Mass Spectrometry Analysis of p8

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

Pacific Grove, California, EEUU

Encuentro; UCSF BBC Joint Retreat; 2006

Universidad de California San Francisco-Graduate Group in Biological and Medical Informatics- Graduate Program in Chemistry and Chemical Biology

p8, an 8 kDa protein was first identified in rat by its induction during the acute phase of pancreatitis. Its mRNA levels are increased in cell lines in response to stress and mitogenic factors, and in vivo after treatment with lipopolysaccharides. Fibroblasts obtained from p8 -/- mice, transformed with a retroviral vector expressing the E1A oncogene and a mutated form of Ras (oncogenic), are unable to grow in soft agar and do not induce tumor formation when injected into nude mice, as do fibroblasts from p8 +/+ mice. Sequence homology analysis in databases indicated that, apart from the known homology between human, rat, mice, Xenopus laevis and Drosophila melanogaster, there is significant homology with pig, cow, fish and arthropods. Further analysis of hp8 showed that it contains a putative bipartite NLS (Nuclear Localization Signal) and remarkably, this NLS is highly conserved in all these species. It also contains a putative NES. As regarding the biochemical properties of p8, it is known that recombinant p8 does not present secondary structure, binds to DNA without preference for a specific sequence, is phosphorylated in vitro by PKA, the phosphorylated form increases its affinity for DNA, and shares chemical properties with the High Mobility Group proteins (HMG) (isoelectric point, charge distribution, mass) (8). It was also seen that p8 can be acetylated in vitro by p300 (9).With immunocytochemistry, we found that p8 presents nuclear localization in sub-confluent cells, but it localizes in the cytoplasm of confluent cells. Further experiments have shown that nuclear import is energy dependent and that the export of p8 does not involve the CRM1 transporter. It was also observed that in sub-confluent cells, arrested in Go, the protein localized in the cytoplasm, and when the cell cycle was restored, p8 migrated back to the nucleus.In the presence of a histone deacetylase inhibitor, Trichostatin A, sub-confluent cells showed cytoplasmic localization of p8.It is remarkable that being small enough to diffuse between nucleus and cytoplasm passively, p8 should posses a NLS and a controlled localization. This result suggests that p8 is forming part of multiprotein complexes and that it could even be the mediator of the translocation of these complexes. In this study we attempt to find these possible partners. We generated a HEK 293 cell line that stably expresses p8 fused to N-terminal histidine and FLAG tags. We have performed tandem affinity purification and mass spectrometry analysis to identify the proteins that are associated to p8, and to detect any post translational modifications on this protein.