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• W1503786870 abstract "The Ret finger protein (RFP) was identified initially as an oncogene product and belongs to a family of proteins that contain a tripartite motif consisting of a RING finger, a B box, and a coiled-coil domain. RFP represses transcription by interacting with Enhancer of Polycomb and is localized to the cytoplasm or nucleus depending on the cell type. Here, we have identified the nuclear export signal (NES) located in the coiled-coil region of RFP. Mutation of this NES or treatment with leptomycin B abrogated the nuclear export of RFP in NIH3T3 cells. In addition, fusion of this NES to other nuclear proteins, such as yeast transcription factor Gal4, resulted in their release into the cytoplasm of NIH3T3 cells. Although the NES function of RFP in HepG2 cells is masked by another domain in RFP or by another protein, 12-O-tetradecanoylphorbol-13-acetate treatment or overexpression of constitutively active protein kinase Cα (PKCα) abrogated masking, leading to the cytoplasmic localization of RFP. Furthermore, treatment of NIH3T3 cells with PKC inhibitors blocked the function of NES, resulting in nuclear localization of RFP. Thus, the nuclear export of RFP is regulated positively by PKC activation. However, RFP was not a direct substrate of PKC, and additional signaling pathways may be involved in the regulation of nuclear export of RFP. The Ret finger protein (RFP) was identified initially as an oncogene product and belongs to a family of proteins that contain a tripartite motif consisting of a RING finger, a B box, and a coiled-coil domain. RFP represses transcription by interacting with Enhancer of Polycomb and is localized to the cytoplasm or nucleus depending on the cell type. Here, we have identified the nuclear export signal (NES) located in the coiled-coil region of RFP. Mutation of this NES or treatment with leptomycin B abrogated the nuclear export of RFP in NIH3T3 cells. In addition, fusion of this NES to other nuclear proteins, such as yeast transcription factor Gal4, resulted in their release into the cytoplasm of NIH3T3 cells. Although the NES function of RFP in HepG2 cells is masked by another domain in RFP or by another protein, 12-O-tetradecanoylphorbol-13-acetate treatment or overexpression of constitutively active protein kinase Cα (PKCα) abrogated masking, leading to the cytoplasmic localization of RFP. Furthermore, treatment of NIH3T3 cells with PKC inhibitors blocked the function of NES, resulting in nuclear localization of RFP. Thus, the nuclear export of RFP is regulated positively by PKC activation. However, RFP was not a direct substrate of PKC, and additional signaling pathways may be involved in the regulation of nuclear export of RFP. RING-B box-coiled-coil enhanced green fluorescent protein promyelocytic leukemia protein Ret finger protein nuclear export signal nuclear localization sequence protein kinase C 12-O-tetradecanoylphorbol-13-acetate leptomycin B green fluorescent protein Jun N-terminal protein kinase DNA binding domain To fulfill their functions in complex organisms, most proteins possess specialized domains that have been conserved throughout evolution. Depending on their overall domain structure, proteins have been grouped into gene families, whose members often share similar functions in cells or organisms. Members of one such gene family, the RING-B box-coiled-coil (RBCC)1 family (1Douarin B.L. Nielsen A.L. Garnier J.-M. Ichinose H. Jeanmougin F. Losson R. Chambon P. EMBO J. 1996; 15: 6701-6715Crossref PubMed Scopus (463) Google Scholar), are characterized by their possession of a tripatic motif consisting of a RING finger (2Saurin A.J. Borden K.L.B. Boddy M.N. Freemont P.S. Trends Biol. Sci. 1996; 21: 208-453Abstract Full Text PDF PubMed Scopus (609) Google Scholar, 3Borden K.L.B. Freemont P.S. Curr. Opin. Struct. Biol. 1996; 6: 395-401Crossref PubMed Scopus (413) Google Scholar), one or two B boxes (4Borden K.L.B. Biochem. Cell Biol. 1998; 76: 351-358Crossref PubMed Scopus (229) Google Scholar), and an α-helical coiled-coil domain (5Lupas A. Trends Biochem. Sci. 1996; 21: 375-382Abstract Full Text PDF PubMed Scopus (1008) Google Scholar). These domains are involved in protein-protein interactions and allow RBCC family members to participate in various cellular processes depending on their subcellular localization. In transformed cells, three members of the RBCC family (PML, transcriptional intermediary factor 1α, and RFP) were found to be oncogenic after their RBCC domains were linked to other proteins by DNA rearrangements. In acute promyelocytic leukemia cells, a reciprocal chromosomal translocation fuses the PML gene with the retinoic acid receptor α gene, resulting in the expression of a PML-RARα fusion protein (6Kakizuka A. Miller Jr., W.H. Umesono K. Warrell Jr., R.P. Frankel S.R. Murty V.V. Dmitrovsky E. Evans R.M. Cell. 1991; 66: 663-674Abstract Full Text PDF PubMed Scopus (1282) Google Scholar, 7Thé H.d. Lavau C. Marchio A. Chomienne C. Degos L. Dejean A. Cell. 1991; 66: 675-684Abstract Full Text PDF PubMed Scopus (1188) Google Scholar) that blocks hematopoiesis at the promyelocytic stage (8Stunnenberg H.G. Garcia-Jimenez C. Betz J.L. Biochim. Biophys. Acta. 1998; 1423: F15-F33Google Scholar,9Melnick A. Light J.D. Blood. 1999; 93: 3167-3215Crossref PubMed Google Scholar). Similarly, the N-terminal RBCC domain of transcriptional intermediary factor 1α recombines with the serine/threonine kinase domain of B-Raf in the T18 oncogene (10Douarin B.L. Zechel C. Garnier J.M. Lutz Y. Tora L. Pierrat B. Heery D. Gronemeyer H. Chambon P. Losson R. EMBO J. 1995; 14: 2020-2033Crossref PubMed Scopus (573) Google Scholar), and the N terminus of the Ret finger protein (RFP) was found to be fused to theret proto-oncogene (11Weering D.H.J.v. Bos J.L. Rec. Results Cancer Res. 1998; 154: 271-281Crossref PubMed Scopus (56) Google Scholar) in transformed NIH3T3 cells (12Takahashi M. Ritz J. Cooper G.M. Cell. 1985; 42: 581-588Abstract Full Text PDF PubMed Scopus (590) Google Scholar,13Takahashi M. Cooper G.M. Mol. Cell. Biol. 1987; 7: 1378-1385Crossref PubMed Scopus (284) Google Scholar). The RBCC motifs are required for the transforming capacities of these oncogenes (14Hasegawa N. Iwashita T. Asai N. Murakami H. Iwata Y. Isomura T. Goto H. Hayakawa T. Takahashi M. Biochem. Biophys. Res. Commun. 1996; 225: 627-631Crossref PubMed Scopus (32) Google Scholar), and oncogenesis may depend on the distinct cellular localizations of the fusion proteins (9Melnick A. Light J.D. Blood. 1999; 93: 3167-3215Crossref PubMed Google Scholar, 15Zhong S. Delva L. Rachez C. Cenciarelli C. Gandini D. Zhang H. Kalantry S. Freedman L.P. Pandolfi P.P. Nat. Genet. 1999; 23: 287-295Crossref PubMed Scopus (115) Google Scholar). After the identification of the oncogenic RFP-Ret fusion protein in NIH3T3 cells, RFP was cloned from human (13Takahashi M. Cooper G.M. Mol. Cell. Biol. 1987; 7: 1378-1385Crossref PubMed Scopus (284) Google Scholar) and mouse (16Cao T. Shannon M. Handel M.A. Etkin L.E. Dev. Genet. 1996; 19: 309-320Crossref PubMed Scopus (44) Google Scholar) and found to be highly expressed in pachytene spermatocytes and round spermatids during spermatogenesis, suggesting that it participates in male germ cell differentiation (16Cao T. Shannon M. Handel M.A. Etkin L.E. Dev. Genet. 1996; 19: 309-320Crossref PubMed Scopus (44) Google Scholar, 17Tezel G. Nagasaka T. Iwahashi N. Asai N. Iwashita T. Sakata K. Takahashi M. Pathol. Int. 1999; 49: 881-886Crossref PubMed Scopus (35) Google Scholar). Depending on the cell type or tissue, RFP is localized either to the cytoplasm or nucleus (16Cao T. Shannon M. Handel M.A. Etkin L.E. Dev. Genet. 1996; 19: 309-320Crossref PubMed Scopus (44) Google Scholar, 17Tezel G. Nagasaka T. Iwahashi N. Asai N. Iwashita T. Sakata K. Takahashi M. Pathol. Int. 1999; 49: 881-886Crossref PubMed Scopus (35) Google Scholar). This localization requires the RBCC motif and homodimerization through the coiled-coil domain (18Cao T. Borden K.L.B. Freemont P.S. Etkin L.D. J. Cell Sci. 1997; 110: 1563-1571Crossref PubMed Google Scholar). In the nucleus, RFP associates with the nuclear matrix (19Isomura T. Tamiya-Koizumi K. Suzuki M. Yoshida S. Taniguchi M. Matsuyama M. Ishigaki T. Sakuma S. Takahashi M. Nucleic Acids Res. 1992; 20: 5305-5310Crossref PubMed Scopus (50) Google Scholar) and is a component of PML nuclear bodies (20Dyck J.A. Maul G.G. Miller Jr., W.H. Chen J.D. Kakizuka A. Evans R.M. Cell. 1994; 76: 333-343Abstract Full Text PDF PubMed Scopus (722) Google Scholar) where it binds directly to PML (21Cao T. Duprez E. Borden K.L.B. Freemont P.S. Etkin L.D. J. Cell Sci. 1998; 111: 1319-1329Crossref PubMed Google Scholar). Furthermore, RFP can interact with theint-6 gene product, another component of PML nuclear bodies (22Morris-Desbois C. Bochard V. Reynaud C. Jalinot P. J. Cell Sci. 1999; 112: 3331-3342Crossref PubMed Google Scholar). Recently, RFP was shown to repress transcription by interacting with Enhancer of Polycomb (23Shimono Y. Murakami H. Hasegawa Y. Takahashi M. J. Biol. Chem. 2000; 275: 39411-39419Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), a member of Polycomb proteins. The Polycomb group proteins were originally identified inDrosophila as being involved in the maintenance of the correct expression pattern of homeotic genes. Polycomb group proteins form a large macromolecular complex and are involved in the epigenetic gene silencing. However, no biological function has yet been defined for RFP in the nucleus or cytoplasm, and mechanisms governing RFP subcellular localization remain largely unknown. The separation of the cytoplasm and nucleus in eukaryotic cells provides an important way to regulate cellular processes through compartmentalization. As was shown for NF-AT (24Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar) or IκBα (25Rodriguez M.S. Thompson J. Hay R.T. Dargemont C. J. Biol. Chem. 1999; 274: 9108-9115Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 26Johnson C. Antwerp D.V. Hope T.J. EMBO J. 1999; 18: 6682-6693Crossref PubMed Google Scholar), nuclear import and export through the nuclear envelope can control gene expression in a signal-dependent manner by restricting the access of transcription factors to their target genes. The exchange between cytoplasm and nucleus occurs through the nuclear pore complex, and this process is mediated in most cases by specific amino acid sequences. Localization sequences exist for nuclear localization (nuclear localization sequence, NLS) (27Jans D.A. Xiao C.-Y. Lam M.H.C. BioEssays. 2000; 22: 532-544Crossref PubMed Scopus (474) Google Scholar, 28Görlich D. Curr. Opin. Cell Biol. 1997; 9: 412-419Crossref PubMed Scopus (255) Google Scholar) and for nuclear export (nuclear export signal, NES) (29Adam S.A. Curr. Opin. Cell Biol. 1999; 11: 402-406Crossref PubMed Scopus (75) Google Scholar, 30Turpin P. Ossareh-Nazari B. Dargemont C. FEBS Lett. 1999; 452: 82-86Crossref PubMed Scopus (51) Google Scholar, 31Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). The leucine-rich NES is specifically recognized by CRM1, which functions as an export receptor mediating the fast release of proteins with this sequence from the nucleus (32Fukuda M. Asano S. Nakamura T. Adachi M. Yoshida M. Yanagida M. Nishida E. Nature. 1997; 390: 308-311Crossref PubMed Scopus (1015) Google Scholar, 33Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1724) Google Scholar, 34Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Crossref PubMed Scopus (619) Google Scholar). Masking of the NLS or NES through their binding to other factors or through post-translational modifications would allow for controlled shuttling of factors between the cytoplasm and the nucleus. Here we have studied the cellular localization of RFP and found that it can shuttle between the cytoplasm and the nucleus. Nuclear export of RFP is dependent on a functional NES, which can be activated by protein kinase C (PKC). The regulation of RFP localization by PKC points to an important role for RFP in the control of cellular differentiation and proliferation. The NIH3T3 and HepG2 cells were maintained at 37 °C in a 5% CO2-containing atmosphere using Dulbecco’s modified Eagle’s medium (Nissui Pharmaceuticals) with 10% bovine calf serum (HyClone) or 10% fetal calf serum (Roche), respectively. 12-O-Tetradecanoylphorbol-13-acetate (TPA), H7, and staurosporine were obtained from Sigma. Leptomycin B (LMB) was a gift from Dr. M. Yoshida. Drug concentrations and incubation times are indicated in the figure legends. Serum concentrations were reduced to 2% when drugs were added to the cell cultures. FLAG-tagged forms of RFP were expressed from pSG5-FLAG-Nt, and Gal4 fusion proteins from pCMX-Gal4 and pG4polyII (35Tora L. White J. Brou C. Tasset D. Webster N. Scheer E. Chambon P. Cell. 1989; 59: 477-487Abstract Full Text PDF PubMed Scopus (881) Google Scholar). For the expression of GFP fusion proteins, the vectors pEGFP-C2, pEGFP-C3, pEGFP-N3, and pEYFP-nuc (CLONTECH) were used. Mutations in the putative NES and PKC phosphorylation sites were introduced by polymerase chain reaction and verified by DNA sequencing. Expression vectors for PKCα were described by Ueda et al. (36Ueda Y. Hirai S. Osada S. Suzuki A. Mizuno K. Ohno S. J. Biol. Chem. 1996; 271: 23512-23519Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). To express the activated Ha-Ras, the SV40 promoter-containing vector pcEXV-1 was used. The SRα promoter-containing vector was used to express Jun N-terminal protein kinase 1 (JNK1). Subcellular localization of RFP and colocalization with other proteins were determined as described by Khanet al. (37Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). For each 3-cm dish, 2 × 105NIH3T3 cells or 4 × 105 HepG2 cells were seeded, and on the next day the cells were transfected with 3 or 1.5 μg of the expression vector using LipofectAMINE (Life Technologies, Inc.). 48 h after transfection, cells were fixed and stained with mouse monoclonal anti-FLAG antibody M2 (Sigma) and rabbit polyclonal antibodies against PKCα (Santa Cruz). Fusion proteins containing the DNA binding domain of Gal4 (Gal4-DBD) were detected by mouse monoclonal anti-Gal4-DBD antibody (Santa Cruz). The signals were visualized by rhodamine- or fluorescein isothiocyanate-conjugated secondary antibodies (Jackson ImmunoResearch), and analyzed by confocal microscopy (Zeiss LSM510). Nuclear DNA was stained with TO-PRO®-3 iodide (Molecular Probes) and used as a nuclear marker. GFP fusion proteins were analyzed in the same way directly after fixation and DNA staining with TO-PRO®-3 iodide. In Figs. Figure 7, Figure 8, Figure 9, two types of staining pattern, which showed the predominantly nuclear or cytoplasmic RFP, were observed, and the cells showing uniform distribution in both nucleus and cytoplasm were not detected. Approximately 50–150 cells were examined, and the percentages of cells with cytoplasmic RFP were calculated. Photographs and numbers from representative experiments are shown in the figures.Figure 8Effect of PKC inhibitors on RFP localization. Panel A, inhibition of TPA-induced nuclear export of RFP by PKC inhibitors in HepG2 cells. HepG2 cells were transfected with the FLAG-tagged RFP expression vector and treated for 1 h with 50 ng/ml TPA or 50 ng/ml TPA plus 200 μm H7 or 20 nm staurosporine. Immunohistochemistry was performed with an anti-FLAG antibody as described in Fig. 1 B, and the percentages of cells with cytoplasmic RFP are shown in the bar graph on theright. Panel B, inhibition of the nuclear export of RFP by PKC inhibitors in NIH3T3 cells. NIH3T3 cells expressing FLAG-tagged RFP were treated similarly with 100 μm H7 or 10 nm staurosporine, and the results are indicated as described above.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9Effect of various signaling pathways on RFP localization. Panel A, PKC levels in NIH3T3 and HepG2 cells. In the left image, whole cell extracts were prepared from NIH3T3 and HepG2 cells, and 50 μg of total protein from each extract was analyzed by Western blot with anti-PKCα and anti-phosphorylated PKCα-specific antibodies. In the right image, PKC activity in NIH3T3 and HepG2 cells was measured, and the average results of three experiments are indicated by a bar graph. Panel B, effect of various signaling pathways on the subcellular localization of RFP. The FLAG-tagged RFP expression vector was cotransfected with the Ha-ras or JNK1 expression vector or control vectors (pcEXV-1 and pSRα0). Immunohistochemistry was performed with an anti-FLAG antibody as described in Fig.1 B, and the percentages of cells with cytoplasmic RFP are shown in the bar graph on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The homodimerization of RFP was studied by the LexA yeast two-hybrid system in the strain L40 (38Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1654) Google Scholar) using the vectors pBTM116 (38Vojtek A.B. Hollenberg S.M. Cooper J.A. Cell. 1993; 74: 205-214Abstract Full Text PDF PubMed Scopus (1654) Google Scholar) for the expression of LexA fusion proteins and pASV3 (39Douarin B.L. Pierrat B. Baur E.v. Chambon P. Losson R. Nucleic Acids Res. 1995; 23: 876-878Crossref PubMed Scopus (71) Google Scholar) for the expression of VP16 fusion proteins. β-Galactosidase assays on individual transformants were carried out as described in Ref. 40Seipel K. Georgiev O. Schaffner W. EMBO J. 1992; 11: 4961-4968Crossref PubMed Scopus (296) Google Scholar. PKC activity in NIH3T3 and HepG2 cells was measured in crude extracts using a PKC assay kit (Calbiochem). Detection of PKCα and a phosphorylated form of PKCα/β (Thr-638) was performed by Western blotting using 50 μg of total protein from NIH3T3 and HepG2 cells, anti-PKCα (C-20, Santa Cruz), or an anti-phospho-PKCα/β (Thr-638) antibody (New England BioLabs). RFP is highly expressed in tumor cell lines, and its subcellular localization depends on cell type. Exogenously expressed mouse RFP is located predominantly in the cytoplasm of NIH3T3 cells, whereas it is localized in the nucleus of HepG2 cells (18Cao T. Borden K.L.B. Freemont P.S. Etkin L.D. J. Cell Sci. 1997; 110: 1563-1571Crossref PubMed Google Scholar). We therefore chose NIH3T3 and HepG2 cells to study mechanisms that control human RFP shuttling between the nucleus and cytoplasm. Expression vectors for FLAG-tagged RFP (Fig.1 A) or an enhanced green fluorescent protein (EGFP)-labeled form of RFP were transfected into NIH3T3 and HepG2 cells, and their localization was analyzed by confocal microscopy using TO-PRO®-3 iodide as a nuclear marker. As expected, both forms of human RFP were found in the cytoplasm of NIH3T3 cells (Fig. 1 B), associated with structures that resembled the endoplasmic reticulum or Golgi apparatus. Depending on the expression level, RFP showed a grainy pattern or clear dot-like structures. Small amounts of RFP were also visible in the nucleus, where they appeared as small dots. In contrast to NIH3T3 cells, both forms of human RFP localized to the nucleus of HepG2 cells (Fig.1 C). Staining was observed for the whole nucleoplasm with some preferences for the perinuclear region. Although RFP was reported to associate with PML nuclear bodies (21Cao T. Duprez E. Borden K.L.B. Freemont P.S. Etkin L.D. J. Cell Sci. 1998; 111: 1319-1329Crossref PubMed Google Scholar), colocalization of RFP and PML nuclear bodies was observed only at low frequency. We next analyzed the mechanisms controlling the cytoplasmic localization of RFP in NIH3T3 cells. Because N-terminally linked EGFP-RFP fusion proteins showed a localization similar to that of FLAG-tagged RFP in both NIH3T3 and HepG2 cell lines (Fig. 1), EGFP-labeled deletion mutants of RFP were used to determine which domains of RFP affected its localization in NIH3T3 cells. Expression vectors encoding EGFP alone or EGFP fusion proteins for N-terminal and C-terminal deletion mutations of RFP were transfected into NIH3T3 cells, and their subcellular localization was analyzed along with a nuclear marker (Fig. 2). The EGFP control protein was localized in both the nucleus and cytoplasm because its small molecular mass of 27 kDa allows it to diffuse freely into the nucleus. However, the RFP fusion proteins used in this study had molecular masses in excess of 40 kDa, which may preclude their passive diffusion into the nucleus (30Turpin P. Ossareh-Nazari B. Dargemont C. FEBS Lett. 1999; 452: 82-86Crossref PubMed Scopus (51) Google Scholar). Although the deletion of the RING finger domain of RFP (RFP96–513) had little effect on RFP localization, further deletion of the RING finger and B box, as in EGFP-RFP(132–513), led to distinct patterns of localization. Although the dot-like structures in the cytoplasm were maintained, increased staining of the nucleoplasm was observed. An additional deletion of the coiled-coil region (EGFP-RFP(315–513)) resulted in the disappearance of the dot-like structures in the cytoplasm and redistribution of RFP throughout the whole cell with a preference for the nucleus. A similar pattern was observed for a mutant lacking the C-terminal part of RFP, which contains the RING finger and the B box (EGFP-RFP(1–132)), although the distribution between the cytoplasm and nucleus was more uniform. Addition of the coiled-coil region to the RING finger and the B box (EGFP-RFP(1–258)) reestablished the localization of RFP as dot-like structures in the cytoplasm. Because the deletion analysis of RFP pointed to an overall importance of the coiled-coil region for RFP localization, a deletion mutant of RFP (EGFP-RFPΔcc), lacking the coiled-coil region, was constructed, and its subcellular localization was analyzed. As expected, the protein was distributed throughout the whole cell with preferential localization to the nucleus. These results indicate that the coiled-coil region is required for the cytoplasmic localization of RFP in NIH3T3 cells. The results described above led us to speculate about the presence of an NES in the coiled-coil region of RFP. In fact, sequence analysis of the coiled-coil region of RFP revealed the putative leucine-rich NES (41Fischer U. Huber J. Boelens W.C. Mattji I.W. Lührmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (976) Google Scholar, 42Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (991) Google Scholar). An alignment of the consensus NES (41Fischer U. Huber J. Boelens W.C. Mattji I.W. Lührmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (976) Google Scholar, 42Wen W. Meinkoth J.L. Tsien R.Y. Taylor S.S. Cell. 1995; 82: 463-473Abstract Full Text PDF PubMed Scopus (991) Google Scholar, 43van Hengel J. Vanhoenacker P. States K. van Roy F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7980-7985Crossref PubMed Scopus (140) Google Scholar, 44Toyoshima F. Moriguchi T. Wada A. Fukuda M. Nishida E. EMBO J. 1998; 17: 2728-2735Crossref PubMed Scopus (276) Google Scholar, 45Engel K. Kotlyarov A. Gaestel M. EMBO J. 1998; 17: 3363-3371Crossref PubMed Scopus (236) Google Scholar) with the putative leucine-rich NES of RFP indicated a perfect homology (Fig.3 A). To examine whether RFP contains a functional NES, the three most conserved leucine residues in the putative NES of RFP (Leu-204, Leu-207, and Leu-209 in Fig.3 A) were mutated to alanines, and the localization of the mutated RFP (RFPmut) as a FLAG-tagged fusion protein was analyzed by immunostaining (Fig. 3 B). RFPmut was located predominantly in the nucleus of NIH3T3 cells, and in some of those cells it localized in dot-like structures similar to PML nuclear bodies. In addition, NIH3T3 cells transfected with the RFP expression vector were treated with the CRM1 inhibitor LMB (31Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 46Wolf B. Sanglier J.J. Wang Y. Chem. Biol. 1997; 4: 139-147Abstract Full Text PDF PubMed Scopus (570) Google Scholar) (Fig. 3 B). Treatment of NIH3T3 cells with LMB enhanced the nuclear localization of RFP. Similar to RFPmut, wild-type RFP in LMB-treated cells could be seen in the whole nucleus or in some cases in dot-like structures. NES function can be studied by linking it to nuclear proteins, such as the DNA binding domain of the yeast transcription factor Gal4 (Gal4-DBD), which changes its subcellular localization from the nucleus to the cytoplasm when fused to an NES (47Hoshino H. Kobayashi A. Yoshida M. Kudo N. Oyake T. Motohashi H. Hayashi N. Yamamoto M. Igarashi K. J. Biol. Chem. 2000; 275: 15370-15376Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). To study the function of the putative NES in RFP, the full-length RFP and the coiled-coil regions of RFP, comprising either normal or mutated versions of the putative NES, were expressed as Gal4-DBD fusion proteins in NIH3T3 cells, and their subcellular localizations were analyzed using anti-Gal4-DBD antibody (Fig.4 A). In contrast to the Gal4-DBD control protein, the wild-type RFP fusion protein was localized preferentially to the cytoplasm, and mutations in the putative NES resulted in relocalization of the Gal4-DBD-RFP fusion protein to the nucleus. Similar results were obtained with Gal4-DBD fused to the coiled-coil regions of RFP. The wild-type coiled-coil region of RFP was able to relocate the Gal4-DBD to the cytoplasm. This relocation was again blocked by point mutations in the putative NES. In the addition to the cytoplasmic and nuclear localization, fusion proteins of the Gal4-DBD with the coiled-coil region of RFP were found in filament-like structures when the fusion proteins were expressed at very high levels (data not shown). Thus, the NES in the coiled-coil region can function alone. To confirm the function of the NES in RFP further, we also fused the RFP protein derivatives to the nuclear form of GFP, which harbors three NLS signals (EGFPnuc). The expression vectors for various EGFPnuc-RFP fusion proteins were transfected into NIH3T3 cells, and their subcellular localizations were analyzed (Fig. 4 B). Similar to the results obtained with the Gal4-DBD fusion proteins, wild-type RFP was again able to relocalize EGFPnuc to the cytoplasm. NES mutations led to nuclear localization of the fusion protein to the dot-like structure that resembled those of PML. In contrast, however, fusions of RFPmut with Gal4-DBD were almost uniformly distributed throughout the nucleoplasm (Fig. 4 A). This difference could be because, unlike EGFPnuc, Gal4-DBD has DNA binding activity. Because RFP was reported to form nuclear dot-like structure under certain conditions (21Cao T. Duprez E. Borden K.L.B. Freemont P.S. Etkin L.D. J. Cell Sci. 1998; 111: 1319-1329Crossref PubMed Google Scholar), the DNA binding activity of Gal4-DBD may disturb the localization of the fusion proteins to the nuclear dot-like structure. Deletion of the coiled-coil region also resulted in nuclear localization of RFP. However, these fusion proteins were distributed throughout an entire nucleoplasm. Thus, the results obtained with two different nuclear proteins (Gal4-DBD and EGFPnuc) have demonstrated that RFP harbors a functional NES in its coiled-coil region. In mouse, RFP homodimerization through the coiled-coil region was reported to be important for RFP localization (18Cao T. Borden K.L.B. Freemont P.S. Etkin L.D. J. Cell Sci. 1997; 110: 1563-1571Crossref PubMed Google Scholar). Human RFP can dimerize in a similar way through its coiled-coil region. 2M. Harbers and S. Ishii, unpublished results. Because the NES is in the coiled-coil region of RFP, we investigated whether point mutations in the NES affect the homodimerization using a yeast two-hybrid system. The results showed that wild-type RFP was able to interact with RFPmut, indicating that the point mutations in the NES did not destroy the overall structure of the coiled-coil domain.2 In contrast to NIH3T3 cells, RFP is localized predominantly in the nucleus in HepG2 cells. To address whether the NES of RFP is functional in HepG2 cells, the Gal4-DBD-RFP fusion proteins were also expressed in HepG2 cells, and their localizations were analyzed as in NIH3T3 cells (Fig.5 A). The coiled-coil region of RFP was able to relocate the Gal4-DBD protein to the cytoplasm, whereas mutations in the NES reestablished the nuclear localization of the Gal4-DBD fusion protein, indicating that the NES of RFP is also functional in HepG2 cells. In contrast, however, the fusion protein containing full-length RFP was still localized in the nucleus, and this was unaffected by point mutations in the putative NES (Gal4-DBD-RFPmut). These results indicate that the activity of NES in full-length RFP is masked by its interaction with other domain(s) in the RFP protein or with other protein(s). In addition to using Gal4-DBD fusion proteins, RFP fused to the nuclear form of GFP (EGFPnuc) was also expressed in HepG2 cells (Fig.5 B). Similar to the results obtained with the Gal4-DBD fusion proteins, full-length RFP failed to relocalize EGFPnuc to the cytoplasm of HepG2 cells." @default.
• W1503786870 created "2016-06-24" @default.
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• W1503786870 creator A5017352127 @default.
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• W1503786870 date "2001-12-01" @default.
• W1503786870 modified "2023-10-14" @default.
• W1503786870 title "Intracellular Localization of the Ret Finger Protein Depends on a Functional Nuclear Export Signal and Protein Kinase C Activation" @default.
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