PERSISTENT KINASE SIGNALS IN PC12 CELL DIFFERENTIATION

PC12 细胞分化中的持续激酶信号

• 批准号: 3469039
• 负责人: LYNN E HEASLEY
• 金额: $ 9.93万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3469039
• 关键词: PC12 cells; SDS polyacrylamide gel electrophoresis; biological signal transduction; cell differentiation; cell growth regulation; enzyme activity; enzyme substrate; fibroblast growth factor; growth factor receptors; immunoprecipitation; neurotrophic factors; phosphorylation; platelet derived growth factor; protein tyrosine kinase; receptor expression; western blottings

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.

这项提案的长期目标是定义特定的效应器 参与生长因子受体调节的酶和蛋白激酶 细胞分化和生长。作为一个模型系统， 嗜铬细胞瘤细胞系对神经生长因子的可逆反应 (NGF)和碱性成纤维细胞生长因子（bFGF）， 在G1期和神经突延伸，而表皮生长因子（EGF）和 胰岛素样生长因子-I（IGF-I）不能诱导分化， 而是发挥适度的促有丝分裂作用。所有这些生长因子 通过膜结合受体酪氨酸激酶发出信号。迄今为止 区分NGF的分化作用的特异性信号， EGF和IGF-I的促有丝分裂作用的bFGF仍然不清楚。 本实验室的最新发现表明，p42/44丝裂原活化 蛋白质（MAP）激酶持续激活，酪氨酸 被生长因子磷酸化，指导分化， 有丝分裂原仅引起该途径的短暂激活。基于此 发现和恒定生长因子暴露的要求 维持分化的PC 12细胞表型，该提案将测试 这一假说认为特异性效应酶的持续激活和 蛋白激酶区分那些诱导 与那些在PC 12细胞中发挥促有丝分裂作用的细胞分化。的 该项目的具体目标是1）鉴定效应酶（GAP， P13-K、PLC γ等）诱导PC 12细胞分化所需的 使用突变的人PDGF受体，该受体缺乏激活一种或多种细胞因子的能力， 更多的效应酶β PDGF受体是一种受体酪氨酸激酶 在亲本PC 12细胞中不存在，但指导可逆的神经突 生长、部分生长停滞和持续性MAP激酶激活 类似于NGF和bFGF稳定转染入细胞。这 允许分子遗传策略来剖析生长的要素， 因子受体信号转导参与PC 12细胞分化。 该建议还试图2）定义NGF，bFGF和 PDGF刺激p42/44 MAP的持续磷酸化和激活 激酶在分化的PC 12细胞中的作用。重组p42 MAP激酶将在 用作蛋白激酶底物以鉴定和测定蛋白激酶 磷酸化并激活MAP激酶。此外，p54 MAP激酶 与p42/44 MAP激酶相关的p34-cdc 2蛋白激酶 将检查神经营养因子的差异调节， 有丝分裂原最后，3）p42 MAP激酶的突变形式，其可能表现出 显性阴性表型将在PC 12细胞中表达，以确定 PC 12细胞生长因子信号转导对p42/44 MAP激酶需求 细胞分化总之，这些目标将开始定义 受体周围的效应物和蛋白激酶网络 PC 12细胞中的酪氨酸激酶刺激信号导致细胞生长 和差异化。