Differential regulation of RNA processing by Akt isoforms

Akt 亚型对 RNA 加工的差异调节

• 批准号: 8889119
• 负责人: PHILIP N. TSICHLIS
• 金额: $ 37.74万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8889119
• 关键词: 1-Phosphatidylinositol 3-Kinase; Address; Agreement; Alternative Splicing; Apoptosis; Area; Binding; Biogenesis; C-terminal; Cell Line; Cell Proliferation; Cell physiology; Cells; Complex; Consensus; DNA Polymerase II; Data; Data Analyses; Databases; Docking; Epithelial; Epithelial Cells; Family; Fibroblast Growth Factor; Fibroblast Growth Factor 2; Fibroblast Growth Factor Receptor 2; Fibroblasts; Gene Targeting; Genes; Genetic Transcription; Histone H3; Histones; Human; Institutes; Laboratories; Link; Lung; Malignant Neoplasms; Malignant neoplasm of lung; Mesenchymal; Metabolism; Molecular; Molecular Chaperones; Non-Small-Cell Lung Carcinoma; Normal Cell; Oncogenic; Pathway interactions; Pattern; Phenotype; Phosphorylation; Play; Polypyrimidine Tract-Binding Protein; Primary Neoplasm; Process; Property; Protein Isoforms; Protein-Serine-Threonine Kinases; Proteins; Publishing; RNA; RNA Processing; RNA Splicing; RNA Transport; RNA polymerase II largest subunit; Regulation; Role; Signal Transduction; Site; Specificity; Specimen; Spices; Spliced Genes; Staging; The Cancer Genome Atlas; Transferase; Tumor Cell Line; Work; Xenograft Model; base; bronchial epithelium; cancer cell; cell motility; design; epithelial to mesenchymal transition; lung Carcinoma; lung tumorigenesis; migration; neoplastic cell; novel; novel diagnostics; novel therapeutics; prevent; public health relevance; receptor; research study; response; stoichiometry; tumor; tumor xenograft; tumorigenesis

 DESCRIPTION (provided by applicant): A phosphoproteomics screen in a set of isogenic lung fibroblast cell lines expressing different Akt isoforms, identified 606 Akt target proteins. Ingenuity pathway network function analysis of the data identified a host of network functions that appear to be differentially regulated by the three isoforms. One of these functions is RNA processing, which was represented by 25 proteins that operate at different stages of RNA biogenesis and are phosphorylated by at least one of the three isoforms. The main focus of this work is on one of these proteins (IWS1), which plays a critical role in the assembly of a transcriptional elongation complex on the C- terminal domain (CTD) of the large subunit of RNA polymerase II. Based on studies we recently published (Sanidas et al Mol Cell 53: 577, 2014), we now know that IWS1 is phosphorylated by Akt3 and Akt1, but not Akt2 at the conserved Ser720/Thr721 site. More important, IWS1 phosphorylation at this site is required for the recruitment of the histone H3 K36 trimethyltransferase SetD2 to the IWS1-dependent transcriptional elongation complex. The inhibition of IWS1 phosphorylation, prevents the recruitment of SetD2 and the trimethylation of histone H3 at K36 in the body of target genes during transcription. This abrogates the recruitment of the histone H3K36me3-interacting protein MRG15 and its binding partner polypyrimidine tract binding protein (PTB) and results in shifts in alternative splicing of target genes, one of which is FGFR-2. Alternative splicing gives rise to two FGFR-2 isoforms, IIIb and IIIc, which are expressed in epithelial and mesenchymal cells respectively and of which only the IIIc isoform encodes a receptor that is recognized by FGF-2. In cancer cells, the switch from IIIb to IIIc is associated with EMT and increased cell motility an invasiveness. The phosphorylation of IWS1 by Akt3 and Akt1 promotes a shift from the IIIb to the IIIc isoform and stimulates the proliferation, migration, invasiveness and oncogenic potential of human tumor cell lines. Some, although not all, of these effects of IWS1 phosphorylation can be fully explained by the alternative splicing of FGFR-2. The role of IWS1 phosphorylation in primary human non-small cell lung carcinomas (NSCLCs), was determined by analyzing the expression and phosphorylation of IWS1 in a set of 24 primary NSCLCs. Twenty one of the 24 NSCLCs were found to express IWS1. More important, the stoichiometry of IWS1 phosphorylation in these tumors was fond to correlate with the FGFR-2 splicing pattern and with Akt phosphorylation and Akt3 expression. These data identify an Akt isoform-dependent regulatory mechanism for RNA processing and demonstrate its role in lung cancer. Our plan is to take advantage of the window to the poorly understood regulation of RNA splicing that is opened by these data, to explore the role of Akt in alternative splicing in normal and tumor cells.

 描述（由申请人提供）：在一组表达不同Akt亚型的同基因肺成纤维细胞系中进行磷酸化蛋白质组学筛选，鉴定了606种Akt靶蛋白。对数据进行的不稳定性途径网络功能分析确定了一系列网络功能，这些功能似乎受到三种亚型的差异调节。这些功能之一是RNA加工，其由25种蛋白质代表，这些蛋白质在RNA生物合成的不同阶段起作用，并且被三种同种型中的至少一种磷酸化。这项工作的主要重点是这些蛋白质之一（IWS 1），它在RNA聚合酶II大亚基的C-末端结构域（CTD）上的转录延伸复合物的组装中起着关键作用。基于我们最近发表的研究（Sanidas et al Mol Cell 53：577，2014），我们现在知道IWS 1在保守的Ser 720/Thr 721位点被Akt 3和Akt 1磷酸化，但不被Akt 2磷酸化。更重要的是，IWS 1磷酸化在这个网站上所需的招募组蛋白H3 K36三甲基转移酶SetD 2的IWS 1依赖的转录延伸复合物。IWS 1磷酸化的抑制阻止了转录过程中靶基因体内SetD 2的募集和组蛋白H3在K36处的三甲基化。这消除了组蛋白H3 K36 me 3相互作用蛋白MRG 15及其结合伴侣多聚嘧啶束结合蛋白（PTB）的募集，并导致靶基因的可变剪接发生变化，其中之一是FGFR-2。选择性剪接产生两种FGFR-2同种型IIIb和IIIc，其分别在上皮细胞和间充质细胞中表达，并且其中仅IIIc同种型编码被FGF-2识别的受体。在癌细胞中，从IIIb到IIIc的转换与EMT和增加的细胞运动性和侵袭性有关。Akt 3和Akt 1对IWS 1的磷酸化促进了从IIIb亚型向IIIc亚型的转变，并刺激了人肿瘤细胞系的增殖、迁移、侵袭和致癌潜力。IWS 1磷酸化的这些作用中的一些（尽管不是全部）可以通过FGFR-2的选择性剪接来完全解释。通过分析一组24个原发性非小细胞肺癌（NSCLC）中IWS 1的表达和磷酸化来确定IWS 1磷酸化在原发性人NSCLC中的作用。24例NSCLC中有21例表达IWS 1。更重要的是，这些肿瘤中IWS 1磷酸化的化学计量学倾向于与FGFR-2剪接模式以及Akt磷酸化和Akt 3表达相关。这些数据确定了Akt亚型依赖的RNA加工调控机制，并证明了其在肺癌中的作用。我们的计划是利用这些数据打开的对RNA剪接调控知之甚少的窗口，探索Akt在正常和肿瘤细胞中选择性剪接中的作用。