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• W2000536319 abstract "Id proteins function as negative regulators for basic helix-loop-helix transcriptional factors that play important roles in cell fate determination. They preferentially associate with ubiquitously expressed E proteins of the basic helix-loop-helix family and prevent them from binding to DNA and activating transcription. Although their small size suggests that Id proteins enter and exit the nucleus by passive diffusion, several studies have indicated that other pathways may regulate their subcellular localization. In this study, we obtained evidence that Id2 has the ability to shuttle between the nucleus and the cytoplasm. When passive diffusion was prevented by fusion with green fluorescent protein (GFP), Id2 was predominantly localized in the cytoplasm. Using GFP fusion constructs, we demonstrated that the C-terminal region is required for cytoplasmic localization. Nuclear accumulation of GFP-Id2 in cells treated with the nuclear export inhibitor leptomycin B suggests that the nuclear export receptor chromosome region maintenance protein 1 mediates the cytoplasmic localization of Id2. Id2 contains two putative leucine-rich nuclear export signals, and the nuclear export signal in the C-terminal region is essential for nuclear export. On the other hand, the helix-loop-helix domain is important for nuclear localization. Finally, experiments using reporter assays revealed an inverse correlation between nuclear export and transcriptional repression via the E-box sequence. Based on all these findings, we propose that nucleo-cytoplasmic shuttling is a novel mechanism for the regulation of Id2 function. Id proteins function as negative regulators for basic helix-loop-helix transcriptional factors that play important roles in cell fate determination. They preferentially associate with ubiquitously expressed E proteins of the basic helix-loop-helix family and prevent them from binding to DNA and activating transcription. Although their small size suggests that Id proteins enter and exit the nucleus by passive diffusion, several studies have indicated that other pathways may regulate their subcellular localization. In this study, we obtained evidence that Id2 has the ability to shuttle between the nucleus and the cytoplasm. When passive diffusion was prevented by fusion with green fluorescent protein (GFP), Id2 was predominantly localized in the cytoplasm. Using GFP fusion constructs, we demonstrated that the C-terminal region is required for cytoplasmic localization. Nuclear accumulation of GFP-Id2 in cells treated with the nuclear export inhibitor leptomycin B suggests that the nuclear export receptor chromosome region maintenance protein 1 mediates the cytoplasmic localization of Id2. Id2 contains two putative leucine-rich nuclear export signals, and the nuclear export signal in the C-terminal region is essential for nuclear export. On the other hand, the helix-loop-helix domain is important for nuclear localization. Finally, experiments using reporter assays revealed an inverse correlation between nuclear export and transcriptional repression via the E-box sequence. Based on all these findings, we propose that nucleo-cytoplasmic shuttling is a novel mechanism for the regulation of Id2 function. Id proteins function as negative regulators of basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; NLS, nuclear localization signal; NES, nuclear export signal; GFP, green fluorescent protein; LMB, leptomycin B; CRM1, chromosome region maintenance protein 1; HLH, helix-loop-helix; TK, thymidine kinase; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; EGFP, enhanced green fluorescent protein.1The abbreviations used are: bHLH, basic helix-loop-helix; NLS, nuclear localization signal; NES, nuclear export signal; GFP, green fluorescent protein; LMB, leptomycin B; CRM1, chromosome region maintenance protein 1; HLH, helix-loop-helix; TK, thymidine kinase; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; EGFP, enhanced green fluorescent protein. transcriptional factors that specifically regulate gene expression during cell fate determination. They play pivotal roles in differentiation, cell cycle control, and cell lineage commitment in both vertebrates and invertebrates (1Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar, 2Ruzinova M.B. Benezra R. Trends Cell Biol. 2003; 13: 410-418Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar). To date, four individual Id genes, Id1–Id4, have been identified in mammals. They display overlapping but distinct expression patterns in a variety of cell types and tissues. Their important and nonoverlapping functions have been demonstrated by extensive analyses of knock-out mice (3Yokota Y. Mansouri A. Mori S. Sugawara S. Adachi S. Nishikawa S. Gruss P. Nature. 1999; 397: 702-706Crossref PubMed Scopus (731) Google Scholar, 4Lyden D. Young A.Z. Zagzag D. Yan W. Gerald W. O'Reilly R. Bader B.L. Hynes R.O. Zhuang Y. Manova K. Benezra R. Nature. 1999; 401: 670-677Crossref PubMed Scopus (772) Google Scholar, 5Yun K. Mantani A. Garel S. Rubenstein J. Israel M.A. Development. 2004; 131: 5441-5448Crossref PubMed Scopus (98) Google Scholar). The functions of Ids are strictly regulated at levels of transcription (6Hara E. Yamaguchi T. Nojima H. Ide T. Campisi J. Okayama H. Oda K. J. Biol. Chem. 1994; 269: 2139-2145Abstract Full Text PDF PubMed Google Scholar, 7Barone M.V. Pepperkok R. Peverali F.A. Philipson L. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4985-4988Crossref PubMed Scopus (205) Google Scholar) and protein stability (8Bounpheng M.A. Dimas J.J. Dodds S.G. Christy B.A. FASEB J. 1999; 13: 2257-2264Crossref PubMed Scopus (108) Google Scholar). Recent studies have revealed additional roles for these proteins in apoptosis, cellular senescence, and tumorigenesis (2Ruzinova M.B. Benezra R. Trends Cell Biol. 2003; 13: 410-418Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 9Fong S. Debs R.J. Desprez P-Y. Trends Mol. Med. 2004; 10: 387-392Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 10Yokota Y. Mori S. J. Cell. Physiol. 2002; 190: 21-28Crossref PubMed Scopus (178) Google Scholar).All members of the Id protein family share a similar overall structure consisting of the highly conserved helix-loop-helix (HLH) domain and the less conserved N- and C-terminal regions. Via their HLH domain, Id proteins preferentially associate with ubiquitously expressed bHLH E proteins such as E2A, E2–2, and HEB and sequester them from tissue-specific bHLH proteins exemplified by MyoD and Mash1 (1Norton J.D. J. Cell Sci. 2000; 113: 3897-3905Crossref PubMed Google Scholar, 2Ruzinova M.B. Benezra R. Trends Cell Biol. 2003; 13: 410-418Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar, 11Norton J.D. Deed R.W. Craggs G. Sablitzky F. Trends Cell Biol. 1998; 8: 58-658Abstract Full Text PDF PubMed Google Scholar). Because Id proteins lack a basic region that is critical for DNA binding, the heterodimer between Id and E proteins fails to bind to an E-box DNA sequence, and consequently the expression of genes possessing the E-box sequence in their regulatory elements is repressed. The affinities of Id2 and Id3 for E proteins are modulated by cell cycle-dependent phosphorylation of the Ser-5 residues in the N-terminal regions (12Hara E. Hall M. Peters G. EMBO J. 1997; 16: 332-342Crossref PubMed Scopus (115) Google Scholar, 13Deed R.W. Hara E. Atherton G.T. Peters G. Norton J.D. Mol. Cell. Biol. 1997; 17: 6815-6821Crossref PubMed Scopus (92) Google Scholar). The N-terminal region of Id2 is also important for apoptotic induction (14Florio M. Hernandez M-C. Yang H. Shu H.-K. Cleveland J.L. Israel M.A. Mol. Cell. Biol. 1998; 18: 5435-5444Crossref PubMed Scopus (106) Google Scholar) and protein degradation (15Fajerman I. Schwartz A.L. Ciechanover A. Biochem. Biophys. Res. Commun. 2004; 314: 505-512Crossref PubMed Scopus (60) Google Scholar). However, little is known about the molecular function of the Id protein C-terminal regions.Appropriate subcellular localization is crucial for the proper function of numerous proteins. Although some proteins are constitutively nuclear, others are actively imported into and exported out of the nucleus, in a signal-dependent or -independent manner (16Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1002) Google Scholar, 17Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-760Crossref PubMed Scopus (1660) Google Scholar). Large proteins shuttle between the nucleus and the cytoplasm through nuclear pore complexes by virtue of their intrinsic nuclear localization signals (NLSs) and nuclear export signals (NESs). The NLSs and NESs are recognized by specific import and export receptors, respectively (18Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (736) Google Scholar, 19Suntharalingam M. Wente S.R. Dev. Cell. 2003; 4: 775-789Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). In contrast, small proteins with molecular masses of less than 40 kDa can freely pass through the nuclear membrane (16Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1002) Google Scholar, 18Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (736) Google Scholar). Because the four members of the Id protein family have molecular masses ranging from 13 to 18 kDa, they are thought to enter and exit the nucleus by passive diffusion. In fact, small epitope-tagged Id2 protein is detected in both the nuclear and cytoplasmic compartments of transiently transfected COS cells (20Ghil S-H. Jeon Y.-J. Suh-Kim H. Exp. Mol. Med. 2002; 34: 367-373Crossref PubMed Scopus (26) Google Scholar).Passive diffusion seems to be one mechanism deciding the subcellular localization of Id proteins, but it is likely that other regulatory pathways also exist. Norton and co-workers (21Deed R.W. Armitage S. Norton J.D. J. Biol. Chem. 1996; 271: 23603-23606Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) previously showed that transiently expressed Id3 accumulated in the nucleus when one of the E2A gene products, E47, was co-expressed. Hence, they concluded that E protein acts as a nuclear chaperone for Id proteins. On the other hand, Samanta and Kessler (22Samanta J. Kessler J.A. Development. 2004; 131: 4131-4142Crossref PubMed Scopus (282) Google Scholar) have recently presented the opposite findings. Their data suggest that Id2 and Id4 would sequester nuclear bHLH proteins to the cytoplasm to promote differentiation of cultured neural progenitors into astrocytes. Furthermore, the subcellular localization of one of the Id protein family members, Id2, has been reported to change from the nucleus to the cytoplasm during neural differentiation into oligodendrocytes (23Wang S. Sdrulla A. Johnson J.E. Yokota Y. Barres B. Neuron. 2001; 29: 603-614Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Nuclear exclusion of Id2 is also observed when myeloid precursors are induced to differentiation (24Tu X. Baffa R. Luke S. Prisco M. Baserga R. Exp. Cell Res. 2003; 288: 119-130Crossref PubMed Scopus (33) Google Scholar).Here we describe the nucleo-cytoplasmic shuttling of Id2. Our results demonstrated that Id2 is predominantly localized in the cytoplasm under conditions in which passive diffusion does not occur. Then we characterize the molecular determinants that govern the predominant cytoplasmic localization and identify a functional leucine-rich NES in the C-terminal region. We also show that Id2 is localized in the nucleus when nuclear export is blocked and that the HLH domain is important for nuclear localization. Finally, our data suggest that nuclear export of Id2 has an inhibitory effect on repression of E-box-mediated transcription.EXPERIMENTAL PROCEDURESPlasmid Constructs—For construction of the N-terminally GFP-tagged Id1–Id4 plasmids, cDNAs encoding full-length mouse Id1–Id4 were inserted into the HindIII/BamHI sites of pEGFP-C1 (Clontech). Deletion and mutant constructs of GFP-tagged Id1–Id4 were generated by PCR amplification using their respective wild-type constructs as templates and appropriate primer pairs including artificial HindIII (for forward primers) and BamHI (for reverse primers) restriction enzyme sites. All of the constructs were verified by DNA sequencing. A tandem-duplicated GFP (GFP × 2) plasmid was constructed by ligating the PCR-amplified BamHI-HindIII fragment of the enhanced green fluorescent protein (EGFP) coding region to the BglII/HindIII sites of pEGFP-C1. Two oligonucleotides, 5′-AGCTTCGCTGACCACCCTGAACACG-GACATCAGCATCCTGTCCTTGCAGGCATCTGAAG-3′ (forward) and 5′-GATCCTTCAGATGCCTGCAAGGACAGGATGCTGATGTCCGTGT-TCAGGGTGGTCAGCGA-3′ (reverse), were annealed and inserted into the HindIII/BamHI sites of the GFP × 2, GFP-Id3, and GFP-Id4 plasmids, resulting in GFP × 2-Id2(103–119), GFP-Id3+Id2(103–119), and GFP-Id4+Id2(103–119), respectively. To generate a plasmid coding for C-terminally FLAG epitope-tagged human E47, an EcoRI-SalI fragment was excised from the plasmid BSKS(+)-E47–2 × FLAG (kindly provided by Dr. M. Sugai) and then cloned into the EcoRI/XhoI sites of pcDNA3.1(+) (Invitrogen). pE7-TK-luc is a thymidine kinase (TK) fire-fly luciferase reporter plasmid harboring seven copies of the E-box sequence and is a derivative of pE7-βA-luc (25Akazawa C. Ishibashi M. Shimizu C. Nakanishi S. Kageyama R. J. Biol. Chem. 1995; 270: 8730-8738Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). pCMV-MyoD was mentioned in our previous study (26Narumi O. Mori S. Boku S. Tsuji Y. Hashimoto N. Nishikawa S. Yokota Y. J. Biol. Chem. 2000; 275: 3510-3521Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar).Cell Culture and DNA Transfection—Mouse NIH3T3 fibroblasts and green monkey kidney COS7 cells were maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% (v/v) fetal bovine serum, streptomycin (100 μg/ml), and penicillin (100 units/ml) at 37 °C in a 5% CO2-humidified atmosphere. The cells were seeded on appropriate plates 18–24 h before transfection. They were transfected at 50–70% confluence using the FuGENE 6 reagent (Roche Applied Science), according to the manufacturer's instructions.Microscopic Analysis—Cells plated on 4-well Lab-Tek chamber slides (Nalge Nunc International) were transfected with 0.5 μg of GFP fusion constructs. Twenty-four hours after transfection, the cells were fixed with 3% formaldehyde in phosphate-buffered saline for 15 min at room temperature and subsequently mounted with the Vecta Shield reagent containing 4′,6-diamidino-2-phenylindole, dihydrochrolide (DAPI) (Molecular Probe) to stain the nuclei. In some experiments, the cells were treated with 5 ng/ml leptomycin B (LMB) for 3 h. LMB was a generous gift from Dr. M. Yoshida (RIKEN Discovery Research Institute, Wako, Japan). Microscopic analysis was conducted using an inverted fluorescence microscope (IX-71; Olympus) equipped with a digital camera (DP70; Olympus). All of the transfections and treatments were performed in at least two separate experiments.Western Blot Analysis and Immunoprecipitation—For Western blot analysis, transfected NIH3T3 cells were suspended in lysis buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 10% glycerol, 1 mm dithiothreitol, and protease inhibitor mixture (Roche Applied Science)), and the cells were incubated for 30 min at 4 °C. The cell debris was removed by centrifugation at 17,000 × g at 4 °C for 20 min, and the supernatant was used as the total cell extract. Twenty micrograms of total proteins were separated by 9–12% SDS-PAGE, and the resolved proteins were transferred onto a nitrocellulose filter (Amersham Biosciences). The filter was blotted with anti-GFP monoclonal antibody (GF200; Nacalai), and immunoreactive bands were visualized using the ECL chemiluminescence system (Amersham Biosciences). For immunoprecipitation, transfected COS7 cells were lysed in immunoprecipitation buffer (25 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.5% Triton X-100, 1 mm EDTA, 10% glycerol, 1 mm dithiothreitol, and protease inhibitor mixture (Roche Applied Science)). The total cell extract was prepared as described above, and 500 μg of total proteins were incubated with anti-FLAG M2-conjugated agarose (Sigma) by rotating for 3 h at 4 °C. Then the immunoprecipitated proteins were subjected to Western blot analysis as described above, using anti-GFP antibody or anti-FLAG M2 antibody (Sigma).Luciferase Assay—Luciferase assays were performed as described previously (26Narumi O. Mori S. Boku S. Tsuji Y. Hashimoto N. Nishikawa S. Yokota Y. J. Biol. Chem. 2000; 275: 3510-3521Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) with minor modifications. Briefly, NIH3T3 cells seeded in 24-well plates were transiently transfected with the various combinations of expression plasmids indicated in each figure legend. As a reporter plasmid, pE7-TK-luc was employed, and the total amount of DNA/well was adjusted to 804 ng by adding pcDNA3.1(+) empty vector. Forty-eight hours after transfection, the cells were lysed, and the luciferase activities were measured using the dual luciferase reporter assay system (Promega) according to the manufacturer's instructions, with a Luminescencer-JHR (ATTO) or Luminescencer-PSN (ATTO). The relative luciferase activity was normalized against the Renilla luciferase activity given by the internal control plasmid, phRL-TK (Promega). The assays were independently carried out at least twice in triplicate. For detection of GFP fusion proteins by Western blot analysis, the cell lysates were concentrated using Microcon YM-10 (Millipore) according to the manufacturer's instructions.RESULTSThe C-terminal Region of Id2 Is Required for Cytoplasmic Localization—To examine the subcellular localization of Id2, we generated a construct wherein the full-length mouse Id2 was N-terminally tagged with GFP (Fig. 1A). The size of the fusion protein was ∼45 kDa (Fig. 1C), and we assumed that it would not undergo passive diffusion. As shown in Fig. 1B, GFP-Id2 was predominantly localized in the cytoplasm when it was transfected into NIH3T3 cells. Similar results were obtained in COS7, 293T, and U2OS cells (data not shown). Unlike GFP-Id2, fluorescently tagged Id3 was previously shown to be diffusely distributed throughout the cells (27O'Toole P.J. Inoue T. Emerson L. Morrison I.E. Mackie A.R. Cherry R.J. Norton J.D. J. Biol. Chem. 2003; 278: 45770-45776Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Thus, the cytoplasmic localization of GFP-Id2 is not cell type-dependent and is not a general effect caused by fusion with a fluorescence tag.The difference in subcellular localization between fluorescently tagged Id2 and Id3 suggests that the former contains a specific domain or signal that facilitates cytoplasmic localization. Because amino acid sequences outside the HLH domains are poorly conserved among the Id protein family (11Norton J.D. Deed R.W. Craggs G. Sablitzky F. Trends Cell Biol. 1998; 8: 58-658Abstract Full Text PDF PubMed Google Scholar), we next examined whether the N- or C-terminal region of Id2 is involved in mediating cytoplasmic localization. Whereas truncation of the N-terminal region had no effect on the subcellular localization, truncation of the C-terminal region resulted in nuclear accumulation of the fusion protein (Fig. 1, A and B). To define the region required for cytoplasmic localization, we generated several C-terminal deletion constructs. Although GFP-Id2(1–101) showed nuclear localization, GFP-Id2(1–119) was located in the cytoplasm, indicating that amino acids between residues 102 and 119 are required for cytoplasmic localization. An internal deletion mutant, GFP-Id2(1–119Δ77–102), exhibited a cytoplasmic localization pattern indistinguishable from that of GFP-Id2(1–119). These results demonstrate that amino acids between residues 103 and 119 are responsible for the cytoplasmic localization. Protein expression of the fusion constructs was confirmed by Western blot analysis using anti-GFP antibody (Fig. 1C).Id2 Is Exported from the Nucleus via a CRM1-dependent Mechanism—Remarkably, the 17-amino acid sequence identified above contains a leucine-rich stretch that well matches the regularly spaced hydrophobic residues in the NES consensus sequence (28Fukuda M. Gotoh I. Gotoh Y. Nishida E. J. Biol. Chem. 1996; 271: 20024-20028Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 29Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 464-473Google Scholar, 30Fisher U. Huber J. Boelens W.C. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (981) Google Scholar). A similar region is present in Id1 but not in Id3 or Id4 (Fig. 2A). The cytoplasmic localization of NES-containing proteins is due to active nuclear export, which is in many cases mediated by the nuclear export receptor CRM1 (chromosome region maintenance protein 1) (16Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1002) Google Scholar, 17Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-760Crossref PubMed Scopus (1660) Google Scholar, 18Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (736) Google Scholar, 19Suntharalingam M. Wente S.R. Dev. Cell. 2003; 4: 775-789Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). To explore the possibility that Id2 is actively exported from the nucleus via a CRM1-dependent mechanism, cells expressing the GFP-Id2 fusion protein were treated with the CRM1-specific nuclear export inhibitor LMB (31Kudo N. Wolff B. Sekimoto T. Schreiner E.P. Yoneda Y. Yanagida M. Horinouchi S. Yoshida M. Exp. Cell Res. 1998; 242: 540-547Crossref PubMed Scopus (704) Google Scholar, 32Kudo N. Matsumori N. Taoka H. Fujiwara D. Schreiner E.P. Wolff B. Yoshida M. Horinouchi S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9112-9117Crossref PubMed Scopus (844) Google Scholar). After treatment of the cells with LMB, GFP-Id2 accumulated in the nucleus, and only a small proportion remained in the cytoplasm (Fig. 2B). This change of localization suggests that CRM1 mediates the nuclear export of Id2.Fig. 2Effects of LMB treatment on subcellular localization of the full-length Id2 and its C-terminal NES. A, the Id2 C-terminal 17-amino acid sequence required for cytoplasmic localization and the corresponding regions of other Id family members were aligned with established NES sequences from mitogen-activated protein kinase kinase (MAPKK) (28Fukuda M. Gotoh I. Gotoh Y. Nishida E. J. Biol. Chem. 1996; 271: 20024-20028Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), PKI-α (29Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 464-473Google Scholar), and HIV Rev (30Fisher U. Huber J. Boelens W.C. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (981) Google Scholar). NES consensus sequence is shown at the bottom, and conserved hydrophobic residues of the aligned sequences are highlighted by boxes. Φ indicates hydrophobic residues including leucine, isoleucine, and valine. B and C, NIH3T3 cells were transiently transfected and cultured for 24 h. Then they were incubated in the absence or presence of LMB (5 ng/ml) for further 3 h. Subcellular localization was examined as described under “Experimental Procedures.” The upper and lower panels are green fluorescence and DAPI staining images, respectively. B, representative images of cells transfected with the plasmid DNA encoding GFP-Id2. LMB treatment led to accumulation of GFP-Id2 in the nucleus. C, representative images of cells transfected with a fusion construct between tandem-duplicated GFP (GFP × 2) and the Id2 C-terminal 17-amino acid sequence that contains a putative NES. The C-terminal 17-amino acid sequence of Id2 has an autonomous nuclear export activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because some isolated NES sequences are still active in nuclear export when transferred to other proteins (33Henderson B.R. Eleftheriou A. Exp. Cell Res. 2000; 256: 213-224Crossref PubMed Scopus (349) Google Scholar), we tested whether the putative NES located in the C-terminal region of Id2 is capable of directing a heterologous protein to the cytoplasm. A single GFP moiety is small enough to enter the nucleus by passive diffusion because its size (26 kDa) is below the exclusion limit of nuclear pores. To avoid passive diffusion of the reporter protein, we generated a tandem-duplicated GFP (GFP × 2) construct and connected it to the C-terminal 17-amino acid sequence of Id2. As reported previously (34Saydam N. Georgiev O. Nakano M.Y. Greber U.F. Schaffner W. J. Biol. Chem. 2001; 276: 25487-25495Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 35Murai N. Murakami Y. Matsufuji S. J. Biol. Chem. 2003; 278: 44791-44798Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 36Kilstrup-Nielsen C. Alessio M. Zappavigna V. EMBO J. 2003; 22: 89-99Crossref PubMed Scopus (53) Google Scholar, 37Karlsson M. Mathers J. Dickinson R.J. Mandl M. Keyse S.M. J. Biol. Chem. 2004; 279: 41882-41891Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), GFP × 2 was uniformly distributed throughout the cells regardless of whether or not they were treated with LMB (Fig. 2C, left panels). In contrast, the fusion protein of GFP × 2-Id2(103–119) was exclusively localized in the cytoplasm of the untreated cells, and LMB effectively blocked this cytoplasmic localization (right panels). These results clearly show that the putative NES of Id2 is autonomously functional in the CRM1-mediated nuclear export.The C-terminal NES of Id2 Is Essential for Nuclear Export— Careful examination of the amino acid sequences revealed the existence of a second leucine-rich region that conforms to the NES consensus motif at the end of the Id2 HLH domain. For simplicity, we refer to the NES sequences in the HLH domain and C-terminal region as NES-1 (residues 65–75) and NES-2 (residues 106–115), respectively (Fig. 3A). To evaluate their contribution to nuclear export, we mutated either the second or fourth conserved hydrophobic residue to alanine in the two NES sequences and examined the effect on the subcellular localization of the GFP-Id2 fusion protein. Although neither mutation in the NES-1 sequence (I69A and L75A) affected the cytoplasmic localization of GFP-Id2, both of the mutations in the NES-2 sequence (I110A and L115A) led to nuclear translocation of the fusion protein (Fig. 3B). Therefore, we concluded that the NES-2 in the C-terminal region is essential for nuclear export of Id2.Fig. 3Effects of mutations in the two NESs on subcellular localization of Id2. A, schematic representation of the full-length Id2 is depicted at the top. Id2 contains two putative NESs, the NES-1 in the end of the HLH domain and the NES-2 in the middle of the C-terminal region, respectively. Asterisks indicate conserved hydrophobic amino acid residues in the NESs. Mutants containing the replaced second and fourth residues by alanine are shown at the bottom. Dashes represent unchanged amino acids. B, representative images of NIH3T3 cells transiently transfected with the mutant constructs shown in A. The upper and lower panels are green fluorescence and DAPI staining images, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The HLH Domain of Id2 Is Important for Nuclear Localization—Treatment with LMB or disruption of NES caused GFP-Id2 to be accumulated in the nucleus rather than diffusely distributed throughout the cell. This implies that Id2 may contain a specific domain or signal that promotes nuclear accumulation when nuclear export is blocked. Because GFP-Id2(1–76) comprising the N-terminal and HLH domains was exclusively localized in the nucleus (Fig. 1), we next analyzed the involvement of the short clusters of basic residues located in the two domains (Fig. 4A), although a classical NLS, a single stretch of basic amino acids or a bipartite sequence of basic residues separated by 10 nonconserved amino acids (16Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1002) Google Scholar, 17Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-760Crossref PubMed Scopus (1660) Google Scholar, 18Macara I.G. Microbiol. Mol. Biol. Rev. 2001; 65: 570-594Crossref PubMed Scopus (736) Google Scholar), is not found there. The nuclear localization of GFP-Id2(1–76) was not altered by the R11A/K12V and R24A/K26A mutations in the N-terminal region, but it was partially impaired by the K45A/K47A mutation and severely compromised by the K57T/K58Q mutation in the HLH domain (Fig. 4B). It can be assumed that the K57T/K58Q mutation would not disrupt the tertiary structure of the HLH domain, because the substituted amino acids were derived from the corresponding residues of Id3 (38Wibley J. Deed R. Jasiok M. Douglas K. Norton J. Biochim. Biophys. Acta. 1996; 1294: 138-146Crossref PubMed Scopus (26) Google Scholar). In fact, we found that the K57T/K58Q mutant retained the ability to associate with E protein (data not shown).Fig. 4The HLH domain of Id2 is important for nuclear localization. A, amino acid sequence of the Id2 N-terminal region (amino acid residues 1–35) and the HLH domain (amino acid residues 36–76). Basic" @default.
• W2000536319 created "2016-06-24" @default.
• W2000536319 creator A5015413462 @default.
• W2000536319 creator A5016247704 @default.
• W2000536319 date "2005-02-01" @default.
• W2000536319 modified "2023-10-07" @default.
• W2000536319 title "Nucleo-cytoplasmic Shuttling of Id2, a Negative Regulator of Basic Helix-Loop-Helix Transcription Factors" @default.
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