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Protein-Arginine Methyltransferase I, The THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 26, Issue of June 30, pp. 19866–19876, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Protein-arginine Methyltransferase I, the Predominant Protein-arginine Methyltransferase in Cells, Interacts with and Is Regulated by Interleukin Enhancer-binding Factor 3* Received for publication, January 5, 2000, and in revised form, March 27, 2000 Published, JBC Papers in Press, April 3, 2000, DOI 10.1074/jbc.M000023200 Jie Tang‡§¶, Peter N. Kaoʈ, and Harvey R. Herschman‡§** From the ‡Molecular Biology Institute and the §Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California 90095 and the ʈDivision of Pulmonary and Critical Care Medicine, Stanford University Medical Center, Stanford, California 94305 Arginine methylation is a common post-translation ferases include many RNA-binding proteins (4), RNA-trans- modification found in many proteins. Protein-arginine porting proteins (4), transcription factors (3, 5), nuclear matrix methyltransferase I (PRMT1) contributes >90% of type I proteins (3), and cytokines (6). Type I arginine protein methyl- protein-arginine methyltransferase activity in cells and ation modifies the activities of transcription factors (3), modu- tissues. To expand our knowledge on the regulation and lates the affinity of nucleic acid-binding proteins for nucleic role of PRMT1 in cells, we used the yeast two-hybrid acids (3), regulates interferon signaling pathways (7), and al- system to identify proteins that interact with PRMT1. ters targeting of nuclear proteins (8). Type II protein-arginine One of the interacting proteins we cloned is interleukin methyltransferases catalyze the formation of both monomethyl- enhancer-binding factor 3 (ILF3), also known as M arginine and symmetric NG,NЈG-dimethylarginine (3). Myelin phase phosphoprotein 4. ILF3 is closely related to nu- basic protein is the only known substrate for type II arginine clear factor 90 (NF90). Using an immunofluorescence methyltransferase activity. Type III protein-arginine methyl- analysis, we determined that ILF3 and PRMT1 co-local- transferase catalyzes the monomethylation of the internal gua- ize in the nucleus. Moreover, PRMT1 and ILF3 co-pre- nidino nitrogen atom to form ␻-NG-monomethylarginine (9). cipitate in immunoprecipitation assays and can be iso- 1 lated together in “pull-down” experiments using Protein-arginine methyltransferase I (PRMT1) was the first recombinant fusion proteins. ILF3 is a robust substrate type I protein-arginine N-methyltransferase in mammalian for methylation by PRMT1 and can modulate PRMT1 cells to be cloned and characterized (10). PRMT1 is the pre- activity in in vitro methylation assays. Deletion studies dominant type I enzyme in tissues and contributes most of the demonstrated that the COOH-terminal region of ILF3, type I protein-arginine methyltransferase activity in mamma- which is rich in arginine, glycine, and serine, is respon- lian cells (11). The calculated molecular mass of the PRMT1 sible for the strong interaction between PRMT1 and polypeptide, based on the cDNA-derived protein sequence, is ILF3 and is the site of ILF3 methylation by PRMT1. 40.5 kDa (10). However, PRMT1 in cells exists as a large Although ILF3 and NF90 are highly similar, they differ complex of 300–400 kDa (10, 12). PRMT1 can interact with a in their carboxyl-terminal regions. Because of this dif- variety of proteins, including TIS21 (10), BTG1 (10), and inter- ference, NF90 does not interact with PRMT1, is a much feron-␣/␤ receptor 1 (7). TIS21 and the related protein BTG1 poorer substrate than ILF3 for PRMT1-dependent meth- are members of a family of proteins thought to be involved in ylation, and does not modulate PRMT1 enzyme activity. negative regulation of cell growth (13, 14). Both TIS21 and BTG1 interact with and regulate the activity of PRMT1 (10). The interaction between PRMT1 and the cytoplasmic domain of Arginine methylation is a common post-translation protein interferon-␣/␤ receptors is proposed to be important to the modification in eucaryotic cells, discovered more than 30 years interferon signaling pathways. ago (1, 2). There are at least three types of protein-arginine We performed a yeast two-hybrid interaction screen for N-methyltransferase activities that transfer methyl groups PRMT1-interacting proteins to identify proteins that might from S-adenosyl-L-methionine to the guanidino group of argi- associate with PRMT1 in cells and to study the regulation of nine residues (3). The formation of monomethylarginine and PRMT1. This screen identified interleukin enhancer-binding G G asymmetric N ,N -dimethylarginine is catalyzed by type I en- factor 3 (ILF3) as a protein that could potentially interact with zymes. Substrates for type I protein-arginine methyltrans- PRMT1. Interleukin enhancer-binding factor proteins belong to a group of proteins that can bind to the antigen receptor re- * This work was supported in part by UCLA Asthma, Allergy, and sponse element 2 (ARRE-2) sequence (15). This group of pro- Immunologic Diseases Center Grant AI34567 from NIAID and NIEHS teins includes ILF1 (15), nuclear factor 45 (NF45, also referred (to H. R. H.) and by National Institutes of Health Grant AI39624 (to as ILF2) (16, 17), and nuclear factor 90 (NF90) (18). ILF3, a P. N. K.). The costs of publication of this article were defrayed in part by protein with an apparent molecular mass of ϳ110 kDa (19), is the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted 1 The abbreviations used are: PRMT, protein-arginine methyltrans- to the GenBankTM/EBI Data Bank with accession number(s) AF220102. ferase; ILF, interleukin enhancer-binding factor; ARRE-2, antigen re- ¶ Postdoctoral trainee supported by United States Public Health ceptor response element-2; NF, nuclear factor; MPP4, M phase phos- Service Institutional Research Award T32 CA09056. phoprotein 4; PCR, polymerase chain reaction; GST, glutathione ** To whom correspondence should be addressed: 341A Molecular S-transferase; PBS, phosphate-buffered saline; PAGE, polyacrylamide Biology Inst., UCLA, 611 Charles E. Young Dr. East, Los Angeles, CA gel electrophoresis; [3H]AdoMet, S-adenosyl-L-[methyl-3H]methionine; 90095. Tel.: 310-825-8735; Fax: 310-825-1447; E-mail: hherschman@ GST-GAR, glutathione S-transferase-fibrillarin glycine arginine do- mednet.ucla.edu. main fusion protein. 19866 This paper is available on line at http://www.jbc.org PRMT1 Interactions with ILF3 19867 closely related in sequence to NF90. In fact, NF90 and ILF3 pBluescript-ILF3 into pcDNA3.1(Ϫ) (Invitrogen). pcDNA3.1(ϩ)- have both been referred to as ILF3 in different reports (16, 18, ILF3⌬C was constructed by subcloning the SalI/NotI fragment from ⌬ 19). However, ILF3 and NF90 are distinct protein species (17, pGEX(SN)-ILF3 C into pBluescript to create a KpnI site at the 5-end and then inserting the KpnI/NotI fragment from pBluescript-ILF3⌬C 19, 20) whose relationships have not been clearly addressed into pcDNA3.1(ϩ). pcDNA3.1(Ϫ)-NF90 was constructed by inserting previously. In this report, the 110-kDa protein is referred to as the EcoRI fragment of NF90 cDNA from pBluescript-NF90 into ILF3, and the 90-kDa protein is termed NF90. pcDNA3.1(Ϫ). Sequences of the cDNA fragments amplified by PCR Mouse ILF3 is encoded by an open reading frame of 911 were confirmed by DNA sequencing. amino acid residues (19). The cDNA of murine ILF3 is encoded Biotinylation of Anti-PRMT1 IgG—IgG proteins from anti-PRMT1 by a single copy gene located on chromosome 9 near D9Mit160 antiserum were purified on a protein A-Sepharose 4B column. Briefly, 1 ml of antiserum was bound to a 1.5-ml protein A-Sepharose 4B (19). ILF3 is cyclically hyperphosphorylated in M phase during column that had been equilibrated with phosphate-buffered saline mitosis and is also referred to as M phase phosphoprotein 4 (PBS). The column was extensively washed with PBS until no protein (MPP4) (21). ILF3 is closely related in sequence to the Xenopus was detectable in the eluate. The absorbed IgG proteins were eluted p122 protein, a double-stranded RNA-binding protein that is with 250 mM glycine (pH 2.5). The eluted proteins were neutralized in also a component of a CCAAT box transcription factor (22), and 2 M Tris-HCl buffer (pH 7.6) and then desalted on a Sephadex G-25 to human NF90 (17). NF90 is virtually identical to ILF3 in the column using 100 mM bicarbonate buffer (pH 8.0). To biotinylate anti-PRMT1 IgG protein, purified IgG (1 mg/ml) in 100 first 600 amino acid residues (17, 19). Like ILF3, NF90 con- mM bicarbonate buffer (pH 8.0) was incubated with 40 ␮lof10mM tains two double-stranded RNA-binding motifs and binds dou- sulfosuccinimidobiotin for4hatroom temperature. Then, IgG proteins ble- and single-stranded RNAs (23). NF90 and NF45 are puri- were separated from sulfosuccinimidobiotin on a Sephadex G-25 col- fied as a dimeric complex that binds specifically to ARRE-2 umn using PBS to elute proteins from the column. IgG proteins were (20), an important cis-acting element found in many cytokine shown to be biotinylated by immunoblotting Rat1 cell lysate with the genes. Both NF90 and NF45 migrate from the nucleus to the biotinylated IgG as the first antibody and using horseradish peroxi- dase-labeled streptavidin as the secondary antibody on Western blots. cytoplasm in mitotic cells (24). NF90 appears to be involved in Immunofluorescent Labeling of ILF3/NF90 and PRMT1—Immuno- transcriptional regulation of promoters that contain nuclear localization of proteins in cells was performed as described previously factor of activated T cells-binding sites (17, 20). In cells, NF90 (12). To double-stain HeLa cells with anti-PRMT1 and anti-ILF3/NF90 can interact with a variety of proteins, including double- antibodies, HeLa cells in four-well chamber slides were washed twice stranded RNA-dependent protein kinase (25) and DNA- with PBS and fixed with 3% paraformaldehyde for 5 min, followed by 10 dependent protein kinase (26).
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