PARTIAL PURIFICATION OF A NOVEL MEK ACTIVATOR IN CULTURED AIRWAY SMOOTH MUSCLE CELLS. ▴ 2304

Abstract

Excess airway smooth muscle proliferation may play a role in the pathogenesis of airflow obstruction in patients with asthma and BPD. We therefore studied the activation pathway of MAP kinase, a family of cytosolic serine/threonine kinases which participate in the transduction of mitogenic signals to the cell nucleus, in cultured bovine tracheal myocytes. The major MAP kinase activation pathway appears to involve sequential activation of Ras, Raf-1 and MAPK/ERK kinase (MEK); alternative pathways exist, however. Previous experiments in bovine tracheal myocytes suggest that MEK-1 activation is required and sufficient for MAP kinase activation in these cells(Pediatr Res 37: 392A, 1995). To examine the contribution of Raf-1 to growth factor-induced MAP kinase activation, we measured Raf-1 and MAP kinase activities following PDGF and EGF stimulation. Raf-1 activity was assessed by an in vitro phosphorylation assay employing recombinant inactive MEK-1 as a substrate; MAP kinase activity was measured by an in-gel kinase assay using myelin basic protein as a substrate. As expected, treatment with PDGF and EGF increased both Raf-1 and MAP kinase activities. However, pre-treatment with forskolin, an activator of adenyl cyclase which inhibits Raf-1 activation via cAMP-dependent protein kinase A, significantly reduced Raf-1 activity but had no effect on the activation of MAP kinase by either growth factor, suggesting that there is a major Raf-1-independent MEK activation pathway in bovine tracheal myocytes. To identify proteins other than Raf-1 which activate MEK-1, lysates from PDGF and forskolin-treated cells were resolved using a Mono-Q FPLC ion-exchange column and phosphorylation activity for MEK-1 assessed by in vitro phosphorylation assay. Despite the inhibition of Raf-1 by forskolin, significant MEK phosphorylation was observed. Peak phosphorylation activity eluted from the column at 160-170 mM NaCl. Western analyses demonstrated that the observed phosphorylation activity was not attributable to MEKK, Raf-1 or MAP kinase, suggesting that the MEK activator is novel. Supported by HL-02731 and HL-54685.