Nuclear Translocation of Insulin Receptor Substrate-1 by the Simian Virus 40 T Antigen and the Activated Type 1 Insulin-like Growth Factor Receptor
2002; Elsevier BV; Volume: 277; Issue: 35 Linguagem: Inglês
10.1074/jbc.m204658200
ISSN1083-351X
AutoresMarco di Prisco, Francesca Santini, Raffaele Baffa, Mingli Liu, Robert Drakas, Anguo Wu, Renato Baserga,
Tópico(s)Protein Degradation and Inhibitors
Resumo32D cells are a murine hemopoietic cell line that undergoes apoptosis upon withdrawal of interleukin-3 (IL-3) from the medium. 32D cells have low levels of the type 1 insulin-like growth factor (IGF-I) receptor and do not express insulin receptor substrate-1 (IRS-1) or IRS-2. Ectopic expression of IRS-1 delays apoptosis but cannot rescue 32D cells from IL-3 dependence. In 32D/IRS-1 cells, IRS-1 is detectable, as expected, in the cytosol/membrane compartment. The SV40 large T antigen is a nuclear protein that, by itself, also fails to protect 32D cells from apoptosis. Co-expression of IRS-1 with the SV40 T antigen in 32D cells results in nuclear translocation of IRS-1 and survival after IL-3 withdrawal. Expression of a human IGF-I receptor in 32D/IRS-1 cells also results in nuclear translocation of IRS-1 and IL-3 independence. The phosphotyrosine-binding domain, but not the pleckstrin domain, is necessary for IRS-1 nuclear translocation. Nuclear translocation of IRS-1 was confirmed in mouse embryo fibroblasts. These results suggest possible new roles for nuclear IRS-1 in IGF-I-mediated growth and anti-apoptotic signaling. 32D cells are a murine hemopoietic cell line that undergoes apoptosis upon withdrawal of interleukin-3 (IL-3) from the medium. 32D cells have low levels of the type 1 insulin-like growth factor (IGF-I) receptor and do not express insulin receptor substrate-1 (IRS-1) or IRS-2. Ectopic expression of IRS-1 delays apoptosis but cannot rescue 32D cells from IL-3 dependence. In 32D/IRS-1 cells, IRS-1 is detectable, as expected, in the cytosol/membrane compartment. The SV40 large T antigen is a nuclear protein that, by itself, also fails to protect 32D cells from apoptosis. Co-expression of IRS-1 with the SV40 T antigen in 32D cells results in nuclear translocation of IRS-1 and survival after IL-3 withdrawal. Expression of a human IGF-I receptor in 32D/IRS-1 cells also results in nuclear translocation of IRS-1 and IL-3 independence. The phosphotyrosine-binding domain, but not the pleckstrin domain, is necessary for IRS-1 nuclear translocation. Nuclear translocation of IRS-1 was confirmed in mouse embryo fibroblasts. These results suggest possible new roles for nuclear IRS-1 in IGF-I-mediated growth and anti-apoptotic signaling. interleukin type 1 insulin-like growth factor IGF-I receptor insulin receptor substrate mouse embryo fibroblast phosphotyrosine-binding pleckstrin homology fetal bovine serum phosphate-buffered saline nuclear localization signal 32D cells are a murine hemopoietic cell line that is interleukin (IL)-3-dependent1for growth (1Valtieri M. Tweardy D.J. Caracciolo D. Johnson K. Mavilio F. Altmann S. Santoli D. Rovera G. J. Immunol. 1987; 138: 3829-3835PubMed Google Scholar). In the absence of IL-3, 32D cells undergo apoptosis (2Askew D.S. Ashmun R.A. Simmons B.C. Cleveland J.L. Oncogene. 1991; 6: 1915-1922PubMed Google Scholar, 3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar, 4Peruzzi F. Prisco M. Dews M. Salomoni P. Grassilli E. Romano G. Calabretta B. Baserga R. Mol. Cell. Biol. 1999; 19: 7203-7215Crossref PubMed Scopus (427) Google Scholar). Parental 32D cells have low levels of IGF-IR, about 2 × 103 receptors/cell (5Prisco M. Peruzzi F. Belletti B. Baserga R. Mol. Cell. Biol. 2001; 21: 5447-5458Crossref PubMed Scopus (30) Google Scholar), and do not express insulin receptor substrate-1 (IRS-1) and IRS-2 (6Wang L.M. Myers Jr., M.G. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (369) Google Scholar, 7Valentinis B. Romano G. Peruzzi F. Morrione A. Prisco M. Soddu S. Cristofanelli B. Sacchi A. Baserga R. J. Biol. Chem. 1999; 274: 12423-12430Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The IRS proteins are major substrates of the IGF-I and insulin receptors and play an important role in the signaling from both receptors (reviewed in Refs. 8White M.F. Mol. Cell. Biochem. 1998; 182: 3-11Crossref PubMed Scopus (623) Google Scholar and 9Blakesley V.A. Butler A.A. Koval A.P. Okubo Y. LeRoith D. The IGF System. Humana Press, Totowa, NJ1999: 143-163Crossref Google Scholar). Expression of a human IGF-IR in 32D cells to about 17 × 103 receptors/cell (32D IGF-IR cells) prevents apoptosis caused by IL-3 withdrawal (4Peruzzi F. Prisco M. Dews M. Salomoni P. Grassilli E. Romano G. Calabretta B. Baserga R. Mol. Cell. Biol. 1999; 19: 7203-7215Crossref PubMed Scopus (427) Google Scholar, 5Prisco M. Peruzzi F. Belletti B. Baserga R. Mol. Cell. Biol. 2001; 21: 5447-5458Crossref PubMed Scopus (30) Google Scholar, 10Dews M. Prisco M. Peruzzi F. Romano G. Morrione A. Baserga R. Endocrinology. 2000; 141: 1289-1300Crossref PubMed Scopus (50) Google Scholar). However, after 48 h of exponential growth, the cells begin to differentiate along the granulocytic pathway (7Valentinis B. Romano G. Peruzzi F. Morrione A. Prisco M. Soddu S. Cristofanelli B. Sacchi A. Baserga R. J. Biol. Chem. 1999; 274: 12423-12430Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Ectopic expression of IRS-1 in 32D IGF-IR cells (32D IGF-IR/IRS-1 cells) inhibits differentiation. The cells become IL-3-independent and form tumors in mice (11Valentinis B. Navarro M. Zanocco-Marani T. Edmonds P. McCormick J. Morrione A. Sacchi A. Romano G. Reiss K. Baserga R. J. Biol. Chem. 2000; 275: 25451-25459Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). In parental 32D cells, expression of only IRS-1 does not prevent apoptosis, although it delays it slightly (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar, 12Zamorano J. Wang H.Y. Wang L.-M. Pierce J.H. Keegan A.D. J. Immunol. 1996; 157: 4926-4934PubMed Google Scholar). The interactions of the IGF axis with the SV40 T antigen (hitherto abbreviated as T antigen) are complex. The SV40 T antigen interacts closely with IRS-1, as demonstrated by reciprocal co-immunoprecipitation. This is true in both MEF (13Zhou-Li F. D'Ambrosio C., Li, S. Surmacz E. Baserga R. Mol. Cell. Biol. 1995; 15: 4232-4239Crossref PubMed Google Scholar) and 32D cells (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar). Truncation of the 250 amino-terminal residues of the T antigen abrogates both its ability to co-transform cells in combination with IRS-1 and its ability to co-precipitate IRS-1 (13Zhou-Li F. D'Ambrosio C., Li, S. Surmacz E. Baserga R. Mol. Cell. Biol. 1995; 15: 4232-4239Crossref PubMed Google Scholar). 32D cells expressing T antigen are not protected from apoptosis induced by IL-3 withdrawal, and they actually die even faster than parental 32D cells (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar). Thus, neither IRS-1 nor T antigen singly can protect parental 32D cells from apoptosis. However, a combination of the two results in the survival of 32D cells after IL-3 withdrawal (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar). IRS-1 is known to interact also with nucleolin (14Burks D.J. Wang J. Towery H. Ishibashi O. Lowe D. Riedel H. White M.F. J. Biol. Chem. 1998; 273: 31061-31067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Both T antigen and nucleolin are predominantly nuclear proteins, although minor fractions of either protein can be detected in the cytosol (15Santos M. Butel J. J. Cell. Biochem. 1982; 19: 127-144Crossref PubMed Scopus (11) Google Scholar, 16Hovanessian A.G. Puvion-Dutilleul F. Nisole S. Svab J. Perret E. Deng J.S. Krust B. Exp. Cell Res. 2000; 261: 312-328Crossref PubMed Scopus (188) Google Scholar), indeed even on the cell surface in the case of nucleolin (17Ginisty H. Sicard H. Roger B. Bouvet P. J. Cell Science. 1999; 112: 761-772PubMed Google Scholar). It has been therefore tacitly assumed that IRS-1, anchored to the receptors, was interacting with the minor cytosolic fractions of either T antigen or nucleolin. A recent report, however, has indicated that under certain conditions IRS-1 can be translocated to the nuclei (18Lassak A. Del Valle L. Peruzzi F. Wang J.Y. Enam S. Croul S. Khalili K. Reiss K. J. Biol. Chem. 2002; 277: 17231-17238Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The situation is further complicated by the fact that T antigen induces 24p3 (19Hraba-Renevey S. Turler H. Kress M. Solomon C. Weill R. Oncogene. 1989; 4: 601-608PubMed Google Scholar). 24p3 is a lipocalin that Devireddy et al. (20Devireddy L.R. Teodoro J.G. Richard F.A. Green M.R. Science. 2001; 293: 829-834Crossref PubMed Scopus (327) Google Scholar) reported to be responsible for the apoptosis caused by IL-3 withdrawal in several types of hemopoietic cell lines, including 32D cells. According to these authors, IGF-I inhibits the transcription of 24p3. A reasonable hypothesis at this point would be that T antigen cannot transform 32D cells because it does not inhibit 24p3 transcription, in fact it superinduces it. Ectopic expression of IRS-1, activated by IGF-I, could inhibit 24p3 transcription and thus rescue 32D/T cells from apoptosis. The purpose of this investigation was to determine the subcellular localization of IRS-1 in 32D-derived cells that survive and growversus cells that do not survive and undergo apoptosis. In addition, we have tested the hypothesis that survival may depend on the inhibition of 24p3 transcription. Using immunohistochemistry and confocal microscopy, we show here that IRS-1 is localized to the cytosol in 32D IRS-1 cells, which undergo apoptosis upon IL-3 withdrawal (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar, 7Valentinis B. Romano G. Peruzzi F. Morrione A. Prisco M. Soddu S. Cristofanelli B. Sacchi A. Baserga R. J. Biol. Chem. 1999; 274: 12423-12430Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 21Navarro M. Baserga R. Endocrinology. 2001; 142: 1073-1081Crossref PubMed Scopus (68) Google Scholar). However, in 32D IRS-1/T cells and in 32D IGF-IR/IRS-1 cells, which survive (and grow) in the absence of IL-3 (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar,7Valentinis B. Romano G. Peruzzi F. Morrione A. Prisco M. Soddu S. Cristofanelli B. Sacchi A. Baserga R. J. Biol. Chem. 1999; 274: 12423-12430Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 11Valentinis B. Navarro M. Zanocco-Marani T. Edmonds P. McCormick J. Morrione A. Sacchi A. Romano G. Reiss K. Baserga R. J. Biol. Chem. 2000; 275: 25451-25459Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), IRS-1 is found also in the nuclei. We have demonstrated the nuclear localization of IRS-1 by a variety of procedures and showed that it also occurs in MEF, where we have confirmed it by subcellular fractionation. In 32D IGF-IR cells, we show that a mutant IRS-1 with a deleted phosphotyrosine-binding (PTB) domain will not translocate to the nuclei, whereas the pleckstrin homology (PH) domain does not seem to be required. We also find that cytosolic IRS-1 inhibits (as predicted) 24p3 transcription, but in the absence of T antigen or an overexpressed IGF-IR, the cells still die, albeit more slowly than the parental 32D cells. When IRS-1 is in combination with either T antigen or an overexpressed IGF-IR, it translocates to the nucleus, and the cells survive. The focus of this paper is to demonstrate rigorously that IRS-1 can translocate to the nuclei of 32D-derived cells and to establish some of the conditions of this translocation. 32D, 32D/T, 32D IRS-1, and 32D IRS-1/T cells have been described by Zhou-Li et al. (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar). 32D IRS-1/IGF-IR cells express both the human wild type IGF-IR and mouse IRS-1 (7Valentinis B. Romano G. Peruzzi F. Morrione A. Prisco M. Soddu S. Cristofanelli B. Sacchi A. Baserga R. J. Biol. Chem. 1999; 274: 12423-12430Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), are IL-3 independent, and form tumors in nude mice (11Valentinis B. Navarro M. Zanocco-Marani T. Edmonds P. McCormick J. Morrione A. Sacchi A. Romano G. Reiss K. Baserga R. J. Biol. Chem. 2000; 275: 25451-25459Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). 32D IGF-IR cells expressing the mutant IRS-1 proteins have been described previously (22Valentinis B. Baserga R. J. Clin. Pathol. Mol Pathol. 2001; 54: 133-137Crossref Scopus (296) Google Scholar, 23Navarro M. Valentinis B. Belletti B. Romano G. Reiss K. Baserga R. Endocrinology. 2001; 142: 5149-5157Crossref PubMed Scopus (19) Google Scholar). They are 32D IGF-IR cells stably transfected with plasmids expressing mutant IRS-1 proteins. The mutants used in these experiments include an IRS-1 with a deletion of the PH domain, a mutant with a deletion of the PTB domain, and another plasmid expressing only the PH/PTB domains (24Belletti B. Prisco M. Morrione A. Valentinis B. Navarro M. Baserga R. J. Biol. Chem. 2001; 276: 13867-13874Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In other experiments, we used R−/GR15 cells, which are MEFs derived from R− cells (25Sell C. Rubini M. Rubin R. Liu J.-P. Efstratiadis A. Baserga R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11217-11221Crossref PubMed Scopus (541) Google Scholar). R−/GR15 cells express substantial levels of human IGF-IR and endogenous IRS-1 (26Romano G. Prisco M. Zanocco-Marani T. Peruzzi F. Valentinis B. Baserga R. J. Cell. Biochem. 1999; 72: 294-310Crossref PubMed Scopus (56) Google Scholar). In some experiments, parental 32D and 32D/T cells were transiently transfected with the pIRS-1 Flag plasmid (see below), using the FuGENE 6 transfection reagent (Roche Molecular Biochemicals), following the manufacturer's instructions. After 48 h, the transfected cells were examined by confocal microscopy. Only transfected cells were positive for the FLAG antibody. pIRS/FLAG was generated from pGR159 MSCV.pac retroviral vector (26Romano G. Prisco M. Zanocco-Marani T. Peruzzi F. Valentinis B. Baserga R. J. Cell. Biochem. 1999; 72: 294-310Crossref PubMed Scopus (56) Google Scholar) by fusing in-frame the wild type mouse IRS-1 sequence with the FLAG sequence (Kodak) at the 3′ end. The IRS-1 sequence fused in-frame with the FLAG epitope at the 3′ end was produced by PCR. The detailed methodology for the construction of the retroviral vector has been described previously (26Romano G. Prisco M. Zanocco-Marani T. Peruzzi F. Valentinis B. Baserga R. J. Cell. Biochem. 1999; 72: 294-310Crossref PubMed Scopus (56) Google Scholar). 24p3 cDNA was obtained by reverse transcription-PCR from 32D cells, 4 h after IL-3 withdrawal. 32D cells were washed three times with Hanks' buffer and seeded at 5 × 105 cells/ml in RPMI medium supplemented with 10% FBS for 4 h. The cells were collected and washed with cold PBS, and RNA was extracted using RNeasy kit from Qiagen. Reverse transcription-PCR was performed with C. hydrogenoformansthermostable polymerase according to the manufacturer's instructions (one-step reverse transcription-PCR system; Roche Molecular Biochemicals). The primers used were: forward 5′-AGACCTAGTAGCTGTGGAAACC-3′ and reverse 5′-GGGGGCATGTATTTATTCAG-3′. The annealing temperature used was 56 °C. The 24p3 cDNA was subcloned in pCR 2.1 vector using the Topo TA cloning kit (Invitrogen) according to the manufacturer's protocol. The sequence of 24p3 cDNA was monitored using T7 and M13 reverse primers and by comparison with the sequence in the BLAST program. 32D and 32D-derived cells (see above) were washed three times with Hanks' buffer and seeded 2 × 105 cells/ml in RPMI medium supplemented with 10% FBS or IGF-1 50 ng/ml on poly-l-lysine coverslips for16 h. The cells were fixed with 3.5% formaldehyde solution for 30 min., washed three times with PBS, permeabilized with 0.3% Triton-X100 solution in PBS for 2 min, and washed three times with PBS. The blocking was done for 30 min using 10% normal donkey serum (Santa Cruz Biotechnology) diluted in PBS, and the slides were incubated for 45 min with the appropriate antibodies. Confocal analysis was performed on a Bio-Rad MRC-600 Ar/Kr laser scanning confocal microscope interfaced to a Zeiss Axiovert 100 microscope with Zeiss Plan-Apo 63× oil immersion objective and a Zeiss 40× objective. The samples were analyzed with simultaneous excitation at 488 and 568 nm with proper filters to visualize fluorescein and rhodamine-lissamine signals. 32D and 32D-derived cells were washed three times with Hanks' buffer and seeded at a density of 5 × 104/2 ml of RPMI supplemented with 10% FBS plus or minus IGF-I (50 ng/ml). The cells were harvested after 16 h, and cytospins were prepared. The fibroblasts were seeded on coverslips (5 × 104 cells/2 ml) in Dulbecco's modified Eagle's medium plus 10% FBS. After the cells had attached to the coverslip, they were shifted to serum-free medium for 24 h and then stimulated with IGF-I (50 ng/ml) for another 6 h. After fixing in 3.7% formaldehyde solution in PBS and permeabilization with 0.2% Triton X-100 in PBS, the immunostaining was carried out using the Histomouse (AEC) kit (Zymed Laboratories, Inc.) following the manufacturer's protocol. The magnifications for the figures presented in this paper were either 400× or 1000×. The cells were seeded at 5 × 105 cells/100 mm plates in Dulbecco's modified Eagle's medium supplemented with 10% FBS. The following day, the cells were shifted to serum-free medium for 48 h and then stimulated with IGF-1 (50 ng/ml) for 6 h. The cells were harvested with cold PBS, collected by scraping, and washed twice in a 15-ml Falcon tube with cold PBS. The cells were resuspended in buffer A (10 mmHepes, pH 7.4, 1 mm EDTA, protease and phosphatase inhibitors from Sigma, 100 mm dithiothreitol, and 0.5% Triton X-100), kept on ice for 10 min, and then homogenized with a tight fitting Dounce and examined under microscope to confirm that the cells were lysed. After centrifugation at 4 °C, 500 ×g for 10 min, the supernatant was collected, centrifuged again at 12,000 × g at 4 °C for 10 min, and collected as the cytoplasmic fraction. The pellet of the first centrifugation was washed at least three times with buffer B (50 mm NaCl, 10 mm Hepes, pH 8, 25% glycerol, 0.1 mm EDTA, protease and phosphatase inhibitors from Sigma, and 100 mm dithiothreitol), resuspended in buffer C (350 mm NaCl, 10 mm Hepes, pH 8, 25% glycerol, 0.1 mm EDTA, protease and phosphatase inhibitors from Sigma, and 100 mm dithiothreitol), and kept rocking for 30 min at 4 °C. After centrifugation at 12,000 × g for 10 min, the supernatant collected represented the nuclear fraction. 20 μg of cytoplasmic and nuclear fractions were separated on a 4–15% gradient gel (Bio-Rad) and transferred to a nitrocellulose membrane. The primary antibodies used were: nucleolin monoclonal antibody, SV40 T antigen monoclonal antibody, Id2 polyclonal antibody, IRS-1(C20) polyclonal antibody with the epitope mapping at the carboxyl terminus of IRS-1, and IRS-1(C19) with the epitope mapping at the amino terminus of IRS-1 from Santa Cruz Biotechnology. Anti-FLAG fluorescein isothiocyanate-conjugated antibody and another anti-IRS-1 polyclonal antibody were from Upstate Biotechnology, Inc. Clathrin polyclonal antibody was a kind gift of Dr. J. H. Keen (Thomas Jefferson University). All of these antibodies were used at a concentration of 5 μg/ml. After washing three times with PBS, the samples were incubated with appropriate secondary antibodies conjugated to rhodamine or fluorescein (Santa Cruz Biotechnology and Jackson ImmunoResearch Laboratories). In some experiments the nuclei were stained with propidium iodide from Molecular Probes. For Western blots, the antibodies used were anti-IRS-1(C20), anti-c-Jun (Santa Cruz Biotechnology), and anti-glyceraldheyde-3-phosphate dehydrogenase (Research Diagnostics, Inc.). Northern blots were carried out using standard techniques using the full-length 24p3 mouse cDNA described above. Parental 32D cells die quickly after IL-3 withdrawal, with most of the cells dead by 24 h (1Valtieri M. Tweardy D.J. Caracciolo D. Johnson K. Mavilio F. Altmann S. Santoli D. Rovera G. J. Immunol. 1987; 138: 3829-3835PubMed Google Scholar, 2Askew D.S. Ashmun R.A. Simmons B.C. Cleveland J.L. Oncogene. 1991; 6: 1915-1922PubMed Google Scholar). Cell death is easily demonstrated by simply counting the number of cells (10Dews M. Prisco M. Peruzzi F. Romano G. Morrione A. Baserga R. Endocrinology. 2000; 141: 1289-1300Crossref PubMed Scopus (50) Google Scholar, 21Navarro M. Baserga R. Endocrinology. 2001; 142: 1073-1081Crossref PubMed Scopus (68) Google Scholar). The evidence that the mode of cell death is apoptosis has been repeatedly documented in previous papers from this and other laboratories. In our first paper (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar), we used fluorescence-activated cell sorter analysis to show that the same cell lines used in the present experiments underwent apoptosis after IL-3 withdrawal. We confirmed apoptosis by the TUNEL method in a subsequent paper (4Peruzzi F. Prisco M. Dews M. Salomoni P. Grassilli E. Romano G. Calabretta B. Baserga R. Mol. Cell. Biol. 1999; 19: 7203-7215Crossref PubMed Scopus (427) Google Scholar). We have monitored the extent of apoptosis in the present experiments, and they exactly reproduced the data of Zhou-Li et al. (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar). Those data had shown that a sizable fraction of 32D/T cells are already dead at 16 h after IL-3 withdrawal, whereas death of 32D IRS-1 cells was delayed and less prominent. The number of apoptotic cells was negligible in 32D IRS-1/T cells or in 32D IGF-IR/IRS-1 cells (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar, 4Peruzzi F. Prisco M. Dews M. Salomoni P. Grassilli E. Romano G. Calabretta B. Baserga R. Mol. Cell. Biol. 1999; 19: 7203-7215Crossref PubMed Scopus (427) Google Scholar, 7Valentinis B. Romano G. Peruzzi F. Morrione A. Prisco M. Soddu S. Cristofanelli B. Sacchi A. Baserga R. J. Biol. Chem. 1999; 274: 12423-12430Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). All 32D-derived cells grow normally if incubated in IL-3 (not shown, but repeatedly shown in previous papers). IRS-1 is known to interact directly with both the insulin and the IGF-I receptors, and the domains required for their interaction have been identified (27Yenush L. Zanella C. Uchida T. Bernal D. White M.F. Mol. Cell. Biol. 1998; 18: 6784-6794Crossref PubMed Scopus (75) Google Scholar). Because of its direct interaction with the receptors, its size, and its downstream signaling, it has been generally assumed that IRS-1 is an exclusively cytosolic (or plasma membrane) protein (for the most recent references, see Refs. 28Razzini G. Ingrosso A. Brancaccio A. Sciacchitano S. Esposito D.L. Falasca M. Mol. Endocrinol. 2000; 14: 823-836Crossref PubMed Scopus (65) Google Scholar and 29Jacobs A.R. LeRoith D. Taylor S.I. J. Biol. Chem. 2001; 276: 40795-40802Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). However, as already mentioned, IRS-1 is known to interact with the SV40 T antigen (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar, 13Zhou-Li F. D'Ambrosio C., Li, S. Surmacz E. Baserga R. Mol. Cell. Biol. 1995; 15: 4232-4239Crossref PubMed Google Scholar) and nucleolin (14Burks D.J. Wang J. Towery H. Ishibashi O. Lowe D. Riedel H. White M.F. J. Biol. Chem. 1998; 273: 31061-31067Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). These two proteins are predominantly nuclear proteins. In addition, a recent report (18Lassak A. Del Valle L. Peruzzi F. Wang J.Y. Enam S. Croul S. Khalili K. Reiss K. J. Biol. Chem. 2002; 277: 17231-17238Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) has shown that IRS-1 can translocate to the nuclei in medulloblastoma cells and cell lines. In an attempt to explain the co-operative effect of SV40 T antigen and IRS-1, we have investigated their localization in 32D cells. Fig. 1 shows confocal and regular microscopy pictures of 32D-derived cells, stained with the appropriate antibodies (see "Experimental Procedures"). 32D cells (and derivatives) growing in IL-3 have the appearance of blast cells, with large nuclei, large and often diffuse nucleoli, and a variable rim of cytoplasm around the large nuclei (7Valentinis B. Romano G. Peruzzi F. Morrione A. Prisco M. Soddu S. Cristofanelli B. Sacchi A. Baserga R. J. Biol. Chem. 1999; 274: 12423-12430Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Fig.1A shows that 32D/T cells are completely negative for IRS-1 staining, as expected from the fact that parental 32D cells do not express IRS-1 (6Wang L.M. Myers Jr., M.G. Sun X.J. Aaronson S.A. White M. Pierce J.H. Science. 1993; 261: 1591-1594Crossref PubMed Scopus (369) Google Scholar, 7Valentinis B. Romano G. Peruzzi F. Morrione A. Prisco M. Soddu S. Cristofanelli B. Sacchi A. Baserga R. J. Biol. Chem. 1999; 274: 12423-12430Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In 32D IRS-1 cells (3Zhou-Li F., Xu, S.-Q. Dews M. Baserga R. Oncogene. 1997; 15: 961-970Crossref PubMed Scopus (49) Google Scholar), IRS-1 is detectable (Santa Cruz antibody) in cells growing in complete medium plus IL-3 (Fig. 1B). The nucleus was stained with hematoxylin, and IRS-1 clearly appears localized in the cytoplasm of these cells. The amount of IRS-1 in the cytosol of individual cells varies, which can be explained by the fact that this is a mixed population. When the amount of IRS-1 is substantial, the color of the nucleus is darker, as one would expect, in immunohistochemistry, from cells in which the nucleus is covered by a thin layer of cytosol. The average amount of IRS-1 in these cells, as detectable by Western blots, is high (7Valentinis B. Romano G. Peruzzi F. Morrione A. Prisco M. Soddu S. Cristofanelli B. Sacchi A. Baserga R. J. Biol. Chem. 1999; 274: 12423-12430Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). These results were confirmed by confocal microscopy. Fig. 1C shows that the T antigen is a nuclear protein in 32D/T cells, the amount detectable in the cytosol (stained for clathrin) being negligible. Parental 32D cells do not stain at all with an antibody to the T antigen (not shown). Fig.1D shows a 32D/IRS-1 cell in which IRS-1 (green) is limited to the cytoplasmic rim, whereas the antibody to nucleolin (red) stains the nucleus diffusely. Fig. 1E shows another example of a 32D/IRS-1 cell, but with the colors reversed (IRS-1 is now stained red and nucleolin green). These experiments were repeated several times, with reproducible results. In contrast, when the same antibodies are used to stain 32D IRS-1/T cells, IRS-1 is now seen predominantly in the nucleus, co-localizing with the T antigen (Fig.2). A halo of IRS-1 can still be seen in the cytosol, but a substantial proportion of IRS-1 is now in the nucleus. The images of A and B of Fig. 2 were obtained using two different antibodies for IRS-1, from different commercial sources (see above). To pinpoint the localization, we show in Fig. 2 isolated cells, but the great majority of 32D IRS-1/T cells showed nuclear co-localization of the two proteins (see also below). The results are consistent with nuclear localization of IRS-1 in 32D cells expressing T antigen. With the results of Fig. 1, we can also state that IRS-1 is not required for the localization in the nuclei of T antigen, whereas T antigen is needed for the nuclear localization of IRS-1. However, the nuclear localization of IRS-1 is not limited to T antigen expressing cells. 32D IRS-1/IGF-IR cells are transformed by any criteria, because they form tumors in nude mice (11Valentinis B. Navarro M. Zanocco-Marani T. Edmonds P. McCormick J. Morrione A. Sacchi A. Romano G. Reiss K. Baserga R. J. Biol. Chem. 2000; 275: 25451-25459Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Confocal microscopy (Fig. 3) shows that IRS-1 in these cells co-localized with nucleolin, predominantly in the nucleus. In these cells, nucleolin gives a diffuse nuclear staining, which is at variance with MEF, where the anti-nucleolin antibodies give a discrete nucleolar-shaped staining. A weak halo of IRS-1 is also detectable in the cytosol. Therefore, ectopically expressed IRS-1 is cytosolic in 32D/IRS-1 cells but mostly nuclear when the cells also express either the T antigen or increased levels of IGF-IR. Because of often justified doubts on the specificity of commercial antibodies (especially in immunohistochemistry), we have attempted to confirm our results by using a FLAG-tagged IRS-1. This is a plasmid in which mouse IRS-1 (30Keller S.R. Aebersold R. Garner C.W. Lienhard G.E. Biocheim. Biophys. Acta. 1993; 1172: 323-326Crossref PubMed Scopus (39) Google Scholar) is tagged with a FLAG epitope at its 3′ end (see "Experimental Procedures"). This plasmid was transiently transfected into 32D/T cells, and the cells were examined 48 h later. Because the efficiency of transfection of parental and derived 32D cells is fairly low, this transient expression experiment offered the advantage of using the untransfected cells as controls for the FLAG epitope. Untransfected cells are negative for the FLAG epitope and stain only with the Id2 antibody (Id2 is a nuclear protein). Fig.4 shows different experiments in which 32D/T ce
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