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Genetically Encoded Protein Phosphorylation in Mammalian Cells.


ABSTRACT: 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 SepRSv1.0/tRNAv1.0CUA pair (created from the Methanococcus maripaludis phosphoseryl-transfer RNA synthetase [MmSepRS]/Methanococcus janaschii [Mj]tRNAGCACys pair) is orthogonal in mammalian cells. We create a eukaryotic elongation factor 1 alpha (EF-1?) variant, EF-1?-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.

SUBMITTER: Beranek V 

PROVIDER: S-EPMC6162345 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Genetically Encoded Protein Phosphorylation in Mammalian Cells.

Beránek Václav V   Reinkemeier Christopher D CD   Zhang Michael S MS   Liang Alexandria D AD   Kym Gene G   Chin Jason W JW  

Cell chemical biology 20180621 9


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<sup>v1.0</sup>/tRNA<sup>v1.0</sup><sub>CUA</sub> pair (created from the Methanococcus maripaludis phosphoseryl-transfer RNA synth  ...[more]

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