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

• Main Authors: Hsieh, Ya-Ting, 謝雅婷
• Other Authors: Lin, Lih-Yuan
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/04899806764596305297

碩士 === 國立清華大學 === 分子與細胞生物研究所 === 101 === 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.