- 學位論文
在脂多醣刺激的RAW264.7中TTP家族蛋白的磷酸化與調節
Phosphorylation and regulation of tristetraprolin (TTP) family proteins in lipopolysaccharide-stimulated RAW264.7
摘要
mRNA穩定性的調節是控制基因表達的關鍵步驟，通常由RNA結合蛋白結合mRNA的3端非轉譯區去進行調控，例如:Tristetraprolin（TTP）蛋白家族成員結合3端非轉譯區中富含AU的區域（ARE），TTP蛋白家族中有四個成員，包含:TTP（ZFP36）、ZFP36L1、ZFP36L2和ZFP36L3其中皆包含保守的CCCH串聯鋅指和C端的NOT1結合結構域用於使含有ARE的mRNA失去穩定性。TTP蛋白家族的功能受其磷酸化的狀態和蛋白質之間的相互作用所影響。 我們先前的研究顯示TTP和Zfp36l1在脂多醣刺激的RAW264.7巨噬細胞中被磷酸化。在這份報告中，我們證明了TTP在受LPS刺激的RAW264.7細胞中被誘導表現，並且在Ser-316的位置被ERK信號通路所磷酸化，這破壞了它與CCR4-NOT脫腺苷酶複合物的相互作用，我們也發現Ser-316的位置會被蛋白磷酸酶2A（PP2A）所去磷酸化。之後我們藉由CRISPR / Cas9的技術在RAW264.7細胞中產生TTP敲除的細胞系。我們接著提供證據證明Zfp36l1的Ser-334（Ser-316的保守殘基）可以被Rsk1、PKA和MK2所磷酸化。此外，我們發現MK2可能不僅能磷酸化Ser-334，而且還有另外的磷酸化位點在Zfp36l1的C末端結構域上。在生化功能方面，Zfp36l1在Ser-334上的磷酸化會降低募集CCR4-NOT1複合物的能力，導致ARE介導的mRNA衰變活性的喪失。此外，我們在已經發表的微陣列數據中鑑定了LPS處理後瞬時上調的基因。通過生物信息學和統計學分析，我們在microRNA數據庫中分析這些基因，並提出一些能參與LPS刺激中扮演基因調控的潛在microRNA。其中之一的miR-27被證明是由LPS刺激所上調的，並通過與Zfp36l1 mRNA的3端非轉譯區結合而導致Zfp36L1的表現量下調。 在我們的研究中，我們揭示了TTP蛋白家族C-末端結構域上共有的磷酸化位點，其可以調節功能活性和影響蛋白質之間的相互作用。此外，miR-27是在LPS刺激的RAW264.7細胞中調節Zfp36L1表達的潛在微RNA。
並列摘要
Regulation of mRNA stability is a critical step to control gene expression. It is usually mediated by the RNA-binding proteins (RNA-BP) that bind to the 3’-untranslated region of mRNA, such as AU-rich elements (AREs). Tristetraprolin (TTP) protein family containing conserved CCCH tandem zinc-finger and C-terminal NOT1-binding domains contributes to the destabilization of ARE-containing mRNA. There are four members, TTP (ZFP36), ZFP36L1, ZFP36L2 and ZFP36L3 in TTP protein family. The function of TTP protein family is affected by its protein phosphorylated states and protein-protein interaction. Our previous study showed that both TTP and Zfp36l1 proteins were phosphorylated in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. In this report, we demonstrated that TTP was induced and phosphorylated at Ser-316 by ERK-signaling pathway in LPS-stimulated RAW264.7 cells, which disrupted its interaction with CCR4-NOT deadenylase complex and dephosphorylated by protein phosphatase 2A (PP2A). We also generate homozygous and heterozygous TTP knock-out cell lines by CRISPR/Cas9 system in RAW264.7 cell. Moreover, we provide evidence to prove that Ser-334 of Zfp36l1, the conserved residue of Ser-316, was phosphorylated by Rsk1, PKA and MK2. We found that MK2 may phosphorylate C-terminal domain of Zfp36l1 not only at Ser-334 but also at an additional site. In the biochemical function, phosphorylation of Zfp36l1 at Ser-334 would decline the ability to recruit the CCR4-NOT1 complex leading to decrease of MKP-1 ARE-mediated mRNA decay activity. Additionally, we identified the transiently up-regulated genes after LPS treatment in published microarray data. Through bioinformatics and statistics, we analyze these genes in microRNA database and suggest some potential microRNAs involved in LPS-stimulated gene regulation. One of them, miR-27, was demonstrated to be up-regulated by LPS and correlated in down-regulation of Zfp36L1 through the 3’UTR of Zfp36l1 mRNA. In our study, we revealed a consensus phosphorylated site on C-terminal domain of TTP protein family, which may regulate the functional activity and protein-protein interaction. Furthermore, miR27 is a potential microRNA that regulates the expression of Zfp36L1 in LPS-stimulated RAW264.7 cell.