Global mapping of CARM1 substrates defines enzyme specificity and substrate recognition

Evgenia Shishkova,Joshua J Coon,Wei Xu,Alexander S Hebert,Nicholas W Kwiecien,Hao Zeng,Fabao Liu

doi:10.1038/ncomms15571

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.

Highlights

Protein arginine methyltransferases (PRMTs) introduce arginine methylation, a post-translational modification with the increasingly eminent role in normal physiology and disease
Our experimental design capitalized on the multiplexing capabilities of tandem mass tags (TMT)[31], a technique that allowed us to simultaneously enrich and analyse asymmetric dimethylarginine (ADMA)-modified peptides from wild type and KO samples, assuring a complete overlap of peptide identifications between the two groups
We identified over 300 unique ADMA sites on 138 different proteins that may be modified by coactivator-associated arginine methyltransferase 1 (CARM1) in vivo; over 90% of them have not been previously reported as substrates of the enzyme

Summary

Introduction

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; few CARM1 substrates are known, and its mechanism of substrate recognition is poorly understood. Aberrant expression and/or enzymatic activity of multiple PRMTs are associated with human cancers[5]; the functional significance of arginine methylation in oncogenic processes is poorly understood. This problem is further confounded by a lack of known cancer-relevant PRMT substrates[6]. Informatic analysis revealed the presence of proline-rich motifs nearby CARM1 methylation sites Both CARM1methylated arginines and the surrounding recognition sequences were frequently targeted by somatic mutations in cancer, likely inducing reduction or complete abolishment of methylation by CARM1. This finding opens new routes in the design of CARM1-specific inhibitors and warrants functional investigation of the N-terminal domains of other PRMT family members in substrate recognition

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Conclusion