Endothelins stimulate tyrosine phosphorylation and activity of p42/mitogen-activated protein kinase in astrocytes.
Endothelins stimulate tyrosine phosphorylation and activity of p42/mitogen-activated protein kinase in astrocytes.
复制标题
内皮素刺激星形胶质细胞中酪氨酸磷酸化和 p42/丝裂原激活蛋白激酶的活性。
作者：
S. Cazaubon;S. Cazaubon;Peter J. Parker;A. Strosberg;P. Couraud
内皮素（ET-1、ET-2、ET-3）通过G蛋白偶联的膜受体传递信号而显示多效性。我们发现ET-1和ET-3在几分钟内刺激小鼠胚胎星形胶质细胞原代培养物中42 kDa蛋白（p42）的酪氨酸磷酸化，但在大鼠星形胶质细胞瘤C6细胞的两个亚克隆中没有。通过细胞提取物的抗磷酸酪氨酸免疫印迹测定的这种效果也在对缓激肽、血小板衍生生长因子、佛波酯佛波12-肉豆蔻酸酯13-乙酸酯和G蛋白激活剂氟铝酸盐的反应中观察到。用百日咳毒素预处理细胞，使Gi/G（o）蛋白失活，不影响这些反应。然而，下调蛋白激酶C完全阻断佛波酯和氟铝酸盐的反应，并至少部分损害了ET-1刺激的p42磷酸化。基于以下数据，我们已经将p42鉴定为p42 mapk，一种促分裂原活化蛋白（MAP）激酶：通过用抗磷酸酪氨酸和抗MAP激酶抗体的连续免疫印迹，（i）观察到p42磷酸化和p42 mapk电泳迁移率的降低的类似动力学，可能对应于其酪氨酸/苏氨酸磷酸化[deVries-Smits，Boudewijn，Burgering，Leevers，马歇尔和博斯（1992）自然（伦敦）357，602-604];（ii）p42和p42 mapk的转换形式在SDS/PAGE上共迁移;（iii）p42 mapk的髓鞘碱性蛋白激酶活性被ET-1刺激，与p42的酪氨酸磷酸化平行。总之，这些研究结果强烈表明，内皮素可以刺激星形胶质细胞中的p42 mapk的酪氨酸磷酸化和激活，通过百日咳毒素不敏感的G蛋白和蛋白激酶C依赖和非依赖的途径。
Endothelins (ET-1, -2, -3) display pleiotropic activities, by signalling through G-protein-coupled membrane receptors. We show here that ET-1 and ET-3 stimulate within minutes the tyrosine phosphorylation of a 42 kDa protein (p42) in primary cultures of mouse embryo astrocytes, but not in any of two subclones of rat astrocytoma C6 cells. This effect, measured by anti-phosphotyrosine immunoblotting of cell extracts, was also observed in response to bradykinin, platelet-derived growth factor, the phorbol ester phorbol 12-myristate 13-acetate and the G-protein activator fluoroaluminate. Pretreatment of cells with pertussis toxin, which inactivates Gi/G(o) proteins, did not affect these responses. However, down-regulation of protein kinase C completely blocked the response to phorbol ester and fluoroaluminate and at least partially impaired the ET-1-stimulated phosphorylation of p42. We have identified p42 as p42mapk, a mitogen-activated protein (MAP) kinase, on the basis of the following data: by sequential immunoblotting with antiphosphotyrosine and anti-MAP kinase antibodies, (i) similar kinetics are observed for p42 phosphorylation and the decrease in p42mapk electrophoretic mobility, likely corresponding to its tyrosine/threonine phosphorylation [de Vries-Smits, Boudewijn, Burgering, Leevers, Marshall and Bos (1992) Nature (London) 357, 602-604]; (ii) p42 and the shifted form of p42mapk co-migrate on SDS/PAGE; (iii) the myelin-basic-protein kinase activity of p42mapk is stimulated by ET-1, in parallel with the tyrosine phosphorylation of p42. In conclusion, these findings strongly suggest that endothelins can stimulate the tyrosine phosphorylation and activation of p42mapk in astrocytes, via pertussis-toxin-insensitive G protein and protein kinase C-dependent and -independent pathways.