Wang Y, Simonson M S, Pouysségur J, Dunn M J
Department of Medicine, Case Western Reserve University, Cleveland, OH.
Biochem J. 1992 Oct 15;287 ( Pt 2)(Pt 2):589-94. doi: 10.1042/bj2870589.
Mitogen-activated protein (MAP) kinases are regarded as switch kinases in the phosphorylation cascade initiated by various agonists. We have investigated whether endothelins (ET), which are constrictor and mitogenic isopeptides, can increase MAP kinase activity in rat mesangial cells, using bovine myelin basic protein (MBP) as a substrate for an in vitro kinase assay. Treatment of quiescent mesangial cells with ET-1 rapidly stimulated a kinase activity which phosphorylated exogenous MBP. This stimulation was dose-dependent, with threshold responses at 1 nM-ET-1. Epidermal growth factor and thrombin also activated this kinase in mesangial cells. We also examined the ET signal transduction pathways leading to activation of MBP kinase. Pertussis toxin had no effect on ET-stimulated MBP kinase activity. Stimulation of protein kinase C by phorbol ester increased MBP kinase activity, and down-regulation of PKC partially inhibited ET-stimulated MBP kinase as well as phorbol ester-stimulated MBP kinase activity. Interestingly, genestein, an inhibitor of protein tyrosine kinases, partially inhibited MBP kinase stimulated by ET but not by phorbol esters. These results suggest that ET stimulates MBP kinase activity in rat mesangial cells via at least two pathways: one which is protein kinase C-dependent and a second one that involves a protein tyrosine kinase. Finally, by raising rabbit antibodies against the two forms of MAP kinase, p44mapk and p42mapk, we demonstrated that both isoforms are expressed in mesangial cells. Antibody alpha 1 Cp42 specifically immunoprecipitated p42mapk and allowed us to demonstrate that ET stimulates MBP kinase activity in the p42mapk immunocomplex. In conclusion, we have provided evidence that, in rat mesangial cells, MAP kinases are rapidly activated by ET-1, a regulatory process that involves at least protein kinase C activation and also a contribution of a tyrosine kinase not yet characterized.
丝裂原活化蛋白（MAP）激酶被视为由各种激动剂引发的磷酸化级联反应中的开关激酶。我们研究了内皮素（ET），一种具有收缩血管和促有丝分裂作用的异肽，是否能增加大鼠系膜细胞中的MAP激酶活性，采用牛髓鞘碱性蛋白（MBP）作为体外激酶测定的底物。用ET-1处理静止的系膜细胞可迅速刺激一种能使外源性MBP磷酸化的激酶活性。这种刺激呈剂量依赖性，在1 nM ET-1时出现阈值反应。表皮生长因子和凝血酶也能激活系膜细胞中的这种激酶。我们还研究了导致MBP激酶激活的ET信号转导途径。百日咳毒素对ET刺激的MBP激酶活性没有影响。佛波酯刺激蛋白激酶C可增加MBP激酶活性，PKC的下调部分抑制了ET刺激的MBP激酶以及佛波酯刺激的MBP激酶活性。有趣的是，蛋白酪氨酸激酶抑制剂染料木黄酮部分抑制了ET刺激的MBP激酶，但不抑制佛波酯刺激的MBP激酶活性。这些结果表明，ET通过至少两条途径刺激大鼠系膜细胞中的MBP激酶活性：一条是依赖蛋白激酶C的途径，另一条涉及一种尚未明确的蛋白酪氨酸激酶。最后，通过制备针对两种形式的MAP激酶p44mapk和p42mapk的兔抗体，我们证明这两种异构体均在系膜细胞中表达。抗体α1 Cp42特异性免疫沉淀p42mapk，并使我们能够证明ET刺激p42mapk免疫复合物中的MBP激酶活性。总之，我们提供了证据表明，在大鼠系膜细胞中，MAP激酶可被ET-1迅速激活，这一调节过程至少涉及蛋白激酶C的激活以及一种尚未明确的酪氨酸激酶的作用。
