Using Unnatural Amino Acid Mutagenesis To Probe the Regulation of PRMT1

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 crosslinking 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.

Protein arginine methyltransferase expression, localization, and activity during disuse-induced skeletal muscle plasticity

“…Interestingly, AMPK had a particularly strong influence on PRMT1 expression and activity. Although tyrosine phosphorylation of PRMT1 reduces its activity (87), our data in skeletal muscle introduce the possibility that AMPK-mediated phosphorylation of the enzyme may enhance its methylating activity. Unlike AMPK, p38 activation status demonstrated limited predictive power, with only PRMT activity showing a significant negative correlation.…”

“…In addition to reduced muscle mass, skeletal muscle atrophy is characterized by decreased muscle fiber CSA and contractile protein content (85,89,91). Other changes include endothelial degradation, increased capillary density, decreased mitochondrial quantity, higher levels of intermuscular adipose tissue and connective tissue, as well as increased expression of type 2 muscle fiber types (85,86,87,89,90,91). Chronic muscle disuse also results in increased insulin resistance, decreased calcium ion concentration, degeneration of neuromuscular junctions, nerve terminal disruption, plus decreased maximal voluntary force production and lower fatigue resistance (85,88,89,91,92,93).…”

Nuclear PRMT1 expression is associated with poor prognosis and chemosensitivity in gastric cancer patients

“…On the other hand, PRTM1 has some splicing variants, and the noncoding variant of PRMT1, PRMT1 variant 2, was reported to be associated with progression of both colon and breast cancer [13,22]. Moreover, it was reported that PRMT1 functions such as substrate recognition and methylation are inhibited by phosphorylation of Tyr291 [23]. The disparity in PRMT1 functional analysis data may be due to the cell-specific balance of substrate methylation status, PRMT1-splicing variants, and PRMT1-phosphorylation status in each cancer cell line.…”

At the Interface of Chemical and Biological Synthesis: An Expanded Genetic Code

“…Site-specific incorporation of pSY allowed for the preparation of high-affinity sulfo-hirudin and various sulfated antibodies to the human immunodeficiency virus (HIV) protein GP120 (Liu et al 2007). Recently, pCMF was used to mimic phosphoTyr291 in protein arginine methyltransferase 1, which plays a role in substrate recognition (Rust et al 2014). A similar strategy has been used to study the function of lysine PTMs within histones.…”