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iASPP preferentially binds p53 proline-rich region and modulates apoptotic function of codon 72–polymorphic p53

Nature Genetics volume 38, pages 1133–1141 (2006)Cite this article

Abstract

iASPP is one of the most evolutionarily conserved inhibitors of p53, whereas ASPP1 and ASPP2 are activators of p53. We show here that, in addition to the DNA-binding domain, the ASPP family members also bind to the proline-rich region of p53, which contains the most common p53 polymorphism at codon 72. Furthermore, the ASPP family members, particularly iASPP, bind to and regulate the activity of p53Pro72 more efficiently than that of p53Arg72. Hence, escape from negative regulation by iASPP is a newly identified mechanism by which p53Arg72 activates apoptosis more efficiently than p53Pro72.

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Figure 1: The SH3 domain of ASPP2 and iASPP interacts with the proline-rich region of p53 with distinct binding affinity.
Figure 2: Selective binding affinity of the ASPP family members for the p53 proline-rich domain.
Figure 3: The ASPP family proteins selectively regulate the transactivation and apoptotic functions of p53Pro72.
Figure 4: Tyr814 in the SH3 domain of iASPP modulates its ability to bind and regulate the activity of p53Pro72.
Figure 5: The expression of the ASPP family members differentially affects the apoptotic potency of p53 polymorphic variants in two different cell lines.
Figure 6: Endogenous iASPP expression levels dictate the activities of p53Arg72 and p53Pro72.
Figure 7: Graph showing the frequency of iASPP overexpression in different categories of tumor samples in comparison with their matched normal samples.
Figure 8

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Acknowledgements

We would like to thank the Ludwig Institute for Cancer Research for supporting the work, E. Slee and D. O'Connor for reading the manuscript and M. Murphy for the WM115 and WM278 cell lines. D.B. is funded by the Association for International Cancer Research. T.C. is a clinical research fellow of Cancer Research UK.

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Author notes

  1. Daniele Bergamaschi

    Present address: Cancer Research UK, Skin Tumour Laboratory, Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London, Queen Mary's School of Medicine and Dentistry, University of London, 4 Newark Street, London, E1 2AT, UK

  2. Yardena Samuels

    Present address: The Sidney Kimmel Comprehensive Cancer Center and The Howard Hughes Medical Institute, The Johns Hopkins University Medical Institutions, Baltimore, Maryland, 21231, USA

Authors and Affiliations

  1. Ludwig Institute for Cancer Research, University College London, 91 Riding House Street, London, W1W 7BS, UK

    Daniele Bergamaschi, Yardena Samuels, Alexandra Sullivan, Marketa Zvelebil, Hilde Breyssens & Xin Lu

  2. Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie (LNCIB), Area Science Park, and Dipartimento di Biochimica Biofisica e Chimica delle Macromolecole, Università di Trieste, Trieste, 34100, Italy

    Andrea Bisso & Giannino Del Sal

  3. Laboratory of Cancer Genetics and Epigenetics, The Breakthrough Toby Robins Breast Cancer Research Centre at The Institute of Cancer Research, Mary-Jean Mitchell Green Building, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB, UK

    Nelofer Syed, Paul Smith & Tim Crook

  4. Department of Medical Oncology, S Croce e Carle Hospital, Cuneo, 12100, Italy

    Milena Gasco

  5. Cancer Research UK, Skin Tumour Laboratory, Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London, Queen Mary's School of Medicine and Dentistry, University of London, 4 Newark Street, London, E1 2AT, UK

    Yardena Samuels

  6. The Sidney Kimmel Comprehensive Cancer Center and The Howard Hughes Medical Institute, The Johns Hopkins University Medical Institutions, Baltimore, Maryland, 21231, USA

    Daniele Bergamaschi

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Contributions

D.B. performed most of the work, including most of the immunoprecipitations of p53/ASPP interaction in vitro and in vivo, all apoptosis assays, part of the transactivation assays and chromatin immunoprecipitation (ChIP). Y.S. was involved in the initial observations and immunoprecipitation in RKO cells as well as part of the transactivation assays. A.S. performed immunoprecipitation of Thio-p53/ASPP interaction and of p53/ASPP complex in melanoma cell lines and performed RT-PCR. M.Z. performed the sequence alignment of SH3 domain-containing proteins and computer modeling of p53/ASPP interaction. H.B. constructed the iASPP and ASPP2 mutants iASPPY814L and ASPP2L1113Y. A.B. constructed the pCDNA3HATNV-Pr72R and pCDNA3HATNV-Pr72P plasmids. G.D.S. supervised the work of A.B. N.S. performed p53 sequence analysis and developed quantitative PCR assays for ASPP1, ASPP2 and iASPP. P.S. performed analysis of p53 arginine and proline polymorphisms and mutations. M.G. analyzed iASPP expression in the panel of human breast cancer samples using quantitative PCR. T.C. microdissected tumor sections, isolated genomic DNA and mRNA from these sections. X.L. was responsible for the overall project, in particular the ideas and strategies.

Corresponding author

Correspondence to Xin Lu.

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Competing interests

The authors do not believe, or are unsure, whether they have competing financial interests as defined in the form “NPG_CFI_form.pdf”. However, in the interest of full disclosure, the authors declare that a patent application has been filed based on the results described in this publication in the name of the Ludwig Institute for Cancer Research, which funded this research.

Supplementary information

Supplementary Fig. 1

Computer modelling to illustrate the importance of Y814 of iASPP and L1113 of ASP2 in contacting codon 72 Pro or Arg of p53. (PDF 127 kb)

Supplementary Fig. 2

Wild-type iASPP preferentially binds to the proline-rich region of p53Pro72, whereas iASPPY814L and ASPP2 preferentially bind to the proline-rich region of p53Arg72. (PDF 27 kb)

Supplementary Table 1

mRNA expression of iASPP in human breast tumor samples expressing either wild-type or mutant p53. (PDF 23 kb)

Supplementary Table 2

Frequency of overexpression of iASPP in tumors with wild-type p53 homozygous for p53Pro72 versus those homozygous for p53Arg72 in the germ line. (PDF 597 kb)

Supplementary Table 3

Primer sequences used for the production of iASPP RNAi, CHIP assays, RT-PCR of various p53 targets and real-time RT-PCR of iASPP in breast tumor and matched normal samples. (PDF 14 kb)

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Bergamaschi, D., Samuels, Y., Sullivan, A. et al. iASPP preferentially binds p53 proline-rich region and modulates apoptotic function of codon 72–polymorphic p53. Nat Genet 38, 1133–1141 (2006). https://doi.org/10.1038/ng1879

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