INJURY-INDUCED PHOSPHORYLATION SITES IN HEPATOCYTE NUCLEAR FACTOR-4 (HNF-4)

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Following serious trauma the body mounts several distinct and specific injury responses. Many of these, for example specific immune responses, or wound or bone repair, can take several days or weeks to reach levels sufficient to establish reversal of a specific condition. In addition, a more nonspecific response, referred to as the Acute Phase Response (APR), is mounted within the first 24 hours following injury. The APR is common to a wide range of traumas, and although changes to the normal physiology of many tissues are observed, significant changes to the liver phenotype occur due to the massive induction of protective proteins by the liver. Whilst the APR is designed for survival, for some critically ill patients it is likely that severe or prolonged activation with its interruption of normal homeostatic function contributes to organ failure and death. Evidence suggests that the APR?s repression of steady-state liver function is a byproduct of its mode of induction. It has also been suggested to be a highly regulated process which shares pathways important to early cell type development, and with some signals associated with the proliferative response. Previous work suggests that injury induced regulation of differentiated genes may be mediated through phosphorylation of liver-specific transcription factors, particularly the Hepatic Nuclear Factor-4 (HNF-4). An improved understanding of the mechanisms regulating this process would have therapeutic importance in support of the APR. The primary aim of this project is the identification and localization of phosphorylation sites in HNF-4 for control and injury-induced models using mass spectrometric techniques. HNF-4 proteins were isolated from control and injury induced male rat liver nuclear cell extracts by immunoprecipitation (IP). Isolated proteins were separated by 1D-SDS-PAGE and subjected to in-gel tryptic digestion. MALDI-TOF mass spectrometry was performed using a Bruker Reflex IV mass spectrometer. Capillary LC-MS/MS studies were performed using a Waters CapLC system interfaced with an Applied Biosystems Q-Star Pulsar I QoTOF MS. CapLC separations were performed on a Waters AtlantisTM C18 100¿m x 150 mm NanoEase column, employing a 50 min gradient of 5-90% acetonitrile, 0.1% formic acid. Data were analysed using Mascot (Matrix Science) and Aldente (SwissProt) database search engines.