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• W2080939902 abstract "Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130–154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein. Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130–154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein. retinoblastoma protein glutathione S-transferase equimolar mixture of dATP, dGTP, dTTP, and dCTP dithiothreitol fluorescein isothiocyanate green fluorescent protein nuclear export signal nuclear localization signal nuclear pore complex(es) wheat germ agglutinin The E2F family of transcription factors is involved in regulation of cell cycle progression and developmental processes such as axis formation, differentiation, and apoptosis (reviewed in Refs. 1Muller H. Helin K. Biochim. Biophys. Acta. 2000; 1470: M1-M12PubMed Google Scholar, 2Nevins J.R. Human Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (743) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (959) Google Scholar). This multigene family includes the E2F proteins (1 through 6), as well as the DP proteins (1 through 3; reviewed in Refs. 1Muller H. Helin K. Biochim. Biophys. Acta. 2000; 1470: M1-M12PubMed Google Scholar, 2Nevins J.R. Human Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (743) Google Scholar, 3Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (959) Google Scholar). E2F factors play a central role in controlling the G → S transition, by regulating transcription of DNA replication enzymes (4Kalma Y. Marash L. Lamed Y. Ginsberg D. Oncogene. 2001; 20: 1379-1387Crossref PubMed Scopus (62) Google Scholar), and cell cycle regulators such as cyclins E and A (reviewed in Refs. 2Nevins J.R. Human Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (743) Google Scholar and 5Ohtani K. Frontiers Biosci. 1999; 4: d793-d804Crossref PubMed Google Scholar). The transcriptional activity of E2F is modulated, in turn, at various levels. One of the most extensively studied mechanisms of regulation involves inhibition of E2F activity through association with pRB1 family proteins (pRB, p107, and p130; reviewed in Ref. 6Classon M. Dyson N. Exp. Cell Res. 2001; 264: 135-147Crossref PubMed Scopus (205) Google Scholar). In particular, formation of E2F·p130 complexes appears to be very important in the permanent cell cycle withdrawal characteristic of terminal differentiation. Recently, it has become apparent that other post-translational mechanisms participate in the control of E2F activity, including phosphorylation (7Peeper D.S. Keblusek P. Helin K. Toebes M. van der Eb A.J. Zantema A. Oncogene. 1995; 10: 39-48PubMed Google Scholar, 8Krek W., Xu, G. Livingston D.M. Cell. 1995; 83: 1149-1158Abstract Full Text PDF PubMed Scopus (316) Google Scholar, 9Lin W.-C. Lin F.-T. Nevins J.R. Genes Dev. 2001; 15: 1833-1844PubMed Google Scholar), acetylation (10Advani S.J. Weichselbaum R.R. Roizman B. J. Virol. 2000; 74: 7842-7850Crossref PubMed Scopus (25) Google Scholar, 11Marzio G. Wagener C. Gutierrez M.I. Cartwright P. Helin K. Giacca M. J. Biol. Chem. 2000; 275: 10887-10892Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 12Martinez-Balbas M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (568) Google Scholar, 13Martinez L.A. Chen Y. Fischer S.M. Conti C.J. Oncogene. 1999; 18: 397-406Crossref PubMed Scopus (61) Google Scholar), and regulation of subcellular localization (14Verona R. Moberg K. Estes S. Starz M. Vernon J.P. Lees J.A. Mol. Cell. Biol. 1997; 17: 7268-7282Crossref PubMed Scopus (181) Google Scholar, 15Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Crossref PubMed Scopus (166) Google Scholar, 16Muller H. Moroni M.C. Vigo E. Petersen B.O. Bartek J. Helin K. Mol. Cell. Biol. 1997; 17: 5508-5520Crossref PubMed Scopus (172) Google Scholar, 17Gill R.M. Hamel P.A. J. Cell Biol. 2000; 148: 1187-1201Crossref PubMed Scopus (38) Google Scholar, 18Gaubatz S. Lees J.A. Lindeman G.J. Livingston D.M. Mol. Cell. Biol. 2001; 21: 1384-1392Crossref PubMed Scopus (84) Google Scholar). In contrast to E2F-1, -2, and -3, which have a consensus NLS and are constitutively nuclear in most cells examined (15Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Crossref PubMed Scopus (166) Google Scholar, 19Magae J., Wu, C.L. Illenye S. Harlow E. Heintz N.H. J. Cell Sci. 1996; 109: 1717-1726Crossref PubMed Google Scholar), E2F-4 shuttles between the cytoplasm and the nucleus, and the relative proportion in each cellular compartment is, at least partially, cell cycle-dependent. Specifically, E2F-4 is predominantly nuclear in quiescent fibroblasts, and moves into the cytoplasm shortly after these cells are stimulated with serum, and as they approach the S phase (15Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Crossref PubMed Scopus (166) Google Scholar). These observations suggested that nuclear E2F-4 mediates transcriptional repression of growth-promoting genes in fibroblasts. In a variety of cell lines, exogenously expressed E2F-4 and E2F-5 are cytoplasmic, but they can be forced to translocate to the nucleus by coexpression of nuclear DP-2 or pRB family proteins. These observations, together with the lack of apparent nuclear localization signals, has led to the proposal that E2F-4 and E2F-5 reach the nucleus mainly by association with nuclear partners such as DP-2 or pRB family proteins (15Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Crossref PubMed Scopus (166) Google Scholar, 18Gaubatz S. Lees J.A. Lindeman G.J. Livingston D.M. Mol. Cell. Biol. 2001; 21: 1384-1392Crossref PubMed Scopus (84) Google Scholar, 19Magae J., Wu, C.L. Illenye S. Harlow E. Heintz N.H. J. Cell Sci. 1996; 109: 1717-1726Crossref PubMed Google Scholar). To date, the subcellular localization of E2F factors has been examined largely in the context of cell cycle progression in fibroblasts or tumorigenic cell lines rather than during irreversible cell cycle arrest associated with terminal differentiation. The latter situation is especially relevant to tissues such as the epidermis, which are constituted by both undifferentiated keratinocytes capable of proliferating, and their terminally differentiated progeny (20Zinkel S. Fuchs E. Semin. Cancer Biol. 1994; 5: 77-90PubMed Google Scholar,21Fuchs E. Curr. Opin. Cell Biol. 1990; 2: 1028-1035Crossref PubMed Scopus (108) Google Scholar). Primary epidermal keratinocytes can be isolated and cultured under conditions that maintain an undifferentiated population. These cells differentiate by culture in medium with extracellular calcium levels > 0.05 mm, to mimic the morphological changes, induction of differentiation markers and irreversible entry into quiescence observed in mature epidermal keratinocytes in situ (22Dotto G.P. Crit. Rev. Oral Biol. Med. 1999; 10: 442-457Crossref PubMed Scopus (94) Google Scholar, 23Yuspa S.H. Hennings H. Tucker R.W. Jaken S. Kilkenny A.E. Roop D.R. Ann. N. Y. Acad. Sci. 1988; 548: 191-196Crossref PubMed Scopus (94) Google Scholar). We have shown that E2F factors are differentially regulated in the epidermis, with E2F-2 and E2F-5 mRNA expression localized, respectively, to undifferentiated and differentiated keratinocytes (24Lindeman G.J. Dagnino L. Gaubatz S., Xu, Y. Bronson R.T. Warren H.B. Livingston D.M. Genes Dev. 1998; 12: 1092-1098Crossref PubMed Scopus (153) Google Scholar, 25Dagnino L. Fry C.J. Bartley S.M. Farnham P. Gallie B.L. Phillips R.A. Cell Growth Differ. 1997; 8: 553-563PubMed Google Scholar). A similar switch in E2F protein expression occurs in primary cultured murine keratinocytes, which express E2F-1, -2, and -3 in the undifferentiated, proliferative state. Induction of differentiation by elevated extracellular Ca+2 causes a reduction in these E2F forms, with a concomitant increase in E2F-5 protein, and no substantial change in E2F-4 levels (26D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Notably, the up-regulation of E2F-5 in differentiating keratinocytes is accompanied by formation of complexes containing E2F-5, p130, and histone deacetylase 1, which appear to be involved in irreversible cell cycle withdrawal (26D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The presence of E2F-5·p130 species in the nucleus of differentiated keratinocytes prompted us to investigate the mechanisms controlling E2F-5 subcellular localization. We now report differential regulation of E2F-4 and E2F-5 subcellular distribution upon induction of keratinocyte maturation. Furthermore, we demonstrate that E2F-5 has distinct domains that determine its subcellular localization. Specifically, E2F-5 has an intrinsic nuclear localization domain, which allows it to translocate into the nucleus independently of interactions with DP or pRB family proteins, and a nuclear export domain, which contributes to cytoplasmic localization mediated by active nuclear export through CRM1. Anti-E2F-4 (sc-866), anti-E2F-5 (sc-999), and monoclonal anti-GST (sc-138) antibodies were purchased from Santa Cruz Biotechnologies. Rabbit Anti-GST- (71–7500) and monoclonal anti-FLAG M5 antibodies were, respectively, from Zymed Laboratories Inc. and Eastman Kodak. Restriction enzymes were from Invitrogen or from New England Biolabs. Digitonin was from Calbiochem. Essential minimal Eagle’s medium without Ca+2 was purchased from BioWhittaker; all other cell culture materials were from Invitrogen. Cy3- and FITC-conjugated goat anti-mouse or goat anti-rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories. FITC-conjugated dextran (Mr 70,000) was purchased from ICN. All other chemicals were purchased from Sigma Chemical Co. HeLa, MCF-7, and HEK293 cells were purchased from the American Type Culture Collection. IMDF cells are a line of spontaneously immortalized dermal fibroblasts that we isolated from the dermis of a 2-day-old CD-1 mouse. All cell lines were maintained in Dulbecco’s modified Eagle’ medium supplemented with 8% fetal bovine serum at 37 °C, in a humidified 5% CO2 atmosphere. Primary mouse keratinocytes were isolated, cultured, and infected with recombinant adenovirus as described (26D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Primary keratinocytes were maintained as proliferating, undifferentiated cells in medium containing 50 μmCa+2 or induced to differentiate in medium containing ≥0.1 mm Ca+2. Transient transfections on HeLa, MCF-7, HEK293, or IMDF cells were conducted by the calcium phosphate method, using 1 μg of DNA/well in a 12-well culture plate. Primary mouse keratinocytes were transfected with LipofectAMINE Plus (Invitrogen). For nuclear export studies, cells were plated on glass coverslips in 12-well culture dishes so that they were 40–50% confluent the next day. At that time, they were transfected with indicated plasmids and, 24–48 h after DNA addition, they were processed for fluorescence microscopy. To inhibit CRM1-mediated nuclear export, transfected cells were cultured with cycloheximide (10 μg/ml, final) and leptomycin B (10 ng/ml, final) for 4 h prior to processing for microscopy. GST-E2F-1 and GST-E2F-2 plasmids were gifts from Drs. P. Hamel (University of Toronto) and D. C. Heimbrook (Merck Research Laboratories). GST-E2F-3 was obtained by excision of a 2-kb BamHI/XbaI fragment from CMV-E2F-3 (27Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin W.G.J. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (413) Google Scholar) and cloning in-frame into pET-41a (Novagen). GST-E2F-4 was produced by cloning a 2.5-kb SalI/EcoRI fragment from CMV-E2F4 (28Beijersbergen R.L. Kerkhoven R.M. Zhu L. Carlee L. Voorhoeve P.M. Bernards R. Genes Dev. 1994; 8: 2680-2690Crossref PubMed Scopus (318) Google Scholar) into pET-41b (Novagen). FLAG-tagged E2F-5 was constructed by cloning in-frame an EcoRI fragment form CMV-E2F-5 corresponding to the mouse E2F-5 cDNA (29Buck V. Allen K.E. Sorensen T. Bybee A. Hijmans E.M. Voorhoeve P.M. Bernards R. La Thangue N.B. Oncogene. 1995; 11: 31-38PubMed Google Scholar) into pBK-CMVΔLacZ-FLAG (a modified pBK-CMV expression vector (Stratagene) lacking the β-galactosidase cDNA), which contained FLAG-encoding sequences. The resulting plasmid (FLAG-E2F-5) encodes the full-length E2F-5 cDNA downstream from the FLAG tag. GFP-E2F-5 was generated by cloning in-frame a 1-kb BamHI/HindIII fragment encoding the tagged E2F-5 cDNA from FLAG-E2F-5 into pEGFP-C2 (CLONTECH). GFP-E2F-5-(Δ315–332), which lacks the pRB family-binding domain, GFP-E2F-5-(105–332), and mutants containing C-terminal deletions were obtained by PCR. GFP-E2F-5-(Δ84–128) was obtained byEcoRV/BglII excision of a 146-bp fragment, followed by Klenow treatment and ligation. GFP-E2F-5-(1–56) was generated excising from GFP-E2F-5 sequences corresponding to amino acids 57–339 and vector ligation. GFP-E2F-5-(1–130) was obtained by excision of a 650-bp BglII/BamHI fragment corresponding to amino acids 131–332 from GFP-E2F-5. Wild type GST-E2F-5 was obtained by cloning in-frame a 1-kbEcoRI fragment from FLAG-E2F-5 into pGEX-KG (a pGEX2T derivative with a polyglycine linker at the thrombin cleavage site). GST-E2F-5-(Δ315–332) was constructed by isolating a 1-kbHindIII/EcoRI fragment corresponding to mouse E2F-5-(Δ315–332) and cloning in-frame into pET-41c. GST-E2F-5-(105–346) was constructed by cloning a 670-bp BglII/HindIII fragment encoding mouse E2F-5 amino acids 105–346 from FLAG-mE2F5 in-frame into pET-41c. GST-E2F-5-(1–56) and GST-E2F-5-(1–179) were constructed by removal from GST-E2F-5-(Δ315–332) of fragments corresponding to E2F-5 amino acids 57–346 and 180–332, respectively, followed by ligation. GST-GFP was constructed by cloning a 738-bpNcoI-EcoRI fragment encoding the EGFP cDNA from pEGFP-C2 (CLONTECH) into pET-41a(+). GST-GFP-NLS was constructed by cloning in-frame a phosphorylated double-stranded oligonucleotide corresponding to the NLS of SV40 large T antigen (PKKKRKV) into GST-GFP. All mutant cDNAs were verified by sequencing using dideoxy methodology. GST fusion proteins containing E2F-1, -2, or -5 sequences were expressed in Escherichia coli BL21(DE3)-RIL (Stratagene), and those containing E2F-3 or E2F-4 sequences were expressed in E. coli BL21(DE3)-RP. All other GST fusion proteins were expressed in E. coli BL21(DE3). To induce protein production, bacteria were cultured at 37 °C in NYZ medium to an optical density of 1 (600 nm) and then supplemented with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside for 2 h. The bacteria were harvested by centrifugation, resuspended in lysis buffer (phosphate-buffered saline, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 4 μg/ml leupeptin, 2 μg/ml aprotinin), and lysed by passing twice through a pre-chilled French press at 1000–1500 p.s.i. After clearing the lysates by centrifugation (14,000 rpm, 15 min, 4 °C), the supernatants were incubated for 30 min at 22 °C with 100 μl of a glutathione-Sepharose slurry (Amersham Biosciences). The bound proteins were washed thrice with ice-cold lysis buffer, followed by elution in 50 mm Tris-HCl (pH 8) containing 10 mmglutathione for 30 min at 4 °C. With the exception of GST-E2F-5, no significant degradation products for each GST fusion protein were detected by SDS-PAGE analysis (not shown). Full-length GST-E2F-5 was purified from partial-length fusion proteins by fast protein liquid chromatography on a SP-Sepharose column. All experiments conducted contained fractions with ≥95% full-length GST-E2F-5. Transport assays were performed essentially as described previously (30Kurisaki A. Kose S. Yoneda Y. Heldin C.-H. Moustakas A. Mol. Biol. Cell. 2001; 12: 1079-1091Crossref PubMed Scopus (152) Google Scholar, 31Adam S.A. Marr R.S. Gerace L. J. Cell Biol. 1990; 111: 807-816Crossref PubMed Scopus (768) Google Scholar). Briefly, HeLa cells were plated on glass coverslips 24–48 h prior to use. The cells were rinsed three times with transport buffer (hereafter termed TB; composed of 20 mm HEPES, pH 7.3, 110 mm KOAc, 2 mmMg(OAc)2, 2 mm DTT, 1 mm EDTA), and permeabilized for 10 min at 4 °C with TB containing 2 mmDTT, 1 mm phenylmethylsulfonyl fluoride, 4 μg/ml leupeptin, 2 μg/ml aprotinin, and 40 μg/ml digitonin. After two washes with TB, the coverslips were immersed in TB supplemented with protease inhibitors and 2 mm DTT, and incubated at room temperature (22–23 °C) for 5 min. Excess buffer was removed, and the permeabilized cells were incubated with transport reaction mixture consisting of complete transport (CT) buffer, HeLa cytosol extract (with protease inhibitors), and the appropriate GST fusion protein for 30 min at the temperature indicated in individual experiments. CT buffer contained TB plus an ATP regeneration system (0.5 mmATP, 0.5 mm GTP, 5 mm creatine phosphate, and 20 units/ml creatine phosphokinase). Assays in the absence of an energy-regenerating system were conducted with TB, no ATP or GTP, and apyrase (10 units/ml). For WGA treatments, permeabilized cells were incubated in the presence of 0.05 mg/ml WGA in TB for 15 min prior to the import reaction. HeLa cytosol extract was prepared as described previously (32Imamoto N. Shimamoto T. Takao T. Tachibana T. Kose S. Matsubae M. Sekimoto T. Shimonishi Y. Yoneda Y. EMBO J. 1995; 14: 3617-3626Crossref PubMed Scopus (271) Google Scholar). Cell permeabilization and absence of nuclear membrane damage were verified by the absence of nuclear diffusion of FITC-labeled dextran (Mr 70,000). After the import assay, cells were rinsed with ice-cold transport buffer, and fixed with 3% paraformaldehyde at 4 °C for 40 min. After rinsing, GST fusion proteins were detected by fluorescence microscopy. To this end, the cells were blocked at room temperature in phosphate-buffered saline with 1% bovine serum albumin for 1 h, rinsed, and probed with an anti-GST antibody for 1 h at room temperature. After three rinses, the cells were probed with the appropriate Cy3- or FITC-labeled secondary antibody at room temperature for 1 h. After removal of the secondary antibody, the cells were rinsed twice, incubated with Hoescht 33258 (10 μg/ml) for 5 min at room temperature, rinsed five times, and mounted. All photomicrographs were obtained with a Leica DMIRBE microscope equipped with an Orca charge-coupled device camera, using Openlab software (Improvision). The proportion of cells exhibiting the presence or absence of the tested GST fusion protein in the nucleus was calculated from examination of 500–1400 cells in random microscope fields, and each experiment was repeated four to nine times with similar results. Induction of terminal differentiation in mouse epidermal keratinocytes is accompanied by increased E2F-5 protein expression and formation of E2F-5·p130 complexes (26D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). These complexes are involved in differentiation-induced keratinocyte exit from the cell cycle. To begin to understand the mechanisms regulating the formation of E2F-5·p130 complexes in differentiated epidermal keratinocytes, we examined by fluorescence microscopy the subcellular distribution of E2F-5 in undifferentiated keratinocytes and in cells induced to differentiate with Ca+2. Whereas E2F-5 in undifferentiated keratinocytes is scarce and largely cytoplasmic, induction of differentiation is accompanied by up-regulation and concentration of E2F-5 in the nucleus (Fig.1), consistent with the established formation of the nuclear E2F-5·p130 species. Interaction with pRB family proteins, such as p130, has been proposed to play a role in regulating E2F-4 and E2F-5 subcellular distribution. Consequently, we next examined whether Ca+2-induced retention of E2F-5 in differentiated keratinocytes requires association with p130. It is well established that interactions of E2F with pRB family proteins are disrupted by several viral oncoproteins. The adenovirus E1A protein binds pRB family proteins, thereby releasing E2F (reviewed in Ref. 33Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1365) Google Scholar). Thus, we examined the subcellular localization of E2F-5 in differentiated keratinocytes in which we induced exogenous expression of E1A by adenovirus-mediated gene transfer and observed maintenance of nuclear distribution of E2F-5 (Fig. 1), indicating that nuclear retention of E2F-5 does not require interactions with pRB family proteins. E2F-5 shares extensive structural homology and biochemical properties with E2F-4, and these two proteins have frequently been grouped together. Furthermore, nuclear localization of E2F-4 has been associated with cell cycle withdrawal and myoblast differentiation (15Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Crossref PubMed Scopus (166) Google Scholar,18Gaubatz S. Lees J.A. Lindeman G.J. Livingston D.M. Mol. Cell. Biol. 2001; 21: 1384-1392Crossref PubMed Scopus (84) Google Scholar, 34Puri P.L. Cimino L. Fulco M. Zimmerman C., La Thangue N.B. Giordano A. Graessmann A. Levrero M. Cancer Res. 1998; 58: 1325-1331PubMed Google Scholar). In contrast to E2F-5, E2F-4 mRNA and protein tend to decrease during differentiation in keratinocytes (25Dagnino L. Fry C.J. Bartley S.M. Farnham P. Gallie B.L. Phillips R.A. Cell Growth Differ. 1997; 8: 553-563PubMed Google Scholar, 26D'Souza S.J.A. Pajak A. Balazsi K. Dagnino L. J. Biol. Chem. 2001; 276: 23531-23538Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To investigate if there are also differences between E2F-4 and E2F-5 regulation of subcellular localization in keratinocytes, we conducted immunofluorescence assays. We observed that E2F-4 always distributes throughout the cell, irrespective of the differentiation status of the keratinocytes (Fig. 1). These patterns of distribution differ from the constitutively nuclear localization of E2F-1, -2, and -3 in keratinocytes, which we have determined by analysis of fractionated cell extracts (data not shown), and is similar to their distribution in other cell types (14Verona R. Moberg K. Estes S. Starz M. Vernon J.P. Lees J.A. Mol. Cell. Biol. 1997; 17: 7268-7282Crossref PubMed Scopus (181) Google Scholar, 15Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Crossref PubMed Scopus (166) Google Scholar, 35Allen K.E. de la Luna S. Kerkhoven R.M. Bernards R. La Thangue N.B. J. Cell Sci. 1997; 110: 2819-2831Crossref PubMed Google Scholar). Given the substantial differences in steady-state subcellular distribution of the various E2F proteins, we first investigated the mechanisms mediating their entry into the nucleus, using GST-E2F fusion proteins in in vitronuclear import assays on digitonin-permeabilized cells. This system has been extensively used to characterize basic nuclear import mechanisms of a variety of proteins. We observed that GST fusion proteins containing sequences corresponding to E2F-1, -2, or -3 localized to the nucleus in ≥97% of permeabilized cells (Fig.2), consistent with the reported existence of a NLS in these proteins (14Verona R. Moberg K. Estes S. Starz M. Vernon J.P. Lees J.A. Mol. Cell. Biol. 1997; 17: 7268-7282Crossref PubMed Scopus (181) Google Scholar, 15Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Crossref PubMed Scopus (166) Google Scholar, 35Allen K.E. de la Luna S. Kerkhoven R.M. Bernards R. La Thangue N.B. J. Cell Sci. 1997; 110: 2819-2831Crossref PubMed Google Scholar). Surprisingly, and in contrast with their predominantly cytoplasmic distribution in many intact cells, E2F-4 and E2F-5 GST fusion proteins were found in the nucleus in about 60% of the cells (Fig. 2). The nuclear distribution of all E2F-containing GST fusion proteins also differed from GST alone, which was largely cytoplasmic, suggesting that sequences in all the E2F proteins examined contain nuclear localization domains. We next used in vitro import assays to examine the mechanisms involved in E2F-5 nuclear entry, contrasting them with those of E2F-2 translocation. We chose E2F-2 as a prototype of E2F proteins containing a consensus NLS. We initially verified that nuclear E2F entry was indeed mediated and not due to simple diffusion. To this end, we determined the sensitivity of E2F nuclear translocation to low temperature, which impairs mediated transport, but has no effect on simple diffusion. We found that both GST-E2F-2 and GST-E2F-5 proteins were unable to enter the nucleus in experiments conducted at 4 °C, confirming the involvement of mediated transport mechanisms in the import step (Fig. 3). Mediated nuclear import through the nuclear pore requires energy. When we conducted import assays in the absence of all sources of energy (that is, no ATP or GTP, and apyrase to inhibit cellular energy regeneration systems), both E2F proteins showed almost exclusively cytoplasmic localization (Fig. 3). This indicates the involvement of import complex formation and translocation of these complexes through nuclear pores. Finally, transport of proteins through the nuclear pore is inactivated by WGA-induced glycosylation. To confirm that E2F translocation to the nucleus indeed involves nuclear pores, we conducted experiments in permeabilized cells pre-treated with WGA, and found that uptake of GST-E2F-2 and GST-E2F-5 was greatly reduced (Fig. 3). Together, these studies demonstrate that E2F proteins exhibit mediated transport into the nucleus, through mechanisms that make use of the nuclear pore. As mentioned above, analysis of the amino acid sequence in E2F-4 and E2F-5 fails to reveal regions with homology to known nuclear localization signals. For this reason, it has been proposed that these E2F factors enter the nucleus by virtue of complex formation with nuclear E2F-interacting proteins such as DP-2 or pRB family members (14Verona R. Moberg K. Estes S. Starz M. Vernon J.P. Lees J.A. Mol. Cell. Biol. 1997; 17: 7268-7282Crossref PubMed Scopus (181) Google Scholar, 35Allen K.E. de la Luna S. Kerkhoven R.M. Bernards R. La Thangue N.B. J. Cell Sci. 1997; 110: 2819-2831Crossref PubMed Google Scholar). However, our in vitro import assays strongly suggested the presence of nucle" @default.
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