PERSISTENT KINASE SIGNALS IN PC12 CELL DIFFERENTIATION

这项提议的长期目标是定义具体的效应者 参与生长因子受体调控的酶和蛋白激酶 细胞分化和生长。作为模型系统的PC12 嗜铬细胞瘤细胞系对神经生长因子的可逆性反应 (NGF)和碱性成纤维细胞生长因子(BFGF)部分生长停滞 在G1和轴突延伸中，表皮生长因子(EGF)和 胰岛素样生长因子-I(IGF-I)不能诱导分化 取而代之的是，发挥适度的促分裂作用。所有这些增长因素 通过膜结合受体酪氨酸激酶传递信号。到目前为止， 区分NGF和NGF分化作用的特定信号 碱性成纤维细胞生长因子是由EGF和IGF-I的促有丝分裂作用产生的，目前仍不清楚。 本实验室的最新发现表明，p42/44有丝分裂原激活 蛋白(MAP)激酶持续激活和酪氨酸 被指导分化的生长因子磷酸化，而 有丝分裂原只能引起该通路的短暂激活。在此基础上 持续接触生长因子的发现和要求 保持分化的PC12细胞表型，这一提议将测试 假设特定效应酶的持续激活和 蛋白激酶区别于那些诱导 与那些在PC12细胞中发挥有丝分裂作用的细胞有区别。这个 该项目的具体目标是L)确定效应酶(GAP， P13-K、PLC伽马等)诱导PC12细胞分化所必需的 使用缺乏激活一个或多个受体的能力的突变型人PDGF受体 更多的效应酶。β-PDGF受体是一种受体酪氨酸激酶 这在亲代PC12细胞中是不存在的，但导向可逆性轴突 生长、部分生长停滞和持续的MAPK激活 与NGF和bFGF稳定导入细胞时相似。这 允许一种分子遗传策略来剖析生长要素 因子受体信号转导参与PC12细胞分化。 该提案还寻求2)定义NGF、bFGF和 PDGF刺激p42/44 MAP持续磷酸化和激活 蛋白激酶在PC12细胞分化中的作用。重组p42 MAP激酶将被 用作蛋白激酶底物以鉴定和测定蛋白激酶 它会磷酸化并激活MAP的激酶。此外，p54的MAP激酶 和与p42/44相关的p34-cdc2蛋白激酶 将检查神经营养因子和神经营养因子的差异调节 有丝分裂原。最后，3)突变形式的p42蛋白激酶可能表现出 显性-阴性表型将在PC12细胞中表达，以确定 PC12生长因子信号转导中对p42/44 MAP激酶的需求 细胞分化。综合起来，这些目标将开始定义 转导受体的效应器和蛋白激酶网络 酪氨酸激酶刺激PC12细胞生长的信号 和差异化。

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.