Transcription-independent ARF regulation in oncogenic stress-mediated p53 responses

Abstract

The tumour suppressor ARF is specifically required for p53 activation under oncogenic stress. Recent studies showed that p53 activation mediated by ARF, but not that induced by DNA damage, acts as a major protection against tumorigenesis in vivo under certain biological settings, suggesting that the ARF-p53 axis has more fundamental functions in tumour suppression than originally thought. Because ARF is a very stable protein in most human cell lines, it has been widely assumed that ARF induction is mediated mainly at the transcriptional level and that activation of the ARF-p53 pathway by oncogenes is a much slower and largely irreversible process by comparison with p53 activation after DNA damage. Here we report that ARF is very unstable in normal human cells but that its degradation is inhibited in cancerous cells. Through biochemical purification, we identified a specific ubiquitin ligase for ARF and named it ULF. ULF interacts with ARF both in vitro and in vivo and promotes the lysine-independent ubiquitylation and degradation of ARF. ULF knockdown stabilizes ARF in normal human cells, triggering ARF-dependent, p53-mediated growth arrest. Moreover, nucleophosmin (NPM) and c-Myc, both of which are commonly overexpressed in cancer cells, are capable of abrogating ULF-mediated ARF ubiquitylation through distinct mechanisms, and thereby promote ARF stabilization in cancer cells. These findings reveal the dynamic feature of the ARF-p53 pathway and suggest that transcription-independent mechanisms are critically involved in ARF regulation during responses to oncogenic stress.

Figure 1. ULF is identified as a major factor for short half-lives of ARF in normal human fibroblast cells

a–c, Western blot analysis of cell extracts from normal human fibroblast cells harvested at 0 or 17 h after treatment with proteasome inhibitor (a), at the indicated time points (h) after treatment with cycloheximide (CHX) (b), or after 17 h of treatment with proteasome inhibitor followed by the addition of cycloheximide (c). d, Diagram of the ULF protein showing several signature motifs. e, Lysates of the NHF-1 cells treated with the different RNAi oligonucleotides were analysed by western blotting with the indicated antibodies. ULF-RNAi-1mut, a point mutation form of ULF-RNAi-1. f, Expression of mRNAs encoding ULF and ARF by RT–PCR from the cells in e.g, Inactivation of ULF by siRNA extends the half-life of endogenous ARF protein in NHF-1 cells.

Figure 2. ULF interacts with ARF

Inactivation of ULF induces ARF-dependent p53 stabilization and cell growth repression in NHF-1 cells a, Co-immunoprecipitation of ARF with ULF or ULF with ARF from NHF-1 cells treated with proteasome inhibitors. IP, immunoprecipitation. b, GST–ARF (lane 3) or GST alone (lane 2) was used in a GST pull-down assay with in vitro translated ³⁵S-labelled ULF. c, Inactivation of ULF by RNAi induces ARF-dependent p53 stabilization. d, NHF-1 cells were labelled and stained with BrdU after RNAi treatment as indicated. e, NHF-1 cells treated with the indicated RNAi oligonucleotides were stained with crystal violet three days after siRNA treatment. f, Inactivation of ULF by RNAi induces G1 arrest in NHF-1 cells. Error bars represent s.d. (n = 3).

Figure 3. ULF-mediated effect on ARF ubiquitylation and degradation is modulated by NPM

a, ARF is ubiquitylated by ULF in vivo. Lysates from transfected 293 cells were immunoprecipitated (IP) with anti-ARF antibody (ab-4), and separated proteins were blotted with antibodies against the HA (top) or monoclonal ARF (middle) antibody. Ectopic ULF in crude lysates were analysed by western blotting (WB) with anti-V5 antibody. b, ARF is ubiquitylated by ULF in vitro. See Methods for detail. c, Subcellular localization of ectopic ULF and endogenous ARF in H1299 cells. d, ARF ubiquitylation mediated by ULF is affected by NPM but not by NPM-c. e, ARF stability regulated by NPM is ULF dependent. Western blot analysis of cell extracts of H1299 cells treated with the indicated RNAi oligonucleotides by the antibodies shown. f, Inactivation of ULF extends the half-life of endogenous ARF protein in NPM-depleted H1299 cells. g, ULF-RNAi-mediated effects are reversed by ULF(R) expression in NPM-depleted H1299 cells.

Figure 4. c-Myc overexpression blocks the interaction between ULF and ARF, which leads to c-Myc-mediated, transcription-independent ARF induction

a, Myc is in the FH (tagged with Flag and HA)–ULF complexes. ULF-stable line, H1299 cells stably transfected with FH–ULF. b, Both wild-type Myc and Myc-ΔBR, but not Myc(1–328), inhibit ARF ubiquitylation mediated by ULF. c, Both wild-type Myc and Myc-ΔBR block the interaction between ULF and ARF. Western blot analysis of cell extracts from the transfected human 293 cells by anti-HA and anti-ARF. d, Both Myc-ΔBR and Myc stabilize ARF; induction of p53 and p21 in NHF-1 cells by adenoviral infection. e, Expression of mRNA encoding ARF by RT–PCR from the cells in d. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. f, Myc-ΔBR extends the half-life of endogenous ARF protein in NHF-1 cells.