AP - 1 transcriptional activity is regulated by a direct association between thioredoxin and Ref - 1 ( redox regulation y disulfide cross - linking y nuclear translocation y mammalian two - hybrid assay )
نویسندگان
چکیده
Thioredoxin (TRX) is a pleiotropic cellular factor that has thiol-mediated redox activity and is important in regulation of cellular processes, including proliferation, apoptosis, and gene expression. The activity of several transcription factors is posttranslationally altered by redox modification(s) of specific cysteine residue(s). One such factor is nuclear factor (NF)-kB, whose DNA-binding activity is markedly augmented by TRX treatment in vitro. Similarly, the DNA-binding activity of activator protein 1 (AP-1) is modified by a DNA repair enzyme, redox factor 1 (Ref-1), which is identical to a DNA repair enzyme, AP endonuclease. Ref-1 activity is in turn modulated by various redox-active compounds, including TRX. We here report the molecular cascade of redox regulation of AP-1 mediated by TRX and Ref-1. Phorbol 12-myristate 13 acetate efficiently translocated TRX into the HeLa cell nucleus where Ref-1 preexists. This process seems to be essential for AP-1 activation by redox modification because co-overexpression of TRX and Ref-1 in COS-7 cells potentiated AP-1 activity only after TRX was transported into the nucleus by phorbol 12-myristate 13 acetate treatment. To prove the direct active site-mediated association between TRX and Ref-1, we generated a series of substitution-mutant cysteine residues of TRX. In both an in vitro diamide-induced cross-linking study and an in vivo mammalian two-hybrid assay we proved that TRX can associate directly with Ref-1 in the nucleus; also, we demonstrated the requirement of cysteine residues in the TRX catalytic center for the potentiation of AP-1 activity. This report presents an example of a cascade in cellular redox regulation. Increasing evidence has indicated that cellular redox status modulates various aspects of cellular events, including proliferation and apoptosis (1). TRX is a small, ubiquitous protein with two redox-active half-cystine residues, -Cys-Gly-Pro-Cys-, in an active center (2–5). TRX also is known as adult T-cell leukemia-derived factor that is involved in human T lymphotropic virus type I leukemogenesis (6, 7). TRX exists either in a reduced or oxidized form and participates in redox reactions through the reversible oxidation of this active center dithiol. TRX serves functions inside (8–10) and outside (11–13) the cell. One of its intracellular functions is the facilitation of protein–nucleic acid interactions (3). In vitro and in vivo experiments showed that TRX augmented the DNA-binding and transcriptional activities of the p50 subunit of nuclear factor (NF)-kB by reducing Cys-62 in its DNA-binding loop (8, 9). Recently, direct physical association of an active-site mutant of TRX and an oligopeptide from NF-kB p50 was demonstrated by a nuclear magnetic resonance (NMR) study in vitro (14); in vivo direct association of TRX–NF-kB remains to be demonstrated. Redox regulation of Jun and Fos molecules has also been implicated. Various antioxidants strongly activate the DNA-binding and transactivation abilities of activator protein 1 (AP-1) complex (1, 15, 16). Interestingly, TRX enhances the DNA-binding activity of Jun and Fos, and this process requires other molecules such as a novel protein called redox factor 1 (Ref-1) (17, 18). Ref-1 was identified in a cell-free system as one of the factors restoring AP-1-DNA binding and was found to be identical to an AP endonuclease (17, 19). There has been no report describing whether TRX and Ref-1 directly associate or require any intervening factor(s) for their interaction. In this paper we studied the association of TRX and Ref-1 through the use of an in vitro cross-linking and a mammalian two-hybrid system, with various mutants of TRX, to investigate the interaction of TRX on AP-1-mediated transcription. MATERIALS AND METHODS Cells and Cell Culture.HeLa and COS-7 cells were cultured in DMEM (GIBCOyBRL) supplemented with 10% fetal calf serum and antibiotics at 95% humidityy5% CO2 in air at 378C. Reagents. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma. Anti-human TRX mAb, 11-mAb, whose epitope has not been determined (10), was produced and provided by Fujirebio (Tokyo). Anti-human Ref-1 rabbit polyclonal antibody (C-20), which recognizes the epitope corresponding to amino acids 299–318, and anti-c-Fos mAb (6–2H-2F), which recognizes the epitope corresponding to the leucine zipper of c-Fos, were purchased from Santa Cruz Biotechnology. Mutagenesis of TRX and Transfection. Mutagenesis of human TRX was performed by a PCR-based technique (20), and authenticity of the sequences was verified by DNA sequencing (Fig. 1). The expression plasmids were introduced into the cells by LipofectAMINE reagent (GIBCOyBRL) according to themanufacturer’s instructions, using Opti-MEM (GIBCOyBRL). Luciferase Assay Using -73y163 Col (Human Collagenase I)-LUC. The 273y163 Col-LUC was constructed with pGL2Basic (Promega) and a fragment from 273y163 Col in pBLCAT3 (21). HeLa cells were plated in 6-well plates at a density of 4 3 105 cells per well. The expression plasmids were introduced into the cells by use of LipofectAMINE reagent. In each transfection, 1mg of the pCDSRa-TRXwild or -TRXC32S/C35S expression plasmid (6) andyor 1 mg of the pRcyCMV-Ref-1 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Copyright q 1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA 0027-8424y97y943633-●$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: TRX, thioredoxin; NF, nuclear factor; AP-1, activator protein-1; Ref-1, redox factor-1; PMA, phorbol 12-myristate 13acetate; 6xHis, hexahistidine; DBD, DNA-binding domain. ‡Present address: Department of Biomedical Genetics, Faculty of Pharmaceutical Sciences, University of Tokyo, Tokyo 113, Japan. §To whom reprint requests should be addressed at: Institute for Virus Research, Kyoto University, 53, Shogoin Kawahara-Cho, Sakyo-Ku, Kyoto 606-01, Japan. e-mail: [email protected].
منابع مشابه
Thioredoxin nuclear translocation and interaction with redox factor-1 activates the activator protein-1 transcription factor in response to ionizing radiation.
Thioredoxin (TRX) is a cytoplasmic, redox-sensitive signaling factor believed to participate in the regulation of nuclear transcription factors mediating cellular responses to environmental stress. Activation of the activator protein (AP)-1 transcription factor is thought to be mediated in part by redox-sensitive interactions between the nuclear signaling protein redox factor-1 (Ref-1) and TRX....
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