CONICET | Buscador de Institutos y Recursos Humanos

Oncogene activation is a widely known tumorigenesis mechanism. The proto-oncogene pituitary tumor transforming gene (PTTG) arises as a key factor due to its frequent overexpression in tumors and by its regulation of cell cycle. Post-translational modifications control PTTG protein: PTTG is modified by ubiquitination, phosphorylation and in particular the SUMOylation enhancer RSUME increases the stability of the PTTG protein, its tumor abundance and prolongs its half-life. In this work we studied the molecular modulation of PTTG protein stability at post-translational level and the role of RSUME.In COS-7 cells we studied the stability of mutated PTTG variants in their possible conjugation sites to SUMO (K25R and K168R) or in the most important site of conjugation to ubiquitin (∆D-box) by western blot (WB) from cellular extracts previously transfected with PTTG wt or mutated. We found that the mutant K25R presents the same protein stability as PTTG wt, and RSUME increases its stability. The K168R mutant has lower stability than PTTG wt and RSUME increases its stability. The ∆D-box is much more stable than PTTG wt and RSUME does not control its stability. By 100μg/mL cycloheximide COS-7 treatment we observed that the K25R and K168R mutants have a shorter half-life than PTTG wt. By co-transfecting cells with RSUME expression vector, we found that RSUME prolongs the half-life of the K25R mutant but has no effect on the half-life of the K168R mutant. In AtT-20 cells (ACTH-secreting murine pituitary line) by quantitative RT-PCR experiments we found that PTTG wt induces the expression of c-Myc, while K25R and K168R mutants showed less induction of c-Myc transcription.We conclude that the ubiquitination ∆D-box site is critical for PTTG stability and does not involve RSUME action, while the SUMO attachment sites are important for the SUMOylation action of RSUME on stability and activity. Supported by ANPCyT and FOCEM (COF 03/11).