Abstract
Stimulation of cell proliferation by mitogens involves tyrosine phosphorylation of proteins at the cell membrane by receptor tyrosine kinases. This promotes formation of multi-protein complexes that can activate the small G-protein, Ras. Activation of Ras, in turn, leads to sequential activation of the following three serine-threonine kinases: Raf, extracellular signal-regulated kinase kinase (MEK), and members of the family of mitogen-activated protein (MAP) kinases. Prior studies have shown that intraperitoneal injection of epidermal growth factor (EGF) leads to rapid activation of hepatic MAP kinases in adult rats but not in late gestation (E19) fetal rats (Boylan, J. M., and Gruppuso, P. A. (1996) Cell Growth & Differ. 7, 1261-1269). The present studies were undertaken to determine the mechanism for this "uncoupling" of the MAP kinase pathway. E19 fetal rats and adult male rats were injected with EGF (0.5 microg/g body weight, intraperitoneally) or with saline. After 15 min, livers were removed and prepared for kinase analyses. EGF injection led to a rapid and marked activation of hepatic Raf and MEK in both fetal and adult rats, whereas MAP kinase activation was minimal in fetal as opposed to adult rats. Examination of the ontogeny of this dissociation of MAP kinase activation from MEK activation showed gradual acquisition of intact signaling as an adult hepatocyte phenotype was attained during the first 4 postnatal weeks. Over this period, MAP kinase content as determined by Western immunoblotting was constant. Recombination experiments using partially purified fetal and adult rat liver MEK and MAP kinase showed intact MAP kinase activation in vitro, indicating that neither enzyme was irreversibly altered in the fetus. In studies using primary cultures of E19 fetal rat hepatocytes, uncoupling of MAP kinase activation from MEK activation could be induced by incubation of fetal hepatocytes for 24 h with a potent fetal hepatocyte mitogen, transforming growth factor-alpha. These findings indicate that a novel negative feedback mechanism for MAP kinase regulation may be active in developing rat hepatocytes.
Highlights
A major signaling pathway through which virtually all known mitogens exert intracellular responses is the mitogenactivated protein (MAP)1 kinase cascade [1, 2]
MAP Kinase Activation in Developing Rat Liver—To confirm our previous observations that MAP kinase activation in fetal liver is uncoupled [8], we immunoprecipitated MAP kinase in fetal liver homogenates from E19 fetal and adult rats given an intraperitoneal injection of phosphate-buffered saline or epidermal growth factor (EGF)
Our prior observation that intraperitoneal injection of late gestation fetal rats in situ with EGF showed only minimal activation of hepatic MAP kinases [8] was initially interpreted as indicating a probable block at the level of Ras activation. This was based on observations from several laboratories indicating that growth factor activation of the MAP kinase pathway can lead to phosphorylation of the Ras guanyl nucleotide exchange protein SOS, thereby down-regulating the MAP kinase pathway (14 –19)
Summary
A major signaling pathway through which virtually all known mitogens exert intracellular responses is the mitogenactivated protein (MAP)1 kinase cascade [1, 2]. Comparative signal transduction experiments using primary cultures of fetal rat hepatocytes have shown that there are no differences in the ability of these two growth factors to stimulate phosphorylation of Shc or activation of MAP kinase [28]. Western immunoblotting of parallel immunoprecipitates (Fig. 1, bottom) showed similar MAP kinase content in fetal and adult samples, indicating that the diminished ability of EGF to stimulate MAP kinase activity in the fetal rat was not due to a decrease in MAP kinase content.
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