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

Abstract

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.

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Figure 1: ULF is identified as a major factor for short half-lives of ARF in normal human fibroblast cells.
Figure 2: ULF interacts with ARF.
Figure 3: ULF-mediated effect on ARF ubiquitylation and degradation is modulated by NPM.
Figure 4: c-Myc overexpression blocks the interaction between ULF and ARF, which leads to c-Myc-mediated, transcription-independent ARF induction.

Accession codes

Data deposits

The full-length ULF sequence is deposited in GenBank under accession number EU489742.

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Acknowledgements

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.

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Correspondence to Wei Gu.

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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

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