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• W2053296976 abstract "Although insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 are known to modulate cell growth by reversibly sequestering extracellular insulin-like growth factors, several reports have suggested that IGFBP-3, and possibly also IGFBP-5, have important insulin-like growth factor-independent effects on cell growth. These effects may be related to the putative nuclear actions of IGFBP-3 and IGFBP-5, which we have recently shown are transported to the nuclei of T47D breast cancer cells. We now describe the mechanism for nuclear import of IGFBP-3 and IGFBP-5. In digitonin-permeabilized cells, where the nuclear envelope remained intact, nuclear translocation of wild-type IGFBP-3 appears to occur by a nuclear localization sequence (NLS)-dependent pathway mediated principally by the importin β nuclear transport factor and requiring both ATP and GTP hydrolysis. Under identical conditions, an NLS mutant form of IGFBP-3, IGFBP-3[228KGRKR → MDGEA], was unable to translocate to the nucleus. In cells where both the plasma membrane and nuclear envelope were permeabilized, wild-type IGFBP-3, but not the mutant form, accumulated in the nucleus, implying that the NLS was also involved in mediating binding to nuclear components. By fusing wild-type and mutant forms of NLS sequences (IGFBP-3 [215–232] and IGFBP-5 [201–218]) to the green fluorescent protein, we identified the critical residues of the NLS necessary and sufficient for nuclear accumulation. Using a Western ligand binding assay, wild-type IGFBP-3 and IGFBP-5, but not an NLS mutant form of IGFBP-3, were shown to be recognized by importin β and the α/β heterodimer but only poorly by importin α. Together these results suggest that the NLSs within the C-terminal domain of IGFBP-3 and IGFBP-5 are required for importin-β-dependent nuclear uptake and probably also accumulation through mediating binding to nuclear components. Although insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-5 are known to modulate cell growth by reversibly sequestering extracellular insulin-like growth factors, several reports have suggested that IGFBP-3, and possibly also IGFBP-5, have important insulin-like growth factor-independent effects on cell growth. These effects may be related to the putative nuclear actions of IGFBP-3 and IGFBP-5, which we have recently shown are transported to the nuclei of T47D breast cancer cells. We now describe the mechanism for nuclear import of IGFBP-3 and IGFBP-5. In digitonin-permeabilized cells, where the nuclear envelope remained intact, nuclear translocation of wild-type IGFBP-3 appears to occur by a nuclear localization sequence (NLS)-dependent pathway mediated principally by the importin β nuclear transport factor and requiring both ATP and GTP hydrolysis. Under identical conditions, an NLS mutant form of IGFBP-3, IGFBP-3[228KGRKR → MDGEA], was unable to translocate to the nucleus. In cells where both the plasma membrane and nuclear envelope were permeabilized, wild-type IGFBP-3, but not the mutant form, accumulated in the nucleus, implying that the NLS was also involved in mediating binding to nuclear components. By fusing wild-type and mutant forms of NLS sequences (IGFBP-3 [215–232] and IGFBP-5 [201–218]) to the green fluorescent protein, we identified the critical residues of the NLS necessary and sufficient for nuclear accumulation. Using a Western ligand binding assay, wild-type IGFBP-3 and IGFBP-5, but not an NLS mutant form of IGFBP-3, were shown to be recognized by importin β and the α/β heterodimer but only poorly by importin α. Together these results suggest that the NLSs within the C-terminal domain of IGFBP-3 and IGFBP-5 are required for importin-β-dependent nuclear uptake and probably also accumulation through mediating binding to nuclear components. insulin-like growth factor insulin-like growth factor-binding protein nuclear localization signal SV40 large tumor antigen β-galactosidase fused to the NLS derived from N1N2 rabbit reticulocyte lysate confocal laser scanning microscopy enhanced green fluorescent protein heterogeneous nuclear ribonucleoprotein fluorescein isothiocyanate guanosine 5′-O-(3-thiotriphosphate) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate bovine serum albumin Chinese hamster ovary The mitogenic effects of insulin-like growth factors (IGFs)1 are modulated by a family of IGF-binding proteins (IGFBPs). Following their secretion into the extracellular environment, the IGFBPs inhibit or stimulate cell growth by regulating access of the extracellular IGFs to the type I IGF receptor (1De Mellow J.S. Baxter R.C. Biochem. Biophys. Res. Commun. 1988; 156: 199-204Crossref PubMed Scopus (488) Google Scholar). However, some IGFBPs, including IGFBP-3, also have effects on cell growth that are type I receptor-independent (2Cohen P. Lamson G. Okajima T. Rosenfeld R.G. Mol. Endocrinol. 1993; 7: 380-386Crossref PubMed Scopus (128) Google Scholar, 3Oh Y. Muller H.L. Lamson G. Rosenfeld R.G. J. Biol. Chem. 1993; 268: 14964-14971Abstract Full Text PDF PubMed Google Scholar). Expression of recombinant human IGFBP-3, for example, has been shown to inhibit the proliferation of murine fibroblasts with a targeted disruption to the type I receptor (4Valentinis B. Bhala A. DeAngelis T. Baserga R. Cohen P. Mol. Endocrinol. 1995; 9: 361-367Crossref PubMed Google Scholar). This growth inhibitory effect was directly related to the induction of apoptosis by IGFBP-3 (5Rajah R. Valentinis B. Cohen P. J. Biol. Chem. 1997; 272: 12181-12188Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). In addition, a number of potent growth-inhibitory and apoptosis-inducing agents such as transforming growth factor β1, retinoic acid, tumor necrosis factor-α, and anti-estrogens also induce IGFBP-3 gene expression (6Martin J.L. Ballesteros M. Baxter R.C. Endocrinology. 1992; 131: 1703-1710Crossref PubMed Scopus (97) Google Scholar, 7Martin J.L. Coverley J.A. Pattison S.T. Baxter R.C. Endocrinology. 1995; 136: 1219-1226Crossref PubMed Google Scholar). These effects on cell growth may be mediated by IGFBP-3 in an IGF-independent manner. There are fewer reports of IGF-independent effects of IGFBP-5; these include its ability to stimulate bone cell growth in the absence of increased IGF-I binding to its receptor (8Mohan S. Nakao Y. Honda Y. Landale E. Leser U. Dony C. Lang K. Baylink D.J. J. Biol. Chem. 1995; 270: 20424-20431Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). The mechanism(s) for the IGF-independent effects of IGFBP-3 and IGFBP-5 are currently unknown but may involve a direct nuclear action.Although some proteins appear to be constitutively nuclear, others enter the nucleus only under defined conditions (9Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (384) Google Scholar). Thus, cells are able to control the activity of nuclear proteins by regulating their nuclear uptake during differentiation and changes in the metabolic state of the cell. The central pore of the nuclear pore complex allows molecules up to 45 kDa to move freely between the nuclear and cytoplasmic compartments (10Dingwall C. Laskey R. Science. 1992; 258: 942-947Crossref PubMed Scopus (160) Google Scholar). For proteins larger than 45 kDa, nuclear transport is generally an active, nuclear localization sequence (NLS)-dependent process that requires specific targeting sequences contained within the primary sequence of the transported protein or a cotransported protein (11Miller M. Park M.K. Hanover J.A. Physiol. Rev. 1991; 71: 909-949Crossref PubMed Scopus (82) Google Scholar).The cell contains multiple signal-dependent pathways for nuclear transport, of which the best characterized requires the cytosolic receptors, importin α and β (12Gorlich D. Kostka S. Kraft R. Dingwall C. Laskey R.A. Hartmann E. Prehn S. Curr. Biol. 1995; 5: 383-392Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar), the monomeric guanine nucleotide-binding protein, Ran (13Gorlich D. Pante N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (527) Google Scholar, 14Moore M.S. Blobel G. Nature. 1993; 365: 661-663Crossref PubMed Scopus (639) Google Scholar, 15Melchior F. Paschal B. Evans J. Gerace L. J. Cell Biol. 1993; 123: 1649-1659Crossref PubMed Scopus (472) Google Scholar), and interacting proteins such as nuclear transport factor 2 (16Moore M.S. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10212-10216Crossref PubMed Scopus (291) Google Scholar, 17Paschal B.M. Gerace L. J. Cell Biol. 1995; 129: 925-937Crossref PubMed Scopus (340) Google Scholar, 18Nehrbass U. Blobel G. Science. 1996; 272: 120-122Crossref PubMed Scopus (149) Google Scholar). Conventionally, the importin α subunit acts as an adapter, binding to the NLS of cytosolic proteins as well as to importin β, which together with Ran effects translocation through the nuclear pore complex. The importin α/β heterodimer recognizes three different classes of NLS: those that contain basic residues arranged as a single stretch (e.g.the NLS of the SV40 large tumor antigen, T-ag) (9Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (384) Google Scholar, 19Kalderon D. Richardson W.D. Markham A.F. Smith A.E. Nature. 1984; 311: 33-38Crossref PubMed Scopus (902) Google Scholar, 20Koepp D.M. Silver P.A. Cell. 1996; 87: 1-4Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar) or as two clusters of basic residues separated by a spacer region (bipartite NLS) (9Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (384) Google Scholar, 21Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1240) Google Scholar) or those resembling the NLS of the yeast homeodomain protein Matα2 (22Hall M.N. Hereford L. Herskowitz I. Cell. 1984; 36: 1057-1065Abstract Full Text PDF PubMed Scopus (216) Google Scholar). Other signal-dependent pathways have been described that include the transport of proteins that bind directly to and are transported by members of the importin β family (in this pathway the adapter, importin α, is not required to effect nuclear transport) (23Chan C.K. Hubner S. Hu W. Jans D.A. Gene Ther. 1998; 5: 1204-1212Crossref PubMed Scopus (97) Google Scholar, 24Lam M.H. Briggs L.J. Hu W. Martin T.J. Gillespie M.T. Jans D.A. J. Biol. Chem. 1999; 274: 7391-7398Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 25Tiganis T. Flint A.J. Adam S.A. Tonks N.K. J. Biol. Chem. 1997; 272: 21548-21557Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and those that do not require soluble cytosolic receptors at all but appear to require ATP (26Efthymiadis A. Briggs L.J. Jans D.A. J. Biol. Chem. 1998; 273: 1623-1628Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 27Fagotto F. Gluck U. Gumbiner B.M. Curr. Biol. 1998; 8: 181-190Abstract Full Text Full Text PDF PubMed Google Scholar, 28Michael W.M. Eder P.S. Dreyfuss G. EMBO J. 1997; 16: 3587-3598Crossref PubMed Scopus (320) Google Scholar).Significantly, in the context of IGF-independent nuclear action, the C-terminal regions of IGFBP-3 and IGFBP-5 contain a domain with strong sequence homology to the bipartite NLS consensus motif (29Radulescu R.T. Trends Biochem. Sci. 1994; 19: 278Abstract Full Text PDF PubMed Scopus (78) Google Scholar). This basic domain is highly conserved in IGFBP-3 and IGFBP-5 from different species, suggesting that it has functional significance. Similar basic sequences have been identified in a number of other secreted proteins and shown to be important for their respective signaling roles. These include platelet-derived growth factor A (30Bejcek B.E. Li D.Y. Deuel T.F. Science. 1989; 245: 1496-1499Crossref PubMed Scopus (127) Google Scholar), acidic fibroblast growth factor (31Imamura T. Engleka K. Zhan X. Tokita Y. Forough R. Roeder D. Jackson A. Maier J.A. Hla T. Maciag T. Science. 1990; 249: 1567-1570Crossref PubMed Scopus (324) Google Scholar), and parathyroid hormone-related protein (32Henderson J.E. Amizuka N. Warshawsky H. Biasotto D. Lanske B.M. Goltzman D. Karaplis A.C. Mol. Cell. Biol. 1995; 15: 4064-4075Crossref PubMed Scopus (304) Google Scholar). We and others have described the nuclear transport of IGFBP-3 and IGFBP-5 in a number of cell lines (33Jaques G. Noll K. Wegmann B. Witten S. Kogan E. Radulescu R.T. Havemann K. Endocrinology. 1997; 138: 1767-1770Crossref PubMed Scopus (124) Google Scholar, 34Li W. Fawcett J. Widmer H.R. Fielder P.J. Rabkin R. Keller G.A. Endocrinology. 1997; 138: 1763-1766Crossref PubMed Scopus (104) Google Scholar, 35Schedlich L.J. Young T.F. Firth S.M. Baxter R.C. J. Biol. Chem. 1998; 273: 18347-18352Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 36Wraight C.J. Liepe I.J. White P.J. Hibbs A.R. Werther G.A. J. Invest. Dermatol. 1998; 111: 239-242Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar).As part of our investigation into the role of nuclear IGFBP-3 and IGFBP-5, the present study examines the mechanisms for their nuclear import. We report that previously identified NLS-like sequences within IGFBP-3 and IGFBP-5 are necessary and sufficient for their nuclear accumulation. IGFBP-3 nuclear import is an energy-dependent process requiring ATP and GTP hydrolysis and mediated by importin β. In addition, IGFBP-3 and IGFBP-5 are both recognized specifically by importin β and the importin α/β heterodimer but not by importin α. Thus, nuclear import of IGFBP-3, and by analogy IGFBP-5, appears to occur by a signal-dependent importin β-mediated pathway. In addition, we show that, possibly mediated by its NLS, IGFBP-3 is capable of interaction with nuclear binding sites, which may play an important role in its nuclear accumulation.DISCUSSIONThis study identifies an 18-amino acid region of IGFBP-3 and IGFBP-5 that is necessary and sufficient for nuclear transport and accumulation. Our results suggest that nuclear import of IGFBP-3, and probably by analogy IGFBP-5, is an energy-dependent process mediated by importin β and following nuclear entry, IGFBP-3 can actively accumulate through binding to detergent-insoluble nuclear components. Mutation of the basic regions within the C-terminal domain of IGFBP-3 and IGFBP-5 attenuates nuclear import and/or accumulation and, as shown for IGFBP-3, reduces importin binding.We have studied nuclear transport of IGFBP-3 using an in vitro nuclear transport assay that allows the role of individual components of the transport system to be examined. As a control, we examined the nuclear uptake of β-galactosidase fused to the N1N2 bipartite NLS (46Hu W. Jans D.A. J. Biol. Chem. 1999; 274: 15820-15827Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In the presence of cytosolic factors and an ATP-regeneration system, full-length wild-type IGFBP-3 was capable of nuclear uptake in all cells where the plasma membrane had been permeabilized, but the nuclear envelope remained intact. Previous studies in intact cells have shown that nuclear transport of IGFBP-3 is detected only in a low percentage of cells in the monolayer (33Jaques G. Noll K. Wegmann B. Witten S. Kogan E. Radulescu R.T. Havemann K. Endocrinology. 1997; 138: 1767-1770Crossref PubMed Scopus (124) Google Scholar, 34Li W. Fawcett J. Widmer H.R. Fielder P.J. Rabkin R. Keller G.A. Endocrinology. 1997; 138: 1763-1766Crossref PubMed Scopus (104) Google Scholar, 35Schedlich L.J. Young T.F. Firth S.M. Baxter R.C. J. Biol. Chem. 1998; 273: 18347-18352Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 36Wraight C.J. Liepe I.J. White P.J. Hibbs A.R. Werther G.A. J. Invest. Dermatol. 1998; 111: 239-242Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Therefore, these data suggest that the plasma membrane is an important regulator of nuclear uptake of IGFBP-3 and consequently also of its subsequent function in the nucleus.Nuclear protein import directed by bipartite NLSs conventionally requires cytosolic factors such as importin α/β, Ran, and nuclear transport factor 2 (44Yoneda Y. J. Biochem. (Tokyo). 1997; 121: 811-817Crossref PubMed Scopus (85) Google Scholar). However, we show here that nuclear transport of IGFBP-3 can occur efficiently in the absence of added soluble transport factors. Analogous observations have been reported for nuclear transport conferred by the HIV-I Tat NLS (26Efthymiadis A. Briggs L.J. Jans D.A. J. Biol. Chem. 1998; 273: 1623-1628Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and the heterogeneous nuclear ribonucleoprotein (hnRNP) K sequence KNS (28Michael W.M. Eder P.S. Dreyfuss G. EMBO J. 1997; 16: 3587-3598Crossref PubMed Scopus (320) Google Scholar). The Wnt signal transduction pathway component β-catenin (27Fagotto F. Gluck U. Gumbiner B.M. Curr. Biol. 1998; 8: 181-190Abstract Full Text Full Text PDF PubMed Google Scholar), which appears to be able to bind directly to nucleoporins, similarly appears not to require soluble factors for nuclear import; interestingly, nuclear transport of β-catenin appears to occur through a Ran-independent pathway (51Yokoya F. Imamoto N. Tachibana T. Yoneda Y. Mol. Biol. Cell. 1999; 10: 1119-1131Crossref PubMed Scopus (203) Google Scholar). However, unlike IGFBP-3, the targeting signals of Tat and hnRNP K represent novel nuclear targeting signals not resembling the T-ag or bipartite NLSs and, furthermore, do not appear to be recognized by importin α/β. Previous studies have found that sufficient importin β, but not importin α, may remain associated with the nuclear pore complex to support a basal level of nuclear import subsequent to digitonin permeabilization (12Gorlich D. Kostka S. Kraft R. Dingwall C. Laskey R.A. Hartmann E. Prehn S. Curr. Biol. 1995; 5: 383-392Abstract Full Text Full Text PDF PubMed Scopus (413) Google Scholar, 47Chi N.C. Adam E.J. Adam S.A. J. Cell Biol. 1995; 130: 265-274Crossref PubMed Scopus (247) Google Scholar, 48Gorlich D. Prehn S. Laskey R.A. Hartmann E. Cell. 1994; 79: 767-778Abstract Full Text PDF PubMed Scopus (599) Google Scholar). Therefore, nuclear import in the absence of added cytosol suggests that the adapter, importin α, is not required for nuclear transport but does not rule out the possibility that importin β alone is mediating uptake. Our findings that inclusion of an antibody specific to importin β, both in the presence and absence of added cytosol, inhibited nuclear import of IGFBP-3, suggests that importin β, but not importin α, is required to sustain basal nuclear import. Similarly, addition of the nonhydrolyzable GTP analogue, GTPγS, to the transport assay significantly reduced nuclear import of IGFBP-3. Together these results suggest that both importin β alone, and GTP hydrolysis are required for efficient transport. Importin β appears to be the sole nuclear targeting signal receptor used by parathyroid hormone-related protein (24Lam M.H. Briggs L.J. Hu W. Martin T.J. Gillespie M.T. Jans D.A. J. Biol. Chem. 1999; 274: 7391-7398Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), T-cell protein tyrosine phosphatase (25Tiganis T. Flint A.J. Adam S.A. Tonks N.K. J. Biol. Chem. 1997; 272: 21548-21557Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar), and the yeast transcription factor, GAL4 (23Chan C.K. Hubner S. Hu W. Jans D.A. Gene Ther. 1998; 5: 1204-1212Crossref PubMed Scopus (97) Google Scholar). Finally, we cannot exclude the possibility that the cytosolic-independent nature of IGFBP-3 nuclear import is effected by other factors that, like importin β, may not be completely solubilized during digitonin permeabilization.Nuclear import mediated by conventional NLSs such as those found in T-ag and retinoblastoma (42Efthymiadis A. Shao H. Hubner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), as well as by novel NLSs of HIV-I Tat (26Efthymiadis A. Briggs L.J. Jans D.A. J. Biol. Chem. 1998; 273: 1623-1628Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and the hnRNP K (28Michael W.M. Eder P.S. Dreyfuss G. EMBO J. 1997; 16: 3587-3598Crossref PubMed Scopus (320) Google Scholar) and hnRNP A1 M9 sequences (52Pollard V.W. Michael W.M. Nakielny S. Siomi M.C. Wang F. Dreyfuss G. Cell. 1996; 86: 985-994Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar), has been shown to be ATP-dependent; cytosolic factor-independent nuclear import of β-catenin has also been shown to require ATP (27Fagotto F. Gluck U. Gumbiner B.M. Curr. Biol. 1998; 8: 181-190Abstract Full Text Full Text PDF PubMed Google Scholar). In the case of HIV-I Tat, ATP hydrolysis is believed to effect its release from cytoplasmic retention factors and enhance binding to nuclear components. Because nuclear import of IGFBP-3 was not observed in the absence of an ATP-regenerating system, IGFBP-3 may also require ATP for cytoplasmic release and/or enhanced nuclear binding. Alternatively, as is the case for many proteins transported to the nucleus, ATP may be involved in other, unknown actions that augment nuclear uptake.In the presence of a permeabilized nuclear envelope, proteins are free to diffuse between the cytoplasmic and nuclear compartments and are only able to accumulate in the nucleus through binding to insoluble nuclear components such as chromatin and lamin (42Efthymiadis A. Shao H. Hubner S. Jans D.A. J. Biol. Chem. 1997; 272: 22134-22139Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Under conditions where the nuclear envelope was permeabilized, IGFBP-3, but not the mutant, IGFBP-3[228KGRKR → MDGEA], was capable of nuclear accumulation. These results support the proposal that, upon entry into the nucleus, IGFBP-3 accumulates through nuclear binding and suggest that residues 228–232 are required for these nuclear interactions. The ability of IGFBP-3 to bind to structures within the nucleus supports the hypothesis that it may have a role in the nucleus, possibly in direct regulation of gene transcription (33Jaques G. Noll K. Wegmann B. Witten S. Kogan E. Radulescu R.T. Havemann K. Endocrinology. 1997; 138: 1767-1770Crossref PubMed Scopus (124) Google Scholar, 34Li W. Fawcett J. Widmer H.R. Fielder P.J. Rabkin R. Keller G.A. Endocrinology. 1997; 138: 1763-1766Crossref PubMed Scopus (104) Google Scholar, 35Schedlich L.J. Young T.F. Firth S.M. Baxter R.C. J. Biol. Chem. 1998; 273: 18347-18352Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 36Wraight C.J. Liepe I.J. White P.J. Hibbs A.R. Werther G.A. J. Invest. Dermatol. 1998; 111: 239-242Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). This has been suggested for granzyme A and B (53Jans D.A. Jans P. Briggs L.J. Sutton V. Trapani J.A. J. Biol. Chem. 1996; 271: 30781-30789Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 54Jans D.A. Briggs L.J. Jans P. Froelich C.J. Parasivam G. Kumar S. Sutton V.R. Trapani J.A. J. Cell Sci. 1998; 111: 2645-2654Crossref PubMed Google Scholar) and parathyroid hormone-related protein (24Lam M.H. Briggs L.J. Hu W. Martin T.J. Gillespie M.T. Jans D.A. J. Biol. Chem. 1999; 274: 7391-7398Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), which can also accumulate in the nucleus even in the absence of an intact nuclear envelope.Fusion of EGFP to isolated motifs has been used to study changes in subcellular distribution directed by these sequences (50Shulga N. Roberts P. Gu Z. Spitz L. Tabb M.M. Nomura M. Goldfarb D.S. J. Cell Biol. 1996; 135: 329-339Crossref PubMed Scopus (186) Google Scholar). Using this approach, we investigated the role of the basic motifs of IGFBP-3 and IGFBP-5 in their nuclear transport. As the fusion proteins are expressed in living CHO cells, the proteins, factors and metabolic pathways present and active in living cells are able to exert their effects on nuclear transport, thus representing an in vivonuclear transport assay. EGFP is small enough to enter the nucleus by passive diffusion, and as expected, the fluorescent signal derived from recombinant EGFP expressed in CHO cells was evenly distributed between the nucleus and cytoplasm. Fusion of the basic region within the C-terminal domain of IGFBP-3 and IGFBP-5 to EGFP caused nuclear accumulation of the fusion protein in greater than 90% of transfected cells, indicating that the basic regions are sufficient for nuclear import. However, these results do not distinguish between active nuclear transport and nuclear binding. Thus, there may be passive diffusion into the nucleus followed by accumulation resulting from interaction between these basic sequences and nuclear binding sites, as well as interaction of the basic residues with importins, leading to active nuclear import.Mutation of the three basic clusters within the putative NLS of IGFBP-3 and IGFBP-5 caused different degrees of attenuation of nuclear transport of the EGFP fusion proteins. As was observed for the mutant form of full-length IGFBP-3, 228KGRKR → MDGEA, the same mutation of both the IGFBP-3 and IGFBP-5 NLS when fused to EGFP, abolished nuclear uptake of EGFP. Mutation of both amino acids within the N-terminal basic cluster had a significant effect on nuclear transport of the EGFP fusion proteins, suggesting that the basic region may represent a classical bipartite NLS similar to that described for other proteins (9Jans D.A. Hubner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (384) Google Scholar, 21Robbins J. Dilworth S.M. Laskey R.A. Dingwall C. Cell. 1991; 64: 615-623Abstract Full Text PDF PubMed Scopus (1240) Google Scholar). However, mutation of the central basic cluster reduced nuclear transport of the fusion proteins by approximately 60%, implying that these basic clusters also influence nuclear accumulation and that the NLS is more complex than a classical bipartite NLS. As discussed above, the ability of the fusion protein to diffuse freely into the nucleus means that a distinction cannot be drawn between enhancement of nuclear import and accumulation because of nuclear binding. Therefore, mutations affecting nuclear transport may relate to either or both effects. However, in vitro studies on the mutant, IGFBP-3[228KGRKR → MDGEA], suggest these sequences are involved in both effects. In this assay the basic sequences derived from IGFBP-5 behaved identically to those derived from IGFBP-3, suggesting that IGFBP-5, which is small enough to diffusion through the nuclear pore complex (molecular mass, 30 kDa), accumulates in the nucleus by a similar mechanism.We found that the importin β subunit recognized both wild-type IGFBP-3 and IGFBP-5 but recognized only to a limited extent the mutant form of IGFBP-3. This suggests that transport of these binding proteins occurs by a signal-mediated pathway, consistent with the observation that importin β is required to effect in vitro nuclear transport of IGFBP-3. Interestingly, all the importin α/β binding to IGFBP-5 could be accounted for by importin β binding, whereas for IGFBP-3 importin β binding appeared significantly lower compared with its binding to the heterodimer. This was not compensated for by an appropriate increase in importin α binding. The explanation for this is unclear but may relate to differing affinities or accessibility of the binding proteins for the importin subunits. Alternatively, importin α may be binding to importin β in this assay and increasing the strength of its interactions with IGFBP-3. However, an essential role for importin α in IGFBP-3 nuclear transport is not supported by our findings that nuclear import occurs in the absence of exogenous cytosol.An interesting question raised by this study is why IGFBP-5, and to a lesser extent IGFBP-3, possess functional NLSs when, because of their size, they have the potential to diffuse from the cytoplasm into the nucleus. There are several reasons why smaller proteins possess NLS-dependent mechanisms for nuclear import. As has been described for interleukin-5 (55Jans D.A. Briggs L.J. Gustin S.E. Jans P. Ford S. Young I.G. FEBS Lett. 1997; 410: 368-372Crossref PubMed Scopus (45) Google Scholar), a functional NLS enables the cotransport of larger non-NLS containing proteins to the nucleus. In analogous fashion, IGFBP-3 or IGFBP-5 may possess functional NLSs to facilitate entry when part of a high molecular mass complex. Thus, they may require an NLS-dependent mechanism when cotransporting IGFs or other signaling molecules to the nucleus. Interestingly, a preliminary report suggests that IGFBP-3 interacts specifically with the retinoic acid X receptor-α (56Cohen P. Liu B. Ferry R. Jansen-Duerr P. Mannhardt B. Clifford J. Lee H. Kurie J. Growth Hormone IGF Res. 1999; 9: 317Google Scholar). IGFBP-3 may thereby modulate the activity of nuclear transcription factors or have a specific signal transduction role in the nucleus. It may also regulate gene expression directly by binding to chromatin as has been reported for basic fibroblast growth factor (57Gualandris A. Coltrini D. Bergonzoni L. Isacchi A. Tenca S. Ginelli B. Presta M. Growth Factors. 1993; 8: 49-60Crossref PubMed Scopus (21) Google Scholar) and the growth hormone receptor (58Lobie P.E. Wood T.J. Chen C.M. Waters M.J. Norstedt G. J. Biol. Chem. 1994; 269: 31735-31746Abstract Full Text PDF PubMed Google Scholar). Apart from the selective use of an NLS-dependent mechanism for the transport of high molecular mass complexes, the kinetics of nuclear import of IGFBP-3 and IGFBP-5 may be enhanced by their ability to interact effectively with importin. A major focus of future work in this laboratory is to distinguish between these possibilities. Understanding of the mechanisms of nuclear import of IGFBP-3 and IGFBP-5 should greatly assist in defining their nuclear functions. The mitogenic" @default.
• W2053296976 created "2016-06-24" @default.
• W2053296976 creator A5025294735 @default.
• W2053296976 creator A5049015437 @default.
• W2053296976 creator A5053618187 @default.
• W2053296976 creator A5054290176 @default.
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• W2053296976 date "2000-08-01" @default.
• W2053296976 modified "2023-10-12" @default.
• W2053296976 title "Nuclear Import of Insulin-like Growth Factor-binding Protein-3 and -5 Is Mediated by the Importin β Subunit" @default.
• W2053296976 cites W1503329918 @default.
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