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• W2013279940 abstract "Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20–40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP (C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1·CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 (residues 106–140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or “docking” of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase. Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20–40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP (C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1·CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 (residues 106–140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or “docking” of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase. IRF-1 (interferon regulatory factor-1) is a transcription factor initially identified as an activator of IFNβ (interferon-β gene) (1Fujita T. Sakakibara J. Sudo Y. Miyamoto M. Kimura Y. Taniguchi T. EMBO J. 1988; 7: 3397-3405Crossref PubMed Scopus (272) Google Scholar), which has subsequently been intimately linked to the antiviral response and the response to DNA damage (2Tanaka N. Ishihara M. Lamphier M.S. Nozawa H. Matsuyama T. Mak T.W. Aizawa S. Tokino T. Oren M. Taniguchi T. Nature. 1996; 382: 816-818Crossref PubMed Scopus (303) Google Scholar, 3Taniguchi T. Ogasawara K. Takaoka A. Tanaka N. Annu. Rev. Immunol. 2001; 19: 623-655Crossref PubMed Scopus (1297) Google Scholar). Additionally, IRF-1 is a tumor suppressor protein, and deletions of IRF-1 are associated with the development of gastric and esophageal tumors as well as some leukemias (4Nozawa H. Oda E. Ueda S. Tamura G. Maesawa C. Muto T. Taniguchi T. Tanaka N. Int. J. Cancer. 1998; 77: 522-527Crossref PubMed Scopus (78) Google Scholar, 5Tamura G. Sakata K. Nishizuka S. Maesawa C. Suzuki Y. Terashima M. Eda Y. Satodate R. J. Pathol. 1996; 180: 371-377Crossref PubMed Scopus (93) Google Scholar, 6Willman C.L. Sever C.E. Pallavicini M.G. Harada H. Tanaka N. Slovak M.L. Yamamoto H. Harada K. Meeker T.C. List A.F. Science. 1993; 259: 968-971Crossref PubMed Scopus (381) Google Scholar). The IRF-1 protein is short lived and has a half-life in the region of 30 min in cultured cells (7Pamment J. Ramsay E. Kelleher M. Dornan D. Ball K.L. Oncogene. 2002; 21: 7776-7785Crossref PubMed Scopus (78) Google Scholar, 8Pion E. Narayan V. Eckert M. Ball K.L. Cell. Signal. 2009; 21: 1479-1487Crossref PubMed Scopus (22) Google Scholar, 9Watanabe N. Sakakibara J. Hovanessian A.G. Taniguchi T. Fujita T. Nucleic Acids Res. 1991; 19: 4421-4428Crossref PubMed Scopus (180) Google Scholar). It is primarily degraded via the 26 S proteasome (8Pion E. Narayan V. Eckert M. Ball K.L. Cell. Signal. 2009; 21: 1479-1487Crossref PubMed Scopus (22) Google Scholar, 10Nakagawa K. Yokosawa H. Eur. J. Biochem. 2000; 267: 1680-1686Crossref PubMed Scopus (73) Google Scholar), and the rate of degradation can be regulated in response to cellular conditions (7Pamment J. Ramsay E. Kelleher M. Dornan D. Ball K.L. Oncogene. 2002; 21: 7776-7785Crossref PubMed Scopus (78) Google Scholar). For example, agents such as ionizing radiation increase steady state levels of the IRF-1 protein through a concerted mechanism that includes a decrease in its rate of degradation (2Tanaka N. Ishihara M. Lamphier M.S. Nozawa H. Matsuyama T. Mak T.W. Aizawa S. Tokino T. Oren M. Taniguchi T. Nature. 1996; 382: 816-818Crossref PubMed Scopus (303) Google Scholar, 7Pamment J. Ramsay E. Kelleher M. Dornan D. Ball K.L. Oncogene. 2002; 21: 7776-7785Crossref PubMed Scopus (78) Google Scholar). Like other proteins degraded via the proteasome, IRF-1 is polyubiquitinated prior to degradation (8Pion E. Narayan V. Eckert M. Ball K.L. Cell. Signal. 2009; 21: 1479-1487Crossref PubMed Scopus (22) Google Scholar, 10Nakagawa K. Yokosawa H. Eur. J. Biochem. 2000; 267: 1680-1686Crossref PubMed Scopus (73) Google Scholar). The ubiquitination process itself involves at least three distinct enzymes. A ubiquitin (Ub) 4The abbreviations used are: UbubiquitinTPRtetratricopeptide repeatpAbpolyclonal antibodyaaamino acids.-activating enzyme, or E1, activates Ub and forms a Ub-thiol ester in an ATP-dependent process subsequently. A ubiquitin-conjugating enzyme (E2), which associates with a ubiquitin ligase (E3), is involved in ubiquitin transfer from the E1 to the substrate through the E3. The E3 and occasionally the E2·E3 complex give specificity to the system because they are involved in substrate recognition (11Pickart C.M. Eddins M.J. Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (1039) Google Scholar). Although IRF-1 has been characterized as a substrate of the ubiquitination system, the E3 ligase(s) involved in IRF-1 ubiquitination have not yet been identified. ubiquitin tetratricopeptide repeat polyclonal antibody amino acids. In the current study, we describe an interaction between IRF-1 and CHIP (C terminus of Hsc70-interacting protein), leading to the identification of CHIP as an E3 ligase for IRF-1. CHIP is thought to provide a link between the protein folding pathway(s) and the pathways within a cell that lead to protein degradation. Structurally, CHIP comprises an N-terminal tetratricopeptide repeat (TPR) domain, through which it can bind to the Hsp70 and Hsp90 families of molecular chaperones; a central charged domain that is required for dimerization but otherwise has a largely unknown function; and a C-terminal U-box structure that binds E2 enzymes and mediates CHIP function as an E3 ubiquitin ligase (12Nikolay R. Wiederkehr T. Rist W. Kramer G. Mayer M.P. Bukau B. J. Biol. Chem. 2004; 279: 2673-2678Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 13Xu Z. Devlin K.I. Ford M.G. Nix J.C. Qin J. Misra S. Biochemistry. 2006; 45: 4749-4759Crossref PubMed Scopus (49) Google Scholar, 14Ballinger C.A. Connell P. Wu Y. Hu Z. Thompson L.J. Yin L.Y. Patterson C. Mol. Cell Biol. 1999; 19: 4535-4545Crossref PubMed Scopus (757) Google Scholar, 15Jiang J. Ballinger C.A. Wu Y. Dai Q. Cyr D.M. Höhfeld J. Patterson C. J. Biol. Chem. 2001; 276: 42938-42944Abstract Full Text Full Text PDF PubMed Scopus (482) Google Scholar). The U-box is structurally similar to the RING (really interesting new gene) domain present in the RING family of E3 Ub ligases, although it is stabilized by intramolecular hydrogen bonds and salt bridges rather than Zn2+ ions (16Aravind L. Koonin E.V. Curr. Biol. 2000; 10: R132-R134Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). It is commonly believed that CHIP binds to Hsp70 and targets misfolded client proteins for degradation, bypassing the need for a direct interaction with its substrate (17Murata S. Minami Y. Minami M. Chiba T. Tanaka K. EMBO Rep. 2001; 2: 1133-1138Crossref PubMed Scopus (469) Google Scholar, 18Connell P. Ballinger C.A. Jiang J. Wu Y. Thompson L.J. Höhfeld J. Patterson C. Nat. Cell Biol. 2001; 3: 93-96Crossref PubMed Scopus (0) Google Scholar). Recently, however, a number of studies have suggested an alternate CHIP ubiquitination pathway in which the substrate binding activity of CHIP may play a key role in determining its specific E3 ligase function (19Shang Y. Zhao X. Xu X. Xin H. Li X. Zhai Y. He D. Jia B. Chen W. Chang Z. Biochem. Biophys. Res. Commun. 2009; 386: 242-246Crossref PubMed Scopus (43) Google Scholar, 20Parsons J.L. Tait P.S. Finch D. Dianova I.I. Allinson S.L. Dianov G.L. Mol. Cell. 2008; 29: 477-487Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Here we present evidence in support of diverse roles for CHIP in the regulation of IRF-1. Although CHIP has a positive effect on IRF-1 protein levels in unstressed cells, in response to specific stresses, such as heat and heavy metal stress, CHIP binds directly to a docking site in the central region of IRF-1 facilitating IRF-1 ubiquitination. Thus, (i) CHIP and IRF-1 can form a stable complex in vitro and in cells that does not require Hsp70, (ii) CHIP binding to an intrinsically disordered domain of IRF-1 is required for its ubiquitination because this domain can act in trans to interact with CHIP and inhibit ubiquitination of IRF-1, and (iii) CHIP·IRF-1 complex formation is regulated in cells exposed to selective stress conditions and correlates with an increase in IRF-1 ubiquitination and a decrease in its steady state levels. Therefore, direct binding of both Hsp70 (21Narayan V. Eckert M. Zylicz A. Zylicz M. Ball K.L. J. Biol. Chem. 2009; 284: 25889-25899Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) and CHIP can regulate IRF-1, highlighting the intimate link between the molecular chaperones and IRF-1 function. Antibodies were used at the concentrations indicated by the supplier and were anti-IRF-1 mAb (catalog no. 612047, BD Biosciences), anti-GFP mAb and pAb (catalog nos. 632380 and 632459, Clontech), anti-GAPDH pAb (catalog no. ab9483, Abcam), anti-FLAG mAb, anti-Myc pAb, anti-GST mAb, anti-β-actin mAb, and anti-vimentin mAb (catalog nos. F3165, C3956, G1160, A5441, and V5255, Sigma), anti-caspase-3 pAb (catalog no. sc7148, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-HP1α mAb (catalog no. 05-689, Millipore), anti-ubiquitin FK-1 and FK-2 (Biomol BML-PW8805-0500 and BML-PW8810-0500), anti-histone H3 and anti-calreticulin mAbs (catalog nos. 5192 and 2891, Cell Signaling), and anti-His mAb (catalog no. 70796-3, Novagen). Anti-CHIP mAb (MBC3) was from Moravian Biotechnology, and anti-Myc mAb was obtained from CRUK. Secondary antibodies were purchased from DakoCytomation. MG-132 (Calbiochem) was dissolved in DMSO to 10 mm and used as indicated. Peptides were from Chiron Mimotopes and were synthesized with a biotin tag and an SGSG spacer at the N terminus. The human IRF-1 sequence was codon-optimized for Escherichia coli expression (Genscript) and inserted into pDEST-15 using Gateway technology (Invitrogen) to generate GST-IRF-1. FLAG-IRF-1 was generated by amplifying an EcoRI-IRF-1-BamHI fragment from pcDNA3-IRF-1 (8Pion E. Narayan V. Eckert M. Ball K.L. Cell. Signal. 2009; 21: 1479-1487Crossref PubMed Scopus (22) Google Scholar) and ligating it into p3xFLAG-Myc-CMV-24 (Sigma). For IRF-1 Δ106–140, the BlpI internal site on human IRF-1 was used. A BlpI-IRF-1(141–325)-BamHI fragment was amplified from FLAG-IRF-1 WT and ligated with FLAG-IRF-1 WT that was digested with BlpI and BamHI to give IRF-1 Δ106–140; this was inserted into the Gateway system by introducing attB1 and attB2 sites according to the manufacturer's instructions. pDEST53-IRF-1 (GFP-IRF-1) was as described previously (8Pion E. Narayan V. Eckert M. Ball K.L. Cell. Signal. 2009; 21: 1479-1487Crossref PubMed Scopus (22) Google Scholar). pET15bmod-CHIP was a kind gift from Prof. Alicja Zylicz and Renata Filipek, and pMBC1-IRF-1(115–140) (GFP-IRF-1(115–140)) was from the Hauser group (22Schaper F. Kirchhoff S. Posern G. Köster M. Oumard A. Sharf R. Levi B.Z. Hauser H. Biochem. J. 1998; 335: 147-157Crossref PubMed Scopus (84) Google Scholar). pcDNA3.1-His/Myc-CHIP WT and domains were kind gifts from Prof. Cam Patterson and Dr. Holly McDonough. GST-IRF-1 expressed in BL21-AI (Invitrogen) was purified using glutathione-Sepharose beads (GE Healthcare) according to the manufacturer's instructions. His-CHIP was expressed in BL21-DE3 and purified using Ni2+-NTA-agarose (Qiagen) according to the manufacturer's instructions. In both cases, Mg2+-ATP washes were incorporated prior to elution to remove any bound chaperones. Additionally, untagged human IRF-1 was expressed and purified in a cell-free environment using the PURExpress in vitro protein synthesis kit (New England Biolabs) according to the manufacturer's instructions. A375, MCF-7, MDA-MB-231, and HeLa cells were cultured in DMEM (Invitrogen), whereas H1299, OVCAR8, HCC-827, DU-145, A549, and ACHN were cultured in RPMI 1640 (Invitrogen). All media were supplemented with 10% (v/v) fetal bovine serum (Autogen Bioclear) and 1% (v/v) penicillin/streptomycin mix (Invitrogen). A375 and HeLa cells were maintained at 10% CO2; all remaining cells were maintained at 5% CO2. Cells were seeded 24 h before transfection. DNA was transfected into the cells using Attractene (Qiagen) and siRNA (OnTargetPlus siRNA pools from Dharmacon; catalogue no. D-001810-10-20 for control siRNA pool and L-007201-00-0020 for CHIP siRNA pool) using Dharmafect (Thermo Scientific) as described in the manufacturer's instructions. If required, cells were treated as follows. For serum starvation, serum was withdrawn from the media at the time of transfection 24 h prior to harvesting; heat shock was carried out at 43 °C for 30 min immediately prior to harvesting unless otherwise indicated; for heavy metal stress, cells were treated with 1 mm ZnCl2 for 90 min. MG-132 treatment (50 μm) was for 4 h prior to harvesting. Post harvesting, cells were lysed in Triton Lysis Buffer (50 mm HEPES, pH 7.5, 0.2% (v/v) Triton X-100, 150 mm NaCl, 10 mm NaF, 2 mm DTT, 0.1 mm EDTA, protease inhibitor mix (20 μg/ml leupeptin, 1 μg/ml aprotinin, 2 μg/ml pepstatin, 1 mm benzamidine, 10 μg/ml soybean trypsin inhibitor, 2 mm pefabloc)) unless otherwise indicated. Immunoblots were performed as described previously (21Narayan V. Eckert M. Zylicz A. Zylicz M. Ball K.L. J. Biol. Chem. 2009; 284: 25889-25899Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). To detect endogenous IRF-1, 75 μg of protein was loaded per lane, whereas 25 μg was sufficient to detect exogenous IRF-1. FLAG-IRF-1 or GFP-IRF-1 (or corresponding empty vector) was transfected into A375 cells as described above. Post-transfection (24 h), cells were harvested and lysed in Triton Lysis Buffer. Following this, the lysates were precleared using Sepharose 4B beads (Sigma). FLAG-tagged complexes from 3 mg of total cellular lysate were purified using anti-FLAG-M2-agarose (35 μl; Sigma) according to the manufacturer's instructions and analyzed by SDS-PAGE/immunoblot. GFP-tagged complexes were isolated by incubating precleared lysate (7.5 mg) with protein G-Sepharose beads (40 μl; GE Healthcare) and anti-GFP pAb (5 μl) overnight at 4 °C. The beads were washed extensively with PBS supplemented with 0.4% Triton X-100 and eluted by heating at 90 °C in SDS-PAGE sample buffer for 5 min. For affinity chromatography, GST-tagged IRF-1 and GST alone were purified as described above; however, bound GST-proteins were not eluted from the glutathione-Sepharose column. Instead, precleared A375 cell lysate (1 mg) was added and incubated for 1 h at 4 °C. The column was washed five times with PBS containing 0.2% (v/v) Triton X-100 and once with Buffer W (100 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1 mm benzamidine). Bound proteins were eluted by heating at 90 °C in sample buffer and analyzed by SDS-PAGE/immunoblot. Purified recombinant His-CHIP (100 ng) was immobilized on a microtiter plate in 0.1 m NaHCO3 buffer (pH 8.6) at 4 °C. Non-reactive sites were blocked using PBS containing 3% BSA. A titration of the protein of interest was added in 1× Reaction Buffer (25 mm HEPES, pH 7.5, 50 mm KCl, 10 mm MgCl2, 5% (v/v) glycerol, 0.1% (v/v) Tween 20, 2 mg/ml BSA) for 1 h at room temperature. After washing in PBS containing 0.1% (v/v) Tween 20, binding was detected using anti-GST and HRP-tagged anti-mouse antibodies, and electrochemical luminescence was quantified using a luminometer (Fluoroskan Ascent FL, Labsystems). For peptide competition assays, His-CHIP (100 ng) was preincubated with a titration of peptide (or DMSO control) in 1× reaction buffer for 10 min at room temperature, after which the mix was incubated for 1 h at room temperature with GST-IRF-1 (100 ng) immobilized on a microtiter plate as above. Washing and detection were as described above, except using anti-His mAb. For peptide binding assays, microtiter plates were coated with streptavidin (1 μg/well), following which enough biotin-tagged peptide to saturate the streptavidin (∼60 pmol) was added. Non-reactive sites were blocked using PBS containing 3% BSA as above. His-CHIP (25 ng) in 1× reaction buffer was added for 1 h at room temperature. Washing and detection were as above using anti-His mAb. In vitro ubiquitination assays were carried out essentially as previously described (23Wallace M. Worrall E. Pettersson S. Hupp T.R. Ball K.L. Mol. Cell. 2006; 23: 251-263Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) except using 25 nm GST-IRF-1 (WT or mutant) as substrate. Reactions were started with His-CHIP (60–360 nm as indicated; usually 60 nm), incubated for 20 min at 30 °C, and analyzed using 4–12% NuPAGE gels in a MOPS buffer system/immunoblot. In vivo ubiquitination assays were carried out as described (8Pion E. Narayan V. Eckert M. Ball K.L. Cell. Signal. 2009; 21: 1479-1487Crossref PubMed Scopus (22) Google Scholar). His/Myc-CHIP (WT or mutant) was expressed in a TNT coupled reticulocyte lysate system (Promega) according to the manufacturer's instructions. The CHIP proteins were isolated using Ni2+-NTA-agarose (Qiagen) and washed extensively according to the manufacturer's recommendations. Mg2+-ATP washes were incorporated to remove any bound chaperones. Post washing, 5 μl of untagged IRF-1 (see “Plasmids and Protein Purification” for details) was added to the column and incubated for 1 h at 4 °C. Columns were then washed extensively with 20 mm Tris, pH 8.0, and 150 mm NaCl containing 0.2% Triton X-100, and bound proteins were eluted in 20 mm Tris, pH 8.0, 150 mm NaCl, and 300 mm imidazole. Eluates were analyzed by SDS-PAGE/immunoblot. Cells were seeded in a 96-well sterile black plate with a clear bottom (Costar) as required. In-Cell WesternTM assays were subsequently performed on a Licor Odyssey SA scanner according to the manufacturer's instructions. Anti-IRF-1 mAb (BD Biosciences) and anti-CHIP mAb (MBC3) were used at 1:500 and 1:50, respectively. The DNA stain (DRAQ5) was used as a control to normalize for cell number, as recommended by the manufacturer. Subcellular fractionation was carried out using the ProteoExtract kit (Calbiochem) or the subcellular protein fractionation kit (Thermo Scientific) as indicated, according to the manufacturer's instructions. We have previously shown that IRF-1 is polyubiquitinated and degraded via the proteasome and that the C terminus of IRF-1, which is required for the efficient ubiquitination of the protein, binds directly to the molecular chaperone Hsp70 (8Pion E. Narayan V. Eckert M. Ball K.L. Cell. Signal. 2009; 21: 1479-1487Crossref PubMed Scopus (22) Google Scholar, 21Narayan V. Eckert M. Zylicz A. Zylicz M. Ball K.L. J. Biol. Chem. 2009; 284: 25889-25899Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Given that the outcome of Hsp70-substrate interactions is governed by a host of co-chaperones, we were interested in extending our previous study (21Narayan V. Eckert M. Zylicz A. Zylicz M. Ball K.L. J. Biol. Chem. 2009; 284: 25889-25899Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) in order to determine whether the co-chaperone and E3 ubiquitin ligase CHIP (24Tsai Y.C. Fishman P.S. Thakor N.V. Oyler G.A. J. Biol. Chem. 2003; 278: 22044-22055Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) played a role in the regulation of IRF-1. When A375 cell lysate was passed through a column prepared by immobilizing GST-IRF-1 on glutathione-Sepharose beads, endogenous CHIP bound specifically to GST-IRF-1 and not to a GST alone control column (Fig. 1A). Additionally, CHIP was co-immunoprecipitated with IRF-1 from A375 cells in which both proteins were overexpressed (Fig. 1B). The above experiments suggest that CHIP and IRF-1 can form a complex; however, they do not address whether the proteins interact directly or whether additional factors, such as Hsp70, are required. In order to examine whether CHIP could bind IRF-1 in the absence of other factors, recombinant proteins purified from E. coli were used. When CHIP was immobilized on a microtiter well and incubated with IRF-1 that was in the mobile phase, it bound specifically to GST-IRF-1 but not GST alone (Fig. 1C). This shows that CHIP has the potential to bind directly to IRF-1 and that Hsp70 or other cellular factors are not required to mediate the interaction. Having established that CHIP can interact with IRF-1 in cells and in a cell-free environment, we sought to map the CHIP binding interface on IRF-1 using a library of biotin-tagged overlapping peptides spanning the entire length of the IRF-1 protein. When the peptides were immobilized on streptavidin-coated microtiter wells and incubated with purified CHIP, the CHIP bound stably to an Arg-Lys-Ser-rich region in the Mf2 (multifunctional 2) domain of IRF-1 (aa 106–140; Fig. 2A, peptides 8 and 9). In addition, CHIP bound to a lesser extent to a number of peptides from the DNA-binding domain of IRF-1 (Fig. 2A, peptides 1, 3, 4, 7). When the Mf2-derived peptide 9 was used in a competition assay to determine if it could reduce the binding of CHIP to full-length IRF-1, peptide 9, but not a control peptide, significantly inhibited CHIP·IRF-1 complex formation (Fig. 2B). The above data suggest that CHIP binds to a complex interface on IRF-1 and that a region from the IRF-1 Mf2 domain, aa 106–140, is sufficient to form a stable interaction and to partially compete with the full-length protein for binding to CHIP. To further establish a requirement for the Mf2 domain of IRF-1 in CHIP binding, a deletion mutant (IRF-1 Δ106–140) was generated and purified from E. coli. A comparison of CHIP binding to GST-tagged full-length and mutant IRF-1 showed a marked loss of binding to the mutant protein lacking the Arg-Lys-Ser motif (Fig. 2C). Fig. 2C (right) shows that the mutant and wild-type protein were normalized on the well. The fact that Mf2-derived peptides do not completely block IRF-1·CHIP complex formation (Fig. 2B) and that IRF-1 Δ106–140 retains partial CHIP binding activity supports the idea that the IRF-1·CHIP interface is complex, involving points of contact in the DNA-binding domain as well as the Mf2 region (Fig. 2A). Together, the results suggest a high affinity interaction between CHIP and aa 106–140 of IRF-1, with additional weaker contact sites in the IRF-1 DNA-binding domain. In order to identify the IRF-1 binding region of CHIP, a series of CHIP mutants lacking its functional domains were used (Fig. 3A) (25McDonough H. Charles P.C. Hilliard E.G. Qian S.B. Min J.N. Portbury A. Cyr D.M. Patterson C. J. Biol. Chem. 2009; 284: 20649-20659Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). When the mutants were transfected into A375 cells together with IRF-1 and subjected to heat stress as described previously (25McDonough H. Charles P.C. Hilliard E.G. Qian S.B. Min J.N. Portbury A. Cyr D.M. Patterson C. J. Biol. Chem. 2009; 284: 20649-20659Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), both full-length CHIP (CHIP WT) and a CHIP mutant lacking the Hsp70 binding domain (CHIP ΔTPR) co-immunoprecipitated with IRF-1 (Fig. 3B, see lanes 4 and 6). In contrast, when either the charged domain (CHIP Δ±) or the U-Box (ΔUbox) was deleted, binding to IRF-1 was below the level of detection (Fig. 3B, compare lanes 8 and 14 with lanes 4, 6, and 12). However, the interpretation of the cell-based assays was complicated by the fact that, (i) different amounts of IRF-1 were pulled down in the presence of the various CHIP constructs, (ii) A375 cells express endogenous CHIP, and (iii) the ΔUbox CHIP mutant runs at the same size as the α-FLAG antibody light chain on SDS-polyacrylamide gels. CHIP has been shown to form dimers in vitro (12Nikolay R. Wiederkehr T. Rist W. Kramer G. Mayer M.P. Bukau B. J. Biol. Chem. 2004; 279: 2673-2678Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) with the interface extending over the U-box and the charged domain (13Xu Z. Devlin K.I. Ford M.G. Nix J.C. Qin J. Misra S. Biochemistry. 2006; 45: 4749-4759Crossref PubMed Scopus (49) Google Scholar), whereas dimerization occurs independently of the TPR domain (12Nikolay R. Wiederkehr T. Rist W. Kramer G. Mayer M.P. Bukau B. J. Biol. Chem. 2004; 279: 2673-2678Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 13Xu Z. Devlin K.I. Ford M.G. Nix J.C. Qin J. Misra S. Biochemistry. 2006; 45: 4749-4759Crossref PubMed Scopus (49) Google Scholar). Thus, the data presented in Fig. 3B may reflect the ability of the various mutants to form complexes with endogenous CHIP and its associated proteins. To address our concerns, a cell-free assay was developed using CHIP constructs purified from a TNT coupled reticulocyte lysate expression system under conditions designed to remove Hsp70, and IRF-1 was generated using the Hsp70-free PURExpress system. All purified protein samples were shown to have Hsp70 levels that were below the levels of detection using immunoblot analysis (data not shown). Consistent with the data obtained using the cell-based assay, when IRF-1 was added to the various CHIP proteins immobilized on Ni2+-NTA agarose, it bound to both the WT and ΔTPR CHIP proteins but not Δ± and ΔUbox deletion mutants (Fig. 3C compare lanes 2 and 3 with lanes 4 and 5). Together the data suggest that (i) IRF-1 binding does not require the TPR domain, (ii) IRF-1 and CHIP can interact independently of Hsp70 function (because Hsp70 cannot bind to the TPR domain mutant), and (iii) IRF-1 binding possibly requires both the charged and U-box domains of CHIP. In order to examine the effects of CHIP on IRF-1 in a cellular environment, A375 cells were transfected with pcDNA3-CHIP alone or pcDNA3-CHIP and pcDNA3-IRF-1 and then fractionated. Interestingly, when exogenous CHIP was present, high molecular weight IRF-1 proteins were detected in the nuclear and cytosolic fractions, indicative of post-translational modification (Fig. 4A, panel 3, compare lanes 4 and 6 with lanes 1 and 3). To determine whether these high molecular weight bands represented ubiquitinated forms of IRF-1, a cell-based ubiquitination assay was utilized (8Pion E. Narayan V. Eckert M. Ball K.L. Cell. Signal. 2009; 21: 1479-1487Crossref PubMed Scopus (22) Google Scholar). H1299 cells were transfected with pcDNA3-IRF-1, His-Ub, and pcDNA3-CHIP as indicated in Fig. 4B. In this assay, although endogenous E3 ligase activity is sufficient for IRF-1 modification, overexpression of CHIP results in a significant increase in the amount of ubiquitinated IRF-1 detected (Fig. 4B, His pulldown, compare lanes 2 and 3). These results demonstrate that CHIP-dependent ubiquitination of IRF-1 can occur in a complex cellular background. In order to determine if direct binding of CHIP to IRF-1 was sufficient to signal ubiquitination or whether additional cellular components, such as Hsp70, were required, a stopped enzyme assay using purified components was developed in which CHIP was rate-limiting (Fig. 5, A and B). Under these conditions, CHIP specifically ubiquitinates GST-IRF-1 but not GST alone (Fig. 5A, compare lanes 6–9 with lanes 2–5). To further dissect the IRF-1 ubiquitination pathway, we used a library of purified E2 ubiquitin-conjugating enzymes to determine which E2s could catalyze CHIP ubiquitination of IRF-1. Our data suggest that E2 enzymes belonging to the UbcH5 family and UbcH6 can cooperate with CHIP to efficiently ubiquitinate IRF-1 in vitro (Fig. 5C). As expected, mutation of the active site Cys (C85A) of the UbcH5 family members completely blocked IRF-1 ubiquitination by CHIP (Fig. 5C, compare lanes 6, 8, and 11 with lanes 5, 7, and 9). Additionally, when GST-IRF-1 (total protein, including ubiquitinated and unmodified IRF-1) was isolated from the ubiquitination reaction mix in which UbcH5a was used as the E2, immunoblot analysis showed that CHIP·UbcH5a can both monoubiquitinate and form polyubiquitin chains on IRF-1 (Fig. 5D). Moreover, CHIP·UbcH5a forms both Lys48-linked and Lys63-linked ubiquitin chains on IRF-1 because Ub mutants with either Lys48 or Lys63 mutated to Arg or with all lysine residues except Lys48 or Lys63 mutated to Arg cause no gross change in the pattern of IRF-1 modification by CHIP (Fig. 5E). The data presented above show that CHIP binds stably to IRF-1 aa 106–140 (Fig. 2A) and that CHIP can efficiently ubiquitinate IRF-1 both in cells and in vitro (Figs. 4B and 5B). However, the experiments do not demonstrate whether the direct binding of CHIP to the Mf2 domain is sufficient to signal ubiquitination of IRF-1. To address this question, we performed a series of cell-ba" @default.
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• W2013279940 title "Docking-dependent Ubiquitination of the Interferon Regulatory Factor-1 Tumor Suppressor Protein by the Ubiquitin Ligase CHIP" @default.
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