| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 98 === Protein arginine methylation is emerging as a pivotal mechanism in regulation of a variety of cellular process including cell growth and differentiation, gene expression and signal transduction. Protein arginine methyltransferase 1 (PRMT1) is the predominant protein arginine methyltransferase in mammalian cells. Vimentin is an intermediate filament protein and was identified as a putative PRMT1 substrate in our previous proteomics screening. Posttranslational modifications such as phosphorylation/dephosphorylation are essential for the functional regulation of the intermediate filament through modulation of solubility, conformation and filament organization. However, methylation of vimentin has not thus far been reported.
To validate our initial finding that vimentin was a putative novel substrate of PRMT1, I first expressed and purified recombinant GST-PRMT1 proteins which was shown to be catalytically active as assayed with two known PRMT1 substrates. In order to directly test whether vimentin could be methylated, I developed a denaturation/renaturation protocol to obtain soluble and purified His-tagged vimentin proteins. An in vitro methylation assay demonstrated that His-tagged vimentin was a substrate of GST-PRMT1. In addition, vimentin was also methylated by mammalian PRMT1 which was immunoprecipitated from HEK293 cells. As a first step toward identifying potential methylation sites, I made two constructs pET6H-vimN and pET6H-vimC for expression of the N-terminal half and the C-terminal half of vimentin. Methylation of vimentin by PRMT1 occurred predominantly in the N-terminal half of vimentin. I immunoprecipitated endogenous vimentin from HEK293 cells and showed that vimentin contained methylarginine. In addition, I found that vimentin were induced and methylated during megakaryocytic differentiation of K562 cells implicating a potential role of vimentin in megakaryocytic differentiation.
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