BACKGROUND/AIMS: Although signal transduction pathways activated by EGF have been extensively studied in cultured cells, few such studies have been done in whole animals. In this study, activation of hepatic kinases, phosphatases, and DNA-binding activity of AP-1 was examined after intraperitoneal injections of either EGF or sodium orthovanadate into mice. METHODS: Cytoplasmic and nuclear proteins, extracted from isolated hepatocytes or whole liver tissue, were immunoprecipitated with either anti-ERK1/2, anti-70S6k, or anti-p90rsk antibodies and kinase activities were measured using specific substrates. Kinase protein levels was evaluated by Western blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay. RESULTS: Systemic administration of EGF induced simultaneous increase in the activities of cytoplasmic and nuclear MAPK, p70S6k, and p90rsk. MAPK and p70S6k were more potently activated in the cytosol while p90rsk activation was more pronounced in the nucleus. Orthovanadate also activated these kinases but to a much lesser degree than EGF. In vitro phosphatase assays showed that neither EGF nor orthovanadate induced measurable changes in phosphatase activities. EGF, but not orthovanadate, activated nuclear AP-1 DNA-binding activity in intact liver, indicating that activation of MAPK, p70S6k, and p90rsk by orthovanadate is not sufficient to activate this transcription factor. CONCLUSION: These observations provide groundwork for future studies to examine the role of EGF-induced kinase cascades and transcription factors in liver regeneration and other growth factor-mediated hepatic processes.
BACKGROUND/AIMS: Although signal transduction pathways activated by EGF have been extensively studied in cultured cells, few such studies have been done in whole animals. In this study, activation of hepatic kinases, phosphatases, and DNA-binding activity of AP-1 was examined after intraperitoneal injections of either EGF or sodium orthovanadate into mice. METHODS: Cytoplasmic and nuclear proteins, extracted from isolated hepatocytes or whole liver tissue, were immunoprecipitated with either anti-ERK1/2, anti-70S6k, or anti-p90rsk antibodies and kinase activities were measured using specific substrates. Kinase protein levels was evaluated by Western blot analysis. AP-1 DNA binding activity was measured by electrophoretic mobility shift assay. RESULTS: Systemic administration of EGF induced simultaneous increase in the activities of cytoplasmic and nuclear MAPK, p70S6k, and p90rsk. MAPK and p70S6k were more potently activated in the cytosol while p90rsk activation was more pronounced in the nucleus. Orthovanadate also activated these kinases but to a much lesser degree than EGF. In vitro phosphatase assays showed that neither EGF nor orthovanadate induced measurable changes in phosphatase activities. EGF, but not orthovanadate, activated nuclear AP-1 DNA-binding activity in intact liver, indicating that activation of MAPK, p70S6k, and p90rsk by orthovanadate is not sufficient to activate this transcription factor. CONCLUSION: These observations provide groundwork for future studies to examine the role of EGF-induced kinase cascades and transcription factors in liver regeneration and other growth factor-mediated hepatic processes.
Authors: Michal Mikula; Pawel Gaj; Karolina Dzwonek; Tymon Rubel; Jakub Karczmarski; Agnieszka Paziewska; Artur Dzwonek; Piotr Bragoszewski; Michal Dadlez; Jerzy Ostrowski Journal: DNA Res Date: 2010-06-29 Impact factor: 4.458