BES2002 Oral Communications Endocrine Neoplasia (8 abstracts)
Disruption of the SH3 domain of pituitary tumor transforming gene (PTTG) reveals distinct mechanisms of FGF-induction and cell transformation
K Boelaert 1 , LA Tannahill 1 , LJ McRobbie 1 , JS Moore 1 , LS Young 2 , MC Sheppard 1 , JA Franklyn 1 , NJL Gittoes 1 & CJ McCabe 1
1Division of Medical Sciences, University of Birmingham, Birmingham, UK; 2CRC Institute for Cancer Studies, University of Birmingham, Birmingham, UK.
PTTG transforms cells in vitro, is tumourigenic in vivo and regulates secretion of fibroblast growth factor-2 (FGF-2). Critical to transactivation of FGF-2 is PTTG's SH3 interacting domain which encodes the gene's sole phosphorylation site. We explored the mechanisms through which PTTG stimulates FGF-2 expression and cell transformation using specific mutations resulting in unphosphorylated PTTG (phos-), a mimic of constitutive phosphorylation (phos+), and a disrupted SH3 domain with normal phosphorylation (SH3-). The effects of these mutations on cell transformation were assessed in colony forming assays and on FGF-2 expression by real time TaqMan RT-PCR following transient transfection in MCF7 cells. Loss of phosphorylation increased PTTG's transforming ability compared with wild type (WT) (phos- = 201 ± 16 colonies/well; WT = 59 ± 2 colonies, n = 13, P < 0.001) whereas constitutive phosphorylation reduced transformation (phos+ = 30 ± 4 colonies, n = 11, P < 0.001). The SH3- mutation showed markedly reduced cell transformation compared with either WT PTTG or with phos- mutants (SH3- = 22 ± 5 colonies, n = 8, P < 0.001 vs WT or phos-). WT PTTG induced a 3-fold induction in FGF-2 compared with vector-only transfected cells (n = 6, P = 0.04). Phos- and phos+ showed similar FGF-2 upregulation to WT PTTG (2.8 and 2.9- fold respectively). In contrast, disruption of the SH3- interacting domain (SH3-) abrogated PTTG stimulation of FGF-2 (n = 6, P = 0.03, SH3- mutation vs WT). EGFP-tagged phos- PTTG demonstrated increased cytoplasmic localisation compared with WT.
We propose that PTTG interacts with a signalling protein to mediate FGF-2 upregulation, and this interaction is independent of phosphorylation. However, phosphorylation appears to acts as a 'switch' for PTTG's transforming ability. As unphosphorylated PTTG shows reduced nuclear entry and increased colony formation, cell transformation may be dependent on the protein's cytoplasmic localisation.
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Endocrine Abstracts
ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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