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...

The mitogen-activated protein kinase (MAP kinase) pathway is thought to play an important role in the actions of neurotrophins. A small molecule inhibitor of the upstream kinase activator of MAP kinase, MAP kinase kinase (MEK) was examined for its effect on the cellular action of nerve growth factor (NGF) in PC-12 pheochromocytoma cells. PD98059 selectively blocks the activity of MEK, inhibiting both the phosphorylation and activation of MAP kinases in vitro. Pretreatment of PC-12 cells with the compound completely blocked the 4-fold increase in MAP kinase activity produced by NGF. Half-maximal inhibition was observed at 2 microM PD98059, with maximal effects at 10-100 microM. The tyrosine phosphorylation of immunoprecipitated MAP kinase was also completely blocked by the compound. In contrast, the compound was without effect on NGF-dependent tyrosine phosphorylation of the pp140trk receptor or its substrate Shc and did not block NGF-dependent activation of phosphatidylinositol 3'-kinase. However, PD98059 completely blocked NGF-induced neurite formation in these cells without altering cell viability. These data indicate that the MAP kinase pathway is absolutely required for NGF-induced neuronal differentiation in PC-12 cells.

A series of rat neuro/glioblastomas all contain the same transforming gene (neu) which induces synthesis of a tumour antigen of relative molecular mass (Mr) 185,000 (p185). The neu oncogene bears homology to erb-B and the tumour antigen, p185, is serologically related to the epidermal growth factor (EGF) receptor. The two proteins, EGF receptor and p185 appear to be distinct, as they coexist in nontransformed Rat-1 cells.

Emerging evidence suggests that inflammation provides a link between obesity and insulin resistance. The noncanonical IκB kinases IKKε and TANK-binding kinase 1 (TBK1) are induced in liver and fat after high fat diet by NF-κB activation, and in turn initiate a program of counter-inflammation that preserves energy storage. Here, we report the discovery of a small molecule inhibitor of these kinases called amlexanox. Treatment of obese mice with amlexanox elevates energy expenditure through increased thermogenesis, producing weight loss, improved insulin sensitivity and decreased steatosis in obese mice. Because of its record of safety in patients, amlexanox may be an interesting candidate for clinical evaluation in the treatment of obesity and related disorders.

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