Genetically Encoded Protein Phosphorylation in Mammalian Cells. Issue 9 (20th September 2018)

• Record Type: Journal Article
• Authors: Beránek, Václav; Reinkemeier, Christopher D.; Zhang, Michael S.; Liang, Alexandria D.; Kym, Gene; Chin, Jason W.
• Is Part Of: Cell chemical biology. Volume 25:Issue 9(2018)
• Page Start: 1067
• Page End: 1074.e5
• Publication Date: 2018-09-20
• Subjects: genetic code expansion -- phosphorylation -- phosphoserine -- mammalian cells -- phosphoserine analog -- synthetic biology; Biochemistry -- Periodicals; 572.05
• DOI: 10.1016/j.chembiol.2018.05.013
• Languages: English
• ISSNs: 2451-9456

Abstract

Summary: Protein phosphorylation regulates diverse processes in eukaryotic cells. Strategies for installing site-specific phosphorylation in target proteins in eukaryotic cells, through routes that are orthogonal to enzymatic post-translational modification, would provide a powerful route for defining the consequences of particular phosphorylations. Here we show that the SepRS v1.0 /tRNA v1.0 CUA pair (created from the Methanococcus maripaludis phosphoseryl-transfer RNA synthetase [ Mm SepRS]/ Methanococcus janaschii [ Mj ]tRNAGCA Cys pair) is orthogonal in mammalian cells. We create a eukaryotic elongation factor 1 alpha (EF-1α) variant, EF-1α-Sep, that enhances phosphoserine incorporation, and combine this with a mutant of eRF1, and manipulations of the cell's phosphoserine biosynthetic pathway, to enable the genetically encoded incorporation of phosphoserine and its non-hydrolyzable phosphonate analog. Using this approach we demonstrate synthetic activation of a protein kinase in mammalian cells. Graphical Abstract: Highlights: SepRS v1.0 /tRNA v1.0 CUA is an orthogonal pair in mammalian cells Phosphoserine is genetically directed into proteins in mammalian cells Phosphonate analog of phosphoserine is stably incorporated in mammalian cells Encoded phosphonate analog enables synthetic kinase activation Abstract : Beránek et al. describe an aminoacyl-tRNA synthetase/tRNA pair that is orthogonal in mammalian cells. They demonstrate that this pair can be used to incorporate phosphoserine and a stable phosphonate analog. By encoding the phosphonate analog in a kinase activation loop, they synthetically activate the kinase.