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: 6450687
• 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: 2001
• 资助国家: 美国
• 起止时间: 2001-04-01 至 2002-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6450687
• 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来研究可逆蛋白磷酸化在信号转导和发育中的作用。磷酸蛋白磷酸酶PP2A是真核生物中主要的丝氨酸/三磷酸酶。该酶被认为在信号转导中发挥重要作用，要么是通过组成性地去磷酸化响应于细胞外刺激而磷酸化的蛋白质，要么是在响应于这种刺激时直接被激活。然而，这一结论是基于该酶在体外对许多蛋白质进行去磷酸化的能力，其特异性较低，或者基于影响许多磷酸酶的抑制剂的作用。我们提出验证PP2A在体内信号转导和发育过程中被磷酸化激活的特定蛋白的去磷酸化中发挥重要作用的假设。真核微生物盘基盘齿柱（Dictyostelium disideum）特别适合这些研究，因为它的基因组样本短，发育周期明确，并且易于通过反向遗传学制造突变体。我们之前已经证明，胞外cAMP通过信号转导诱导基因，激活盘基骨菌的细胞表面受体。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PP2A （PP2Ac）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显性负突变，并通过位点特异性诱变构建其他负突变。突变体在发育上似乎有缺陷。我们计划确认和扩展这些研究，寻找可能被PP2A去磷酸化的底物蛋白或可能在响应信号时与酶相互作用的蛋白。我们还计划通过位点特异性诱变PP2Ac构建更多的抗冈田酸突变体。我们建议利用这些突变体来精确地确定PP2A参与发育或生长的点以及被激活的途径。据推测，PP2A可以使一种重要的信号酶，蛋白激酶B （PKB）去磷酸化，这一酶被广泛研究。哺乳动物PKB基因是一种原癌基因，也参与细胞凋亡的释放。我们已经敲除了盘基骨菌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阴性和冈田酸突变体来验证PP2A调节PKB信号转导活性的假设。这些研究应该更清楚地定义PP2A在体内信号传导中的相互作用。此外，该机构应该从与学生和教师以及所提供的技术的互动中受益。