CONICET | Buscador de Institutos y Recursos Humanos

Proto-oncogene pituitary tumor transforming gene (PTTG) is a cell cycle regulator whose overexpression was correlated with malignant status and poor prognosis in many tumors. Securin PTTG upregulation is a hallmark of pituitary adenomas. We described that high PTTG protein levels are induced by the RWD-containing SUMOylation enhancer (RWDD3 or RSUME), a protein originally identified in the same pituitary tumor cell line in which PTTG was also cloned. In this work, we explore the mechanism of stabilization of PTTG focusing in a tight control of protein levels by post-translational modifications. YAPA, a RSUME mutant with loss of activity as SUMOylation enhancer, showed by western blot (WB) a decreased action on the stability of PTTG. Accordingly, we found diminished protein levels of PTTG in the presence of Gam1 (a viral enzyme that inhibits SUMOylation), even in the presence of RSUME. By inmunoprecipitation (IP) assays in COS-7 we observed that PTTG is conjugated to SUMO-1 and RSUME enhances this SUMOylation. We validated PTTG SUMOylation at endogenous level in pituitary lactosomatotroph tumoral GH4 cells and HeLa cells, and in vitro SUMOylation experiments. RSUME knockdown with a specific small hairpin RNA (shRNA), has not effect on PTTG protein levels when the proteasome degradation is inhibited. By IP of ubiquitinated proteins we observed that both RSUME and SUMO-1 (with less potency) decrease ubiquitin conjugation to PTTG. RSUME knockdown has the opposite effect on PTTG ubiquitination. Gam1 restored PTTG ubiquitination levels by preventing SUMOylation, even in the presence of RSUME. We conclude that PTTG tight regulation and stabilization by SUMOylation, which reduces its degradation by means of the ubiquitin proteasome system, accounts for its abundance in tumoral cells.Supported by ANPCyT, CONICET, UBA and FOCEM (COF 03/11).