Abstract
Our understanding of the post-translational processes involved in regulating the interferon regulatory factor-1 (IRF-1) tumor suppressor protein is limited. The introduction of mutations within the C-terminal Mf1 domain (amino acids 301-325) impacts on IRF-1-mediated gene repression and growth suppression as well as the rate of IRF-1 degradation. However, nothing is known about the proteins that interact with this region to modulate IRF-1 function. A biochemical screen for Mf1-interacting proteins has identified an LXXLL motif that is required for binding of Hsp70 family members and cooperation with Hsp90 to regulate IRF-1 turnover and activity. These conclusions are supported by the finding that Hsp90 inhibitors suppress IRF-1-dependent transcription shortly after treatment, although at later time points inhibition of Hsp90 leads to an Hsp70-dependent depletion of nuclear IRF-1. Conversely, the half-life of IRF-1 is increased by Hsp90 in an ATPase-dependent manner leading to the accumulation of nuclear but not cytoplasmic IRF-1. This study begins to elucidate the role of the Mf1 domain of IRF-1 in orchestrating the recruitment of regulatory factors that can impact on both its turnover and transcriptional activity.
Highlights
Interferon regulatory factor-1 (IRF-1),3 the founding member of the interferon regulatory factor family, is a transcription factor that regulates a diverse range of target genes during the response to stimuli such as pathogen infection [1], DNA damage [2, 3], and hypoxia [4]
The loss of IRF-1 can cooperate with c-Ha-ras [5] in cellular transformation; it becomes up-regulated in cells that bear oncogenic lesions [6], and deletions of IRF-1 are associated with the development of gastric and esophageal tumors, as well as some leukemias [7,8,9]
The Mf1 is involved in multiple regulatory processes [15, 17], nothing is currently known about the mechanism of action of this region and how, for example, cellular factors interact with the Mf1 domain to modulate IRF-1-dependent gene expression and growth repressor activity or to promote IRF-1 turnover
Summary
Antibodies, and Peptides—Antibodies were used at 1 g/ml and were anti-IRF-1 and anti-GFP (BD Biosciences), anti-GAPDH (Abcam), anti-FLAG and anti-GST (Sigma), antiChk (G-4), anti-caspase-3 (Santa Cruz Biotechnology), and anti-Hp1␣ (Upstate). Enough biotinylated peptide to saturate the streptavidin-agarose bead binding sites was used (Sigma) and incubated with the beads for 1 h at room temperature, and the column was washed three times with Buffer W (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM benzamidine) to remove unbound peptide. After washing extensively in phosphate-buffered saline containing 0.1% (v/v) Tween 20, binding was detected using anti-GST and horseradish peroxidasetagged anti-mouse antibodies, and electrochemical luminescence was quantified using a luminometer (Labsystems; Fluoroskan Ascent FL). Size Exclusion Chromatography—A375 cells were lysed in fast protein liquid chromatography Lysis Buffer (20 mM Hepes, pH 7.5, 0.25 M NaCl, 10% (w/v) sucrose, 10% (v/v) glycerol, 0.1% (v/v) Triton X-100, 5 mM NaF, 2 mM -glycerophosphate, 1 mM dithiothreitol, 20 g/ml leupeptin, 1 g/ml aprotinin, 2 g/ml pepstatin, 1 mM benzamidine, 10 g/ml soybean trypsin inhibitor, 2 mM Pefabloc, 1.6 mM EGTA) and passed through a 0.45-m filter.
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