Delineating the Biology of Translational Repressor 4E-BP1

描述翻译抑制子 4E-BP1 的生物学

• 批准号: 10629541
• 负责人: Amanda Garner
• 金额: $ 7.76万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10629541
• 关键词: Affect; Automobile Driving; Binding; Binding Proteins; Biological Assay; Biological Process; Biology; CDK4 gene; Cell Cycle Progression; Cellular biology; Clinical; Complex; Disease; EIF4EBP1 gene; Eukaryotic Initiation Factors; FRAP1 gene; Gatekeeping; Genetic Translation; Growth; Knowledge; Link; Malignant Neoplasms; Mediating; Messenger RNA; Metabolic Diseases; Molecular; Neurodegenerative Disorders; Orphan; Peptide Initiation Factors; Phosphorylation; Phosphotransferases; Protein Biosynthesis; Proteins; Reporting; Role; Serine; Signal Transduction; Site; Specificity; Substrate Interaction; Threonine; Transcript; Translation Process; Translational Regulation; Translations; Work; base; chemoproteomics; new therapeutic target; protein function

Eukaryotic translation initiation factor (eIF4E)-binding protein 1 (4E-BP1) is an intrinsically disordered protein that functions as the gate-keeper of cap-dependent mRNA translation, the process by which mRNA transcripts containing a m7G cap at their 5’ terminus are converted into protein. As cap-containing mRNAs predominantly encode for growth and survival factors, the role of 4E-BP1 in cell biology is significant and aberrant 4E-BP1 activity has been linked to cancer, neurodegenerative diseases and metabolic disorders among others. 4E-BP1 is regulated by phosphorylation: hypophosphorylated 4E-BP1 binds strongly to eIF4E, the m7G cap-binding translation initiation factor, to inhibit translation, while hyperphosphorylated 4E-BP1 releases eIF4E to initiate translation. To date, the only kinase known to affect 4E-BP1 phosphorylation is mechanistic target of rapamycin complex 1 (mTORC1); however, reports have demonstrated that other unknown kinases can also phosphorylate 4E-BP1 to stimulate cap-dependent translation, particularly in cases of mTORC1 inhibition. In order to identify these kinases, we have developed an unbiased, chemoproteomic approach for identifying high confidence kinase-substrate interactions with phosphosite specificity. Using this assay, we have uncovered the role of cyclin- dependent kinase 4 (CDK4), a clinically validated kinase important for cell cycle progression, in driving cap- dependent translation via phosphorylation of 4E-BP1. Importantly, this work constitutes the first example of successful kinase discovery using an activity-based, kinase-directed probe. As 4E-BP1 is phosphorylated at as many as 13 unique sites, we hypothesize that many other kinases signal to and regulate 4E-BP1. Additionally, despite the critical role of 4E-BP1 phosphorylation in protein synthesis, few studies have been disclosed regarding the biological function of each of its phosphorylated serine and threonine residues, including its orphan sites known to be unaffected by mTORC1. To fill in these knowledge gaps, the Specific Aims of this proposal are as follows: (1) To determine the molecular details of CDK4-mediated 4E-BP1 hyperphosphorylation; (2) To determine the functional and mechanistic significance of CDK4-mediated 4E-BP1 hyperphosphorylation; and (3) To identify and validate additional kinases acting on 4E-BP1 using chemoproteomics. Through these studies, we will not only further enhance our knowledge of 4E-BP1-mediated translational regulation, but also illuminate new druggable targets for treatment of the many diseases associated with aberrant cap-dependent translation.

真核翻译起始因子结合蛋白1（4 E-BP 1）是一种本质上无序的蛋白质 作为帽依赖性mRNA翻译的守门人，mRNA转录的过程 在它们的5'末端含有m7 G帽的蛋白质被转化为蛋白质。由于含帽mRNA主要 4 E-BP 1编码生长和存活因子，在细胞生物学中的作用是显著和异常的4 E-BP 1 活性与癌症、神经退行性疾病和代谢紊乱等有关。4E-BP1 受磷酸化调节：低磷酸化的4 E-BP 1与eIF 4 E强烈结合，m7 G帽结合 翻译起始因子，以抑制翻译，而过度磷酸化的4 E-BP 1释放eIF 4 E，以启动 翻译.迄今为止，已知影响4 E-BP 1磷酸化的唯一激酶是雷帕霉素的机制靶点 复合物1（mTORC 1）;然而，报告表明，其他未知的激酶也可以磷酸化 4 E-BP 1刺激帽依赖性翻译，特别是在mTORC 1抑制的情况下。以便识别 这些激酶，我们已经开发出一种公正的，化学蛋白质组学的方法，用于确定高置信度 具有磷酸化位点特异性的激酶-底物相互作用。使用这种检测，我们发现了细胞周期蛋白的作用， 依赖性激酶4（CDK 4），一种临床验证的对细胞周期进展重要的激酶，在驱动帽- 通过4 E-BP 1的磷酸化依赖翻译。重要的是，这项工作构成了第一个例子， 使用基于活性的激酶导向探针成功发现激酶。As 4 E-BP 1被磷酸化， 多达13个独特的位点，我们假设许多其他激酶的信号和调节4 E-BP 1。此外，本发明还 尽管4 E-BP 1磷酸化在蛋白质合成中起关键作用，但很少有研究被公开 关于其每个磷酸化丝氨酸和苏氨酸残基的生物学功能，包括其孤儿残基， 已知不受mTORC 1影响的部位。为了填补这些知识空白，本提案的具体目标 目的：（1）研究CDK 4介导的4 E-BP 1过度磷酸化的分子机制;（2）研究CDK 4介导的4 E-BP 1过度磷酸化的分子机制。 确定CDK 4介导的4 E-BP 1过度磷酸化的功能和机制意义;和（3） 使用化学蛋白质组学鉴定和验证作用于4 E-BP 1的其他激酶。通过这些研究， 我们不仅将进一步提高我们对4 E-BP 1介导的翻译调控的认识， 用于治疗与异常帽依赖性翻译相关的许多疾病的新的可药用靶标。