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• W2023382624 abstract "The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg57 and Arg59, two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization. The HMGA1a protein belongs to the high mobility group A (HMGA) family of architectural nuclear factors, a group of proteins that plays an important role in chromatin dynamics. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes, such as transcriptional regulation, viral integration, DNA repair, RNA processing, and chromatin remodeling. The activity of HMGA proteins is finely modulated by a variety of post-translational modifications. Arginine methylation was recently demonstrated to occur on HMGA1a protein, and it correlates with the apoptotic process and neoplastic progression. Methyltransferases responsible for these modifications are unknown. Here we show that the protein arginine methyltransferase PRMT6 specifically methylates HMGA1a protein both in vitro and in vivo. By mass spectrometry, the sites of methylation were unambiguously mapped to Arg57 and Arg59, two residues which are embedded in the second AT-hook, a region critical for both protein-DNA and protein-protein interactions and whose modification may cause profound alterations in the HMGA network. The in vivo association of HMGA and PRMT6 place this yet functionally uncharacterized methyltransferase in the well established functional context of the chromatin structure organization. HMGA1a belongs, together with its isoform HMGA1b and the highly related protein HMGA2, to the high mobility group A (HMGA) 3The abbreviations used are: HMGA, high mobility group A; PRMT, protein arginine methyltransferase; PTM, post-translational modification; IFN-β, interferon β; CREB, cAMP-response element-binding protein; HIV, human immunodeficiency virus; HA, hemagglutinin; GFP, green fluorescent protein; GST, glutathione S-transferase; NPM, nucleophosmin; PABP1, poly(A)-binding protein 1; LC-MS, liquid chromatography-mass spectrometry; MS/MS, tandem mass spectrometry; HEK, human embryonic kidney; [3H]AdoMet, 3H-labeled S-adenosyl-l-methionine; GAR, glycine arginine-rich.3The abbreviations used are: HMGA, high mobility group A; PRMT, protein arginine methyltransferase; PTM, post-translational modification; IFN-β, interferon β; CREB, cAMP-response element-binding protein; HIV, human immunodeficiency virus; HA, hemagglutinin; GFP, green fluorescent protein; GST, glutathione S-transferase; NPM, nucleophosmin; PABP1, poly(A)-binding protein 1; LC-MS, liquid chromatography-mass spectrometry; MS/MS, tandem mass spectrometry; HEK, human embryonic kidney; [3H]AdoMet, 3H-labeled S-adenosyl-l-methionine; GAR, glycine arginine-rich. family of non-histone chromosomal proteins (1.Johnson K.R. Lehn D.A. Elton T.S. Barr P.J. Reeves R. J. Biol. Chem. 1988; 263: 18338-18342Abstract Full Text PDF PubMed Google Scholar, 2.Manfioletti G. Giancotti V. Bandiera A. Buratti E. Sautiere P. Cary P. Crane-Robinson C. Coles B. Goodwin G. Nucleic Acids Res. 1991; 19: 6793-6797Crossref PubMed Scopus (166) Google Scholar). HMGA family members are considered proto-oncogenes and, when overexpressed in cell line models, are able to transform cells or increase their malignancy. In addition, transgenic mice overexpressing HMGA develop different tumors (3.Sgarra R. Rustighi A. Tessari M.A. Di Bernardo J. Altamura S. Fusco A. Manfioletti G. Giancotti V. FEBS Lett. 2004; 574: 1-8Crossref PubMed Scopus (188) Google Scholar, 4.Fedele M. Battista S. Manfioletti G. Croce C.M. Giancotti V. Fusco A. Carcinogenesis. 2001; 22: 1583-1591Crossref PubMed Scopus (106) Google Scholar, 5.Tallini G. Dal Cin P. Adv. Anat. Pathol. 1999; 6: 237-246Crossref PubMed Scopus (118) Google Scholar).HMGA proteins contain about 100 amino acid residues and have three DNA-binding domains called AT-hooks that mediate their ability to interact with the narrow minor groove of AT-rich DNA sequences (6.Reeves R. Gene. 2001; 277: 63-81Crossref PubMed Scopus (467) Google Scholar). By binding to DNA and/or transcription factors, HMGA proteins can organize the assembly of nucleoprotein-DNA transcriptional complexes (called enhanceosomes) at the level of enhancers or promoters activating or repressing transcription of a large number of mammalian genes. For this reason, they are referred to as architectural transcription factors (6.Reeves R. Gene. 2001; 277: 63-81Crossref PubMed Scopus (467) Google Scholar, 7.Reeves R. Beckerbauer L. Biochim. Biophys. Acta. 2001; 1519: 13-29Crossref PubMed Scopus (318) Google Scholar). In addition to transcriptional regulation, HMGA proteins are involved in other nuclear processes, such as viral integration, RNA processing, DNA repair, and chromatin structural organization and remodeling, indicating HMGA as highly connected nodes in the chromatin protein network (3.Sgarra R. Rustighi A. Tessari M.A. Di Bernardo J. Altamura S. Fusco A. Manfioletti G. Giancotti V. FEBS Lett. 2004; 574: 1-8Crossref PubMed Scopus (188) Google Scholar, 6.Reeves R. Gene. 2001; 277: 63-81Crossref PubMed Scopus (467) Google Scholar, 8.Sgarra R. Tessari M.A. Di Bernardo J. Rustighi A. Zago P. Liberatori S. Armini A. Bini L. Giancotti V. Manfioletti G. Proteomics. 2005; 5: 1494-1506Crossref PubMed Scopus (41) Google Scholar).HMGA proteins are subjected to a variety of post-translational modifications (PTMs) that modulate their multi-interacting property with both DNA and proteins (9.Edberg D.D. Adkins J.N. Springer D.L. Reeves R. J. Biol. Chem. 2005; 280: 8961-8973Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 10.Edberg D.D. Bruce J.E. Siems W.F. Reeves R. Biochemistry. 2004; 43: 11500-11515Crossref PubMed Scopus (46) Google Scholar, 11.Sgarra R. Diana F. Bellarosa C. Dekleva V. Rustighi A. Toller M. Manfioletti G. Giancotti V. Biochemistry. 2003; 42: 3575-3585Crossref PubMed Scopus (45) Google Scholar, 12.Sgarra R. Diana F. Rustighi A. Manfioletti G. Giancotti V. Cell Death Diff. 2003; 10: 386-389Crossref PubMed Scopus (24) Google Scholar, 13.Banks G.C. Li Y. Reeves R. Biochemistry. 2000; 39: 8333-8346Crossref PubMed Scopus (69) Google Scholar, 14.Diana F. Sgarra R. Manfioletti G. Rustighi A. Poletto D. Sciortino M.T. Mastino A. Giancotti V. J. Biol. Chem. 2001; 276: 11354-11361Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 15.Schwanbeck R. Manfioletti G. Wisniewski J.R. J. Biol. Chem. 2000; 275: 1793-1801Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). HMGA1a and HMGA1b are constitutively phosphorylated by casein kinase 2 at the two or three serine residues of the C-terminal end (16.Ferranti P. Malorni A. Marino G. Pucci P. Goodwin G.H. Manfioletti G. Giancotti V. J. Biol. Chem. 1992; 267: 22486-22489Abstract Full Text PDF PubMed Google Scholar). Cell cycle-dependent phosphorylation by p34/cdc2 kinase has been detected on Thr52 and Thr77 (numbers refer to human HMGA1a) residues, flanking the second AT-hook and shown to cause a strong decrease in the DNA-binding affinity (17.Nissen M.S. Langan T.A. Reeves R. J. Biol. Chem. 1991; 266: 19945-19952Abstract Full Text PDF PubMed Google Scholar, 18.Reeves R. Langan T.A. Nissen M.S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1671-1675Crossref PubMed Scopus (115) Google Scholar). Likewise p34/cdc2, phosphorylation by protein kinase C on Thr20, Ser43, and Ser63 caused a significant reduction of DNA-binding affinity (13.Banks G.C. Li Y. Reeves R. Biochemistry. 2000; 39: 8333-8346Crossref PubMed Scopus (69) Google Scholar). Acetylation of Lys64 and Lys70 are critical for the stability of the enhanceosome assembled on the IFN-β gene (19.Munshi N. Agalioti T. Lomvardas S. Merika M. Chen G. Thanos D. Science. 2001; 293: 1133-1136Crossref PubMed Scopus (182) Google Scholar, 20.Yie J. Senger K. Thanos D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13108-13113Crossref PubMed Scopus (98) Google Scholar). In particular, acetylation of Lys70 by p300 CBP-associated factor (PCAF)/GCN5 results in the stabilization, whereas that of Lys64 by p300/CBP (CREB-binding protein) causes destabilization of the enhanceosome. Methylation is the most recent HMGA PTM reported (9.Edberg D.D. Adkins J.N. Springer D.L. Reeves R. J. Biol. Chem. 2005; 280: 8961-8973Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 10.Edberg D.D. Bruce J.E. Siems W.F. Reeves R. Biochemistry. 2004; 43: 11500-11515Crossref PubMed Scopus (46) Google Scholar, 11.Sgarra R. Diana F. Bellarosa C. Dekleva V. Rustighi A. Toller M. Manfioletti G. Giancotti V. Biochemistry. 2003; 42: 3575-3585Crossref PubMed Scopus (45) Google Scholar, 12.Sgarra R. Diana F. Rustighi A. Manfioletti G. Giancotti V. Cell Death Diff. 2003; 10: 386-389Crossref PubMed Scopus (24) Google Scholar, 13.Banks G.C. Li Y. Reeves R. Biochemistry. 2000; 39: 8333-8346Crossref PubMed Scopus (69) Google Scholar, 14.Diana F. Sgarra R. Manfioletti G. Rustighi A. Poletto D. Sciortino M.T. Mastino A. Giancotti V. J. Biol. Chem. 2001; 276: 11354-11361Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Arg25 within the first AT-hook of HMGA1a has been found methylated (monomethylated) in tumor cell lines, reaching in some samples up to 50% of total HMGA1a protein content. Methylation on this residue is modulated during the apoptotic process reaching highest levels at later stages, with the formation of apoptotic bodies (14.Diana F. Sgarra R. Manfioletti G. Rustighi A. Poletto D. Sciortino M.T. Mastino A. Giancotti V. J. Biol. Chem. 2001; 276: 11354-11361Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). More recently, dimethylation on the same Arg25 residue has been reported by another group (21.Zou Y. Wang Y. Biochemistry. 2005; 44: 6293-6301Crossref PubMed Scopus (31) Google Scholar). An extensive study on both HMGA1a and HMGA1b proteins reported a high level of PTMs, and in particular, dimethylation of arginine and lysine residues was increased in breast cancer cells with higher metastatic potential (9.Edberg D.D. Adkins J.N. Springer D.L. Reeves R. J. Biol. Chem. 2005; 280: 8961-8973Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 10.Edberg D.D. Bruce J.E. Siems W.F. Reeves R. Biochemistry. 2004; 43: 11500-11515Crossref PubMed Scopus (46) Google Scholar). Altogether, these data lead authors to suggest the existence of a PTM “code” for HMGA proteins similar to that reported for histones (10.Edberg D.D. Bruce J.E. Siems W.F. Reeves R. Biochemistry. 2004; 43: 11500-11515Crossref PubMed Scopus (46) Google Scholar, 11.Sgarra R. Diana F. Bellarosa C. Dekleva V. Rustighi A. Toller M. Manfioletti G. Giancotti V. Biochemistry. 2003; 42: 3575-3585Crossref PubMed Scopus (45) Google Scholar). Although the kinases and acetyltransferases that are able to modify HMGA proteins have been identified, enzymes responsible for arginine and lysine methylation are just starting to be discovered (22.Miranda T.B. Webb K.J. Edberg D.D. Reeves R. Clarke S. Biochem. Biophys. Res. Commun. 2005; 336: 831-835Crossref PubMed Scopus (52) Google Scholar).Protein (lysine and arginine) methylation is an emerging type of PTM that has added a new dimension to the signal transduction field (23.Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (330) Google Scholar, 24.Bedford M.T. Richard S. Mol. Cell. 2005; 18: 263-272Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). At variance from histones, where the prevalent modification is lysine methylation (25.Bannister J.A. Kouzarides T. Nature. 2005; 436: 1103-1106Crossref PubMed Scopus (384) Google Scholar), in HMGA proteins, arginine methylation seems particularly relevant (9.Edberg D.D. Adkins J.N. Springer D.L. Reeves R. J. Biol. Chem. 2005; 280: 8961-8973Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 11.Sgarra R. Diana F. Bellarosa C. Dekleva V. Rustighi A. Toller M. Manfioletti G. Giancotti V. Biochemistry. 2003; 42: 3575-3585Crossref PubMed Scopus (45) Google Scholar). Arginine methylation has been implicated in signal transduction, RNA metabolism, transcriptional regulation, and DNA repair (23.Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2005; 26: 147-170Crossref PubMed Scopus (330) Google Scholar, 24.Bedford M.T. Richard S. Mol. Cell. 2005; 18: 263-272Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). Arginine residues are methylated by protein arginine methyltransferases (PRMTs). To date, two distinct PRMT activities have been found in mammalian cells; both types catalyze the formation of ω-NG-monomethyl arginine as an intermediate. Type I PRMT activity is defined by the formation of asymmetric ω-NG,NG-dimethylarginine residues, whereas type II activity is defined by the formation of symmetric ω-NG,N′G-dimethylarginine residues. Currently, known type I enzymes include PRMT1, PRMT3, coactivator-associated arginine methyltransferase 1 (CARM1)/PRMT4, PRMT6, and PRMT8 (24.Bedford M.T. Richard S. Mol. Cell. 2005; 18: 263-272Abstract Full Text Full Text PDF PubMed Scopus (904) Google Scholar). No activity has been demonstrated for PRMT2 (26.Scott H.S. Antonarakis S.E. Lalioti M.D. Rossier C. Silver P.A. Henry M.F. Genomics. 1998; 38: 330-340Crossref Scopus (143) Google Scholar, 27.Zhang X. Cheng X. Structure (Camb.). 2003; 11: 509-520Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). The only type II PRMTs identified to date are the Janus kinase-binding protein JBP1/PRMT5 (28.Branscombe T.L. Frankel A. Lee J.H. Cook J.R. Yang Z. Pestka S. Clarke S. J. Biol. Chem. 2001; 276: 32971-32976Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar) and PRMT7 (29.Lee J.H. Cook J.R. Yang Z.H. Mirochnitchenko O. Gunderson S.I. Felix A.M. Nerth N. Hoffmann R. Pestka S. J. Biol. Chem. 2005; 280: 3656-3664Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 30.Miranda T.B. Miranda M. Frankel A. Clarke S. J. Biol. Chem. 2004; 279: 22902-22907Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar).In this manuscript, we have reported the identification of PRMT6 as a protein methyltransferase able to efficiently methylate in vitro and in vivo HMGA1a. PRMT6 has been recently identified as a type I PRMT enzyme with an exclusive nuclear localization (31.Frankel A. Yadav N. Lee J. Branscombe T.L. Clarke S. Bedford M.T. J. Biol. Chem. 2002; 277: 3537-3543Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). PRMT6 in vivo cellular substrates are unknown. In fact, if we exclude its automethylation activity, the only substrate identified so far is the viral Tat human immunodeficiency virus (HIV) protein (32.Boulanger M.C. Liang C. Russell R.S. Lin R. Bedford M.T. Wainberg M.A. Richard S. J. Virol. 2005; 79: 124-131Crossref PubMed Scopus (155) Google Scholar). Tat-modified Arg residues have not been identified; thus its substrate specificity remains unknown. We report that PRMT6 methylates HMGA1a at the level of Arg57 and Arg59 within the second AT-hook domain. This is a critical region for HMGA1a function, which has been shown to have the highest affinity for DNA binding and also to be involved in protein-protein interaction, thus implying an important role for arginine methylation in modulating HMGA functions. In addition, this study, identifying the residues modified by PRMT6, lays down the basis for future studies aimed at establishing a consensus for its substrate specificity.EXPERIMENTAL PROCEDURESPlasmids—Plasmids pARHMGA1a, pARHMGA1a-(1–80), pARHMGA1a-(1–52), pARHMGA1a-(35–107), pARHMGA1a-(46–107) expressing the wild-type and deletion mutants of human HMGA1a proteins were generated by PCR using the human HMGA1a cDNA as the template. The primers used for expression vector construction were as follows: A5–1, 5′-AGG AGA TAT ACA TAT GAG TGA GTC GAG CT-3′; A5–35, 5′-GCA AGC AGC ATA TGG TGA GTC CCG GGA CA; A5–46, 5′-TGG TAG GGC ATA TGA AGG AGC CCA GCG A-3′; A3–107, 5′-GCA GCC CGG ATC CTT ATC ACT GCT CCT CCT C-3′; A3–80, 5′-GGG TCT GCC GGA TCC TTA TCA TCC TGG AGT TGT-3′; and A3–52, 5′-GCC CCG AGG GGA TCC TTA TCA TGG CAC TTC GCT-3′. The PCR products for deletion constructs at the C terminus were obtained using primer A5–1 in combination with the different A3 primers, whereas products for deletion constructs at the N terminus were obtained using A5–35 and A5–46 in combination with A3–107. The PCR products were cloned between the NdeI and BamHI sites of the bacterial expression vector pAR3038 under the bacteriophage T7 promoter. The resulting clones were verified by sequencing. Plasmids pARHMGA1aR25A, pARHMGA1aR57A, pARHMGA1aR59A, pARHMGA1aR57A,R59A were obtained using the QuikChange site-directed mutagenesis kit by Stratagene. The mutagenesis was performed via standard Stratagene protocol using as template the pARHMGA1a plasmid, except for pARHMGA1aR57A,R59A in which the template used was pARHMGA1aR59A, and using the following primers: R5759A (forward), 5′-CAC CTA AGA GAC CTG CGG GCG CAC CAA AGG-3′; R5759A (reverse), 5′-CCT TTG GTG CGC CCG CAG GTC TCT TAG GTG-3′; R57A (forward), 5′-CAC CTA AGA GAC CTG CGG GCC GAC CAA AGG-3′; R57A (reverse), 5′-CCT TTG GTC GGC CCG CAG GTC TCT TAG GTG-3′; R59A (forward), 5′-GAG ACC TCG GGG CGC ACC AAA GGG AAG C-3′; R59A (reverse), 5′-GCT TCC CTT TGG TGC GCC CCG AGG TCT C-3′; R25A (forward), 5′-AGA AGC GGG GCG CGG GCA GGC CGC-3′; R25A (reverse), 5′-GCG GCC TGC CCG CGC CCC GCT TCT-3′. Plasmids pcDNA3HA-PRMT6 and pcDNA3HA-PRMT1, expressing proteins in-frame with the hemagglutinin (HA) epitope, were obtained by cloning the open reading frames of PRMT6-GFP and PRMT1-GFP (31.Frankel A. Yadav N. Lee J. Branscombe T.L. Clarke S. Bedford M.T. J. Biol. Chem. 2002; 277: 3537-3543Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar) into the BamHI/EcoRI sites and the EcoRI site of the pcDNA3HA vector, respectively. Plasmid pcDNA3HA-HMGA1a was obtained via subcloning using BamHI/EcoRI restriction sites from pcDNAI-HMGA1a (a gift of Dr. Thanos, Alexander Fleming Biomedical Sciences Research Cener, Vari, Greece), whereas the vector pcDNA3HA-nucleophosmin (NPM) was obtained via PCR from the PINCO-HA-NPM retroviral vector (a gift of Dr. Pelicci European Institute of Oncology, Milan, Italy) using the following primers including the BamHI/NotI sites: NPM (forward), 5′-GGC AGG GAT CCA TGG AAG ATT CGA T-3′ and NPM (reverse), 5′-TTA AAG CGG CCG CTT AAA GAG ACT TCC-3′ in the corresponding sites of pcDNA3HA. The plasmids pGEX6P1-PABP1 and pGEX2T-GAR, expressing the poly(A)-binding protein 1 (PABP1) and a protein arginine methyltransferase substrate composed of the first 148 amino acids (glycine arginine-rich domain) of the human fibrillarin protein (both in fusion with the glutathione S-transferase (GST)) have been described previously (33.Lee J. Bedford M.T. EMBO Rep. 2002; 3: 268-273Crossref PubMed Scopus (183) Google Scholar, 34.Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar).Recombinant Protein Expression and Purification—Recombinant HMGA proteins were expressed, extracted, purified, and analyzed by mass spectrometry as previously described (35.Mantovani F. Covaceuszach S. Rustighi A. Sgarra R. Heath C. Goodwin G.H. Manfioletti G. Nucleic Acids Res. 1998; 26: 1433-1439Crossref PubMed Scopus (63) Google Scholar). HMGA1a-(45–75) was a degradation product obtained during the preparation of HMGA1a-(45–106). GST fusion proteins were produced and purified as already described (31.Frankel A. Yadav N. Lee J. Branscombe T.L. Clarke S. Bedford M.T. J. Biol. Chem. 2002; 277: 3537-3543Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar).Mass Spectrometry Analyses—LC-MS analyses on PRMT6-methylated HMGA1a were carried out with an API 1 mass spectrometer (PerkinElmer SCIEX) as previously described (14.Diana F. Sgarra R. Manfioletti G. Rustighi A. Poletto D. Sciortino M.T. Mastino A. Giancotti V. J. Biol. Chem. 2001; 276: 11354-11361Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Mass values are reported as Da ± 1. To identify the methylation sites, 10 μg of PRMT6-methylated HMGA1a were digested with 0.5 μg of Endoproteinase Lys-C sequencing grade (Roche Applied Science) for 18 h at 37 °C. HMGA1a fragments were analyzed by LC-MS as previously described (14.Diana F. Sgarra R. Manfioletti G. Rustighi A. Poletto D. Sciortino M.T. Mastino A. Giancotti V. J. Biol. Chem. 2001; 276: 11354-11361Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) using a Waters Symmetry C18 3.5 μm, 1.0 × 150-mm column. All the LC-MS analyses performed with the API 1 mass spectrometer were carried out splitting the flow outside the chromatographic column allowing in this way to both record mass spectra and collect protein/peptide fractions for subsequent analyses. Tandem mass spectrometry (MS/MS) measurements were performed on a Q-TOF Micro mass spectrometer (Micromass, UK) equipped with a Z-spray nanoflow electrospray ionization interface. Mass spectra of the peptide digests of HMGA1a were acquired using the nano-electrospray source operating at capillary, cone, and extractor voltages of 1400, 30, and 1 V, respectively (positive ion mode). For the MS analyses, samples were dissolved in a 1:1 solution of acetonitrile:water containing 1% formic acid. Nanoelectrospray ionization capillaries were prepared in-house from borosilicate glass tubes of 1 mm outer diameter and 0.78 mm inner diameter (Harvard Apparatus, Holliston, MA) using a Flaming/Brown P-80 PC micropipette puller (Sutter Instruments, Hercules, CA) and gold coated using an Edwards S-150B sputter coater (Edwards High Vacuum, West Sussex, UK). MS/MS analyses of the peptide-(55–61) and its methylated forms were performed utilizing the same parameters of the MS instrument as above, using argon as the collision gas and a collision energy setting of 18 V. Instrument control and data acquisition and processing were achieved using the MassLynx software (Micromass, UK). The relative ratio of the monomethylated peptides was calculated as the ratio between the relative peak intensity of the monomethylated peptide (1-Me) and the relative peak intensity of the unmethylated (0-Me) plus the monomethylated peptide (0-Me + 1-Me). Peptide relative intensity ratio = 1-Me/(0-Me + 1-Me).Blot Overlay—Blot overlay experiments were performed essentially as previously described (8.Sgarra R. Tessari M.A. Di Bernardo J. Rustighi A. Zago P. Liberatori S. Armini A. Bini L. Giancotti V. Manfioletti G. Proteomics. 2005; 5: 1494-1506Crossref PubMed Scopus (41) Google Scholar). Subconfluent HEK293 cells seeded on 100 mm-diameter Petri dishes were transfected with pcDNA3HA, pcDNA3HA-PRMT6, or pcDNA3HA-NPM by the conventional calcium phosphate procedure. Thirty-six hours later, the cells were washed with ice-cold phosphate-buffered saline and then harvested in 1 ml of ice-cold lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0, 1% Nonidet P-40) supplemented with protease inhibitors. Lysis was performed at 4 °C for 20 min. The lysates were then clarified by centrifugation and utilized as probes in the blot overlay experiments. Blot overlay membranes were incubated with an α-HA primary antibody (Santa Cruz Biotechnology) and bound primary antibodies visualized by enhanced chemiluminescence.Co-immunoprecipitation—Subconfluent HEK293 cells were transfected with the indicated vector as described above and immunoprecipitation was performed as previously described (36.Tessari M.A. Gostissa M. Altamura S. Sgarra R. Rustighi A. Salvagno C. Caretti G. Imbriano C. Mantovani R. Del Sal G. Giancotti V. Manfioletti G. Mol. Cell. Biol. 2003; 23: 9104-9116Crossref PubMed Scopus (120) Google Scholar).In Vitro Methylation—In vitro methyltransferase assay, electrophoresis, and fluorography of methylation reactions were carried out as previously described (31.Frankel A. Yadav N. Lee J. Branscombe T.L. Clarke S. Bedford M.T. J. Biol. Chem. 2002; 277: 3537-3543Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar).In Vivo Methylation—Subconfluent HEK293 cells seeded on 100 mm diameter Petri dishes were transfected with the indicated vectors as described above. Protein synthesis inhibition and in vivo methylation were carried out as previously described (33.Lee J. Bedford M.T. EMBO Rep. 2002; 3: 268-273Crossref PubMed Scopus (183) Google Scholar). Cells were washed with ice-cold phosphate-buffered saline and then harvested and lysed in 1 ml of ice-cold lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0, 1% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 5 mm NaF, 1 mm Na3VO4) supplemented with protease inhibitors (Sigma). Cell lysates were quantified using a standard Bradford method and 1 mg incubated with 8 mg of α-HA primary antibody (Sigma) prebound to 50 ml of protein A-Sepharose (Amersham Biosciences). The beads were then washed three times in ice-cold lysis buffer, and the bound proteins were solubilized by the addition of SDS sample buffer. Proteins were then separated by SDS-PAGE and transferred to nitrocellulose membranes. Western blot analyses were performed by standard procedures with an anti-HA primary antibody (Sigma) and enhanced chemiluminescence visualization. Radioactivity ([35S]methionine and methyl-3H) was visualized by fluorography; membranes were soaked in NAMP100 Amplify (Amersham Biosciences), air dried, and exposed to films at –80 °C for two months.RESULTSHMGA1a Is a Substrate of PRMT6—HMGA1a protein is methylated at the level of several arginine residues (9.Edberg D.D. Adkins J.N. Springer D.L. Reeves R. J. Biol. Chem. 2005; 280: 8961-8973Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 11.Sgarra R. Diana F. Bellarosa C. Dekleva V. Rustighi A. Toller M. Manfioletti G. Giancotti V. Biochemistry. 2003; 42: 3575-3585Crossref PubMed Scopus (45) Google Scholar). Because methyltransferases responsible for this post-translational modification were completely unknown, we decided to screen arginine methyltransferases for their ability to methylate HMGA1a. To this aim, pure recombinant HMGA1a protein, together with its isoform HMGA1b and the highly related HMGA2, were incubated with the different recombinant PRMTs in the presence of 3H-labeled S-adenosyl-l-methionine ([3H]AdoMet) as a methyl donor. Methylated proteins were separated by SDS-PAGE and visualized by fluorography. All of the PRMTs (PRMT1, PRMT3, PRMT4, PRMT6, and PRMT7) that maintain their enzymatic activity as recombinant proteins were tested. Fig. 1 clearly shows that PRMT6 is the only arginine methyltransferase that is able to efficiently methylate HMGA1a and also the other HMGA proteins. Interestingly, PRMT6 has an exclusive nuclear localization. Among the other methyltransferases tested, only PRMT1 and PRMT3 were able to weakly methylate HMGA2 (but not HMGA1a and HMGA1b). Recombinant GST-GAR was included as a positive control for PRMT1, PRMT3, and PRMT6, whereas GST-PABP1 is the positive control for PRMT4 (31.Frankel A. Yadav N. Lee J. Branscombe T.L. Clarke S. Bedford M.T. J. Biol. Chem. 2002; 277: 3537-3543Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). The newly described PRMT7 (29.Lee J.H. Cook J.R. Yang Z.H. Mirochnitchenko O. Gunderson S.I. Felix A.M. Nerth N. Hoffmann R. Pestka S. J. Biol. Chem. 2005; 280: 3656-3664Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 30.Miranda T.B. Miranda M. Frankel A. Clarke S. J. Biol. Chem. 2004; 279: 22902-22907Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) was tested at a later stage, but did not display any activity on HMGA proteins (data not shown).PRMT6 Methylates Arg Residues within the Second AT-hook of HMGA1a—Because PRMTs are able to methylate short peptide sequences (33.Lee J. Bedford M.T. EMBO Rep. 2002; 3: 268-273Crossref PubMed Scopus (183) Google Scholar, 37.Schurter B.T. Koh S.S. Chen D. Bunick G.J. Harp J.M. Hanson B.L. Henschen Edman A. Mackay D.R. Stallcup M.R. Aswad D.W. Biochemistry. 2001; 40: 5747-5756Crossref PubMed Scopus (275) Google Scholar, 38.Cimato T.R. Tang J. Xu Y. Guarnaccia C. Herschman H.R. Pongor S. Aletta J.M. J. Neurosci. Res. 2002; 67: 435-442Crossref PubMed Scopus (62) Google Scholar), to map the region of HMGA1a that is required for PRMT6 methylation, several HMGA1a deletion mutants were used. In addition, because methylation of Arg25 has been previously reported to occur at high levels in HMGA1a, point-mutated HMGA1aR25A was included. In vitro methylation reactions were performed as described above. Only the deletion of the second AT-hook region of HMGA1a (amino acids 52–7" @default.
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• W2023382624 title "The AT-hook of the Chromatin Architectural Transcription Factor High Mobility Group A1a Is Arginine-methylated by Protein Arginine Methyltransferase 6" @default.
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