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Show HONORS COLLEGE SPRING 2013 KariAnne Rencher Johnson Jared Rutter 236 IDENTIFICATION OF PASK SUBSTRATES AND/OR INTERACTING PROTEINS KariAnne Rencher Johnson (Jared Rutter, Sarah Fogarty) Department of Biochemistry University of Utah PAS kinase (PASK) is an evolutionary conserved serine/threonine kinase that appears to have a role in the regulation of cellular energy metabolism. It has previously been shown that mice lacking PASK (PASK-/- mice) are resistant to the development of hepatic steatosis (lipid accumulation in the liver), glucose intolerance, insulin resistance and obesity when on a high fat diet. These results suggest that PASK could be a potential target for the treatment of type II diabetes and obesity. The function of PASK at the cellular level, however, is unknown. The objective of this project is to identify substrates and/or interacting partners of PASK in order to better understand its biological significance and connection to metabolic diseases. In order to achieve this objective, FLAG and HA dually-tagged constructs expressing wild type (WT) and kinase inactive (Kl) PASK have been generated in a viral expression vector. These constructs were initially characterized in h u m a n embryonic kidney (HEK293T) cells. Both W T and Kl PASK could be immunoprecipitated using anti-FLAG and anti-HA antibodies and demonstrated the expected kinase activities (normal for W T and zero for Kl) in a radioactive peptide-based assay. A small-scale tandem affinity purification (TAP) of W T PASK was performed and purified protein could be visualized as a distinct band on a Coomassie-stained gel. HepG2 cells have been successfully infected with viral particles encoding N-terminally tagged WT or Kl PASK. TAP purification was optimized for this cell line. After purification, PASK-associated proteins were identified by mass spectrometry. The THRAP3 protein (a protein involved in splicing) was identified as a potential interacting protein and is n o w being confirmed as such by co-immunoprecipitation experiments. In vitro kinase assays will then be used to investigate THRAP3's potential role as a PASK substrate and truncated versions of THRAP3 will be used to m a p potential phosphorylation sites. |
