Hepatic epidermal growth factor-regulated mitogen-activated protein kinasekinase kinase activity in the rat: lack of identity with known forms of Raf and MEKK

Mitogenic signaling involves protein kinases that phosphorylate the mitogen -activated protein kinase (MAPK) activator, MEK. In rats, basal hepatic MEK kinase activity is low in vivo in both adult rats and late gestation fetal rats, and is markedly stimulated by intraperitoneal administration of epid ermal growth factor (EGF), The level of stimulated MEK phosphorylating acti vity is approximately 15 times higher in fetal liver than in adult liver. T o identify regulated forms of the two categories of MEK kinase, Raf and MEK K, Western immunoblotting, immunoprecipitation kinase assays and immunodepl etion studies were performed, Western immunoblotting confirmed that Raf-1, A-Raf, B-Raf, MEKK1 and MEKK2 were present at similar levels in E19 and adu lt liver. However, specific immunoprecipitation kinase assays did not detec t any kinases that could account for marked EGF sensitivity or the higher l evel of activity in E19 fetuses. Immunodepletion studies produced a marked reduction in immunoreactive Raf/MEKK content and activity, but a minimal de crease in the ability of chromatography fractions to phosphorylate and acti vate recombinant MEK-1. Our results indicate that hepatic, EGF-sensitive ME K kinase activity may reside with a previously unidentified and physiologic ally relevant form of Raf and/or MEKK. (C) 2000 Federation of European Bioc hemical Societies.