title: 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 (Ser595, Ser598, Thr601 and Thr603) in the C-terminus of CARM1. Mutation of all four sites [CARM1 quadruple mutant (CARM1QM)] markedly decreased O-GlcNAcylation, but did not affect protein stability, dimerization or cellular localization of CARM1. Moreover, CARM1QM elicits similar co-activator activity as CARM1 wild-type (CARM1WT) on a few transcription factors known to be activated by CARM1. However, O-GlcNAc-depleted 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 CARM1WT. Our findings suggest that O-GlcNAcylation of CARM1 at its C-terminus is an important determinant for CARM1 substrate specificity.
authors.affiliation: *McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, U.S.A.
authors.forename: Purin
authors.initials: P
authors.lastname: Charoensuksai
authors.affiliation: *McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, U.S.A.
authors.forename: Peter
authors.initials: P
authors.lastname: Kuhn
authors.affiliation: *McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, U.S.A.
authors.forename: Lu
authors.initials: L
authors.lastname: Wang
authors.affiliation: *McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, U.S.A.
authors.forename: Nathan
authors.initials: N
authors.lastname: Sherer
authors.affiliation: *McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, U.S.A.
authors.forename: Wei
authors.initials: W
authors.lastname: Xu
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D057809:HEK293 Cells; D006801:Humans; D061986:MCF-7 Cells; D051379:Mice; D008969:Molecular Sequence Data; D011484:Protein-Arginine N-Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D011484:Protein-Arginine N-Methyltransferases; C119966:coactivator-associated arginine methyltransferase 1; D000117:Acetylglucosamine