Interleukin-1␤ (IL-1␤) is cytotoxic to rat pancreatic ␤-cells by inhibiting glucose oxidation, causing DNA damage and inducing apoptosis. Nitric oxide (NO) is a necessary but not sufficient mediator of these effects. IL-1␤ induced kinase activity toward Elk-1, activation transcription factor 2, c-Jun, and heat shock protein 25 in rat islets. By Western blotting with phosphospecific antibodies and by immunocomplex kinase assay, IL-1␤ was shown to activate extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38) in islets and rat insulinoma cells. Specific ERK1/2 and p38 inhibitors individually reduced but in combination blocked IL-1␤-mediated islet NO synthesis, and reverse transcription-polymerase chain reaction of inducible NO synthase mRNA showed that ERK1/2 and p38 controlled IL-1␤-induced islet inducible NO synthase expression at the transcriptional level. Hyperosmolarity caused phosphorylation of Elk-1, activation transcription factor 2, and heat shock protein 25 and activation of ERK1/2 and p38 in islets comparable to that induced by IL-1␤ but did not lead to NO synthesis. Inhibition of p38 but not of ERK1/2 attenuated IL-1␤-mediated inhibition of glucose-stimulated insulin release. We conclude that ERK1/2 and p38 activation is necessary but not sufficient for IL-1␤-mediated ␤-cell NO synthesis and that p38 is involved in signaling of NO-independent effects of IL-1␤ in ␤-cells.

Homophilic binding in trans of the neural cell adhesion molecule (NCAM) mediates adhesion between cells and leads, via activation of intracellular signaling cascades, to neurite outgrowth in primary neurons as well as in the neuronal cell line PC12. NCAM mediates neurite extension in PC12 cells by two principal routes of signaling: NCAM/Fyn and NCAM/fibroblast growth factor receptor (FGFR), respectively. Previous studies have shown that activation of mitogen-activated protein kinases is a pivotal point of convergence in NCAM signaling, but the mechanisms behind this activation are not clear. Here, we investigated the involvement of adaptor proteins in NCAM and fibroblast growth factor 2 (FGF2)-mediated neurite outgrowth in the PC12-E2 cell line. We found that both FGFR substrate-2 and Grb2 play important roles in NCAM as well as in FGF2-stimulated events. In contrast, the docking protein ShcA was pivotal to neurite outgrowth induced by NCAM, but not by FGF2, in PC12 cells. Moreover, in rat cerebellar granule neurons, phosphorylation of ShcA was stimulated by an NCAM mimicking peptide, but not by FGF2. This activation was blocked by inhibitors of both FGFR and Fyn, indicating that NCAM activates FGFR signaling in a manner distinct from FGF2 stimulation, and regulates ShcA phosphorylation by the concerted efforts of the NCAM/FGFR as well as the NCAM/ Fyn signaling pathway.

Treatment of cells with insulin results in activation of signal transduction pathways involved in metabolic as well as mitogenic responses. The MAP kinase pathway is in this context of considerable importance and has been investigated intensively. Tyrosine phosphorylation of SHC through the insulin receptor has been shown to activate MAP kinase in a GRB2-SOS-Ras-dependent manner. The GRB-2-SOS complex interacts also with tyrosine phosphorylated IRS-i and contributes to MAP kinase activation (Skolnik et al., 1993, Myers et al., 1994. A third alternative to activate MAP kinase in a Ras-dependent manner involves binding of the tyrosine specific phosphatase SHPTP2 to tyrosine phosphorylated IRS-I (Noguchi et al. 1994), however, the precise mechanism is still unclear. A fourth signal transduction pathway leading to MAP kinase activation may involve PI-3 kinase which is activated in response to insulin and various growth factors. This enzyme phosphorylates the D-3 position of the inositol ring in phosphatidylinositol leading to the second messengers PI3P, P13,4P2 and P13,4,5P3. It has ecently been shown that P13.4,5P3 acts as an agonist for the PKC isoform Ç and Pi-3 kinase positively regulates PKC 2.. PKC has the potential to phosphorylate and activate Raf, which in turn activates MEK resulting in MAP kinase activation. A fifth possible mechanism involves PLCy which hydrolyses in response to various growth factors PIP2 into DAG and IP3. lP3 causes the release of Ca2 from intracellular stores and Ca together with membrane bound DAG activates certain PKC isoforms, implying that they also acitvate MAP kinase in a Raf and Mek dependent manner. Whether and how PLC1 might be involved in raising the intracellular Ca and DAG level in response to insulin is, however, not clear.We have investigated the involvement of GRB2, SHPTP2, pi-3 kinase PLCy and various PKC isoforms in insulin-mediated activation of MAP kinase by using transiently transfected A-293 fibroblasts as a model system.Treatment of untransfected 293 cells with insulin results in a transient activation of MAP kinase which peaks at 10 minutes and declines thereafter within 60 minutes to almost basal levels. In order to confirm the involvement of GRB2 and SHPTP2 in insulin mediated activation of MAP kinase the GRB2 SH2 domain mutant R86A and the catalytically inactive SHPTP2 mutant C459A were tested for their ability to block MAP kinase activation in response to insulin. Indeed, the mutant versions of GRB2 and SHPTP2 reduce MAP kinase activation dramatically confirming their involvement in insulin induced MAP kinase activation. 0v-erexpression of wild type GRB2 had no inhibitory and no activating effect, while SHPTP2 in an expression level dependent manner positively regulates MAP kinase activation. Having shown that GRB2 and SHPTP2 are important mediators of MAP kinase activation in response to insulin also in A-293 fibroblasts, we next investigated the effect of overexpressed wild type PLC'y and catalytically inactive PLCy H335F, H380F (Smith et al., 1994). Overex...