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Molecular and Cellular Biology, January 2007, p. 120-134, Vol. 27, No. 1
0270-7306/07/$08.00+0     doi:10.1128/MCB.00815-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The Activity and Stability of the Transcriptional Coactivator p/CIP/SRC-3 Are Regulated by CARM1-Dependent Methylation

Department of Oncology, London Regional Cancer Program,¹ Department of Physiology and Pharmacology,² Department of Microbiology and Immunology, The University of Western Ontario, London, Ontario, Canada,³ University of Texas M. D. Anderson Cancer Center, Department of Carcinogenesis, Smithville, Texas 78957⁴

Received 8 May 2006/ Returned for modification 18 June 2006/ Accepted 5 October 2006

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-³H]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 carboxy-terminal 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.

Published ahead of print on 16 October 2006.

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