The tumour suppressor ARF is specifically required for p53 activation under oncogenic stress1,2,3,4,5,6. 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 settings7,8, 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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
The full-length ULF sequence is deposited in GenBank under accession number EU489742.
Lowe, S. W. & Sherr, C. J. Tumor suppression by Ink4a-Arf: progress and puzzles. Curr. Opin. Genet. Dev. 13, 77–83 (2003)
Gil, J. & Peters, G. Regulation of the INK4b-ARF-INK4a tumour suppressor locus: all for one or one for all. Nature Rev. Mol. Cell Biol. 7, 667–677 (2006)
Matheu, A., Maraver, A. & Serrano, M. The Arf/p53 pathway in cancer and aging. Cancer Res. 68, 6031–6034 (2008)
Brooks, C. L. & Gu, W. p53 ubiquitination: Mdm2 and beyond. Mol. Cell 21, 307–315 (2006)
Sherr, C. J. Divorcing ARF and p53: an unsettled case. Nature Rev. Cancer 6, 663–673 (2006)
Kruse, J.-P. & Gu, W. Modes of p53 regulation. Cell 137, 609–622 (2009)
Christophorou, M. A., Ringshausen, I., Finch, A. J., Swigart, L. B. & Evan, G. I. The pathological response to DNA damage does not contribute to p53-mediated tumour suppression. Nature 443, 214–217 (2006)
Martins, C. P., Brown-Swigart, L. & Evan, G. I. Modeling the therapeutic efficacy of p53 restoration in tumors. Cell 127, 1323–1334 (2006)
Kuo, M. L., den Besten, W., Bertwistle, D., Roussel, M. F. & Sherr, C. J. N-terminal polyubiquitination and degradation of the Arf tumor suppressor. Genes Dev. 18, 1862–1874 (2004)
Itahana, K. et al. Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation. Mol. Cell 12, 1151–1164 (2003)
Korgaonkar, C. et al. Nucleophosmin (B23) targets ARF to nucleoli and inhibits its function. Mol. Cell. Biol. 25, 1258–1271 (2005)
Brady, S. N., Yu, Y., Maggi, L. B. & Weber, J. D. ARF impedes NPM/B23 shuttling in an Mdm2-sensitive tumor suppressor pathway. Mol. Cell. Biol. 24, 9327–9338 (2004)
Bertwistle, D., Sugimoto, M. & Sherr, C. J. Physical and functional interactions of the Arf tumor suppressor protein with nucleophosmin/B23. Mol. Cell. Biol. 24, 985–996 (2004)
Chen, D. L. et al. ARF-BP1/mule is a critical mediator of the ARF tumor suppressor. Cell 121, 1071–1083 (2005)
Moulin, S., Llanos, S., Kim, S. H. & Peters, G. Binding to nucleophosmin determines the localization of human and chicken ARF but not its impact on p53. Oncogene 27, 2382–2389 (2008)
Colombo, E. et al. Nucleophosmin is required for DNA integrity and p19Arf protein stability. Mol. Cell. Biol. 25, 8874–8886 (2005)
Grisendi, S. et al. Role of nucleophosmin in embryonic development and tumorigenesis. Nature 437, 147–153 (2005)
Lee, J. W., Choi, H. S., Gyuris, J., Brent, R. & Moore, D. D. Two classes of proteins dependent on either the presence or absence of thyroid hormone for interaction with the thyroid hormone receptor. Mol. Endocrinol. 9, 243–254 (1995)
den Besten, W., Kuo, M. L., Williams, R. T. & Sherr, C. J. Myeloid leukemia-associated nucleophosmin mutants perturb p53-dependent and independent activities of the Arf tumor suppressor protein. Cell Cycle 4, 1593–1598 (2005)
Colombo, E. et al. Delocalization and destabilization of the Arf tumor suppressor by the leukemia-associated NPM mutant. Cancer Res. 66, 3044–3050 (2006)
Cheng, K. et al. The leukemia-associated cytoplasmic nucleophosmin mutant is an oncogene with paradoxical functions: Arf inactivation and induction of cellular senescence. Oncogene 26, 7391–7400 (2007)
Feuerstein, N. & Mond, J. J. ‘Numatrin,’ a nuclear matrix protein associated with induction of proliferation in B lymphocytes. J. Biol. Chem. 262, 11389–11397 (1987)
Nozawa, Y., vanBelzen, N., vanderMade, A. C. J., Dinjens, W. N. M. & Bosman, F. T. Expression of nucleophosmin/B23 in normal and neoplastic colorectal mucosa. J. Pathol. 178, 48–52 (1996)
Falini, B. et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N. Engl. J. Med. 352, 254–266 (2005)
Quentmeier, H. et al. Cell line OCI/AML3 bears exon-12 NPM gene mutation-A and cytoplasmic expression of nucleophosmin. Leukemia 19, 1760–1767 (2005)
Grisendi, S., Mecucci, C., Falini, B. & Pandolfi, P. P. Nucleophosmin and cancer. Nature Rev. Cancer 6, 493–505 (2006)
Saporita, A. J., Maggi, L. B., Apicelli, A. J. & Weber, J. D. Therapeutic targets in the ARF tumor suppressor pathway. Curr. Med. Chem. 14, 1815–1827 (2007)
Finak, G. et al. Stromal gene expression predicts clinical outcome in breast cancer. Nature Med. 14, 518–527 (2008)
Buchholz, M. et al. Transcriptome analysis of microdissected pancreatic intraepithelial neoplastic lesions. Oncogene 44, 6626–6636 (2005)
Zindy, F. et al. Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization. Genes Dev. 12, 2424–2433 (1998)
Abide, W. M. & Gu, W. p53-Dependent and p53-independent activation of autophagy by ARF. Cancer Res. 68, 352–357 (2006)
Li, M. et al. Mono- versus polyubiquitination: differential control of p53 fate by Mdm2. Science 302, 1972–1975 (2003)
We thank R. Baer and R. Dalla-Favera for critical comments on this study; B. Falini and J. B. Yoon for providing reagents; W. Z. Zhang for helping in mass spectrometry analysis; E. McIntush for developing the anti-ULF antibody. This study was supported by grants from National Institutes of Health/National Cancer Institute, the Leukemia and Lymphoma Society and NSFC-30628028. W.G. is an Ellison Medical Foundation Senior Scholar in aging.
Author Contributions The experiments were conceived and designed by D.C. and W.G. Experiments were performed mainly by D.C. Protein identification, mass spectrometric analysis and cloning were performed by D.C., J.S., W.Z. and J.Q. The paper was written by D.C. and W.G.
About this article
Cite this article
Chen, D., Shan, J., Zhu, WG. et al. Transcription-independent ARF regulation in oncogenic stress-mediated p53 responses. Nature 464, 624–627 (2010). https://doi.org/10.1038/nature08820
Cell Reports (2020)
Scientific Reports (2020)
Lentivirus-Mediated Overexpression of SIVA-1 Reverses Cisplatin Resistance in Gastric Cancer in vitro
Cell Biochemistry and Biophysics (2020)
International Journal of Molecular Sciences (2020)