Genetically Encoded Protein Phosphorylation in Mammalian Cells

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 [MmSepRS]/Methanococcus janaschii [Mj]tRNA(GCA)(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.