Identification of Protein Complexes associated to p8, a protein related to tumor progression

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

Mar del Plata, Argentina

Congreso; XLIII Reunión Anual SAIB; 2007

Sociedad Argentina de Investigacion Bioquimica

Identification of Protein Complexes associated to p8, a protein related to tumor progression M.P. Valacco1,2, C. Varone1, J.L. Iovanna1, S. Moreno1, A.L. Burlingame2. 1Departamento de Química Biológica, Facultad de  Ciencias Exactas y Naturales, UBA, Buenos Aires, Argentina. 2Mass Spectrometry Facility, UCSF. p8 is an 8 kDa protein which was first identified in rat due to its induction during the acute phase of pancreatitis. It was later characterized in mice and humans. Various functions related to cell growth control and stress have been attributed to p8 since its mRNA levels are increased in cell lines in response to stress and mitogenic factors. An important role in tumor progression was also assigned to p8 since it has been shown that, when fibroblasts obtained from p8 +/+ mice are transformed with a retroviral vector expressing oncogene E1A, they are able to induce tumor formation when injected into nude mice; while transformed fibroblasts derived from p8 -/-  mice do not present these tumorigenic properties.It is known that p8 binds DNA without preference for a specific sequence, it can be phosphorylated by PKA, and acetylated by p300 in vitro; it shares biochemical properties with the High Mobility Group Proteins.Through sequence homology analysis in databases p8 was identified in many different species of higher eukaryotes. The sequence contains a highly conserved region, which corresponds to a putative bipartite NLS (Nuclear Localization Signal). Immunocytochemistry experiments, show that p8 presents nuclear localization in sub-confluent cells, but it localizes in the cytoplasm of confluent cells. It was also observed that nuclear import of p8 is energy dependent, and that its sub-cellular localization also changes with cell cycle stage and acetylation state of the cells. It is remarkable that being small enough to diffuse between nucleus and cytoplasm passively, p8 should posses a NLS and a strictly controlled sub-cellular localization. This suggests 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. In order to do this, 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 digested with either trypsin or chymotrypsin and analyzed by tandem mass spectrometry to identify the proteins that are associated to p8, and to detect any possible post translational modifications on this protein. 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 p8s PTMs and to verify the specificity of the identified partners.