Delineating the Biology of Translational Repressor 4E-BP1

真核翻译起始因子 (eIF4E) 结合蛋白 1 (4E-BP1) 是一种本质上无序的蛋白质 作为帽依赖性 mRNA 翻译（mRNA 转录过程）的看门人 5' 末端含有 m7G 帽的蛋白质被转化为蛋白质。由于主要含有帽子的 mRNA 编码生长和存活因子，4E-BP1 在细胞生物学中的作用非常重要且异常 4E-BP1 活动与癌症、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 4E-BP1 与 eIF4E（m7G 帽结合）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。迄今为止，唯一已知影响 4E-BP1 磷酸化的激酶是雷帕霉素的机制靶点 复合物 1 (mTORC1);然而，报告表明其他未知激酶也可以磷酸化 4E-BP1 刺激帽依赖性翻译，特别是在 mTORC1 抑制的情况下。为了识别 这些激酶，我们开发了一种公正的化学蛋白质组学方法来识别高可信度 具有磷酸位点特异性的激酶-底物相互作用。使用该测定，我们发现了细胞周期蛋白的作用 依赖性激酶 4 (CDK4) 是一种经过临床验证的激酶，对细胞周期进展非常重要，可驱动帽 通过 4E-BP1 磷酸化进行依赖性翻译。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。由于 4E-BP1 被磷酸化为 由于有多达 13 个独特位点，我们假设许多其他激酶向 4E-BP1 发出信号并对其进行调节。另外， 尽管 4E-BP1 磷酸化在蛋白质合成中发挥着关键作用，但很少有研究被披露 关于其每个磷酸化丝氨酸和苏氨酸残基（包括其孤儿残基）的生物学功能 已知不受 mTORC1 影响的位点。为了填补这些知识空白，本提案的具体目标 具体如下：（1）确定CDK4介导的4E-BP1过度磷酸化的分子细节； (2) 至 确定 CDK4 介导的 4E-BP1 过度磷酸化的功能和机制意义；和（3） 使用化学蛋白质组学来识别和验证作用于 4E-BP1 的其他激酶。通过这些研究， 我们不仅将进一步增强我们对 4E-BP1 介导的翻译调控的了解，而且还将阐明 用于治疗与异常帽依赖性翻译相关的许多疾病的新药物靶标。

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.