Delineating the Biology of Translational Repressor 4E-BP1

真核细胞翻译起始因子结合蛋白1(4E-BP1)是一种天然无序的蛋白质 这是帽依赖的信使核糖核酸翻译的守门人，信使核糖核酸转录的过程 在其5‘末端含有m7G帽的蛋白质被转化为蛋白质。作为主要含帽的mRNA 编码生长和生存因子的4E-BP1在细胞生物学中的作用是显著的和异常的 活动与癌症、神经退行性疾病和代谢紊乱等疾病有关。4E-BP1 受磷酸化调节：低磷酸化的4E-BP1与m7G帽结合的eIF4E强烈结合 翻译起始因子，抑制翻译，而过度磷酸化的4E-BP1释放eIF4E启动 翻译。到目前为止，已知的唯一影响4E-BP1磷酸化的激酶是雷帕霉素的机械靶点 复合体1(MTORC1)；然而，报道表明，其他未知的激酶也可以磷酸化 4E-BP1刺激帽子依赖的翻译，特别是在mTORC1抑制的情况下。为了辨认 这些激酶，我们开发了一种无偏见的化学蛋白质组方法来识别高置信度 具有亚磷酸盐专一性的激酶-底物相互作用。使用这种分析方法，我们已经发现了细胞周期蛋白的作用- 依赖激酶4(CDK4)是一种临床验证的对细胞周期进程至关重要的激酶，在推动细胞周期进程中发挥重要作用。 4E-BP1的磷酸化依赖翻译。重要的是，这项工作构成了 使用基于活性的、以激酶为导向的探针成功地发现了激酶。AS 4E-BP1在AS上被磷酸化 多达13个独特的位置，我们假设，许多其他的激酶信号和调节4E-BP1。另外， 尽管4E-BP1的磷酸化在蛋白质合成中起着关键作用，但很少有研究披露 关于它的每个磷酸化丝氨酸和苏氨酸残基的生物学功能，包括它的孤儿 已知不受mTORC1影响的站点。为了填补这些知识空白，这项提议的具体目的 具体如下：(1)确定CDK4介导的4E-BP1过度磷酸化的分子细节；(2) 确定CDK4介导的4E-BP1过度磷酸化的功能和机制意义； 利用化学蛋白质组学鉴定和验证作用于4E-BP1的其他激酶。通过这些研究， 我们不仅将进一步提高我们对4E-BP1介导的翻译调控的认识，而且还将阐明 治疗与异常帽子依赖翻译相关的许多疾病的新的可药物靶点。

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.