PERSISTENT KINASE SIGNALS IN PC12 CELL DIFFERENTIATION

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The long-term goal of this proposal is to define the specific effector enzymes and protein kinases involved in growth factor receptor-regulated cell differentiation and growth. As a model system the PC12 pheochromocytoma cell line reversibly responds to nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) with partial growth arrest in G1 and neurite extension while epidermal growth factor (EGF) and insulin-like growth factor-I (IGF-I) fail to induce differentiation and instead, exert modest mitogenic actions. All of these growth factors signal through membrane-bound receptor tyrosine kinases. To date, the specific signals that distinguish the differentiation action of NGF and bFGF from the mitogenic actions of EGF and IGF-I remain poorly defined. Recent findings in this lab indicate that the p42/44 mitogen-activated protein (MAP) kinases are persistently activated and tyrosine phosphorylated by growth factors that direct differentiation while mitogens cause only transient activation of the pathway. Based on this finding and the requirement for constant growth factor exposure to maintain the differentiated PC12 cell phenotype, this proposal will test the hypothesis that persistent activation of specific effector enzymes and protein kinases discriminates those growth factors that induce differentiation from those that exert mitogenic actions in PC12 cells. The specific aims of the project are to l) identify the effector enzymes (GAP, P13-K, PLCgamma, etc.) required for induction of PC12 cell differentiation using mutant human PDGF receptors that lack the ability to activate one or more effector enzymes. The betaPDGF receptor is a receptor tyrosine kinase that is absent in parental PC12 cells, but directs reversible neurite outgrowth, partial growth arrest and persistent MAP kinase activation similar to NGF and bFGF when stably transfected into the cells. This permits a molecular genetic strategy to dissect the elements of growth factor receptor signal transduction involved in PC12 cell differentiation. This proposal also seeks to 2) define the mechanism by which NGF, bFGF and PDGF stimulate persistent phosphorylation and activation of the p42/44 MAP kinases in differentiating PC12 cells. Recombinant p42 MAP kinase will be used as a protein kinase substrate to identify and assay protein kinases that phosphorylate and activate the MAP kinases. Also, the p54 MAP kinase and p34-cdc2 protein kinases which are related to the p42/44 MAP kinases will be examined for differential regulation by neurotrophic factors and mitogens. Finally, 3) mutated forms of p42 MAP kinase that may exhibit dominant-negative phenotypes will be expressed in PC12 cells to ascertain the requirement for p42/44 MAP kinases in growth factor signalling of PC12 cell differentiation. Together, these aims will begin to define the network of effectors and protein kinases that transduce the receptor tyrosine kinase-stimulated signals in PC12 cells resulting in cell growth and differentiation.