Using unnatural amino acid mutagenesis to probe the regulation of PRMT1.
Department of Biochemistry and Molecular Pharmacology
Medical Subject Headings
Amino Acid Substitution; Benzophenones; Cross-Linking Reagents; Histones; Humans; Immunoprecipitation; K562 Cells; Methylation; *Mutagenesis, Site-Directed; Phenylalanine; Phosphorylation; Protein Processing, Post-Translational; Protein-Arginine N-Methyltransferases; Repressor Proteins; Substrate Specificity; Tyrosine
Biochemistry | Enzymes and Coenzymes | Medicinal-Pharmaceutical Chemistry | Therapeutics
Protein arginine methyltransferase 1 (PRMT1)-dependent methylation contributes to the onset and progression of numerous diseases (e.g., cancer, heart disease, ALS); however, the regulatory mechanisms that control PRMT1 activity are relatively unexplored. We therefore set out to decipher how phosphorylation regulates PRMT1 activity. Curated mass spectrometry data identified Tyr291, a residue adjacent to the conserved THW loop, as being phosphorylated. Natural and unnatural amino acid mutagenesis, including the incorporation of p-carboxymethyl-l-phenylalanine (pCmF) as a phosphotyrosine mimic, were used to show that Tyr291 phosphorylation alters the substrate specificity of PRMT1. Additionally, p-benzoyl-l-phenylalanine (pBpF) was incorporated at the Tyr291 position, and cross-linking experiments with K562 cell extracts identified several proteins (e.g., hnRNPA1 and hnRNP H3) that bind specifically to this site. Moreover, we also demonstrate that Tyr291 phosphorylation impairs PRMT1's ability to bind and methylate both proteins. In total, these studies demonstrate that Tyr291 phosphorylation alters both PRMT1 substrate specificity and protein-protein interactions.
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Citation: ACS Chem Biol. 2014 Mar 21;9(3):649-55. doi: 10.1021/cb400859z. Epub 2014 Jan 6. Link to article on publisher's site
Rust, Heather L.; Subramanian, Venkataraman; West, Graham M.; Young, Douglas D.; Schultz, Peter G.; and Thompson, Paul R., "Using unnatural amino acid mutagenesis to probe the regulation of PRMT1." (2014). Thompson Lab Publications. 17.