Subcellular localization and transport of p8

RICARDO NEME TAUIL,; MARÍA PÍA VALACCO; CEDRIC MALICET; CECILIA VARONE; JUAN IOVANNA; SILVIA MORENO

Pinamar, Provincia de Buenos Aires, Argentina

Congreso; XLI Reunión Anual de la Sociedad Argentina Investigaciones Bioquímicas (SAIB); 2005

SUBCELLULAR LOCALIZATION AND TRANSPORT OF P8 Neme Tauil R M*; Valacco M P*; Malicet C§; Varone C*; Iovanna J§; Moreno S*.  *Quím. Biológica, FCEyN, UBA, Buenos Aires, Argentina, § U624 INSERM, Marsella, Francia rmneme@qb.fcen.uba.ar 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. Immunocytochemistry showed that p8 localizes to the nucleus in subconfluent 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 flourescent protein (GFP) to the nucleus. The histone deacetylases inhibitor trichostatin A provokes delocalisation, 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 RanBP1 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 RanBP1 directly interact with p8. In RanQ69L, a mutant unable to hydrolyse GTP, the interaction is lost, suggesting a crucial site for complex formation.