ENDOTHELIN RAPIDLY STIMULATES MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVITY IN RAT MESANGIAL CELLS
ENDOTHELIN RAPIDLY STIMULATES MITOGEN-ACTIVATED PROTEIN-KINASE ACTIVITY IN RAT MESANGIAL CELLS
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DOI：
10.1042/bj2870589
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发表时间：
1992-10-15
影响因子：
4.1
通讯作者：
DUNN, MJ
中科院分区：
文献类型：
--
作者：
WANG, YZ;SIMONSON, MS;DUNN, MJ
丝裂原活化蛋白(MAP)激酶被认为是由多种激动剂启动的磷酸化级联反应中的开关蛋白。我们以牛髓鞘碱性蛋白(MBP)为底物，研究了收缩和促有丝分裂的异肽--内皮素(ET)是否能增加大鼠肾小球系膜细胞MAPK的活性。用ET-1处理静止的肾小球系膜细胞可迅速刺激一种使外源性MBP磷酸化的激酶活性。这种刺激是剂量依赖的，阈值反应为1 nM-ET-1。表皮生长因子和凝血酶也激活了系膜细胞中的这一激酶。我们还研究了导致MBP激酶激活的ET信号转导通路。百日咳毒素对ET刺激的MBP激酶活性无影响。佛波酯刺激蛋白激酶C可增加MBP的活性，下调PKC可部分抑制ET刺激的MBP和佛波酯刺激的MBP的活性。有趣的是，蛋白酪氨酸激酶的抑制剂genstein能部分抑制ET刺激的MBP激酶，但不能抑制佛波酯的刺激。这些结果表明，ET至少通过两条途径刺激大鼠肾小球系膜细胞的MBP激酶活性：一条途径依赖于蛋白激酶C，另一条途径涉及蛋白酪氨酸激酶。最后，通过提高兔抗p44mapk和p42mapk两种形式的MAPK的抗体，我们证明了这两种异构体都在系膜细胞中表达。抗体α1 Cp42特异性地免疫沉淀p42mapk，使我们能够证明ET刺激p42mapk免疫复合体中的MBP激酶活性。总之，我们提供的证据表明，在大鼠肾小球系膜细胞中，MAP激酶可被ET-1迅速激活，这一调控过程至少涉及蛋白激酶C的激活，也是一种尚未确定的酪氨酸激酶的作用。
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 alpha1 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.