PHOSPHORYLATION SITES IN ISOFORMS OF INITIATION FACTOR EIF4E IN CELEGANS

CELEGANS 引发因子 EIF4E 异构体中的磷酸化位点

• 批准号: 7724219
• 负责人: ROBERT E. RHOADS
• 金额: $ 0.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7724219
• 关键词: Animal Model; Animals; Biochemical; Biochemical Genetics; Biological; Caenorhabditis elegans; Complex; Computer Retrieval of Information on Scientific Projects Database; Data; Development; Funding; Goals; Grant; Half-Life; Homologous Gene; Institution; Knock-out; Knowledge; Mammals; Mass Spectrum Analysis; Messenger RNA; Organism; Peptide Initiation Factors; Phenotype; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiological; Process; Production; Protein Biosynthesis; Protein Isoforms; RNA Cap-Binding Proteins; Rate; Regulation; Research; Research Personnel; Resources; Ribosomes; Role; Signal Pathway; Site; Source; Tissues; Transgenic Organisms; Translating; United States National Institutes of Health; prevent

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long-term goals are to understand the biochemical mechanisms, physiological regulation, and biological roles of the mRNA cap-binding protein eIF4E, an initiation factor involved in the recruitment of mRNA to the ribosome. This process determines the rate of protein synthesis, the spectrum of mRNAs translated, and the rate of mRNA turnover. This specific project is centered on the physiological role of eIF4E phosphorylation. Although the rate of protein synthesis and the phosphorylation of eIF4E are highly correlated, and although eIF4E phosphorylation has been evolutionarily conserved in all animals examined, the biochemical consequences of eIF4E phosphorylation remain elusive and controversial. The most definitive data are likely to come from genetically tractable animal models like C. elegans. We have discovered and extensively characterized five isoforms of eIF4E in C. elegans, termed IFE-1, IFE-2, etc., including the production of knockout strains for all five isoforms. In one case, we have been able to rescue the wild-type phenotype by transgenic expression of the missing IFE. In mammals, eIF4E is phosphorylated at a single site in the C-terminus and by two kinases termed Mnk1 and Mnk2. These are tethered to eIF4G, a component of translational initiation complexes. We propose to determine the phosphorylation sites in each of the five IFEs by mass spectrometry. Then we will alter the site in one particular IFE to prevent phosphorylation. We will determine the result of expressing the modified form in knockout worms with regard to overall rate of protein synthesis, tissue and developmental expression of the IFE, half-life of IFE, spectrum of mRNAs translated, and overall phenotype of the organism. Knowledge of the phosphorylation sites will also assist us in finding the kinase(s) responsible for IFE phosphorylation and specifically determine whether a C. elegans homologue of Mnk is responsible. Since we have discovered the kinase(s) responsible, we can study their upstream signaling pathways using biochemical and genetic approaches.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 长期目标是了解mRNA帽结合蛋白eIF 4 E的生化机制，生理调节和生物学作用，eIF 4 E是一种参与mRNA向核糖体募集的起始因子。 这一过程决定了蛋白质合成的速率、mRNA翻译的范围和mRNA周转的速率。 这个特定的项目是集中在eIF 4 E磷酸化的生理作用。 尽管蛋白质合成速率和eIF 4 E磷酸化高度相关，尽管eIF 4 E磷酸化在所有研究的动物中进化上保守，但eIF 4 E磷酸化的生化后果仍然难以捉摸且有争议。 最权威的数据可能来自遗传学上易于处理的动物模型，如C。优雅的 我们已经在C.线虫，称为IFE-1、IFE-2等，包括产生所有五种同种型的敲除菌株。 在一种情况下，我们已经能够拯救野生型表型的缺失IFE的转基因表达。 在哺乳动物中，eIF 4 E在C末端的单个位点被称为Mnk 1和Mnk 2的两种激酶磷酸化。 它们与eIF 4G（翻译起始复合物的一种组分）相连。 我们建议通过质谱法确定五个IFE中每个IFE的磷酸化位点。 然后，我们将改变一个特定IFE中的位点以防止磷酸化。 我们将确定在基因敲除蠕虫中表达修饰形式的结果，包括蛋白质合成的总体速率、IFE的组织和发育表达、IFE的半衰期、翻译的mRNA谱和生物体的总体表型。 磷酸化位点的知识也将帮助我们找到负责IFE磷酸化的激酶，特别是确定C。与Mnk同源的elegans同源物负责。 由于我们已经发现了负责的激酶，我们可以使用生物化学和遗传学方法研究它们的上游信号通路。