MEK1 IS REQUIRED FOR PDGF-INDUCED ERK ACTIVATION AND DNA-SYNTHESIS INTRACHEAL MYOCYTES

We tested whether activation of mitogen-activated protein kinase/extra cellular signal-regulated kinase kinase-1 (MEK1) is required and suffi cient for extracellular signal-regulated kinase (ERK) activation in ai rway smooth muscle cells. First, we transiently cotransfected bovine t racheal myocytes with an epitope-tagged ERK2 and a dominant-negative o r a constitutively active form of the gene encoding MEK1 and assessed ERK2 activation by in vitro phosphorylation assay. Expression of the d ominant-negative MEK1 inhibited platelet-derived growth factor (PDGF)- induced ERK2 activation, whereas expression of the constitutively acti ve MEK1 induced ERK2 activation, suggesting that MEK1 is required and sufficient for ERK activation in these cells. Next, we assessed the ef fect of PD-98059, a synthetic MEK inhibitor, on PDGF-induced MEK1 and ERK activation. PD-98059 (10 mu M) 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 t hat PD-98059 is a specific inhibitor of MEK in this system. Finally, P D-98059 reduced PDGF-induced [H-3]thymidine incorporation in a concent ration-dependent manner, suggesting that catalytic activation of MEK1 and ERKs is required for DNA synthesis. We conclude that MEK1 is requi red for PDGF-induced ERK activation in bovine tracheal myocytes and th at MEK1 and ERKs are required for PDGF-induced DNA synthesis in these cells.