Karpova A Y, Abe M K, Li J, Liu P T, Rhee J M, Kuo W L, Hershenson M B
Department of Pediatrics, University of Chicago, Illinois 60637, USA.
Am J Physiol. 1997 Mar;272(3 Pt 1):L558-65. doi: 10.1152/ajplung.1997.272.3.L558.
We tested whether activation of mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase-1 (MEK1) is required and sufficient for extracellular signal-regulated kinase (ERK) activation in airway smooth muscle cells. First, we transiently cotransfected bovine tracheal myocytes with an epitope-tagged ERK2 and a dominant-negative or a constitutively active form of the gene encoding MEK1 and assessed ERK2 activation by in vitro phosphorylation assay. Expression of the dominant-negative MEK1 inhibited platelet-derived growth factor (PDGF)-induced ERK2 activation, whereas expression of the constitutively active MEK1 induced ERK2 activation, suggesting that MEK1 is required and sufficient for ERK activation in these cells. Next, we assessed the effect of PD-98059, a synthetic MEK inhibitor, on PDGF-induced MEK1 and ERK activation. PD-98059 (10 microM) inhibited MEK1 and ERK activation, confirming that MEK1 is required for ERK activation in bovine tracheal myocytes. PD-98059 had no effect on Src or Raf-1 activity, evidence that PD-98059 is a specific inhibitor of MEK in this system. Finally, PD-98059 reduced PDGF-induced [(3)H]thymidine incorporation in a concentration-dependent manner, suggesting that catalytic activation of MEK1 and ERKs is required for DNA synthesis. We conclude that MEK1 is required for PDGF-induced ERK activation in bovine tracheal myocytes and that MEK1 and ERKs are required for PDGF-induced DNA synthesis in these cells.
我们测试了丝裂原活化蛋白激酶/细胞外信号调节激酶激酶-1(MEK1)的激活对于气道平滑肌细胞中细胞外信号调节激酶(ERK)的激活是否必要且充分。首先,我们将带有表位标签的ERK2与MEK1编码基因的显性负性或组成型活性形式一起瞬时共转染牛气管肌细胞,并通过体外磷酸化试验评估ERK2的激活情况。显性负性MEK1的表达抑制了血小板衍生生长因子(PDGF)诱导ERK2的激活,而组成型活性MEK1的表达则诱导了ERK2的激活,这表明MEK1对于这些细胞中ERK的激活是必要且充分的。接下来,我们评估了合成的MEK抑制剂PD-98059对PDGF诱导的MEK1和ERK激活的影响。PD-98059(10微摩尔)抑制了MEK1和ERK的激活,证实了MEK1是牛气管肌细胞中ERK激活所必需的。PD-98059对Src或Raf-1活性没有影响,这证明PD-98059在该系统中是MEK的特异性抑制剂。最后,PD-98059以浓度依赖的方式降低了PDGF诱导的[³H]胸苷掺入,表明MEK1和ERK的催化激活是DNA合成所必需的。我们得出结论,MEK1是PDGF诱导牛气管肌细胞中ERK激活所必需的,并且MEK1和ERK是PDGF诱导这些细胞中DNA合成所必需的。