Wang Y, Pouysségur J, Dunn M J
Department of Medicine, Case Western Reserve University, Cleveland, Ohio.
J Cardiovasc Pharmacol. 1993;22 Suppl 8:S164-7. doi: 10.1097/00005344-199322008-00044.
We have reported that endothelin-1 (ET-1), which is a constrictor and mitogenic peptide, can increase mitogen-activated protein kinase p42 (p42mapk) activity in rat mesangial cells. In this study, we investigate the mechanism of activation of p42mapk. Treatment of quiescent mesangial cells with 10(-7) M ET-1 biphasically stimulated p42mapk activity. The kinetics of the immunoprecipitated p42mapk activity induced by ET-1 showed a maximal 3.5- to 4.5-fold stimulation 5 min after the addition of the agonist to the cell cultures and a smaller, 2.5-fold increase of activity between 2 and 6 h after ET challenge. Neither peak of p42mapk activity induced by ET-1 was inhibited by pretreatment of the cells with either cycloheximide to inhibit protein synthesis or actinomycin D to retard transcription. Analysis by immunoblot showed that p42mapk was not affected by these pretreatments. In addition, the kinetics of phosphorylation of p42mapk showed a significant 32P incorporation into p42 at 5, 30, and 240 min after ET stimulation. Because phosphorylation on tyrosine and threonine residues of the enzyme is necessary for activation of the kinase, we believe that the phosphorylation of the p42mapk rather than transcriptional or translational induction is responsible for the activation of p42mapk in mesangial cells stimulated with ET-1.
我们曾报道过，内皮素 -1（ET -1）作为一种具有收缩血管和促有丝分裂作用的肽，能够增强大鼠系膜细胞中丝裂原活化蛋白激酶p42（p42mapk）的活性。在本研究中，我们探究了p42mapk的激活机制。用10(-7) M ET -1处理静止的系膜细胞会双相刺激p42mapk的活性。ET -1诱导的免疫沉淀p42mapk活性动力学显示，在向细胞培养物中添加激动剂后5分钟，活性最大可提高3.5至4.5倍，在ET刺激后2至6小时，活性有较小幅度的增加，为2.5倍。用环己酰亚胺抑制蛋白质合成或用放线菌素D延缓转录对细胞进行预处理，均不能抑制ET -1诱导的p42mapk活性峰值。免疫印迹分析表明，这些预处理对p42mapk没有影响。此外，p42mapk的磷酸化动力学显示，在ET刺激后5、30和240分钟，有显著的32P掺入p42。由于该酶酪氨酸和苏氨酸残基的磷酸化是激酶激活所必需的，我们认为p42mapk的磷酸化而非转录或翻译诱导是ET -1刺激的系膜细胞中p42mapk激活的原因。
