O-GlcNAcylation of co-activator-associated arginine methyltransferase 1 regulates its protein substrate specificity

Abstract

Co-activator-associated arginine methyltransferase 1 (CARM1) asymmetrically di-methylates proteins on arginine residues. CARM1 was previously known to be modified through O-linked- β -N -acetylglucosaminidation (O-GlcNAcylation). However, the site(s) of O-GlcNAcylation were not mapped and the effects of O-GlcNAcylation on biological functions of CARM1 were undetermined. In the present study, we describe the comprehensive mapping of CARM1 post-translational modification (PTM) using top-down MS. We found that all detectable recombinant CARM1 expressed in human embryonic kidney (HEK293T) cells is automethylated as we previously reported and that about 50% of this automethylated CARM1 contains a single O-linked-β -N -acetylglucosamine (O-GlcNAc) moiety [31]. The O-GlcNAc moiety was mapped by MS to four possible sites (Ser 595, Ser 598, Thr 601 and Thr 603) in the C-terminus of CARM1. Mutation of all four sites [CARM1 quadruple mutant (CARM1 QM)] markedly decreased O-GlcNAcylation, but did not affect protein stability, dimerization or cellular localization of CARM1. Moreover, CARM1 QM elicits similar co-activator activity as CARM1 wild-type (CARM1 WT) on a few transcription factors known to be activated by CARM1. However, O-GlcNAcdepleted CARM1 generated by wheat germ agglutinin (WGA) enrichment, O-GlcNAcase (OGA) treatment and mutation of putative O-GlcNAcylation sites displays different substrate specificity from that of CARM1 WT. Our findings suggest that O-GlcNAcylation of CARM1 at its C-terminus is an important determinant for CARM1 substrate specificity.