Subcellular Localization and Mechanisms of Nucleocytoplasmic Trafficking of Steroid Receptor Coactivator-1
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m300730200
ISSN1083-351X
AutoresLarbi Amazit, Youssef Alj, Rakesh K. Tyagi, Anne Chauchereau, Hugues Loosfelt, Christophe Pichon, Jacques Pantel, Emmanuelle Foulon-Guinchard, Philippe Leclerc, Edwin Milgröm, Anne Guiochon‐Mantel,
Tópico(s)Steroid Chemistry and Biochemistry
ResumoSteroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation. Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation. Steroid hormone receptors belong to the superfamily of nuclear receptors (1Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6292) Google Scholar, 2Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar). Although these proteins are mainly localized in the nucleus, they are constantly shuttling between the nucleus and the cytoplasm (3Hager G.L. Lim C.S. Elbi C. Baumann C.T. J. Steroid. Biochem. Mol. Biol. 2000; 74: 249-254Crossref PubMed Scopus (111) Google Scholar, 4Guiochon-Mantel A. Delabre K. Lescop P. Milgrom E. J. Steroid. Biochem. Mol. Biol. 1996; 56: 3-9Crossref PubMed Scopus (80) Google Scholar, 5DeFranco D.B. Cell Biochem. Biophys. 1999; 30: 1-24Crossref PubMed Scopus (38) Google Scholar). Their nuclear localization signals (NLS) 1The abbreviations used are: NLS, nuclear localization signal; NES, nuclear export signal; AD, activation domain; CAT, chloramphenicol acetyltransferase; CHX, cycloheximide; DMEM, Dulbecco's modified Eagle's medium; DsRed, (Discosoma sp.) red fluorescent protein; EGFP, enhanced green fluorescent protein; FCS, fetal calf serum; HA, hemagglutinin; LMB, leptomycin B; PBS, phosphate-buffered saline; PR, progesterone receptor; R5020, 17,21-dimethyl-19-norpregna-4,9-dien-3,20-dione; SRC, steroid receptor coactivator; bHLH, basic helix-loop-helix; HIF, hypoxia-inducible factor; aa, amino acid(s); PAS, Per Arnt-Sim motif.1The abbreviations used are: NLS, nuclear localization signal; NES, nuclear export signal; AD, activation domain; CAT, chloramphenicol acetyltransferase; CHX, cycloheximide; DMEM, Dulbecco's modified Eagle's medium; DsRed, (Discosoma sp.) red fluorescent protein; EGFP, enhanced green fluorescent protein; FCS, fetal calf serum; HA, hemagglutinin; LMB, leptomycin B; PBS, phosphate-buffered saline; PR, progesterone receptor; R5020, 17,21-dimethyl-19-norpregna-4,9-dien-3,20-dione; SRC, steroid receptor coactivator; bHLH, basic helix-loop-helix; HIF, hypoxia-inducible factor; aa, amino acid(s); PAS, Per Arnt-Sim motif. have been characterized as complex amino acid sequences encompassing the DNA binding domain and part of the hinge region in the ligand binding domain (6Picard D. Yamamoto K.R. EMBO J. 1987; 6: 3333-3340Crossref PubMed Scopus (722) Google Scholar, 7Guiochon-Mantel A. Loosfelt H. Lescop P. Sar S. Atger M. Perrot-Applanat M. Milgrom E. Cell. 1989; 57: 1147-1154Abstract Full Text PDF PubMed Scopus (242) Google Scholar, 8Picard D. Kumar V. Chambon P. Yamamoto K.R. Cell Regul. 1990; 1: 291-299Crossref PubMed Scopus (210) Google Scholar, 9Ylikomi T. Bocquel M.T. Berry M. Gronemeyer H. Chambon P. EMBO J. 1992; 11: 3681-3694Crossref PubMed Scopus (251) Google Scholar, 10Simental J.A. Sar M. Lane M.V. French F.S. Wilson E.M. J. Biol. Chem. 1991; 266: 510-518Abstract Full Text PDF PubMed Google Scholar, 11Laudet V. Gronemeyer H. The Nuclear Receptor, Facts Book. 1st ed. Academic Press, San Diego2001Google Scholar). Nuclear receptors activate transcription by recruiting coactivators and cointegrators (12Robyr D. Wolffe A.P. Wahli W. Mol. Endocrinol. 2000; 14: 329-347Crossref PubMed Scopus (325) Google Scholar, 13McKenna N.J. Lanz R.B. O'Malley B.W. Endocr. Rev. 1999; 20: 321-344Crossref PubMed Scopus (1639) Google Scholar), which in turn recruit histone acetyltransferases to acetylate histones. Acetylation of the nucleosomes generates a loose chromatin form that allows the assembly of the transcription initiation complex on the target gene promoter (14Sterner D.E. Berger S.L. Microbiol. Mol. Biol. Rev. 2000; 64: 435-459Crossref PubMed Scopus (1385) Google Scholar, 15McKenna N.J. Xu J. Nawaz Z. Tsai S.Y. Tsai M.J. O'Malley B.W. J. Steroid. Biochem. Mol. Biol. 1999; 69: 3-12Crossref PubMed Scopus (362) Google Scholar, 16Collingwood T.N. Urnov F.D. Wolffe A.P. J. Mol. Endocrinol. 1999; 23: 255-275Crossref PubMed Scopus (265) Google Scholar). On the contrary, corepressor binding leads to deacetylation of histones and inhibition of gene transcription (15McKenna N.J. Xu J. Nawaz Z. Tsai S.Y. Tsai M.J. O'Malley B.W. J. Steroid. Biochem. Mol. Biol. 1999; 69: 3-12Crossref PubMed Scopus (362) Google Scholar, 16Collingwood T.N. Urnov F.D. Wolffe A.P. J. Mol. Endocrinol. 1999; 23: 255-275Crossref PubMed Scopus (265) Google Scholar, 17Shibata H. Spencer T.E. Onate S.A. Jenster G. Tsai S.Y. Tsai M.J. O'Malley B.W. Recent Prog. Horm. Res. 1997; 52: 141-164PubMed Google Scholar, 18Xu L. Glass C.K. Rosenfeld M.G. Curr. Opin. Genet. Dev. 1999; 9: 140-147Crossref PubMed Scopus (808) Google Scholar).Little is known about the subcellular localization and dynamic changes in subcellular distribution of steroid receptor coregulators. Available data are still contradictory, and these variations may be attributed to different cell culture conditions, cell differentiation state, and different detection methods (19Wu R.C. Qin J. Hashimoto Y. Wong J. Xu J. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell Biol. 2002; 22: 3549-3561Crossref PubMed Scopus (234) Google Scholar, 20Chen S.L. Wang S.C. Hosking B. Muscat G.E. Mol. Endocrinol. 2001; 15: 783-796Crossref PubMed Google Scholar, 21Kim H.J. Yi J.Y. Sung H.S. Moore D.D. Jhun B.H. Lee Y.C. Lee J.W. Mol. Cell Biol. 1999; 19: 6323-6332Crossref PubMed Scopus (52) Google Scholar, 22Wang Z. Rose D.W. Hermanson O. Liu F. Herman T. Wu W. Szeto D. Gleiberman A. Krones A. Pratt K. Rosenfeld R. Glass C.K. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13549-13554Crossref PubMed Scopus (170) Google Scholar, 23Stenoien D.L. Mancini M.G. Patel K. Allegretto E.A. Smith C.L. Mancini M.A. Mol. Endocrinol. 2000; 14: 518-534PubMed Google Scholar, 24Zilliacus J. Holter E. Wakui H. Tazawa H. Treuter E. Gustafsson J.A. Mol. Endocrinol. 2001; 15: 501-511Crossref PubMed Scopus (64) Google Scholar). Most of these observations were obtained by expressing fluorescent chimeric proteins in live cells (23Stenoien D.L. Mancini M.G. Patel K. Allegretto E.A. Smith C.L. Mancini M.A. Mol. Endocrinol. 2000; 14: 518-534PubMed Google Scholar, 25Stenoien D.L. Patel K. Mancini M.G. Dutertre M. Smith C.L. O'Malley B.W. Mancini M.A. Nat. Cell Biol. 2001; 3: 15-23Crossref PubMed Scopus (336) Google Scholar). The advantage of this approach is to allow in vivo detection of the protein and thus the study of intracellular trafficking dynamics. However, the data obtained with this method need to be substantiated with immunodetection experiments to avoid potential pitfalls due to the fluorescent tag. Indeed, the nature of the fluorescent tag (EGFP, DsRed1, DsRed2, etc.) and its position in the expressed protein (N- or C-terminal) can modify the conformation of the protein and thus interfere with its subcellular localization, trafficking, and transcription function (26Söling A. Simm A. Rainov N.G. FEBS Lett. 2002; 527: 153-158Crossref PubMed Scopus (22) Google Scholar).The present study documents the subcellular localization and the dynamics of intracellular trafficking of the steroid receptor coactivator 1 (SRC-1) as a prototype of nuclear receptors coactivators (27Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2042) Google Scholar, 28Takeshita A. Yen P.M. Misiti S. Cardona G.R. Liu Y. Chin W.W. Endocrinology. 1996; 137: 3594-3597Crossref PubMed Scopus (145) Google Scholar). It belongs to the p160 family of coactivators that also includes SRC-2/GRIP1/TIF2 (29Hong H. Kohli K. Trivedi A. Johnson D.L. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4948-4952Crossref PubMed Scopus (611) Google Scholar, 30Voegel J.J. Heine M.J. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (947) Google Scholar) and SRC-3/AIB1/ACTR/RAC3/TRAM1/p/CIP (31Suen C.S. Berrodin T.J. Mastroeni R. Cheskis B.J. Lyttle C.R. Frail D.E. J. Biol. Chem. 1998; 273: 27645-27653Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 32Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Crossref PubMed Scopus (1422) Google Scholar, 33Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1253) Google Scholar, 34Li H. Gomes P.J. Chen J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8479-8484Crossref PubMed Scopus (499) Google Scholar, 35Takeshita A. Cardona G.R. Koibuchi N. Suen C.S. Chin W.W. J. Biol. Chem. 1997; 272: 27629-27634Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 36Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1102) Google Scholar). This family of coactivators is characterized by the presence of several conserved functional domains: a bHLH-PAS N-terminal domain, a CBP interacting domain (AD1), a glutamine-rich region, a C-terminal activation domain (AD2), and several LXXLL boxes involved in nuclear receptor binding. Isoforms of SRC-1 have been described, SRC-1a and SRC-1e, which differ in their C-terminal region (37Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1916) Google Scholar, 38Hayashi Y. Ohmori S. Ito T. Seo H. Biochem. Biophys. Res. Commun. 1997; 236: 83-87Crossref PubMed Scopus (51) Google Scholar, 39Kalkhoven E. Valentine J.E. Heery D.M. Parker M.G. EMBO J. 1998; 17: 232-243Crossref PubMed Scopus (273) Google Scholar). Presently, the minor amount of information on the subcellular localization of SRC-1 is controversial (20Chen S.L. Wang S.C. Hosking B. Muscat G.E. Mol. Endocrinol. 2001; 15: 783-796Crossref PubMed Google Scholar, 23Stenoien D.L. Mancini M.G. Patel K. Allegretto E.A. Smith C.L. Mancini M.A. Mol. Endocrinol. 2000; 14: 518-534PubMed Google Scholar, 40Nazareth L.V. Stenoien D.L. Bingman 3rd, W.E. James A.J. Wu C. Zhang Y. Edwards D.P. Mancini M. Marcelli M. Lamb D.J. Weigel N.L. Mol. Endocrinol. 1999; 13: 2065-2075Crossref PubMed Google Scholar). We have used several fluorescent tags for live cell imaging in addition to molecular and immunological approaches. In this report we show that SRC-1 is trafficking between the nucleus and the cytoplasm. We postulate that nucleocytoplasmic trafficking of SRC-1 has a role in modulating the transcriptional activity of PR in particular and steroid receptors in general, thereby controlling molecular events of hormone action.EXPERIMENTAL PROCEDURESPlasmids: Nomenclature—Plasmid derivatives denoted with the Δ symbol lack the coactivator segment delineated by the numbered amino acids. Plasmids encoding the wild-type human SRC-1 (pSG5-SRC-1 and pSG5-HA-SRC-1) and deletion mutants (pSG5-HA-SRC-1Δ(1–567), pSG5-HA-SRC-1Δ(1–781)) have been previously described (41Chauchereau A. Georgiakaki M. Perrin-Wolff M. Milgrom E. Loosfelt H. J. Biol. Chem. 2000; 275: 8540-8548Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Expression vectors pSG5-HA-SRC-1Δ(1–1198) and pSG5-HA-SRC-Δ(1–177) were obtained by ligation after digestion of pSG5-HA-SRC-1 by NotI/MscI and XmaI/PstI, respectively. Expression vectors pSG5-HA-SRC-1Δ(216–1440) and pSG5-HA-SRC-1Δ(784–1440) were obtained after insertion of a stop codon in the 5′HindIII and 5′BamHI sites of pSG5-HA-SRC-1, respectively. PCR was used to generate the expression vector for the SRC-1 mutant pSG5-HA-SRC-1Δ(785–1038) by inserting a BamH1 site in position 2343 corresponding to the amino acid 782. For the generation of the mutant pSG5-HA-SRC-1Δ(988–1440) a PCR fragment was prepared containing BamHI/MscI extremities and a stop codon in the position 2962 corresponding to the amino acid 988. For these two constructs, the deletion was obtained by replacing the wild-type fragment BamHI/MscI by the BamHI/MscI-digested PCR fragment. PCR-based site-directed mutagenesis of pSG5-HA-SRC-1 was used to create deletion mutants: pSG5-HA-Δ(18–36)-SRC-1 (named Δ(NLS)SRC-1 in the text), pSG5-HA-SRC-1Δ(865–876), pSG5-HA-SRC-1Δ(948–960), pSG5-HA-SRC-1Δ(948–969), pSG5-HA-SRC-1Δ(865–876, 948–960), and pSG5-HA-SRC-1Δ(990–1060). pSG5-HA-GFP-SRC-1 and pSG5-HA-GFP-Δ(NLS)-SRC-1 plasmids were generated by PCR amplification of the EGFP sequence from pEGFP-C1 vector (Clontech, Palo Alto, CA) using the primers 5′-EGFP (5′-ACTGGCGCGCCTATGGTGAGCAAGGGCGAGGA-3′) and 3′-EGFP (5′-AGCGCGGCCGCCGGACTTGTACAGCTCGTCCA-3′) to create an EGFP encoding fragment with 5′AscI and 3′NotI sites used for subcloning into pSG5-HA-SRC-1 and pSG5-HA-Δ(NLS)-SRC-1 vectors, respectively. HA-DsRed-PR was generated by PCR amplification of the DsRed1 sequence from pDsRed1-C1 vector (Clontech) using the primers 5′DsRed (5′-ACTGGCGCTAGCATGGTGCGCTCCTCCAAGAA-3′) and 3′DsRed (5′-GATGTCGAATTCGAGCAGGAACAGGTGGTGGC-3′) to create a DsRed1 encoding fragment with 5′NheI and 3′EcoRI sites used for subcloning into pSG5-HA-rPR vector, encoding rabbit progesterone receptor. Each construct was verified by sequencing analysis. PR encoding vector and (PRE)2-TATA-CAT reporter plasmids have been previously described (42Guiochon-Mantel A. Savouret J.F. Quignon F. Delabre K. Milgrom E. de Thé H. Mol. Endocrinol. 1995; 9: 1791-1803PubMed Google Scholar). pKSV-PRΔ(25–103, 547–662) expression vector encodes a cytoplasmic PR mutant (named Δ(NLS)PR in the text) and has been previously described (43Tyagi R.K. Amazit L. Lescop P. Milgrom E. Guiochon-Mantel A. Mol. Endocrinol. 1998; 12: 1684-1695Crossref PubMed Scopus (71) Google Scholar).Monoclonal and Polyclonal Anti-SRC-1 Antibodies—Rabbit polyclonal and murine monoclonal antibodies were produced against the N-terminal amino acids (1–361) of the human SRC-1 according to a previously described protocol (44Loosfelt H. Pichon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (175) Google Scholar). Two polyclonal antibodies and five monoclonal antibodies (anti-SRC-1 S1 to S5) were generated and analyzed by enzyme-linked immunosorbent assay. The specificity of antibodies was studied as previously described (44Loosfelt H. Pichon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (175) Google Scholar), including detection of SRC-1 expressed by transfection of mammalian cells, using Western blot and immunocytochemical techniques. The antibodies generated a very weak signal against the endogenous protein in non-transfected cells. In these studies, we used the higher titer polyclonal antibody (1:4000) and the S4 monoclonal antibody (IgG1K) at the concentration of 5 μg/ml.Cell Culture—COS-7, CV1, HeLa, and L cells were grown in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Gaithersburg, MD) containing 10% fetal calf serum (FCS) (Invitrogen), and supplemented with l-glutamine (2 mm), sodium pyruvate (1 mm), and antibiotics (Invitrogen). BHK21 cells were grown under the same conditions except for temperature (33 °C) and atmosphere (10% CO2 and 90% air). The generation of mouse L cells stably expressing SRC-1 was performed as described previously (45Guiochon-Mantel A. Lescop P. Christin-Maitre S. Loosfelt H. Perrot-Applanat M. Milgrom E. EMBO J. 1991; 10: 3851-3859Crossref PubMed Scopus (246) Google Scholar). For hormonal regulation experiments, cells were grown in the presence of 10% steroid-depleted FCS. Where indicated cycloheximide (CHX) (Sigma) was used at a concentration of 10 μg/ml. This concentration suppressed 95% of protein synthesis (46Guiochon-Mantel A. Delabre K. Lescop P. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7179-7183Crossref PubMed Scopus (78) Google Scholar, 47Nardulli A.M. Katzenellenbogen B.S. Endocrinology. 1986; 119: 2038-2046Crossref PubMed Scopus (76) Google Scholar, 48Madsen P. Nielsen S. Celis J.E. J. Cell Biol. 1986; 103: 2083-2089Crossref PubMed Scopus (20) Google Scholar). Progesterone (Sigma) and R5020 (17,21-dimethyl-19-norpregna-4,9-dien-3,20-dione) were used at a concentration of 10 nm. Leptomycin B (LMB) was used at a concentration of 40 nm (43Tyagi R.K. Amazit L. Lescop P. Milgrom E. Guiochon-Mantel A. Mol. Endocrinol. 1998; 12: 1684-1695Crossref PubMed Scopus (71) Google Scholar, 49Wolff B. Sanglier J.J. Wang Y. Chem. Biol. 1997; 4: 139-147Abstract Full Text PDF PubMed Scopus (572) Google Scholar). L cell lines permanently expressing pSG5-HA-SRC-1 and pSG5-GFP-SRC-1 were obtained as previously described (45Guiochon-Mantel A. Lescop P. Christin-Maitre S. Loosfelt H. Perrot-Applanat M. Milgrom E. EMBO J. 1991; 10: 3851-3859Crossref PubMed Scopus (246) Google Scholar).Cell Cycle—BHK21 cells transfected in duplicate with SRC-1-encoding vector were first maintained for 36 h in isoleucine-deficient minimum essential medium supplemented with 5% charcoal-stripped and dialyzed FCS to arrest cells in the G1 phase (50Tobey R.A. Ley K.D. J. Cell Biol. 1970; 46: 151-157Crossref PubMed Scopus (127) Google Scholar). Cells were then released from cell cycle arrest and switched to complete DMEM containing 1.5 mm hydroxyurea for 12 h to resynchronize the cells in early S phase (51Tobey R.A. Methods Cell Biol. 1973; 6: 67-112Crossref PubMed Scopus (40) Google Scholar). For G2 synchrony (52Tobey R.A. Oishi N. Crissman H.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5104-5108Crossref PubMed Scopus (62) Google Scholar), cells were released from hydroxyurea and cultured in DMEM containing Hoechst 33342 (Calbiochem) at 1.5 μg/ml for 12 h. One set of cells was analyzed by immunochemistry (see below), and another was used for cell cycle analysis. Cell cycle analysis was carried out by flow cytometry. Briefly, cells were collected by trypsinization, pelleted, and resuspended in phosphate buffered saline (PBS) to a final concentration of ∼106 cells/ml. Two volumes of cold absolute ethanol were added, and the samples were stored at –20 °C until the day of analysis. During analysis, cells were pelleted and resuspended in PBS. These ethanol-fixed cells were incubated in a staining solution containing 50 μg/ml propidium iodide and 100 μg/ml RNase A (Stratagene) in PBS as described previously (53Deitch A.D. Law H. DeVere White R. J. Histochem. Cytochem. 1982; 30: 967-972Crossref PubMed Scopus (128) Google Scholar). The cells were analyzed after 1 h of incubation in darkness at 4 °C.Transfection and Analysis of CAT Activity—Transfections of CV1 cells were performed in 6-well dishes by the standard calcium phosphate precipitation method as described previously (41Chauchereau A. Georgiakaki M. Perrin-Wolff M. Milgrom E. Loosfelt H. J. Biol. Chem. 2000; 275: 8540-8548Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). CAT activity was measured with the CAT enzyme-linked immunosorbent assay kit (Roche Applied Science). Protein concentrations were determined by using the BCA protein assay (Pierce), and CAT activity was corrected for protein content. The results are the means ± S.E. of three separate experiments.Immunodetection and Confocal Microscopy—Cells were seeded on glass coverslips placed in 6-well dishes (Costar). Transfections were performed with the indicated expression vectors using LipofectAMINE according to the manufacturer's recommendations (Invitrogen). For GFP-Δ(NLS)-SRC-1/DsRed-PR and Δ(NLS)-PR/HA-SRC-1 cotransport experiments, the corresponding expression plasmids were transfected at a ratio of 1:5. Cells were fixed and processed as previously described (7Guiochon-Mantel A. Loosfelt H. Lescop P. Sar S. Atger M. Perrot-Applanat M. Milgrom E. Cell. 1989; 57: 1147-1154Abstract Full Text PDF PubMed Scopus (242) Google Scholar). For PR detection, we used the Mi60 monoclonal antibody (54Logeat F. Vu Hai M.T. Fournier A. Legrain P. Buttin G. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6456-6459Crossref PubMed Scopus (118) Google Scholar) at a final concentration of 8 μg/ml. Rat monoclonal anti-HA epitope antibody 3F10 (Roche Diagnostic) was used at 2 μg/ml. All secondary antibodies were used at a dilution of 1:400. The conjugated secondary antibodies goat Alexa 488, goat Alexa 546, and goat Alexa 594 (Molecular Probes) and sheep Cy3 (Sigma) were used to detect anti-PR Mi60 monoclonal, anti-SRC-1 polyclonal, anti-HA (3F10) monoclonal, and anti-SRC-1 (S4) monoclonal antibodies, respectively. In some experiments, a nucleic acid marker was added during secondary antibody incubation (SYTOX Green, Molecular Probes) at 200 nm.The subcellular localization of the coactivator was scored in at least 100 cells under each experimental condition. Staining was considered as nuclear when it was exclusively nuclear (N) or stronger in the nucleus than in the cytoplasm (N > C). In all other cases, it was considered cytoplasmic (if C = N, if C > N and if C). Fluorescent cells were observed and scanned with the LSM410 system (Carl Zeiss, Jena, Germany) assembled on an inverted microscope Axiovert 135M. Digitalized pictures were analyzed by Zeiss LSM application.RESULTSSRC-1 Is Localized in Both the Cytoplasm and the Nucleus— We transfected COS-7 cells with an expression vector encoding SRC-1 to examine its subcellular distribution. Immunocytochemical detection (40 h post-transfection) using a specific anti-SRC-1 antibody showed the protein to be present in both the cytoplasm and the nucleus in about 50% of cells (Fig. 1, a–c). In the other cells, labeling was restricted either to the nucleus or to the cytoplasm. In both compartments, SRC-1 was associated with corpuscular structures. Localization of transcriptional regulators in nuclear speckles has previously been described, but the presence of similar structures in the cytoplasm is an unusual observation (55Baumann C.T. Ma H. Wolford R. Reyes J.C. Maruvada P. Lim C. Yen P.M. Stallcup M.R. Hager G.L. Mol. Endocrinol. 2001; 15: 485-500Crossref PubMed Scopus (86) Google Scholar, 56Wu X. Li H. Park E.J. Chen J.D. J. Biol. Chem. 2001; 276: 24177-24185Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Furthermore, some detection techniques have been shown to produce artifactual data. Thus, we examined the possibility that our observation might be an artifact due to fixation, immunocytochemistry, or transfection. Identical localization of SRC-1 was observed even when we varied the fixation methods (ethanol, paraformaldehyde, and methanol), antibodies (monoclonal or polyclonal anti-SRC-1 antibodies, anti-HA antibody detection of a HA-SRC-1 construct), cell types (CV1, L, SW13, and C33), and transfection methods (calcium phosphate and LipofectAMINE). The same pattern of localization was also observed in living cells after transfection with GFP-SRC-1 (Fig. 1b). The possibility that overexpression of the protein might be responsible for this pattern has also been considered. However, by varying the amount of the vector (from 1 to 2000 ng), we did not observe difference in the localization of SRC-1 (Fig. 2A). Similar results were also observed with established L cell lines permanently expressing either SRC-1 or GFP-SRC-1 fusion protein (Fig. 2B, panel a).Fig. 2SRC-1 subcellular localization is not affected by its expression level. A, COS-7 cells were transiently transfected with different amounts of vector encoding SRC-1: 1 ng (a), 5 ng (b), 100 ng (c), 500 ng (d), and 2000 ng (e). Forty hours post-transfection, the cells were fixed, treated for immunodetection with anti-SRC-1 antibody, and observed by confocal microscopy. Bar, 5 μm. B, L cells were fixed, treated for immunodetection, and observed by confocal microscopy. a, L cells permanently expressing SRC-1 were analyzed by immunofluorescence with anti-SRC-1 antibody. b, L cells were treated for immunodetection with a non-specific antibody. c, L cells were treated for immunodetection with anti-SRC-1 antibody. SRC-1 can be either nuclear or nuclear and cytoplasmic in different cells. Bar, 10 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Detection of endogenous SRC-1 in non-transfected cells was at the limit of sensitivity of the immunocytochemical method. Diffuse nucleocytoplasmic labeling was observed (Fig. 2B, panel c). Thus, based on our observations, SRC-1 localizes either in the nucleus or in the cytoplasm or in both compartments in different cells.Nuclear Cytoplasmic Trafficking of SRC-1—We next tried to understand the heterogeneous localization of SRC-1. One possibility was that SRC-1 localization varied in a cell cycle phase-dependent manner. BHK21 cells were transfected with an SRC-1 expression vector and arrested at different stages of the cell cycle (G1, S, and G2/M). Fluorescence-activated cell sorting analysis of the arrested cells failed to demonstrate a correlation between SRC-1 subcellular localization and cell cycle phase (Fig. 3).Fig. 3SRC-1 subcellular localization is not modified during the cell cycle. BHK21 cells transfected with SRC-1 encoding vector were synchronized in G1 and cultured in defined conditions to observe the different cell cycle phases as described under "Experimental Procedures." Cell cycle phases were determined by cytofluorimetry (G1, early S, and G2). At each point, the subcellular localization of SRC-1 was scored by immunodetection as described under "Experimental Procedures." Nuclear labeling: open bars. Cytoplasmic labeling: closed bars. The results are means ± S.D. of three independent determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Another possible explanation for the heterogeneity of immunolabeling of SRC-1 was that it reflected a dynamic situation, the protein being localized initially in one of the cellular compartments and secondarily moving to the other. We could thus observe cells at different stages of such a cycle. To investigate this possibility, we transfected COS-7 cells with SRC-1 expression vector and fixed the cells at different time points after transfection (Fig. 4). A short time after transfection (13 h), SRC-1 was exclusively nuclear and concentrated into speckles. With increasing time following transfection (20, 26, and 32 h), a growing proportion of cells with primarily cytoplasmic labeling of SRC-1 was observed (14, 22, and 64%, respectively). Forty hours after transfection, SRC-1 was mostly located in cytoplasmic speckles (78% of cells) (Fig. 4c).Fig. 4SRC-1 initially localizes in the nu
Referência(s)