Uncoupling of Hepatic, Epidermal Growth Factor-mediated Mitogen-activated Protein Kinase Activation in the Fetal Rat

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 mitogenactivated 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 g/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-␣. These findings indicate that a novel negative feedback mechanism for MAP kinase regulation may be active in developing rat hepatocytes.A major signaling pathway through which virtually all known mitogens exert intracellular responses is the mitogenactivated protein (MAP) 1 kinase cascade (1, 2). A primary mechanism by which growth factor receptor tyrosine kinases activate MAP kinases involves tyrosine phosphorylation of an adaptor protein, Shc. Shc binds to a second adaptor protein, Grb2, by SH2/3 interactions. This complex incorporates a guanyl nucleotide exchange factor, Sos, which interacts with and activates Ras. The GTP-bound Ras recruits Raf kinase to the cell membrane and initiates a protein kinase cascade that phosphorylates and activates a MAP kinase kinase termed MEK (MAP kinase or extracellular signal-regulated kinase kinase). MEK in turn phosphorylates MAP kinases on both tyrosyl and threonyl sites. Active MAP kinases translocate to the nucleus where they phosphorylate other serine-threonine kinases and transcription factors which can medi...

Regulation of fetal liver growth in a model of diet restriction in the pregnant rat

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Ribosomal Protein S6 Phosphorylation and Function during Late Gestation Liver Development in the Rat

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