal distribution in transfected cells (Fig. 4B f) demonstrating that there wasn’t an active localization signal. Thus, we demonstrated that in the PAS-B domain of MET there is one NES and one NLS. 3.5. A search for the NES signal in the C-terminal fragment of MET As shown above the C-terminal part of MET encompassing residues 509–716 localized strictly in the cytoplasm (see Fig. 2B h), although initially in silico analyses with the NetNes1.1 server did not predict any NES activity residing in this area (not shown). To

further narrow down the region responsible for cytoplasmic retention, we decided to divide the C-terminal part into two fragments, one containing residues 509–694 (Fig. 5A a) and one with 695–716 (Fig. 5A b). Knowing that a typical consensus NES is rich in L residues and that the sequence of the last amino acid residues from 695 to 716 contains many residues of L (not shown), we expected these fragments to accumulate in the cytoplasm. Surprisingly, the results contradicted our assumption. The localization of expressed YFP–MET509–694 was cytoplasmic (Fig. 5B a), whereas YFP-connected to fragment YFP–MET695–716 exhibited fluorescence in both compartments of the cells (Fig. 5B b). A comparison of known NES sequences and the MET C-terminal part with CLUSTAL_X helped us to identify a fragment in the region of interest which presented some similarity to the retinoid X receptor NES (Fig. 5C). We examined this sequence by altering the amino acid residues we suspected were part of the hypothesized NES signal and created the point mutant YFP–MET508–716/R550A/V552G/ L555A (Fig. 5A c). Unfortunately, the obtained results didn’t satisfy our hypothesis as the expressed mutant was still located strictly in the cytoplasm of the cell (Fig. 5B c). These data indicate that either the analyzed sequence was not an active NES or the altered amino acid residues were not necessary for the suggested signal activity. So, we were not able to identify a sequence which directs MET/ 508–716 to the cytoplasm, and this needs further research. 3.6. The orchestration of the MET protein localization signal and the influence of juvenile hormone As we detected more than one NLS and one NES in MET, we decided to investigate the interplay of these localization signals. We prepared a series of constructs with point mutations in the NLS and NES sequences. To simplify the system we truncated the C-terminal fragment to avoid the effect of an unidentified NES. We also truncated the N-terminal fragment since it didn’t appear to contain a sequence involved in subcellular trafficking. The MET derivatives were then N-terminally tagged with YFP and their subcellular localization was analyzed using confocal microscopy. A schematic representation of the mutants is shown in Fig. 6A a–c. As

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shown in Fig. 6B a, the deletion mutant without a point mutation (Fig. 6A a) accumulated in the nucleus, which indicates that the NLS residing in the PAS-A domain of MET (98-RRRKK-102; NLS-1 see Fig. 7) apparently has a dominant character. This can be deduced from other experiments where the disruption of NLS-1 in the construct represented in Fig. 6A b allowed us to observe cytoplasmic localization (Fig. 6B b) coming from active NES’s (129LTDTLMQLLDCCFL-139; NES-1 and 446-VRWVIVALRQM-456; NES-2 see Fig. 7) present in the examined mutant that were dominant over the remaining NLS activity (NLS-2 see Fig. 7). We then substituted V446 and I450 (NES in PAS-B) with A residues in the construct mentioned above, again disrupting the NLS-1 (Fig. 6A c). The subsequent mutant protein again displayed nuclear localization indicating the dominant nature of NLS-2 (see Fig. 7) located in the PAS-B domain (Fig. 6B c). To further investigate whether the disruption of NLS-1 (98RRRKK-102) in the full-length protein would give analogous results to those presented above, we decided to prepare an N-terminally tagged construct with K98 and L102 substituted with A (Fig. 6A e). Subcellular localization was again analyzed using confocal microscopy. For the full-length mutant whose NLS-1 in the bHLH-PAS-A area had been switched off, as had been similarly observed for the deletion mutant with the K98 and L102 mutation

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(Fig. 6B b), the distribution of the expressed protein was definitely cytoplasmic (Fig. 6B e). Most likely, the NES activity (in PAS-A and PAS-B) was enhanced by the additional presence of an unidentified sequence in the C-terminal fragment (see Section 3.5). Previous studies Pursley et al. (2000) and Miura et al. (2005) have shown that MET localizes in the nucleus both in the presence and in the absence of juvenile hormone (JH). We were interested to see if the wild type MET and its NLS mutant in PAS-A (NLS-1, 98RRRKK-102) containing a hidden NLS localized in PAS-B (NLS-2, 482-TKFLEVDRGSNKVHSF-498) would be distributed similarly with and without JH. The results of our research for wild type MET (Fig. 6A d) were in complete agreement with previously published data (Miura et al., 2005; Pursley et al., 2000). The expressed protein localized predominantly in the nucleus, both in the absence (Fig. 1A) and in the presence of the hormone (Fig. 6B d). As discussed above, the MET mutant in PAS-A with hidden NLS activity in PAS-B (NLS-2) (Fig. 6A e) was localized in the cytoplasm in the absence of JH (Fig. 6B e). However, in the presence of JH this MET derivative (Fig. 6A f) was observed predominantly in the nuclei of analyzed cells (Fig. 6B f). Apparently, JH might have induced a change in the MET structure which eliminated the dominating NES activity in MET and activated NLS-2 localized in PAS-B. Together, the obtained results indicate that there might be both hor-

Fig. 6. Influence of JH on the subcellular localization of the wild type and mutant MET. Regions of MET are depicted using different patterns. The length of each domain in the diagram is arbitrary. (B) Confocal images of the chimeric proteins depicted in (A). Bar, 10 lm. The distribution of fusion proteins was determined in COS-7 cells. Juvenile hormone concentration was 10 6 M. For more details see text.

Fig. 7. Schematic representation of NLS and NES signals residing within the MET protein. Bold and underlined letters in sequences indicate residues which were mutated in this study. For more details see text.

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mone-dependent and hormone-independent elements in MET that control its subcellular traffic. The first NLS (NLS-1) near the PAS-A domain is a dominant nucleus import signal in MET. The NLS-1 activity does not seem to require the presence of JH. The second NLS we identified in the PAS-B domain seems to be JH-dependent.

4. Discussion D. melanogaster MET protein belongs to the bHLH-PAS family of transcription factors. The subcellular localization of such proteins is crucial to the performance of their physiological roles. In the present study we have demonstrated that the wild type MET protein resides primarily in the nucleus of transfected cells, even though some cells showed a cytoplasmic pattern of distribution. These observations have led us to the hypothesis that MET contains both an NLS and an NES. The results of the experiments we performed have proven the presence of a monopartite Simian Virus 40 T large antigen type NLS (NLS-1, Fig. 7) (Kalderon et al., 1984) that is mapped to amino acid residues 98–102 (RRRKK) and closely associated with bHLH. JH does not seem to be required for the NLS to be active. As presented in the introduction, MET is able to interact with itself as well as with other proteins. In the case of localization of an NLS or NES close to the surface of protein interaction with other proteins, the signals may be masked. The dominant character of the NLS-1 signal could result from its localization outside of the core PAS-A domain, which is typically responsible for the dimerization of proteins belonging to the bHLH-PAS family, and inside the loop which connects bHLH and PAS-A. Conversely, the signal might be modulated by partner-induced conformational changes. In addition, the MET protein contains a second NLS (NLS2, Fig. 7) in the terminal part of the PAS-B region (482TKFLEVDRGSNKVHSF-498) which might be JH-dependent. This seems to be consistent with reports indicating that the PAS-B domain of bHLH-PAS proteins is often responsible for ligand binding. Our search for the presence of an NES signal revealed two NES’s similar to the recognized consensus (LX(1–3)LX(2–3)LXL of the Rev-like NES. The first one (NES-1; 126-LTDTLMQLLDCCFL-139, Fig. 7) was localized in the PAS-A domain, which as discussed above, is usually responsible for the dimerization of bHLH-PAS proteins. Thus, this NES-1 function could depend on the presence of partner proteins that are necessary to mask or unmask its activity. The second NES (NES-2; 446-VRWVIVALRQM-456, Fig. 7) was localized in the central part of the PAS-B domain. Interestingly, NLS-1 was placed next to NES-1, and NLS-2 was placed next to NES-2, which suggests that their activity may be orchestrated by some modulating factors like the interaction of partner proteins with PAS domains and/or the binding of JH by the PAS-B domain. Additionally, the presence of some unidentified NES activity (NES-3) in the C-terminal part of MET can’t be excluded. However, we were not able to specify the sequence of a third potential NES (Fig. 7). The bHLH-PAS proteins have to be localized in the nucleus to carry out their role as transcriptional regulators. To date, many different mechanisms for nuclear localization have been reported. For example, ARNT contains a bipartite NLS in the bHLH region and localizes in the nucleus (Eguchi et al., 1997). AhR possesses both an NLS and NES in the bHLH domain and a second NES in the PAS-B domain. In this case, AhR is able to shuttle between the cytoplasm and the nucleus and its TCDD ligand has to be bound in order for AhR to be retained in the nucleus (Berg and Pongratz, 2001; Ikuta et al., 1998). HIF-1a contains one NLS in the bHLH domain and one NLS in the C-terminal part, which is responsible for transporting HIF-1a to the nucleus when exposed to hypoxia. It was shown that the PAS domain harbors a structure repressing nuclear import mediated by the NLS in bHLH and that removal of this part

resulted in uncoupling the protein from hypoxia, but more precise experiments were not performed at that time (Kallio et al., 1998). Neuronal PAS domain protein (NPAS1), a transcriptional repressor, contains an NES in the PAS-B domain and is localized in the cytoplasm. After heterodimerization with ARNT, NPAS1 is transported to the nucleus (Teh et al., 2006). Here, we have shown for the first time, that MET can translocate from the cytoplasm to the nucleus, and this process is mediated through a mosaic of NLSs and NESs in a JH-dependent and/or JHindependent manner. Interestingly, we have not detected an NLS signal inside the bHLH domain, which is typical in other bHLHPAS proteins, and the amount of basic amino acids in MET in the basic bHLH domain is not that high in comparison to typical bHLH proteins. The 98-RRRKK-102 sequence we have identified as a dominant NLS localized close to the PAS-A domain is unique for MET (Blast data not shown). The presence of an NES in PAS-A encompassing the LXXLL motif, described as mediating the protein–protein interaction of transcriptional cofactors with nuclear receptors (Plevin et al., 2005) is very intriguing. At this point there has been no documentation on the interaction of MET with another protein with the LXXLL motif; however, we can’t preclude this as a possibility. Ikuta et al. (2002) has shown for AhR that the LXXLL[50–54] motif in contrast to the LXXLL[224–228] motif is important for regulating the localization and transcriptional activation of AhR. In contrast to AhR and HIF-1a, which are not able to homodimerise, MET is able to form homodimers and heterodimers like Tango (Drosophila homolog of Arnt) without interacting with it. Thus, it seems that MET belongs to another, probably new, group of bHLH-PAS transcription factors. Miura et al. (2005) indicated that MET possesses a transactivation domain whose function is dependent on the presence of JH. They also suggest that unliganded MET may function as a transcriptional repressor. Until now no homolog for MET has been found in vertebrates. The present study has enabled us to show that MET is shuttling protein and the final intracellular localization of MET probably depends on a new and complex mode of multi-step regulation determined by the combined impact of nuclear localization signals, whose activity can be modified by other proteins/partners, JH, and post translational factors. Additional detailed research is needed on this subject. Acknowledgements The technical assistance of Mrs. Barbara Czuba-Pełech, Eng., Jadwiga Oczos´, M.Sc. and Mrs. Mirosława Ostrowska, Eng. is gratefully acknowledged. This work has been supported by the Wrocław University of Technology. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.mce.2011.06.035. References Ashok, M., Turner, C., Wilson, T.G., 1998. Insect juvenile hormone resistance gene homology with the bHLH-PAS family of transcriptional regulators. Proc. Natl. Acad. Sci. USA 95, 2761–2766. Beckstead, R.B., Lam, G., Thummel, C.S., 2007. Specific transcriptional responses to juvenile hormone and ecdysone in Drosophila. Insect Biochem. Mol. Biol. 37, 570–578. Berg, P., Pongratz, I., 2001. Differential usage of nuclear export sequences regulates intracellular localization of the dioxin (aryl hydrocarbon) receptor. J. Biol. Chem. 276, 43231–43238. Bitra, K., Palli, S.R., 2009. Interaction of proteins involved in ecdysone and juvenile hormone signal transduction. Arch. Insect Biochem. Physiol. 70, 90–105. Bogerd, H.P., Fridell, R.A., Benson, R.E., Hua, J., Cullen, B.R., 1996. Protein sequence requirements for function of the human T-cell leukemia virus type 1 Rex

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