Role of a Novel Mitotic 4E-BP1 Protein Isoform in Cellular Transformation

摘要 4 E-BP 1是癌细胞帽依赖性蛋白翻译的主要看门人。它是直接针对 mTOR通路在癌症中经常失调。我们已经发现CDK 1/CYCB 1替代了 mTOR在有丝分裂期间磷酸化4 E-BP 1，在丝氨酸（S）83处产生新的磷酸化标记， 当mTOR磷酸化4 E-BP 1时不存在。4 E-BP 1的突变形式不能在S83处磷酸化 部分逆转由默克尔细胞多瘤病毒（MCV）小T癌蛋白引起的细胞转化。这 该项目的重点是研究一种新的依赖于CDK-1但不依赖于mTOR的4 E-BP 1调控蛋白。 通路该建议的中心假设是S83磷酸化调节独特的蛋白质的翻译。 促进有丝分裂特异性蛋白质表达的mRNA的子集。在目的I中，突变体（S83 A）和 将磷酸化模拟物（S83 D）4 EBP 1蛋白导入EIF 4 EBP 1缺失细胞，以及从EIF 4 EBP 1缺失细胞中发育MEF。 将使用EIF 4 EBP 1 S83 A敲入小鼠来测定S83磷酸化对基础细胞的影响。 稳态，包括细胞周期分析、增殖和蛋白质合成。在目标II中，我们将确定 有丝分裂4 E-BP 1通过两个互补的磷酸化， 方法：有丝分裂阻滞细胞的核糖体分析，RNA免疫沉淀和测序 （RIPseq）。在Aim III中，我们将使用活细胞成像工具来跟踪和量化活细胞中的翻译动力学。 最后，在目的IV中，我们将探索一种独特的4 E-BP 1失调的敲入突变小鼠模型。我们的研究 将促进我们对4 E-BP 1的有丝分裂特异性过度磷酸化形式的基本理解 在正常循环细胞中的功能，以及它在癌细胞中的失调如何有助于人类 恶性肿瘤。

ABSTRACT 4E-BP1 is the primary gatekeeper for cancer cell, cap-dependent protein translation. It is directly targeted by the mTOR pathway that is frequently dysregulated in cancer. We have found that CDK1/CYCB1 substitutes for mTOR during mitosis to phosphorylate 4E-BP1 generating a novel phosphorylation mark at serine (S) 83 that is not present when mTOR phosphorylates 4E-BP1. A mutant form of 4E-BP1 unable to be phosphorylated at S83 partially reverses cell transformation caused by the Merkel cell polyomavirus (MCV) small T oncoprotein. This project is focused on investigating a novel CDK-1-dependent but mTOR-independent 4E-BP1 regulatory pathway. The central hypothesis for this proposal is that S83 phosphorylation modulates translation of a unique subset of mRNAs to facilitate mitosis-specific protein expression. In Aim I, substitution of a mutant (S83A) and the phosphomimetic (S83D) 4EBP1 proteins into EIF4EBP1 null cells, as well as developing MEFs from the EIF4EBP1 S83A knock-in mice will be used to assay the effects of S83 phosphorylation on basic cell homeostasis, including cell cycle analysis, proliferation and protein synthesis. In Aim II, we will identify differentially translated mRNAs as a result of mitotic 4E-BP1 phosphorylation through two complementary approaches: ribosomal profiling of mitosis-arrested cells, and RNA immunoprecipitation and sequencing (RIPseq). In Aim III we will use live-cell imaging tools to track and quantify dynamics of translation in live cells. Finally in Aim IV, we will explore a unique knock-in mutant mouse model for 4E-BP1 dysregulation. Our studies will advance our fundamental understanding of how a mitosis-specific hyperphosphorylated form of 4E-BP1 functions in normally cycling cells and how its dysregulation in cancer cells may contribute to human malignancies.