This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.p8, an 8 kDa protein, was first identified in 1997 by its induction during the acute phase of pancreatitis, and was further characterized in mouse and human. Its mRNA levels are increased in cell lines in response to stress and mitogenic factors, and in malignant processes such as breast, pancreatic and thyroid cancer. Various contradicting roles related to cell growth control and apoptosis have been proposed for p8, but up to date, the specific functions and pathways of p8 have not been defined. It has been observed that p8 is involved in tumor progression, 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. It has been shown that p8 interacts with and can be acetylated by the transcriptional coactivator p300 in vitro, and it can be phosphorylated by PKA in vitro. p8 contains a functional NLS. In different mammalian cell lines p8 migrates between nucleus and cytoplasm 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 final objective of the p8 project is to study the action mechanism of the protein. The objective of the present project is to purify a Histidine-Flag tagged p8 from cell extracts and use the different mass spectrometry techniques available in the Mass Spectrometry Facility to identify the post translational modifications of p8 and to characterize the protein complexes to which p8 is associated. This will provide information about the possible functions of p8 as well as the pathways in which it is involved and this could be a key step in the comprehension of tumor progression and cancer treatment.
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