In vitro Methylation Assay to Study Protein Arginine Methylation

Rama Kamesh Bikkavilli; Sreedevi Avasarala; Michelle Van Scoyk; Manoj Kumar Karuppusamy Rathinam; Jordi Tauler; Stanley Borowicz; Robert A. Winn

doi:10.3791/51997

Method Article

In vitro Methylation Assay to Study Protein Arginine Methylation

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DOI:

10.3791/51997

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October 5th, 2014

Rama Kamesh Bikkavilli¹ , Sreedevi Avasarala¹ , Michelle Van Scoyk¹ , Manoj Kumar Karuppusamy Rathinam¹ , Jordi Tauler¹ , Stanley Borowicz¹^,² , Robert A. Winn¹^,³

¹Department of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, ²Department of Hematology and Oncology, University of Illinois at Chicago, ³Jesse Brown Veterans Affairs Medical Center

Summary

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Abstract

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-³H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-³H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

Introduction

Protein methylation was first described in 1968¹. It was not until the first cloning of PRMT1 in 1996, that researchers began to appreciate the importance of this post-translational modification². Interestingly, about 2% of arginine residues in the proteins of nuclear extracts are methylated³, indicating the abundance of this modification. Arginine is a positively charged amino acid with a basic side chain and the nitrogen/s within the side chains of arginine can be post-translationally modified via the addition of a methyl group, a process known as arginine methylation^4-6. Arginine methylation is catalyzed by a class of enz....