Uncoupling of hepatic, epidermal growth factor-mediated mitogen-activated protein kinase activation in the fetal rat
Uncoupling of hepatic, epidermal growth factor-mediated mitogen-activated protein kinase activation in the fetal rat
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DOI：
10.1074/jbc.273.6.3784
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发表时间：
1998-02-06
影响因子：
4.8
通讯作者：
Gruppuso, PA
中科院分区：
文献类型：
--
作者：
Boylan, JM;Gruppuso, PA
有丝分裂原刺激细胞增殖涉及受体酪氨酸激酶使细胞膜上的蛋白质酪氨酸磷酸化。这促进了可以激活小G蛋白RAS的多蛋白复合体的形成。RAS的激活依次导致以下三种丝氨酸-苏氨酸激酶的激活：RAF、细胞外信号调节的激酶(MEK)和丝裂原活化蛋白(MAP)激酶家族的成员。先前的研究表明，在成年大鼠体内注射表皮生长因子(EGF)能迅速激活肝脏MAP激酶，但在妊娠晚期(E19)胎鼠(Boylan，J.M.和Gruppuso，P.A.，1996)细胞生长和差异不明显。7、1261-1269)。本研究旨在确定MAP激酶通路这种“解偶联”的机制。E19胎鼠和成年雄性大鼠腹腔注射EGF(0.5mgg/g体重)或生理盐水。15min后，取肝脏进行蛋白激酶分析，注射EGF后，胎鼠和成年鼠的肝脏Raf和MEK迅速而显著地激活，而胎鼠的MAPK活性比成年鼠低。对这种分离的MAPK激活和MEK激活的个体发生的检查表明，在出生后的头4周，随着成年肝细胞表型的获得，完整的信号逐渐获得。在此期间，用免疫印迹法检测MAPK的含量是恒定的，用部分纯化的胎鼠和成年大鼠肝脏进行的重组实验显示MAPK活性在体外是完整的，表明这两种酶在胚胎中都没有不可逆转的变化。在E19胎鼠肝细胞原代培养的研究中，胎肝细胞与强大的胎肝细胞有丝分裂原转化生长因子-α孵育24小时，可以诱导MAPK激活与MEK激活的解偶联。这些发现表明，在发育中的大鼠肝细胞中，一种新的负反馈机制可能是MAP激酶调节的活跃机制。
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 mu 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-alpha. These findings indicate that a novel negative feedback mechanism for MAP kinase regulation may be active in developing rat hepatocytes.