GANGLIOSIDE SIGNALING CASCADES FOR GLIOMA GROWTH

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DESCRIPTION: (adapted from the investigator's abstract) Gangliosides are potent modulators of cell growth and could have profound effects on glioma proliferation. The general hypothesis is that gangliosides can stimulate glioma proliferation by activating specific signal transduction pathways. This is based on their finding that exogenous treatment of U-1242 MG glioma cells with ganglioside GM I stimulated DNA synthesis by a signaling mechanism involving the rapid activation of the extracellular signal-regulated protein kinase 2 (Erk2) and p70 S6 kinase (p70s6k). They will use use these cells as a model for studying the mechanisms by which gangliosides stimulate signal transduction, and believes the studies should clarify the role of gangliosides in cell proliferation and this is important in human brain tumors. In Specific Aim 1 he will characterize ganglioside interactions at and within the cell surface and relate these to activation of the Erk2 and p70s6k signaling cascades. He will use metabolic inhibitors to test whether endogenous ganglioside biosynthesis is important for signaling by exogenous GM 1. He will also test whether activation of Erk2 correlates with the stable association of GM l with cell surface proteins or with the insertion of GM I into the membrane. Based on studies with wortmannin, a specific inhibitor of phosphatidylinositol 3'-kinase, (PI3K), he proposes that GM I activates PI3K. In Specific Aim 2 he will measure GM1stimulated in vivo production of 3-phosphoinositides to estimate PI3K activation and determine the effects of tyrosine kinase inhibitors and G-protein inhibitors on this activity. In part b he will identify GMI-stimulated molecules that regulate PI3K using biochemical, immunological and molecular approaches. In part c he will create cells that are defective for PI3K activation and use them for studying GM1 -mediated activation of Erk2 and p70 s6k. GM1 stimulates Erk2 by a mechanism involving activation of the Raf-1 oncoprotein as well as stimulates p70s6k by an uncharacterized pathway. In Specific Aim 3, he will use genetic and biochemical approaches to examine whether GM1 stimulates Ras, a well known activator of Raf-1. He will also use novel Raf mutants and phosphoamino acid profiles to to identify novel GM1-stimulated regulation of Raf. He will also use in vitro kinase assays to test whether GM1 activates p70s6k through the serine/threonine kinase Akt.