PHOSPHOPROTEIN PHOSPHATASE PP2A IN EUKARYOTIC SIGNAL TRANSDUCTION

真核信号转导中的磷酸蛋白磷酸酶 PP2A

• 批准号: 6313791
• 负责人: ROBERT P DOTTIN
• 金额: $ 5.95万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6313791
• 关键词: Dictyostelium; biological signal transduction; enzyme activity; epitope mapping; eukaryote; immunoprecipitation; northern blottings; nucleic acid sequence; okadaic acid; phenotype; phosphoprotein phosphatase; phosphorylation; protein kinase; protein structure function; receptor binding; site directed mutagenesis; transfection; western blottings; yeast two hybrid system

We propose to investigate, by molecular genetic approaches, the role of reversible protein phosphorylation in signal transduction and development. The phospho-protein phospho-protein phosphatase PP2A is the major ser/thre phosphatase in eukaryotes. The enzyme is assumed to play an important role in signal transduction either by constitutively dephosphorylating proteins that are phosphorylated in response to extracellular stimuli, or becoming activated directly in response to such stimulation. However, this conclusion is based on the ability of the enzyme to dephosphorylate many proteins in vitro, where its specificity is low, or on the action of inhibitors that affect many phosphatases. We propose to test the hypothesis that PP2A play an important role in vivo in the dephosphorylation of specific proteins which are activated by phosphorylation during signal transduction and development. The eukaryotic microorganism Dictyostelium discoideum, is particularly suitable for these studies, because of its sample genome, short, well- defined developmental cycle and ease of making mutants by reverse genetics. We had previously shown that extracellular cAMP, which activates cell surface receptors in Dictyostelium, induces genes by signal transduction. The proteins involved in the pathways are highly conserved. In preliminary studies we cloned the gene for the catalytic subunit PP2A (PP2Ac) and transfected it intro wild type cells. We discovered dominant negative mutations by the transfection technique, and constructed others by site specific mutagenesis. The mutants appear to be defective in development. We plan to confirm and extend these studies, to look for substrate proteins that may dephosphorylated by PP2A or proteins that may interact with the enzyme in response to signaling. We also plan to construct more okadaic acid resistant mutants by site specific mutagenesis of PP2Ac. We propose to exploit these mutants to determine precisely the points at which PP2A is involved in development or growth and the pathways that are activated. PP2A is presumed to dephosphorylate an important and signaling enzyme, protein kinase B (PKB) which is intensively studied. The mammalian PKB gene is a proto-oncogene and is also involved in the release from apoptosis. We have knocked out the Dictyostelium PKB gene and observed that it affects development. We propose to test the hypothesis that PP2A regulates the signal transduction activities of PKB by use of the dominant negative and okadaic acid mutants described above. The studies should define more clearly the interactions that involve PP2A in signaling in vivo. In addition the institution should benefit from the interactions that quality arises from this research with students and faculty and the technologies provided.

我们建议通过分子遗传学方法研究可逆性蛋白磷酸化在信号转导和发育中的作用。磷酸蛋白磷酸酶PP 2A是真核生物中主要的丝氨酸/苏氨酸磷酸酶。该酶被认为在信号转导中起重要作用，通过组成性去磷酸化响应于细胞外刺激的磷酸化蛋白质，或直接响应于这种刺激而被激活。然而，这一结论是基于酶在体外使许多蛋白质脱磷酸化的能力，其中其特异性较低，或者基于影响许多磷酸酶的抑制剂的作用。我们建议测试的假设，PP 2A在体内发挥重要作用，在信号转导和发展过程中的磷酸化激活的特定蛋白质的去磷酸化。真核微生物盘基网柄藻特别适合于这些研究，因为其样品基因组、短的、明确定义的发育周期和易于通过反向遗传学产生突变体。我们以前已经表明，细胞外cAMP，激活细胞表面受体在网骨藻，诱导基因的信号转导。参与这些途径的蛋白质是高度保守的。在初步研究中，我们克隆了催化亚基PP 2A（PP 2Ac）的基因，并将其转染到野生型细胞中。我们通过转染技术发现了显性负突变，并通过定点突变构建了其他突变。突变体似乎在发育上有缺陷。我们计划确认并扩展这些研究，寻找可能被PP 2A去磷酸化的底物蛋白或可能与酶相互作用以响应信号的蛋白。我们还计划通过PP 2Ac的定点突变构建更多的冈田酸抗性突变体。我们建议利用这些突变体来精确地确定PP 2A参与发育或生长的点以及被激活的途径。PP 2A被认为是一种重要的信号酶，蛋白激酶B（PKB），这是深入研究去磷酸化。哺乳动物PKB基因是一种原癌基因，也参与细胞凋亡的释放。我们已经敲除了Dictyosteoprotein PKB基因，并观察到它会影响发育。我们建议通过使用上述显性负突变体和冈田酸突变体来检验PP 2A调节PKB信号转导活性的假设。这些研究应该更清楚地定义PP 2A在体内信号传导中的相互作用。此外，该机构应受益于互动，质量产生于这项研究与学生和教师和提供的技术。