Biochem. J. (2015) 466, 587–599 (Printed in Great Britain) doi:10.1042/BJ20141072 587 O-GlcNAcylation of co-activator-associated arginine methyltransferase 1 regulates its protein substrate specificity Purin Charoensuksai*, Peter Kuhn*†, Lu Wang*, Nathan Sherer* and Wei Xu*1 *McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705, U.S.A. †Biological Sciences Department, Edgewood College, Madison, WI 53711, U.S.A. Co-activator-associated arginine methyltransferase 1 (CARM1) (CARM1QM)] markedly decreased O-GlcNAcylation, but did asymmetrically di-methylates proteins on arginine residues. not affect protein stability, dimerization or cellular localization CARM1 was previously known to be modified through O-linked- of CARM1. Moreover, CARM1QM elicits similar co-activator β-N-acetylglucosaminidation (O-GlcNAcylation). However, the activity as CARM1 wild-type (CARM1WT) on a few transcription site(s) of O-GlcNAcylation were not mapped and the effects factors known to be activated by CARM1. However, O-GlcNAc- of O-GlcNAcylation on biological functions of CARM1 depleted CARM1 generated by wheat germ agglutinin (WGA) were undetermined. In the present study, we describe enrichment, O-GlcNAcase (OGA) treatment and mutation of the comprehensive mapping of CARM1 post-translational putative O-GlcNAcylation sites displays different substrate modification (PTM) using top-down MS. We found that all specificity from that of CARM1WT. Our findings suggest that detectable recombinant CARM1 expressed in human embryonic O-GlcNAcylation of CARM1 at its C-terminus is an important kidney (HEK293T) cells is automethylated as we previously determinant for CARM1 substrate specificity. reported and that about 50% of this automethylated CARM1 contains a single O-linked-β-N-acetylglucosamine (O-GlcNAc) Key words: co-activator-associated arginine methyltransferase 1 moiety [31]. The O-GlcNAc moiety was mapped by MS to four (CARM1), co-activator, mass spectrometry (MS), protein methyl- possible sites (Ser595,Ser598,Thr601 and Thr603) in the C-terminus ation, O-linked-β-N-acetylglucosaminidation (O-GlcNAcyla- of CARM1. Mutation of all four sites [CARM1 quadruple mutant tion), transcription. INTRODUCTION methylate diversified substrates allows it to orchestrate a broad spectrum of biological processes. However, little is known about Co-activator-associated arginine methyltransferase 1 (CARM1), how the methyltransferase activity and substrate specificity of also known as protein arginine methyltransferase4 (PRMT4), is a CARM1 are regulated. type I PRMT that asymmetrically dimethylates arginine on target Proteins in the PRMT family share a common domain proteins. CARM1 was first identified as an interacting partner organization. The core region contains a methyltransferase of the glucocorticoid receptor interacting protein 1 (GRIP1) co- catalytic site, methyl donor S-adenosyl methionine (SAM)- activator protein that enhanced transcriptional activity of several binding pocket and dimerization arm. Although the catalytic steroid hormone receptors [1]. Subsequent studies showed that cores are highly conserved among PRMTs, CARM1 possesses CARM1 activates many cancer-relevant transcription factors a unique C-terminus [9]. This C-terminal region is not essential including PPARγ (peroxisome proliferator-activated receptor for CARM1’s methyltransferase activity in vitro;however, γ ), p53, NF-κB (nuclear factor kappa-light-chain-enhancer of it exhibits an autonomous transcriptional activation function activated B-cells), E2F1 (E2F transcription factor 1) and β- whose mechanism remains unclear. The C-terminal region can catenin [1–5]. The methyltransferase activity of CARM1 has also mediate protein–protein interaction. For example, TIF1α been extensively studied. CARM1 methylates and modulates the (transcription intermediary factor 1 α)/TRIM24 (tripartite motif- functions of a plethora of proteins. The first CARM1 substrate containing 24) was reported to interact with this domain [10]. identified was histone H3 [1]. CARM1-specific histone H3 O-GlcNAcylation (O-linked-β-N-acetylglucosaminidation), methylation at Arg17 has been linked to gene activation and is found on >1000 nuclear and cytoplasmic proteins, is considered part of the ‘histone code’ [6]. Other substrates of characterized by the addition of N-acetyl-D-glucosamine to CARM1 include BAF155 (BRG1-associated factor 155), SmB serine and threonine residues of target proteins [11–14]. O- (small nuclear ribonucleoprotein polypeptide B), SAP49 (splicing GlcNAcylation is a reversible post-translational modification factor 3B subunit 4), U1C (U1 small nuclear RNP specific C), (PTM) governed by two enzymes: O-GlcNAc (O-linked-β-N- CA150 (co-activator of 150 kDa), HuR (Hu antigen R), HuD (Hu acetylglucosamine) transferase (OGT) [15,16] and O-GlcNAcase antigen D), TARPP (thymocyte cAMP-regulated phosphoprotein) (OGA) [17] (reviewed in [18–21]). UDP–GlcNAc, the GlcNAc and PABP1 [poly(A)-binding protein 1], many of which are donor molecule, is generated in the hexosamine biosynthesis involved in mRNA processing and transcription elongation [7,8]. pathway. Protein O-GlcNAcylation has been shown to regulate The ability of CARM1 to activate many transcription factors and diverse protein functions including stability, localization, Abbreviations: CAD, collisionally-activated dissociation; CARM1, co-activator-associated arginine methyltransferase 1; CARM1QM, CARM1 quadruple mutant; CARM1WT, CARM1 wild-type; CHX, cycloheximide; DMEM, Dulbecco’s modified Eagle’s medium; E2, oestradiol; ECD, electron-capture dissociation; ERα, oestrogen receptor α; GRIP1, glucocorticoid receptor interacting protein 1; HEK, human embryonic kidney; OGA, O-GlcNAcase; O- GlcNAc, O-linked-β-N-acetylglucosamine; O-GlcNAcylation, O-linked-β-N-acetylglucosaminidation; OGT, O-GlcNAc transferase; PABP1, poly(A)-binding protein 1; PBST, phosphate-buffered saline with Tween 20; PPARγ, peroxisome proliferator-activated receptor γ; PRMT, protein arginine methyltransferase; PTM, post-translational modification; TIF1α, transcription intermediary factor 1 α; WGA, wheat germ agglutinin; ZFN, zinc finger nuclease. 1 To whom correspondence should be addressed (email [email protected]). c The Authors Journal compilation c 2015 Biochemical Society 588 P. Charoensuksai and others protein–protein interaction, transcriptional activity, enzymatic 6 h. The reaction was then resolved by SDS/PAGE and activity and substrate specificity (reviewed in [18,22–24]). stained with Coomassie Brilliant Blue [0.05 (w/v):50:10:40; Using top-down MS, we found that nearly 100% of CARM1 Coomassie Brilliant Blue R-250/methanol/acetic acid/H2O] is automethylated in vivo and that 50% of this automethylated overnight followed by destaining with dye-free buffer for 2– CARM1 exhibits a 203 Da mass shift, indicative of mono- 4 h. Gels were then incubated with Amplify scintillation fluid O-GlcNAcylation. We mapped Ser595,Ser598,Thr601 and Thr603 (GE Healthcare Life Sciences) for 20 min prior to drying as major O-GlcNAcylation sites located in the C-terminus of on a Whatman paper and exposing to an X-ray film. For CARM1 protein, implying that CARM1 could exist as a mixture 2D electrophoresis, 2.5 μg of purified CARM1 was incubated −/− of mono-O-GlcNAcylated forms. O-GlcNAcylation of CARM1 with 500 μgofCal51CARM1 cell lysates. The reaction was did not appear to alter stability, nuclear–cytoplasmic distribution, resolved using isoelectric focusing pH 4–8. 2D electrophoresis dimerization capability and co-activator activity of CARM1 on was performed by Kendrick Labs. a few tested transcription factors. However, O-GlcNAcylation of CARM1 affects its substrate specificity. Our findings reveal that O-GlcNAcylation of the CARM1 C-terminus regulates substrate Enrichment of O-GlcNAcylated CARM1 with wheat germ agglutinin specificity and thus may affect a variety of CARM1 functions resin depending on substrate methylation. Enrichment of O-GlcNAcylated CARM1 was adapted from the protocol described by Zachara et al. [27]. Purified CARM1 EXPERIMENTAL (20 μg) was incubated with 100 μl of WGA (wheat germ agglutinin) agarose bead (Vector Labs) for 1 h on ice. After The expression and purification of recombinant CARM1 centrifugation, the flow-through fraction was collected. The Full-length mouse CARM1 cDNA was cloned into Halo– column was then washed with WGA wash buffer (25 mM Tris, tag vector, pFC14K (Promega), as described previously 300 mM NaCl, 5 mM CaCl2 and 1 mM MgCl2) followed by [25]. CARM1S595A, CARM1S598A, CARM1T601A, CARM1T603A and elution with WGA elution buffer (0.2% v/v Nonidet P40 and CARM1QM (CARM1 quadruple mutant) were generated by site- 1MN-acetyl-D-glucosamine). directed mutagenesis using pFC14K–CARM1 construct as a template. CARM1CTD (C-terminal domain deleted CARM1) was generated by PCR cloning of mouse CARM1 encoding Generation of CARM1 (WT or QM) stably expressing cell lines amino acid 1–553 into a pFC14K Flexi vector. The expression and purification of Halo–tagged recombinant CARM1 were CARM1-null human embryonic kidney (HEK293T), MCF7 (ERa performed as previously described [25]. positive breast cancer cell line) and MDA-MB-231 (triple- negative breast cancer cell line) cell lines were described in [28]. CARM1-null Cal51 cell lines were generated using Top-down MS the same method as described in [28]. Flag–tagged mouse The high-resolution FT-ICR (Fourier transform-ion cyclotron CARM1WT (CARM1 wild-type) or CARM1QM was cloned into resonance) MS analysis