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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 长期的目标是了解mRNA帽结合蛋白eIF4E的生化机制、生理调节和生物学作用，eIF4E是一种参与mRNA向核糖体募集的起始因子。这个过程决定了蛋白质合成的速度，翻译的mRNAs的光谱，以及mRNAs的周转率。这个特定的项目集中在eIF4E磷酸化的生理作用上。虽然蛋白质合成速率和eIF4E的磷酸化高度相关，尽管eIF4E的磷酸化在所有被研究的动物中在进化上都是保守的，但eIF4E磷酸化的生物化学后果仍然是难以捉摸和有争议的。最确定的数据可能来自易遗传的动物模型，比如线虫。我们已经在线虫中发现并广泛鉴定了五种eIF4E亚型，命名为IFE-1，IFE-2等，包括产生这五种亚型的基因敲除菌株。在一个案例中，我们已经能够通过转基因表达缺失的IFE来挽救野生型表型。在哺乳动物中，eIF4E在C末端的单个位置被称为Mnk1和Mnk2的两个激酶磷酸化。它们被拴在翻译起始复合体的一个组成部分eIF4G上。我们建议用质谱法确定五个IFE中每一个的磷酸化位点。然后，我们将改变一个特定的IFE中的位点，以防止磷酸化。我们将从蛋白质合成的总体速度、IFE的组织和发育表达、IFE的半衰期、翻译的mRNAs谱以及生物体的整体表型来确定在敲除蠕虫中表达修饰形式的结果。对磷酸化位点的了解也将有助于我们找到负责IFE磷酸化的激酶(S)，并具体确定是否有线虫MNK的同源物负责。既然我们已经发现了负责的激酶(S)，我们就可以用生化和遗传学的方法来研究它们的上游信号通路。