Partial characterization of a novel mitogen-activated protein kinase extracellular signal-regulated kinase activator in airway smooth-muscle cells

We demonstrated previously that in bovine tracheal myocytes, pretreatment w ith either forskolin or histamine significantly reduces both platelet-deriv ed growth factor (PDGF)- and epidermal growth factor-induced Raf-1 activati on but fails to inhibit extracellular signal-regulated kinase (ERK) activat ion substantially, evidence of a Raf-1-independent ERK activation pathway. To identify Raf-1-independent upstream signaling intermediates of mitogen-a ctivated protein kinase/ERK kinase-1 (MEK1), the dual-function kinase requi red and sufficient for ERK activation in these cells, lysates from forskoli n and PDGF-treated bovine tracheal myocytes were resolved using ion exchang e chromatography. Kinase activity for MEK1 was assessed by in vitro phospho rylation assay. in all experiments, the major peak of MEK1 phosphorylation activity was detected in fractions 18 through 26 (80 to 160 mM NaCl), with the peak fraction eluting at a NaCl concentration of 140 mM. The ability of these fractions to activate MEK1 was confirmed by examining the phosphoryl ation of myelin basic protein, a known substrate for ERKs, in the presence of functional MEK1 and ERK1. Fractions containing kinase activity were also probed with antibodies against MEK kinase-1, Raf-1, A-Raf, B-Raf, Mos, and Tpl-2. None of these proteins was detected in fractions containing peak ki nase activity, suggesting the presence of a novel PDGF-stimulated, forskoli n-insensitive MEK1 kinase. Further separation of fractions holding peak MEK phosphorylation activity by gel filtration suggested an apparent molecular mass of 40 to 45 kD. We conclude that PDGF-induced activation of MEK1 in b ovine tracheal myocytes is mediated at least in part by a novel kinase.