PBF overexpression causes increased p53 ubiquitination and degradation via MDM2

Gavin Ryan, Martin Read, Robert Seed, Neil Sharma, Vicki Smith, Perkin Kwan, Jayne Franklyn, Andrew Turnell, Christopher McCabe, Kristien Boelaert

Research output: Contribution to journalAbstractpeer-review

Abstract

The PTTG-binding factor (PBF) is a relatively uncharacterized gene,
which is overexpressed in thyroid tumors. Targeted overexpression of
PBF in murine thyroids results in repressed iodide uptake, increased
thyroid hormone retention, and significant hyperplasia. Previous research
has shown that PBF can bind p53, reducing stimulation of its
downstream target genes by competitively binding. Reduced p53
stability was observed when PBF was overexpressed in K1 and TPC-1
thyroid papillary cancer cell lines through increased p53 ubiquitination
and subsequent degradation by the proteasome. However, the
exact mechanism of this remains unknown.
GST pull-down assays and coimmunoprecipitation assays were
used to determine direct protein binding. Half-life assays were used
with and without the p53-MDM2-binding competitive inhibitor,
Nutlin-3, to investigate protein stability. Ubiquitination was determined
via a ubiquitin antibody to immunoprecipitated p53. Immunofluorescence
was used to investigate protein subcellular
localization.
Pull-down assays demonstrated direct binding between PBF and
MDM2, the principal negative regulator of p53. Nutlin-3 inhibition of
p53-MDM2 binding blocked the increased degradation of p53 observed
when PBF was overexpressed. However, there was no change
in p53-MDM2 binding stringency, determined by coimmunoprecipitation,
when PBF expression was knocked down by siRNA treatment
in K1 and TPC-1 cells. Furthermore, MDM2 subcellular localization
was unchanged when PBF was overexpressed. This suggests that
another mechanism, other than increased binding or altered subcellular
localization, is responsible for the increased MDM2-mediated
ubiquitination of p53 elicited by PBF overexpression.
Taken together, these data demonstrate a novel interaction and
method of p53 regulation by PBF, mediated by MDM2. Modulation of
p53 stability by PBF may be critical in the DNA damage response.
Given that PBF potently represses radioiodide uptake, these findings
may be particularly pertinent to anaplastic thyroid carcinoma, where
p53 is frequently inactivated, and tumors are resistant to radioiodine
therapy.
Original languageEnglish
Article numberP143
JournalThyroid
Volume22(1_Suppl)
Publication statusPublished - 2012
Event82nd Annual Meeting of the American Thyroid Association - Quebec City, Canada
Duration: 19 Sept 201223 Sept 2012

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