SUBCELLULAR LOCALIZATION AND TRANSPORT OFP8

NEME TAUIL, R., VALACCO, P.MALICET, C., VARONE, C., IOVANNA JL, Y MORENO, S.

Pinamar, Buenos Aires

Congreso; 10° Reunion Internacional de PAB;B y XLI Reunion Nacional de SAIB; 2005

PABMB y SAIB

P8 is an 8kDa protein induced in human pancreatitis. It is expressed in response to stress, could be involved in tumour development and act as a growth factor. It is phosphorylated in vitro by PKA and acetylated by p300. Immunoeytochemistry showed that p810calizes to the nucleus in subcont1uent cell cultures and throughout the whole cell under superconfluence. p8 was found to have a functional nuclear localisation signal, necessary and sufficient to localise the green t1ourescent protein (GFP) to the nucleus. The histone deacetylases inhibitor trichostatin A provokesdelocalisation, suggesting a role for acetylation. Through the Cytotrap two-hybrid system we could identify several partners of p8, among which we chose to study the small GTPase Ran as well as its binding protein RanBPl since they are involved in nuclear transport, spindle assembly and post-mitotic nuclear envelope reassembly. Ran shifts between GDP and GTP-bound states thanks to accessory proteins, such as RanBP1. They where expressed in E. coli as GST fusion proteins. Pull down technique confirmed that both Ran (GDP or GTP-bound) and RanBRl directIy interact wÜh p8. In RanQ69L, a mutant unable to hydrolyse GTP, the interaction is lost, suggesting a crucial site for complex formation.