The guanosine triphosphate (GTP)-binding protein Ras functions in regulating growth and differentiation; however, little is known about the protein interactions that bring about its biological activity. Wild-type Ras or mutant forms of Ras were covalently attached to an insoluble matrix and then used to examine the interaction of signaling proteins with Ras. Forms of Ras activated either by mutation (Gly12Val) or by binding of the GTP analog, guanylyl-imidodiphosphate (GMP-PNP) interacted specifically with Raf-1 whereas an effector domain mutant, Ile36Ala, failed to interact with Raf-1. Mitogen-activated protein kinase (MAP kinase) activity was only associated with activated forms of Ras. The specific interaction of activated Ras with active MAP kinase kinase (MAPKK) was confirmed by direct assays. Thus the forming of complexes containing MAPKK activity and Raf-1 protein are dependent upon the activity of Ras.

The tumor suppressor protein PTEN is mutated in glioblastoma multiform brain tumors, resulting in deregulated signaling through the phosphoinositide 3-kinase (PI3K)-protein kinase B (PKB) pathway, which is critical for maintaining proliferation and survival. We have examined the relative roles of the two major phospholipid products of PI3K activity, phosphatidylinositol 3,4-biphosphate [PtdIns(3,4)P2] and phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], in the regulation of PKB activity in glioblastoma cells containing high levels of both of these lipids due to defective PTEN expression. Reexpression of PTEN or treatment with the PI3K inhibitor LY294002 abolished the levels of both PtdIns(3,4)P2 and PtdIns(3,4,5)P3, reduced phosphorylation of PKB on Thr308 and Ser473, and inhibited PKB activity. Overexpression of SHIP-2 abolished the levels of PtdIns(3,4,5)P3, whereas PtdIns(3,4)P2 levels remained high. However, PKB phosphorylation and activity were reduced to the same extent as they were with PTEN expression. PTEN and SHIP-2 also significantly decreased the amount of PKB associated with cell membranes. Reduction of SHIP-2 levels using antisense oligonucleotides increased PKB activity. SHIP-2 became tyrosine phosphorylated following stimulation by growth factors, but this did not significantly alter its phosphatase activity or ability to antagonize PKB activation. Finally we found that SHIP-2, like PTEN, caused a potent cell cycle arrest in G 1 in glioblastoma cells, which is associated with an increase in the stability of expression of the cell cycle inhibitor p27 KIP1 . Our results suggest that SHIP-2 plays a negative role in regulating the PI3K-PKB pathway.

Nerve growth factor (NGF) activates the mitogen-activated protein (MAP) kinase cascade through a p21r'-dependent signal transduction pathway in PC12 cells. The linkage between p2l'as and MEKI was investigated to identify those elements which participate in the regulation of MEK1 activity. We have screened for MEK activators using a coupled assay in which the MAP kinase cascade has been reconstituted in vitro. We report that we have detected a single NGF-stimulated MEK-activating activity which has been identified as B-Raf. PC12 cells express both B-Raf and c-Rafl; however, the MEK-activating activity was found only in fractions containing B-Raf. c-Rafl-containing fractions did not exhibit a MEK-activating activity. Gel filtration analysis revealed that the B-Raf eluted with an apparent Mr of 250,000 to 300,000, indicating that it is present within a stable complex with other unidentified proteins. Immunoprecipitation with B-Raf-specific antisera quantitatively precipitated all MEK activator activity from these fractions. We also demonstrate that B-Raf, as well as c-Rafl, directly interacted with activated p2l' immobilized on silica beads. NGF treatment of the cells had no effect on the ability of B-Raf or c-Rafl to bind to activated p2l'. These data indicate that this interaction was not dependent upon the activation state of these enzymes; however, MEK kinase activity was found to be associated with p2l' following incubation with NGF-treated samples at levels higher than those obtained from unstimulated cells. These data provide direct evidence that NGF-stimulated B-Raf is responsible for the activation of the MAP kinase cascade in PC12 cells, whereas c-Rafl activity was not found to function within this pathway.Nerve growth factor (NGF) is responsible for the survival and differentiation of distinct subsets of neurons both within the central nervous system and in the periphery. The clonal rat pheochromocytoma cells, PC12, have proven to be a valuable model in which to investigate NGF action (15). These cells respond to NGF by cessation of division and then differentiate into a sympathetic-like neuronal phenotype. NGF initiates its actions through interaction with the proto-oncogene trk (trkA) (5, 25). TrkA possesses an intrinsic protein tyrosine kinase activity that is activated upon NGF binding, resulting in receptor autophosphorylation and phosphorylation of other proteins which participate in the signal transduction process. The detailed mechanisms through which NGF elicits its specific biochemical effects have been elusive; however, it is clear that the transmission of signals from the cell surface involves the activation of the G protein p2lras. This is followed by the serial activation of other protein kinases, driving a cascade of protein phosphorylation that mediates the specific biochemical events characteristic of NGF action through the phosphorylation of cytosolic proteins and nuclear transcription factors (7,10,18,66 (8,14,33,38

In order to identify novel substrates involved in insulin receptor signaling, a yeast two-hybrid 3T3-L1 adipocyte cDNA library was screened with the cytoplasmic domain of the human insulin receptor as bait. Here we describe the isolation and characterization of an interacting protein, APS, which contains pleckstrin homology and Src homology 2 domains and several potential tyrosine phosphorylation sites. APS mRNA and protein are expressed primarily in skeletal muscle, heart, and adipose tissue, and in differentiated 3T3-L1 adipocytes. We show that APS associates with phosphotyrosines situated within the activation loop of the insulin receptor via the APS Src homology 2 domain. Insulin stimulation of 3T3-L1 adipocytes resulted in rapid tyrosine phosphorylation of endogenous APS on tyrosine 618, whereas platelet-derived growth factor treatment resulted in no APS phosphorylation. In summary, we have identified a new insulin receptor substrate that is primarily expressed in insulin-responsive tissues and in 3T3-L1 adipocytes whose phosphorylation shows insulin receptor specificity. These findings suggest a potential role for APS in insulin-regulated metabolic signaling pathways.Insulin binding to its receptor results in tyrosine autophosphorylation of the ␤-subunit. This results in an active insulin receptor that is able to phosphorylate several cytoplasmic proteins on tyrosine residues. The tyrosine-phosphorylated insulin receptor interacts with downstream docking proteins that appear to be essential for insulin signaling. Phosphorylation of these adapter proteins induces their association with proteins containing SH2 1 domains, resulting in activation of a variety of downstream signaling pathways. Examples of such adapter proteins include the insulin receptor substrates (IRS) 1, 2, 3, and 4 and the SHC proto-oncogene product. These adapter proteins appear to link the insulin receptor to at least two signaling pathways, the phosphatidylinositol 3-kinase (PI3K) pathway and the Ras-mitogen-activated protein kinase pathway. It remains unclear whether these adapters constitute the full complement of signaling intermediates utilized by the insulin receptor. In fact, much recent evidence has begun to accumulate to suggest that the known signaling pathways that emanate from the IR may be insufficient to fully explain insulin-mediated metabolic regulation, and in particular the regulation of GLUT4 translocation.One of the most important signaling pathways that appears to be required for many metabolic effects of insulin is the activation of PI3K. Binding of the p85 regulatory subunit of PI3K to tyrosine-phosphorylated IRS proteins leads to activation of the lipid kinase activity of the p110 catalytic subunit of this enzyme (1, 2). The metabolic effects of insulin that require PI3K activity include GLUT4 translocation, glucose uptake, activation of fatty acid synthase and glycogen synthase, and stimulation of amino acid transport and protein synthesis (3-5). All of these actions were inhibited either by pharmacological inhib...

Recent studies have demonstrated the existence of a physical complex containing p2lras (RAS), p74raf-4 (RAF-1), and MEK-1. Although it is clear that formation of this complex depends on the activation state of RAS, it is not known whether this complex is regulated by the activation state of the cell and whether MEK-2 is also present in the complex. To analyze the regulation and specificity of this complex, we utilized immobilized RAS to probe lysates of cultured NIH 3T3 fibroblasts and analyzed the proteins complexing with RAS following serum starvation or stimulation. Complex formation among RAS, RAF-1, and MEK-1 was dependent only on RAS:GMP-PNP and not on cell stimulation. Incubations of lysates with immobilized RAS depleted all RAF-1 from the lysate but bound only a small fraction of cytosolic MEK-1, and further MEK-1 could bind immobilized RAS only if exogenous RAF-1 was added to the lysate. This indicates that binding of MEK-1 to RAS depends on the presence of RAF-1 or an equivalent protein. In contrast to MEK-1, MEK-2 was not detected in the RAS signalling complex. A proline-rich region of MEK-1 containing a phosphorylation site appears to be essential for signalling complex formation. Consistent with the preferential binding of MEK-1 to RAS:RAF-1, the basal activity of MEK-1 in v-ras-transformed cells was found to be elevated sixfold, whereas MEK-2 was elevated only twofold, suggesting that the RAS signalling pathway favors MEK-1 activation. p2lras (RAS) is a membrane-associated guanine nucleotidebinding protein which is active when bound to GTP and inactive when bound to GDP (31). Activation of RAS occurs in response to numerous agonists associated with growth and differentiation, reflecting the importance of RAS as a molecular switch regulating diverse cellular responses (22). The ratio of GTP to GDP can be regulated either by controlling the rate of nucleotide exchange or by regulating the rate of GTP hydrolysis (10, 31). Activation of RAS is sufficient to stimulate a kinase cascade leading to the activation of mitogen-activated protein (MAP) kinases (40, 41); in most cases, activation of RAS is also necessary for activation of MAP kinases (9,26,36).Recent studies have clarified some of the components which lie downstream from RAS in the regulation of MAP kinases. The MAP kinases are dually phosphorylated on threonine and tyrosine by MAP kinase kinases (MKKs or MEKs) (1, 2, 44), and at least two MEKs, MEK-1 and MEK-2, are known to be able to catalyze this phosphorylation in vitro (3,7,16,27,33,42,43,46). MEK-1 is a phosphoprotein, regulated by serine/ threonine phosphorylation, and a substantial body of evidence suggests that p74 ra-(RAF-1) is capable of catalyzing this activating phosphorylation (8,14,17,21,25,29). Although v-RAF can phosphorylate MEK-2 in vitro (42), it is as yet unclear how MEK-2 is regulated in vivo and whether there are functional or regulatory differences between MEK-1 and MEK-2.The role of RAS in the regulation of the MAP kinase cascade is thought to involve direct physical ass...