Departments of †Chemistry, ‡Mass Spectrometry and Proteomics, and §Molecular Therapeutics, The Scripps Research Institute , 130 Scripps Way, Jupiter, Florida 33458, United States.
ACS Chem Biol. 2014 Mar 21;9(3):649-55. doi: 10.1021/cb400859z. Epub 2014 Jan 6.
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.
蛋白质精氨酸甲基转移酶 1（PRMT1）依赖性甲基化有助于许多疾病（如癌症、心脏病、ALS）的发生和发展；然而，控制 PRMT1 活性的调节机制相对未知。因此，我们着手破译磷酸化如何调节 PRMT1 活性。精心策划的质谱数据鉴定出紧邻保守 THW 环的 Tyr291 残基被磷酸化。天然和非天然氨基酸诱变，包括掺入 p-羧甲基-l-苯丙氨酸（pCmF）作为磷酸酪氨酸模拟物，用于表明 Tyr291 磷酸化改变了 PRMT1 的底物特异性。此外，在 Tyr291 位置掺入 p-苯甲酰基-l-苯丙氨酸（pBpF），并与 K562 细胞提取物进行交联实验鉴定出几种特异性结合该位点的蛋白质（例如 hnRNPA1 和 hnRNP H3）。此外，我们还证明 Tyr291 磷酸化会损害 PRMT1 结合和甲基化这两种蛋白质的能力。总的来说，这些研究表明 Tyr291 磷酸化改变了 PRMT1 的底物特异性和蛋白质-蛋白质相互作用。
