IDENTIFICATION OF PROTEIN COMPLEXES ASSOCIATED TO P8, A PROTEIN RELATED TO TUMOR PROGRESSION

VALACCO, M.P.; VARONE, C.; IOVANNA, J.L.; MORENO, S.; BURLINGAME, A.

Mar del Plata, Buenos Aires

Congreso; 43 Reunion Anual SAIB; 2007

SAIB

p8 is an 8 kDa protein involved in tumor progression. In different mammalian cell lines p8 migrates between nucleus and cytoplasm, via a functional NLS, depending on cell culture density, ATP availability, cell cycle arrest and deacetylase inhibition. It is remarkable that being small enough to diffuse between nucleus and cytoplasm passively, p8 should possess an NLS and a controlled localization. These results suggest that p8 is forming part of multiprotein complexes and that it could even be the mediator of the translocation of these complexes. The aim of this study is to identify the members of these complexes. A HEK 293 cell line, that stably expresses p8 fused to N-terminal histidine-FLAG tags, was generated. Tandem affinity purification, using anti-flag antibodies and metal affinity chromatography (Nickel and Cobalt) was performed. The purified complexes were proteolitically digested and analyzed by tandem mass spectrometry to identify p8 associated proteins and to detect any possible post translational modifications on p8. This approach has allowed us to identify a small group of putative partners of p8, the most interesting one being Ying Yang 1, a ubiquitous transcription factor which has no NLS but presents the same sub-cellular localization pattern as p8. Further studies are in progress in order to find p8’s PTMs and to verify the specificity of the identified partners.