Using Unnatural Amino Acid Mutagenesis To Probe the Regulation of PRMT1

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 phosphor...

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Bibliographic Details
Published in:ACS chemical biology 2014-03, Vol.9 (3), p.649-655
Main Authors: Rust, Heather L, Subramanian, Venkataraman, West, Graham M, Young, Douglas D, Schultz, Peter G, Thompson, Paul R
Format: Article
Language:English
Subjects:
Amino Acid Substitution
Benzophenones - metabolism
Cross-Linking Reagents - chemistry
Histones - metabolism
Humans
Immunoprecipitation
K562 Cells
Methylation
Mutagenesis, Site-Directed
Phenylalanine - analogs & derivatives
Phenylalanine - genetics
Phenylalanine - metabolism
Phosphorylation
Protein Processing, Post-Translational
Protein-Arginine N-Methyltransferases - genetics
Protein-Arginine N-Methyltransferases - metabolism
Repressor Proteins - genetics
Repressor Proteins - metabolism
Substrate Specificity
Tyrosine - genetics
Tyrosine - metabolism
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Summary: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.
ISSN:1554-8929
1554-8937
DOI:10.1021/cb400859z