| Coactivator-associated arginine methyltransferase 1 (CARM1 or PRMT4) is a member of the cellular protein arginine methyltransferase (PRMT) family, which catalyzes the formation of asymmetrically dimethylated arginine (ADMA). CARM1 has been described as a coactivator for a number of transcription factors, underscoring its potential in the regulation of diverse cellular processes. Overexpression of CARM1 has been observed in various cancer types. Depletion of CARM1 in cancerous cells could result in cell cycle arrest and apoptosis, implying that CARM1 might be a molecular target for cancer treatment. The biological functions of CARM1 are regulated by many post-translational modifications (PTMs). Recently, the addition of O-linked N-acetylglucosamine (O-GlcNAc) to CARM1 was reported. However, the functional consequences of this novel PTM have yet to be described. The goal of my study is to elucidate the functional impact of O-GlcNAcylation on CARM1. O-GlcNAcylation was first confirmed to exist on the endogenous CARM1 in MCF7 cells. The O-GlcNAcylation sites were mapped by mass spectrometry to residues 594-606, containing four possible sites (S595, S598, T601 and T603) in the C-terminus of CARM1. Mutation of all four sites (quadruple mutant, CARM1QM) depleted CARM1 O-GlcNAcylation. CARM1QM was subsequently used to represent non-O-GlcNAcylated CARM1. CARM1QM did not alter protein stability, dimerization capability, cellular localization, or co-activator activity of CARM1 towards a few transcription factors. Interestingly, loss of O-GlcNAcylation, either by mutating CARM1 at putative O-GlcNAcylation sites or by removal of the O-GlcNAc moiety enzymatically, resulted in an alteration of CARM1 substrate specificity. This work demonstrates that O-GlcNAcylation of CARM1 at its C-terminus is an important determinant for CARM1 substrate specificity. In addition to phosphorylation and automethylation, this work identifies O-GlcNAcylation as another PTM that modulates CARM1 function. Thus, regulation of O-GlcNAcylation, a nutrient-sensitive sugar modification, might provide a new means to tune the biological activities of CARM1 in response to nutrients and cellular metabolites. |
