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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 长期目标是了解mRNA帽结合蛋白EIF4E的生化机制，生理调节以及生物学作用，这是将mRNA募集到核糖体中涉及的起始因子。 该过程决定了蛋白质合成的速率，被翻译的mRNA谱和mRNA更新速率。 该特定项目集中在EIF4E磷酸化的生理作用上。 尽管蛋白质合成的速率和EIF4E的磷酸化速率高度相关，尽管在所有检查的动物中，EIF4E磷酸化在进化上都是保守的，但EIF4E磷酸化的生化后果仍然难以捉摸和有争议。 最明确的数据可能来自诸如秀丽隐杆线虫等遗传障碍动物模型。 我们已经发现并广泛地表征了秀丽隐杆线虫中EIF4E的五种同工型，称为IFE-1，IFE-2等，包括所有五种同工型的敲除菌株的产生。 在一种情况下，我们已经能够通过缺失IFE的转基因表达来挽救野生型表型。 在哺乳动物中，EIF4E在C末端的单个位点和两个称为MNK1和MNK2的激酶进行磷酸化。 这些被束缚于EIF4G，这是翻译起始复合物的组成部分。 我们建议通过质谱来确定五个IFE的磷酸化位点。 然后，我们将在一个特定的IFE中更改该位点以防止磷酸化。 我们将确定在敲除蠕虫中表达修饰形式的结果，这些蛋白质合成，组织和发育表达的总体速率，IFE的半衰期，IFE的半衰期，被翻译的mRNA光谱以及生物体的总体表型。 对磷酸化位点的了解还将有助于我们找到负责IFE磷酸化的激酶，并明确确定MNK的秀丽隐杆线虫同源物是否负责。 由于我们发现了负责任的激酶，因此我们可以使用生化和遗传方法研究其上游信号通路。