金屬感應轉錄因子與PTEN交互作用區域之潛在磷酸化胺基酸的分析

Analysis of potential phosphorylation sites in the MTF-1 domain interacting with PTEN

謝雅婷(Ya-Ting Hsieh)

指導教授 ： 林立元

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2013年)

https://doi.org/10.6843/NTHU.2013.00168

摘要

金屬硫蛋白(MT)在哺乳類細胞中負責調節重金屬離子的含量，將細胞所需的離子維持在適當的濃度，並且螯合對細胞有毒害的金屬離子以減少細胞受損的情形。當細胞受到金屬離子刺激時，金屬感應轉錄因子(MTF-1)會與位於MT基因啟動子的金屬感應序列(MRE)結合，活化MT表現。雖然目前研究對於MTF-1的活化機制尚未十分了解，後修飾作用已被報導為調控其功能的重要因素之一。實驗室先前的研究發現腫瘤抑制因子PTEN與MTF-1會進行交互作用，且發現作用之位置在於MTF-1 acidic domain上，因此我們想了解PTEN對於acidic domain序列中胺基酸的磷酸化狀態是否有影響。我們首先將acidic domain中的serine及threonine進行點突變模擬胺基酸的磷酸化與否，以報導基因活性觀察磷酸化狀態是否影響到MTF-1的轉錄活性，發現單一位置的胺基酸突變並未造成顯著的影響。將該domain中唯一的tyrosine進行點突變後，同樣未顯著影響MTF-1之轉錄活性，亦並未影響MTF-1與PTEN的交互作用。最後我們在大腸桿菌中分別表現PTEN及MTF-1的acidic domain，發現即使該domain未被磷酸化也會與PTEN進行交互作用。

關鍵字

金屬感應轉錄因子 ； PTEN ； 磷酸化

並列摘要

In mammalian cells, metallothionein (MT) functions in regulating the abundance of heavy metal ions, modulating essential metal ions and chelating those hazardous to avoid causing cell damages. Upon stimulation, metal responsive transcription factor-1 (MTF-1) binds to the metal response element (MRE) located on the promoter of MT gene, and induce activation. However, the detailed mechanism of how MTF-1 is regulated is still unknown. Post-translational modification has been reported as one of the key modulations for the function of MTF-1. Previous studies from our lab have confirmed interaction between MTF-1 and the tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), the interaction occurred at the acidic domain of MTF-1. Therefore, we attempted to identify the role of PTEN on the phosphorylation state of the acidic domain. First, we performed phospho-mimic experiments on the serines and threonines in the acidic domain with point mutation, and determined transcriptional activity of MTF-1 by reporter gene assay. We found that the mutation at single amino acid is insufficient to cause any effects. We then mutated the only tyrosine in the acidic domain, and obtained the same results: the transcriptional activity of MTF-1 was unaffected by the mutation, and neither was the interaction between MTF-1 and PTEN. Expression of PTEN and the acidic domain individually in E. coli followed by in vitro interaction assay revealed that the acidic domain is capable of interaction with PTEN regardless of its phosphorylation state. The result suggests that PTEN-MTF-1 interaction is independent of the phosphorylation status of the proteins.

並列關鍵字

MTF-1 ； PTEN ； phosphorylation

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