A synthetic inhibitor of the mitogen-activated protein kinase cascade.

Abstract

Many diverse extracellular stimuli-including growth factors, hormones, osmolar shock, stress, and elevated temperatureresult in activation of phosphorylation cascades utilizing mitogen-activated protein kinases (MAPKs) (1-8). MAPKs (sometimes called extracellular signal-regulated kinases, or ERKs) comprise a family of related protein kinases that are themselves activated by phosphorylation on threonine and tyrosine residues. The MAPK-activating enzymes (MAPK/ ERK kinases, or MEKs) are unusual in their ability to catalyze phosphorylation on both threonine and tyrosine residues (9, 10). MEKs are in turn activated by phosphorylation on serine residues by upstream kinases. These MEK kinases, which appear to require activation by the ras protooncogene product (11, 12), include members of the Raf family (13-15), a mammalian homologue of the yeast STEll gene product (16), the tpll2 prc_ oncogene product (17), and a growth-factor sensitive enz--:e derived from PC12 rat pheochromocytoma cells (18). However, the precise specificity of these kinases in vivo is unclear, since some of them may participate in cascades leading to activation of the related stress-activated protein kinases (19,20).While the MAPK pathway is activated under many circumstances in tissue culture cells, the exact role of this pathway in vivo remains undefined. Approaches using dominant negative interfering mutant constructs of MEK have indicated that this pathway is required for nerve growth factor-dependent differentiation of PC12 cells. Furthermore, expression of constitutively activated mutants has resulted in transformation (21,22). We sought a more widely applicable method to determine the physiological role of this pathway by identifying selective inhibitors of specific components of the MAPK cascade. MATERIALS AND METHODSIn Vitro Kinase Assay. Incorporation of 32p into myelin basic protein (MBP) was assayed in the presence of glutathione S-transferase (GST) fusion proteins containing the 44-kDa MAPK (GST-MAPK) or the 45-kDa MEK (GST-MEK1). For direct evaluation of MEK activity, 10 ,tg of GST-MEK1 was incubated with 5 ,ug of a GST fusion protein containing 44-kDa MAPKwith a lysine-to-alanine mutation at position 71 (GST-MAPK-KA). This mutation eliminates kinase activity of MAPK, so that only kinase activity attributed to the added MEK remains. Similar incubations were performed with 5 ,ug of a fusion protein containing artificially partially activated MEK with serine-to-glutamate mutations at positions 218 and 222 (GST-MEK-2E). These assays utilized the same buffer and incubation conditions as described above. Phosphorylated MAPK-KA was resolved by SDS/10% PAGE and detected by autoradiography.Immunoprecipitation and Imminoblot Analysis. Tyrosinephosphorylated MAPK-KA was determined by using the same incubation protocol as for phosphorylation, but without radiolabeled, ATP. After electrophoresis, proteins on the gel were transferred to a nitrocellulose membrane, and nonspecific binding sites on the membrane were blocked by incubation with 1% ova...

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