Global mapping of CARM1 substrates defines enzyme specificity and substrate recognition

Abstract

Protein arginine methyltransferases (PRMTs) introduce arginine methylation, a post-translational modification with the increasingly eminent role in normal physiology and disease. PRMT4 or coactivator-associated arginine methyltransferase 1 (CARM1) is a propitious target for cancer therapy; however, few CARM1 substrates are known, and its mechanism of substrate recognition is poorly understood. Here we employed a quantitative mass spectrometry approach to globally profile CARM1 substrates in breast cancer cell lines. We identified >130 CARM1 protein substrates and validated in vitro >90% of sites they encompass. Bioinformatics analyses reveal enrichment of proline-containing motifs, in which both methylation sites and their proximal sequences are frequently targeted by somatic mutations in cancer. Finally, we demonstrate that the N-terminus of CARM1 is involved in substrate recognition and nearly indispensable for substrate methylation. We propose that development of CARM1-specific inhibitors should focus on its N-terminus and predict that other PRMTs may employ similar mechanism for substrate recognition.

The emerging role of mediator complex subunit 12 in tumorigenesis and response to chemotherapeutics

“…37 We have identified MED12 as a substrate for coactivator-associated arginine methyltransferase 1 (CARM1), which asymmetrically dimethylates protein substrates on the arginine residues. 38 We have demonstrated that the expression levels of CARM1 and MED12 in cancers are positively correlated. In addition, their high expression often predicts a better prognosis in patients with breast cancer who receive chemotherapy.…”

“…Protein arginine methylation catalyzed by protein arginine methyltransferases in mammalian cells shares many attributes with other covalent modifications . We have identified MED12 as a substrate for coactivator‐associated arginine methyltransferase 1 (CARM1), which asymmetrically dimethylates protein substrates on the arginine residues . We have demonstrated that the expression levels of CARM1 and MED12 in cancers are positively correlated.…”

“…In addition, their high expression often predicts a better prognosis in patients with breast cancer who receive chemotherapy. We identified 2 MED12 methylation sites: arginine 1862 (R1862) and arginine 1912 (R1912) . By searching the COSMIC database for mutations on R1862 and R1912, we found that R1862 was mutated in a case of lung carcinoma, and a somatic, homozygous mutation at R1912 was identified in a melanoma from a patient who developed resistance to combined treatment with RAF and mitogen‐activated protein kinase (MEK) inhibitors.…”

Identification and in silico structural analysis of Gallus gallus protein arginine methyltransferase 4 (PRMT4)

“…). Given that the PH domain has recently been found to be responsible for substrate recognition and methylation of most PRMT4 substrates in human cells , we investigated here the sequence variations and conservations of chicken versus other vertebrate PH domains to elucidate its structural connection to the catalytic core domain and how this might translate to its essential enzymatic functions.…”

“…Interestingly, our in silico structural comparison of the N‐terminal PH domain of chicken and murine PRMT4 identified strictly conserved amino acids that contribute to a newly predicted interaction interface between the PH and the catalytic domain representing the first forecast of their relative spatial arrangement. Furthermore, these findings suggest a structural basis for the recently reported essential functions of the PH domain in substrate recognition and methylation by PRMT4 . Given the strict transspecies conservation of the amino acids within the PH domain mediating the interaction toward the catalytic core, we propose that targeting this interface with small molecules could be a promising strategy for the design of PRMT4‐selective inhibitors.…”