Cynthia R. Coffill1*, Patricia A.J. Muller2*, Hue Kian Oh1, Suat Peng Neo1, Kelly A. Hogue1, Chit Fang Cheok3, Karen H. Vousden2, David P. Lane3, Walter P. Blackstock1 and Jayantha Gunaratne1
1 - Institute of Molecular and Cell Biology, Singapore
2 - The Beatson Institute for Cancer Research, Glasgow, UK
3 - p53 Laboratory (A-STAR), Immunos, Singapore
*These authors contributed equally to this work.
Published in EMBO Rep. Jun 1, 2012 (doi: 10.1038/embor.2012.74)
The invasiveness of tumour cells depends on changes in cell shape, polarity and migration. Mutant p53 induces enhanced tumour metastasis in mice, and human cells overexpressing p53R273H have aberrant polarity and increased invasiveness, demonstrating the 'gain of function' of mutant p53 in carcinogenesis. We hypothesize that p53R273H interacts with mutant p53-specific binding partners that control polarity, migration or invasion. Here we analyze the p53R273H interactome using stable isotope labelling by amino acids in cell culture and quantitative mass spectrometry, and identify at least 15 new potential mutant p53-specific binding partners. The interaction of p53R273H with one of them-nardilysin (NRD1)-promotes an invasive response to heparin binding-epidermal growth factor-like growth factor that is p53R273H-dependant but does not require Rab coupling protein or p63. Advanced proteomics has thus allowed the detection of a new mechanism of p53-driven invasion.
A-D. Identification of NRD1 as a p53R273H-specific interactor using SILAC. (A) Triple-label SILAC experiment of H1299 p53-null cells that were transfected with vector, wt-p53 or p53R273H, grown in media with three different stable isotopes. Western blots show whole-cell lysate p53 levels (B) Differential protein identification in wt-p53 versus p53R273H immunoprecipitation. The summed peptide intensity distribution was plotted against the corresponding protein fold change (SILAC ratio) following immunoprecipitation. The red data points indicate highly significant SILAC ratios (P<1 × 10−11) and blue represents the main population of 1:1 nonspecific interacting proteins (P>0.05). Those data points furthest to the right represent those proteins that preferentially interact with p53R273H with the highest confidence level, while those proteins on the left are augmented in the wt-p53 sample. (C) A representative mass spectrum showing relative abundance versus mass-to-charge ratio (m/z) of NRD1 SILAC peptide pairs. (D) Detection of NRD1 following p53R273H-specific immunoprecipitation and western blot analysis from lysates of human tumour cells expressing endogenous mutant p53.
E-F. Endogenous mutant p53 and NRD1 invasion. Three-dimensional invasion studies show that U251 (mutant p53R273K) cells invaded in a mutant p53- and NRD1-dependent manner towards HB–EGF (E), whereas MDA MB231 (mutant p53R280K) cells that express the p53R280K mutation invaded in a p53-dependent and NRD1-independent manner (F).
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