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

摘要 4E-BP1是癌细胞帽子依赖蛋白翻译的主要守门人。它直接成为 在癌症中经常失控的mTOR通路。我们发现CDK1/CyCB1可以替代 有丝分裂期间mTOR使4E-BP1磷酸化，在丝氨酸(S)83位产生一个新的磷酸化标记，即 当mTOR使4E-BP1磷酸化时不存在。4E-BP1突变形式在S83处不能被磷酸化 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CDK-1但不依赖mTOR的4E-BP1调控蛋白 路径。这一提议的中心假设是S83的磷酸化调节一个独特的 促进有丝分裂特异性蛋白表达的mRNAs的子集。在目标I中，替换突变体(S83A)和 将拟磷脂(S83D)4EBP1蛋白导入EIF4EBP1缺失细胞，以及从EIF4EBP1缺失细胞中产生MEF EIF4EBP1 S83A敲入小鼠将用于检测S83磷酸化对碱性细胞的影响 动态平衡，包括细胞周期分析、增殖和蛋白质合成。在AIM II中，我们将确定 有丝分裂4E-BP1通过互补的两种途径磷酸化的差异翻译的mRNAs 方法：有丝分裂停滞细胞的核糖体图谱，以及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翻译的动态。 最后，在目标IV中，我们将探索一种独特的敲入突变小鼠模型，以解决4E-BP1调控异常。我们的研究 将促进我们对有丝分裂特异的4E-BP1过度磷酸化形式如何 正常循环细胞的功能及其在癌细胞中的失调可能对人类 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.