The activity and stability of the transcriptional coactivator p/CIP/SRC-3 are regulated by CARM1-dependent methylation.
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 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.
Naeem
Hina
H
Cancer Research Laboratories, London Regional Cancer Program, London, Ontario, Canada N6A 4L6.
Cheng
Donghang
D
Zhao
Qingshi
Q
Underhill
Caroline
C
Tini
Marc
M
Bedford
Marc T
MT
Torchia
Joseph
J
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D019175:DNA Methylation; D018076:DNA, Complementary / Q000378:metabolism; D005786:Gene Expression Regulation; D006367:HeLa Cells; D051548:Histone Acetyltransferases / Q000502:physiology*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D056921:Nuclear Receptor Coactivator 3; D011484:Protein-Arginine N-Methyltransferases / Q000502:physiology*; D012436:S-Adenosylmethionine / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000502:physiology*; D014158:Transcription, Genetic
D018076:DNA, Complementary; D015534:Trans-Activators; D012436:S-Adenosylmethionine; D011484:Protein-Arginine N-Methyltransferases; C119966:coactivator-associated arginine methyltransferase 1; D051548:Histone Acetyltransferases; C538866:NCOA3 protein, human; C538867:Ncoa3 protein, mouse; D056921:Nuclear Receptor Coactivator 3