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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Activation of phosphatidylinositol 3Ј-kinase (PI 3-kinase) 1 appears to play a pivotal role in signal transduction by growth factors and insulin (1, 2). PI 3-kinase has the unusual property of phosphorylating phosphatidyinositides on the 3Ј position generating phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) in vivo (3). These two products are thought to act as second messengers in processes such as mitogenesis (4, 5), oncogenic transformation (6), apoptosis (7), and various types of membrane trafficking (8, 9). Specifically, PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 bind to the pleckstrin homology (PH) domains of the Akt/PKB protein kinase and of the PDK1 protein kinase which activates Akt/PKB (10 -15). Binding of PtdIns(3,4,5)P 3 activates PDK1 and PtdIns(3,4)P 2 has been reported to preferentially activate Akt/PKB (12-16). In addition, PtdIns(3,4)P 2 and PtdIns (3,4,5)P 3 activate several isoforms of protein kinase C (17), and in vitro binding of PtdIns(3,4,5)P 3 to the SH2 domains of p85 and Src SH2 has been demonstrated (18).The production of some forms of phosphoinositides depends on the activities of inositol polyphosphate-5-phosphatases. Many different species of these enzymes exist, with a recently described member, SHIP (for SH2 domain-containing inositol 5-phosphatase), being implicated in receptor signaling in hematopoietic cells) (19 -21). SHIP is tyrosine phosphorylated in response to treatment of cells with erythropoietin, interleukin-2, interleukin-3, macrophage colony-stimulating factor, B cell receptor cross-linking, and T cell activation (22); however, in most cases, phosphorylation does not appear to affect activity. SHIP has an SH2 domain, multiple proline-rich sites representing possible sites of interaction with SH3 domains, two NPXY motifs, and associates with Shc, Grb2, and SHP-2 under certain conditions (23-25). The 5Ј-phosphatase activity of SHIP is specific for phosphatidyinositols and inositol which are phosphorylated at the 3Ј position (23). In some systems, SHIP appears to negatively regulate cell growth (21) or induce apoptosis (25), perhaps counteracting growth factor signals. It is not clear whether tyrosine phosphorylation of SHIP promotes or diminishes these inhibitory effects.Since PI 3-kinase appears to play a fundamental role in signal transduction in all mammalian cell types and SHIP expression is limited to hematopoietic cells, we examined whether the more widely expressed SHIP-related protein 51C/ SHIP2 (26, 27) is involved in signaling from receptor tyrosine kinases in other cells. EXPERIMENTAL PROCEDURESReagents-Rabbit antisera were raised against a glutathione S-transferase fusion protein containing carboxyl-terminal residues 1105-1213 of the SHIP2 sequence (27) as described previously (28 Cell Culture-SH-SY5Y human neuroblastoma cells were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. PC12 cells (a kind gift from D. Maysinger, McGill University) were grown in Dulbecco's modif...

In a previous study, we found that the SHIP2 protein became tyrosine phosphorylated and associated with the Shc adapter protein in response to the treatment of cells with growth factors and insulin (T. Habib, J. A. Hejna, R. E. Moses, and S. J. Decker, J. Biol. Chem. 273:18605-18609, 1998). We describe here a novel interaction between SHIP2 and the p130Cas adapter protein, a mediator of actin cytoskeleton organization. SHIP2 and p130Cas association was detected in anti-SHIP2 immunoprecipitates from several cell types. Reattachment of trypsinized cells stimulated tyrosine phosphorylation of SHIP2 and increased the formation of a complex containing SHIP2 and a faster-migrating tyrosine-phosphorylated form of p130Cas . The fastermigrating form of p130Cas was no longer recognized by antibodies to the amino terminus of p130 Cas and appeared to be generated through proteolysis. Interaction of the SHIP2 protein with the various forms of p130Cas was mediated primarily through the SH2 domain of SHIP2. Immunofluorescence studies indicated that SHIP2 localized to focal contacts and to lamellipodia. Increased adhesion was observed in HeLa cells transiently expressing exogenous WT-SHIP2. These effects were not seen with SHIP2 possessing a mutation in the SH2 domain (R47G). Transfection of a catalytic domain deletion mutant of SHIP2 (⌬RV) inhibited cell spreading. Taken together, our studies suggest an important role for SHIP2 in adhesion and spreading.Products of phosphatidylinositol (PI) metabolism are important second messengers in cellular signaling pathways (1,11,70,76). Activation of PI 3Ј-kinase, which phosphorylates the 3Ј position of the inositol ring of PI, is a critical event in growth factor, insulin, and G protein-mediated signal transduction (14,25,49). In addition, PI 3Ј-kinase plays an important role in the regulation of adhesion and migration (68). PI 3Ј-kinase activation localizes to cell-cell and cell-matrix adhesion sites in epithelial cells, as well as to membrane ruffles in fibroblasts (78). Inhibition of PI 3Ј-kinase attenuated integrin-mediated adhesion and migration in several cell types, while expression of the catalytic p110 subunit of PI 3Ј-kinase enhanced cell adhesion (18,22,29,31,33,34,52,80). Moreover, p85, the regulatory subunit of PI 3Ј-kinase, interacted with proteins regulating adhesion and migration such as focal adhesion kinase (FAK) and p130Crk -associated substrate (p130 Cas ) (3,8,43).In vivo, the major substrate for PI 3Ј-kinase is phosphatidylinositol-4,5-bisphosphate [PI-(4,5)-P2] leading to the formation of phosphatidylinositol-3,4,5-trisphosphate [PI-(3,4,5)-P3] (63). Significant pools of phosphatidylinositol-3,4-bisphosphate [PI-(3,4)-P2] are also generated following PI 3Ј-kinase activation primarily through dephosphorylation of PI-(3,4,5)-P3 by 5Ј inositol phosphatases (27). PI-(3,4,5)-P3 and PI-(3,4)-P2 specifically interact with pleckstrin homology (PH) domains of proteins, regulating activity or intracellular localization of cellular enzymes such as Akt/PKB and its upstream k...

It is shown that RalA is regulated by a Ral GAP complex (RGC 1/2) in insulin action and links PI 3-kinase signaling to RalA activation. Akt phosphorylates the complex and inhibits its function, resulting in increased RalA activity and glucose uptake.