The transcriptional coactivator p/CIP(SRC-3/AIB1/ACTR/RAC3) binds liganded nuclear hormone receptors and facilitates transcription by directly recruiting accessory factors such as acetyltransferase CBP/p300 and the coactivator arginine methyltransferase CARM1. In the present study, we have established that recombinant p/CIP (p300/CBP interacting protein) is robustly methylated by CARM1 in vitro but not by other protein arginine methyltransferase family members. Metabolic labeling of MCF-7 breast cancer cells with S-adenosyl-L-[methyl-3 H]methionine and immunoblotting using dimethyl arginine-specific antibodies demonstrated that p/CIP is specifically methylated in intact cells. In addition, methylation of full-length p/CIP is not supported by extracts derived from CARM1 ؊/؊ mouse embryo fibroblasts, indicating that CARM1 is required for p/CIP methylation. Using mass spectrometry, we have identified three CARM1-dependent methylation sites located in a glutamine-rich region within the carboxy terminus of p/CIP which are conserved among all steroid receptor coactivator proteins. These results were confirmed by in vitro methylation of p/CIP using carboxyterminal truncation mutants and synthetic peptides as substrates for CARM1. Analysis of methylation site mutants revealed that arginine methylation causes an increase in full-length p/CIP turnover as a result of enhanced degradation. Additionally, methylation negatively impacts transcription via a second mechanism by impairing the ability of p/CIP to associate with CBP. Collectively, our data highlight coactivator methylation as an important regulatory mechanism in hormonal signaling.
Protein arginine methylation has emerged as an important mechanism for regulating the functions of proteins involved in diverse aspects of gene regulation such as transcriptional activation and repression, mRNA processing and nuclear-cytoplasmic shuttling. This modification is catalyzed by the PRMT family of enzymes which utilize intracellular S-adenosyl methionine as a cofactor to dimethylate-specific arginines found within many target proteins.The establishment of in vitro biochemical assays as well as the development of modification-specific antibodies, and more recently mass spectrometry, have increased our understanding of the mechanism of catalysis of the PRMT family of enzymes. In the following discussion, we present some of the more commonly used in vivo and in vitro techniques which can be utilized to study the mechanism of arginine methylation and its role in transcription.
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