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.

The protein kinase inhibitors staurosporine and K252A inhibit some of the cellular actions of nerve growth factor (NGF). To explore the molecular mechanisms involved, we test the ability of these agents to block one of the earliest cellular responses to NGF, protein tyrosine phosphorylation. Concentrations of 10-100 nM staurosporine and K252A inhibit NGF-dependent tyrosine phosphorylation in PC12 cells and inhibit trk oncogene-dependent tyrosine phosphorylation in trk-transformed NIH3T3 (trk-3T3 cells). In contrast, these compounds are without effect on epidermal growth factor (EGF)-stimulated tyrosine phosphorylation in PC12 cells. NGF-stimulated tyrosine phosphorylation of the pp140c-trk NGF receptor and tyrosine phosphorylation of pp70trk are also inhibited by similar concentrations of staurosporine and K252A, whereas tyrosine phosphorylation of the EGF receptor, insulin receptor, and v-src is not affected. Both staurosporine and K252A inhibit the autophosphorylation of pp70trk on tyrosine residues in an in vitro immune complex kinase reaction. Incubation of trk-3T3 cells with 10 nM staurosporine causes rounded transformed cells to revert to a normal flattened phenotype, whereas src-transformed cells are unaffected by this agent. These data suggest that staurosporine and K252A specifically inhibit the trk tyrosine kinase activity through a direct mechanism, probably accounting for the attenuation by these agents of the cellular actions of NGF.

Background and purpose: GPR119 is a G protein-coupled receptor that is preferentially expressed in islet cells and mediates insulin secretion. Oleoyl-lysophosphatidylcholine and oleoylethanolamide (OEA) act as endogenous ligands for this receptor, whereas PSN375963 and PSN632408 are two recently reported synthetic agonists. In this study, we explored mechanisms underlying GPR119-induced insulin secretion. In addition, we assessed the potential utility of the synthetic agonists as tools for exploring GPR119 biology. Experimental approach: We examined natural and synthetic GPR119 agonist activity at GPR119 in MIN6c4 and RINm5f insulinoma cells. We evaluated insulin secretion, intracellular calcium ½Ca 2þ i , ion channel involvement and levels of cAMP. Key results: We report that increases in insulin secretion induced by OEA were associated with increased cAMP and a potentiation of glucose-stimulated increases in ½Ca 2þi . We also demonstrate that ATP-sensitive K þ and voltage-dependent calcium channels were required for GPR119-mediated increases in glucose-stimulated insulin secretion. In contrast to OEA, the synthetic GPR119 agonist PSN375963 and PSN632408 have divergent effects on insulin secretion, cAMP and intracellular calcium in MIN6c4 cells. Conclusions and implications:The endogenous ligand OEA signals through GPR119 in a manner similar to glucagon-like peptide-1 (GLP-1) and its receptor with respect to insulin secretion, ½Ca 2þ i and cAMP. In addition, PSN375963 and PSN632408 substantially differ from OEA and from one another.