Role of a new pituitary gene in post-translational protein modification.

CARBIA-NAGASHIMA A.,; GEREZ, J.; PEREZ-CASTRO, C.; PAEZ-PEREDA, M.; SILBERSTEIN, S.; STALLA, G.K.; HOLSBOER, F.; ARZT, E.

Rosario, 12 al 15 de noviembre de 2006.

Congreso; XLII Reunión Anual de la SAIB; 2006

SAIB

Conjugation of the small ubiquitin-like modifier SUMO regulates cellular processes including protein trafficking and degradation, chromatin structure and regulation of transcription. In this work, we characterize R-SUME (for RWD-containing Sumoylation Enhancer), a new RWD domain-containing gene cloned from a pituitary cell line with an increased tumorigenic potential. In cultured cell lines, R-SUME localizes to both nucleus and cytoplasm and enhances SUMO-1, 2 and 3 conjugation as observed by Western blot analys. This effect is dependant of Ubc9, the SUMO conjugase, as it is abrogated whern a dominant negative form of Ubc9 is co-expressed. R-SUME co-localizes with Ubc9 in the nucleus when analyzed by fluorescent confocal microscopy. Moreover, in pull-down experniments, recombinant R-SUME interacts with a GST-Ubc9 fusion protein, even in the absence of SUMO suggesting a direct interaction which is also observed when GST-Ubc9 is used to precipitate a cell extract containing R-SUME. In addition, R-SUME increases the sumoylation of IκB, a known SUMO target, in vivo and in vitro (whereas a structural mutant of R-SUME inhibits sumoylation), leading to an inhibition of NF-κB transcriptional activity (κB-LUC reporter assay; 40% inhibition p < 0,05). Together, these results indicate an important role of R-SUME regulating SUMO conjugation, and therefore many critical regulatory pathways.