Genetically Encoded Protein Phosphorylation in Mammalian Cells

Václav Beránek,Christopher D Reinkemeier,Michael S Zhang,Alexandria D Liang,Gene Kym,Jason W Chin

doi:10.1016/j.chembiol.2018.05.013

Václav Beránek, Christopher D Reinkemeier + Show 4 more

Open Access

https://doi.org/10.1016/j.chembiol.2018.05.013

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Abstract

SummaryProtein 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-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.

Highlights

Protein phosphorylation is a key post-translational modification that expands the complexity of protein function, and regulates diverse biological processes in eukaryotic systems (Manning et al, 2002)
We demonstrated that by manipulating phosphoserine biosynthesis in E. coli, a non-hydrolyzable, phosphonate analog of phosphoserine (2) could be incorporated into proteins using the SepRSv1.0/tRNAv1.0CUA pair (Rogerson et al, 2015), and that the pair can be further evolved to incorporate phosphothreonine into proteins in E. coli (Zhang et al, 2017)
Expressing SepRSv1.0/tRNAv1.0CUA in Human Cells We first demonstrated that the SepRSv1.0/tRNAv1.0CUA pair can be expressed in mammalian cells

Summary

Introduction

Protein phosphorylation is a key post-translational modification that expands the complexity of protein function, and regulates diverse biological processes in eukaryotic systems (Manning et al, 2002). We demonstrated that phosphoserine could be efficiently incorporated into proteins in E. coli using an evolved SepRS/tRNACUA pair (Rogerson et al, 2015) This pair, in which SepRS and the anticodon stem and anticodon loop of tRNACUA were evolved to function efficiently, referred to as the SepRSv1.0/tRNAv1.0CUA pair, has been used to produce a number of site- phosphorylated proteins for structural and functional studies (Rogerson et al, 2015; Huguenin-Dezot et al, 2016; Burgess et al, 2018; Dickson et al, 2018). We demonstrated that by manipulating phosphoserine biosynthesis in E. coli, a non-hydrolyzable, phosphonate analog of phosphoserine (2) could be incorporated into proteins using the SepRSv1.0/tRNAv1.0CUA pair (Rogerson et al, 2015), and that the pair can be further evolved to incorporate phosphothreonine into proteins in E. coli (Zhang et al, 2017)

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Discussion

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