Endocrine Abstracts (2006) 11 P492

PTTG binding factor (PBF) can transform cells independently of interaction with PTTG

AL Stratford, K Boelaert, DS Kim, JA Franklyn & CJ McCabe

University of Birmingham, Birmingham, United Kingdom.

We have previously shown PTTG and PBF to be over-expressed in differentiated thyroid cancer and to be prognostic indicators for recurrence. Subsequently we reported PBF to be a transforming gene in vitro and tumourigenic in vivo. Since over-expression of PTTG results in the same findings, we examined whether PBF-induced tumourigenesis was an independent effect, or else a result of increased PTTG activity. Two HA-tagged mutants of PBF were generated, firstly substituting the five basic residues of the nuclear localisation signal (NLS-) and secondly deleting the C-terminal 30 amino acids responsible for PTTG interaction (C-term). Both PBF mutants were unable to interact with PTTG as demonstrated by immunoprecipitation, and using subcellular fractionation we were able to show that neither mutant could enter the nucleus. Stable over-expression of PBF and both mutants in NIH3T3 cells resulted in anchorage-independent growth in soft agar, with the formation of large and abundant colonies compared with vector-only (VO) (VO=9.1 colonies ±1.4; PBF=201±33.2, P<0.001; NLS- 159±33.2, P<0.001; C-term 177±23.4, P<0.001). There was no significant difference in the number of colonies between wild-type PBF and the two mutants. A mutant of PTTG, in which the PBF interaction domain (amino acids 123–154) was deleted (BD-), was over-expressed in NIH3T3 cells and its ability to transform cells assessed in the same assay. Over-expression of wild-type PTTG resulted in the formation of large colonies compared to VO; however removal of the region which interacts with PBF abrogated its ability to transform cells (PTTG=1501±91, P<0.001; BD-=15.7±2.84, P=n/s). We conclude, therefore, that PBF is able to transform cells independently of PTTG, whereas PTTG-induced cell transformation may be dependent on interaction with PBF.

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