Genetically Encoded Protein Phosphorylation in Mammalian Cells

Area of Science:

• Molecular Biology
• Biochemistry
• Synthetic Biology

Background:

• Protein phosphorylation is a crucial post-translational modification regulating numerous cellular processes in eukaryotes.
• Current methods for studying phosphorylation often lack site-specificity and orthogonality to endogenous pathways.
• Developing methods for site-specific phosphorylation is essential for understanding its functional consequences.

Purpose of the Study:

• To establish an orthogonal system for site-specific phosphoserine incorporation in mammalian cells.
• To enable the genetic encoding of phosphoserine and its analogs for studying phosphorylation.
• To demonstrate the utility of this system by synthetically activating a protein kinase.

Main Methods:

• Utilized an engineered Methanococcus maripaludis phosphoseryl-transfer RNA synthetase (MmSepRS)/Methanococcus janaschii tRNA pair (SepRS^v1.0/tRNA^v1.0_CUA) for orthogonality in mammalian cells.
• Created a eukaryotic elongation factor 1 alpha (EF-1α) variant (EF-1α-Sep) to enhance phosphoserine incorporation.
• Combined EF-1α-Sep with a mutant eRF1 and modified the phosphoserine biosynthetic pathway for genetic encoding.

Main Results:

• Demonstrated the orthogonality of the SepRS^v1.0/tRNA^v1.0_CUA pair in mammalian cells.
• Successfully achieved genetically encoded incorporation of phosphoserine and a non-hydrolyzable phosphonate analog.
• Showcased synthetic activation of a protein kinase using the developed phosphoserine incorporation system.

Conclusions:

• The developed orthogonal system provides a powerful tool for site-specific protein phosphorylation in mammalian cells.
• This method facilitates the study of phosphorylation's role in cellular processes and signaling.
• The ability to genetically encode phosphoserine analogs opens new avenues for chemical biology and drug discovery.