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ACS Chem Biol. 2014 Mar 21;9(3):649-55. doi: 10.1021/cb400859z. Epub 2014 Jan 6.

Using unnatural amino acid mutagenesis to probe the regulation of PRMT1.

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1
Departments of †Chemistry, ‡Mass Spectrometry and Proteomics, and §Molecular Therapeutics, The Scripps Research Institute , 130 Scripps Way, Jupiter, Florida 33458, United States.

Abstract

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.

PMID:
24358983
PMCID:
PMC4505744
DOI:
10.1021/cb400859z
[Indexed for MEDLINE]
Free PMC Article

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