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Asymmetric ArginineDimethylation ofHeterogeneous NuclearRibonucleoprotein Kby Protein-arginineMethyltransferase 1Inhibits ItsInteraction withc-Src Antje Ostareck‐LedererDirk H. OstareckKarl Peter Rücknagel2006 год

Asymmetric Arginine Dimethylation of Heterogeneous Nuclear Ribonucleoprotein K by Protein-arginine Methyltransferase 1 Inhibits Its Interaction with c-Src
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Аннотация: Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric NG,NG-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions. Arginine methylation is a post-translational modification found in many RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein K (hnRNP K) from HeLa cells was shown, by mass spectrometry and Edman degradation, to contain asymmetric NG,NG-dimethylarginine at five positions in its amino acid sequence (Arg256, Arg258, Arg268, Arg296, and Arg299). Whereas these five residues were quantitatively modified, Arg303 was asymmetrically dimethylated in <33% of hnRNP K and Arg287 was monomethylated in <10% of the protein. All other arginine residues were unmethylated. Protein-arginine methyltransferase 1 was identified as the only enzyme methylating hnRNP K in vitro and in vivo. An hnRNP K variant in which the five quantitatively modified arginine residues had been substituted was not methylated. Methylation of arginine residues by protein-arginine methyltransferase 1 did not influence the RNA-binding activity, the translation inhibitory function, or the cellular localization of hnRNP K but reduced the interaction of hnRNP K with the tyrosine kinase c-Src. This led to an inhibition of c-Src activation and hnRNP K phosphorylation. These findings support the role of arginine methylation in the regulation of protein-protein interactions. Arginine dimethylation is a common post-translational modification in eukaryotes (1Bedford M.T. Richard S. Mol. Cell. 2005; 18: 263-272Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar, 2Gary J.D. Clarke S. Prog. Nucleic Acids Res. 1998; 61: 65-133Crossref PubMed Google Scholar, 3Jenuwein T. Allis C.D. Science. 2001; 293: 1074-1080Crossref PubMed Scopus (7666) Google Scholar, 4Lee D.Y. Teyssier C. Strahl B.D. Stallcup M.R. Endocr. Rev. 2004; 2: 147-170Google Scholar, 5McBride A.E. Silver P. Cell. 2001; 106: 5-8Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar). The enzymes responsible for this modification are the protein-arginine methyltransferases (PRMTs). 3The abbreviations used are: PRMT, protein-arginine methyltransferase; hnRNP K, heterogeneous ribonucleoprotein K; ES, embryonic stem; Hrp1, nuclear polyadenylated RNA-binding protein 4 (Nap4p); Sam68, Src associated in mitosis 68-kDa protein; PABPN1, mammalian nuclear poly(A)-binding protein 1; r15-LOX, reticulocyte-15-lipoxygenase; DICE, differentiation control element; L2, protein L2 of human papillomavirus type 16; GST, glutathione S-transferase; LIF, leukemia inhibitory factor; GST-GAR, GST fusion protein containing the glycine- and arginine-rich N-terminal region of fibrillarin; HPLC, high pressure liquid chromatography; MALDI-TOF MS, matrix-assisted laser desorption ionization-time of flight mass spectrometry; ESI MS, electrospray ionization-mass spectrometry; SAM, S-adenosylmethionine; SH3-domain, Src homology-3 domain; Src, tyrosine kinase of the Rous sarcoma virus; WW, denotes two conserved tryptophan residues within this domain; Erk, extracellular signal-regulated kinase; GFP, green fluorescent protein; MS/MS, tandem mass spectrometry. They are classified in two groups (2Gary J.D. Clarke S. Prog. Nucleic Acids Res. 1998; 61: 65-133Crossref PubMed Google Scholar). Type I enzymes promote the formation of asymmetric NG,NG-dimethylated arginine. The known mammalian type I enzymes are PRMT1, PRMT2, PRMT3, PRMT4/coactivator-associated arginine methyltransferase 1, PRMT6, and the recently discovered brain-specific PRMT8 (6Lin W.J. Gary J.D. Yang M.C. Clarke S. Hershman H.R. J. Biol. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar, 7Qui C. Chang J. Zhu Y. Yeldani A.V. Rao S.M. Zhu Y.J. J. Biol. Chem. 2002; 277: 28624-28630Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Tang J. Gary J.D. Clarke S. Hershman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 9Schurter 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 (279) Google Scholar, 10Frankel 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 (282) Google Scholar, 11Lee J. Sayegh J. Daniel J. Clarke S. Bedford M.T. J. Biol. Chem. 2005; 280: 32890-32896Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Type II enzymes catalyze the symmetric NG,N′G-dimethylation of arginine residues. PRMT5 and PRMT7 are the mammalian type II enzymes described so far (12Branscombe T.L. Frankel A. Lee J.H. Cook J.R. Yang Z.Z. Pestka S. Clarke S. J. Biol. Chem. 2001; 276: 32971-32976Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 13Miranda T.B. Miranda M. Frankel A. Clarke S. J. Biol. Chem. 2004; 279: 22902-22907Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 14Lee J.H. Cook J.R. Yang Z.H. Mirochnitchenko O. Gunderson S.I. Felix A.M. Herth N. Hoffmann R. Pestka S. J. Biol. Chem. 2005; 280: 3656-3664Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). PRMT1, which is predominantly localized to the cytoplasm (15Herrmann F. Lee J. Bedford M.T. Fackelmayer F.O. J. Biol. Chem. 2005; 280: 38005-38010Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), is thought to account for the generation of ∼85% of asymmetric dimethylarginine residues (16Tang J. Kao P.N. Hershman H.R. J. Biol. Chem. 2000; 275: 19866-19876Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar) and is required for early post-implantation development in mice but not for the viability of embryonic stem cells (17Pawlak M.R. Scherer C.A. Chen J. Roshon M.J. Ruley H.E. Mol. Cell. Biol. 2000; 20: 4859-4869Crossref PubMed Scopus (279) Google Scholar). Arginine methylation affects several cellular processes, including histone function and transcriptional regulation (18An W. Kim J. Roeder R.G. Cell. 2004; 117: 735-748Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 19Covic M. Hassa P.O. Saccani S. Buerki C. Meier N.I. Lombardi C. Imhof R. Bedford M.T. Natoli G. Hottiger M.O. EMBO J. 2004; 24: 85-96Crossref PubMed Scopus (182) Google Scholar, 20Huang S. Litt M. Felsenfeld G. Genes Dev. 2005; 19: 1885-1893Crossref PubMed Scopus (179) Google Scholar, 21Rezai-Zadeh N. Zhang X. Namour F. Fejer G. Wen Y.D. Yao Y.L. Gyory I. Wright K. Seto E. Genes Dev. 2003; 17: 1019-1029Crossref PubMed Scopus (149) Google Scholar, 22Xu W. Chen H. Du K. Asahara H. Tini M. Emerson B.M. Montminy M. Evans R.M. Science. 2001; 294: 2507-2511Crossref PubMed Scopus (337) Google Scholar), RNA processing (23Brahms H. Meheus L. de Brabandere V. Fischer U. Lührmann R. RNA. 2001; 7: 1531-1542Crossref PubMed Scopus (300) Google Scholar, 24Meister G. Fischer U. EMBO J. 2002; 21: 5853-5863Crossref PubMed Scopus (162) Google Scholar, 25Friesen W.J. Paushkin S. Wyce A. Massenet S. Pesiridis G.S. Van Duyne G. Mann M. Dreyfuss G. Mol. Cell. Biol. 2001; 21: 8289-8300Crossref PubMed Scopus (314) Google Scholar), signal transduction (26Abramovich C. Yakobson B. Chebath J. Revel M. EMBO J. 1997; 16: 260-266Crossref PubMed Scopus (153) Google Scholar, 27Chen W. Daines M.O. Hershey G.K. J. Immunol. 2004; 172: 6744-6750Crossref PubMed Scopus (53) Google Scholar, 28Hsu I.W. Hsu M. Li C. Chuang T.W. Lin R.I. Tarn W.Y. J. Biol. Chem. 2005; 280: 34507-34512Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 29Mowen K.A. Tang J. Zhu W. Schurter B.T. Shuai K. Herschman H.R. David M. Cell. 2001; 104: 731-741Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar), and intracellular localization of proteins (30Shen E.C. Henry M.F. Weiss V.H. Valentinin S.R. Silver P.A. Lee M.S. Genes Dev. 1998; 12: 679-691Crossref PubMed Scopus (251) Google Scholar, 31McBride A.E. Weiss V.H. Kim H.K. Hogle J.M. Silver P.A. J. Biol. Chem. 2000; 275: 3128-3136Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 32McBride A.E. Cook J.T. Stemmler E.A. Rutledge K.L. McGrath K.A. Rubens J.A. J. Biol. Chem. 2005; 280: 30888-30898Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 33Green D.M. Marfatia K.A. Crafton E.B. Zhang X. Cheng X. Corbett A.H. J. Biol. Chem. 2002; 277: 7752-7760Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 34Xu C. Henry M.F. Mol. Cell. Biol. 2004; 24: 10742-10756Crossref PubMed Scopus (46) Google Scholar, 35Yun C.Y. Fu X.D. J. Cell Biol. 2000; 150: 707-717Crossref PubMed Scopus (137) Google Scholar). Many proteins involved in RNA metabolism like hnRNP A1 (36Kim S. Merrill B.M. Rajpurohit R. Kumar A. Stone K.L. Papov V.V. Schneiders J.M. Szer W. Wilson S.H. Paik W.K. Williams K.R. Biochemistry. 1997; 36: 5185-5192Crossref PubMed Scopus (107) Google Scholar), hnRNP A2 (37Nichols R.C. Wang X.W. Tang J. Hamilton B.J. High F.A. Herschman H.R. Rigby W.F.C. Exp. Cell Res. 2000; 256: 522-532Crossref PubMed Scopus (145) Google Scholar), Sam68 (38Côté J. Boisvert F.M. Boulanger M.C. Bedford M.T. Richard S. Mol. Biol. Cell. 2003; 14: 274-287Crossref PubMed Scopus (212) Google Scholar), and SAF-A (hnRNP U) (39Herrmann F. Bossert M. Schwander A. Akgun E. Fackelmayer F.O. J. Biol. Chem. 2004; 279: 48774-48779Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) contain regions with clustered arginine residues in Arg-Gly-Gly motifs (RGG box) or RG repeats. These arginine residues are typically asymmetrically dimethylated. In addition, asymmetric arginine dimethylation has also been found in clustered RXR motifs (40Smith J.J. Rücknagel K.P. Schierhorn A. Tang J. Nemeth A. Linder M. Herschman H.R. Wahle E. J. Biol. Chem. 1999; 274: 13229-13234Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar) and other sequences. Therefore, the prediction of a methylated arginine is difficult. Furthermore, the enzyme responsible for the dimethylation of a particular protein is unknown in many cases, and the substrate specificities of the different methyltransferases remain poorly characterized. The exact knowledge of the methylated arginine residues and their quantitative distribution as well as the identification of the relevant methyltransferases is a prerequisite for the functional analysis of arginine methylation. HnRNP K belongs to the family of heterogeneous nuclear RNPs that participate in the processing of pre-mRNAs and in the export of mRNAs from the nucleus. An N-terminal bipartite nuclear-localization signal and an hnRNP K-specific nuclear shuttling signal confer the capacity for bi-directional transport across the nuclear envelop (41Michael W.M. Choi M. Dreyfuss G. Cell. 1995; 83: 415-422Abstract Full Text PDF PubMed Scopus (470) Google Scholar, 42Michael W.M. Eder P.S. Dreyfuss G. EMBO J. 1997; 16: 3587-3598Crossref PubMed Scopus (327) Google Scholar). The cytoplasmic accumulation of hnRNP K is mediated by its Erk-dependent serine phosphorylation (43Habelhah H. Shah K. Huang L. Ostareck-Lederer A. Burlingame A.L. Shokat K.M. Hentze M.W. Ronai Z. Nat. Cell Biol. 2001; 3: 325-330Crossref PubMed Scopus (250) Google Scholar). In the cytoplasm hnRNP K functions in the post-transcriptional regulation of gene expression. For example, hnRNP K, together with hnRNP E1 and hnRNP E2, regulates human papilloma virus type 16 L2 capsid protein synthesis in the course of cellular differentiation by binding to the L2 mRNA 3′-untranslated region (44Collier B. Goobar-Larsson L. Sokolowski M. Schwartz S. J. Biol. Chem. 1998; 273: 22648-22656Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Binding of hnRNP K, hnRNP E1, and hnRNP E2 to the internal ribosome entry site of the c-myc mRNA enhances the translation of the c-myc proto-oncogene (45Evans J.R. Mitchell S.A. Spriggs K.A. Ostrowski J. Bomsztyk K. Ostareck D.H. Willis A.E. Oncogene. 2003; 22: 8012-8020Crossref PubMed Scopus (186) Google Scholar). Furthermore, hnRNP K is involved in the regulation of reticulocyte 15-lipoxygenase (r15-LOX) mRNA translation. r15-LOX, a key enzyme in erythroid cell differentiation, participates in the breakdown of mitochondria in mature reticulocytes, which is a prerequisite for erythrocyte formation. Its premature expression in erythroid precursor cells is temporally restricted by translational silencing, mediated by hnRNP K and hnRNP E1 binding, individually or together, to the differentiation control element (DICE) in the r15-LOX mRNA 3′-untranslated region. The hnRNP K/E1-DICE complex blocks 80 S ribosome assembly (46Ostareck D.H. Ostareck-Lederer A. Wilm M. Thiele B.J. Mann M. Hentze M.W. Cell. 1997; 89: 597-606Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 47Ostareck D.H. Ostareck-Lederer A. Shatsky I.N. Hentze M.W. Cell. 2001; 104: 281-290Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). The silencing mechanism can be recapitulated in vitro in rabbit reticulocyte lysate or wheat germ extract and in transfected HeLa cells (46Ostareck D.H. Ostareck-Lederer A. Wilm M. Thiele B.J. Mann M. Hentze M.W. Cell. 1997; 89: 597-606Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 47Ostareck D.H. Ostareck-Lederer A. Shatsky I.N. Hentze M.W. Cell. 2001; 104: 281-290Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Interestingly, hnRNP K has been found to bind and to activate the tyrosine kinase c-Src specifically. Activated c-Src, in turn, phosphorylates hnRNP K, which leads to an inhibition of its DICE-binding activity and abolishes inhibition of mRNA translation by hnRNP K. In contrast, hnRNP E1 is neither an activator nor a substrate of c-Src (48Ostareck-Lederer A. Ostareck D.H. Cans C. Neubauer G. Bomsztyk K. Superti-Furga G. Hentze M.W. Mol. Cell. Biol. 2002; 22: 4535-4543Crossref PubMed Scopus (195) Google Scholar). HnRNP K contains three proline-rich domains, allowing an interaction with the Src homology domain 3 (SH3) of c-Src (49Taylor S.J. Shalloway D. Nature. 1994; 368: 867-871Crossref PubMed Scopus (371) Google Scholar, 50Van Seuningen I. Ostrowski J. Bustelo X.R. Sleath P.R. Bomsztyk K. J. Biol. Chem. 1995; 270: 26976-26985Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 51Weng Z. Thomas S.M. Rickles J.J. Taylor J.A. Brauer A.W. Seidel-Dugan C. Michael W.M. Dreyfuss G. Brugge J.S. Mol. Cell. Biol. 1994; 14: 4509-4521Crossref PubMed Scopus (206) Google Scholar). In addition, hnRNP K bears five clustered Arg-Gly-Gly (RGG) motifs within the proline-rich domains (52Matunis M.J. Michael W.M. Dreyfuss G. Mol. Cell. Biol. 1992; 12: 164-171Crossref PubMed Scopus (238) Google Scholar). HnRNP K, like other hnRNP proteins, is methylated in HeLa cells and lymphoblastoid cells (53Liu Q. Dreyfuss G. Mol. Cell. Biol. 1995; 15: 2800-2808Crossref PubMed Scopus (270) Google Scholar, 54Huang H.M. Tam M.F. Tam T.C.S. Chen D.H. Hsieh M. Li C. J. Biochem. 2002; 132: 813-818Crossref PubMed Scopus (13) Google Scholar), but neither the methylation sites nor the specific methyltransferase or the functional significance of arginine methylation are known. In this report, we have identified the methylated arginine residues in hnRNP K by mass spectrometry and Edman sequencing and quantitatively determined the methylation state. We provide biochemical and genetic evidence that PRMT1 is the only methyltransferase that methylates hnRNP K in vitro and in vivo. An hnRNP K variant in which all completely modified arginines were substituted is neither methylated by recombinant PRMT1 nor in HeLa cells. Methylation of arginine residues in hnRNP K did not influence its DICE-binding activity, its translation inhibitory function, or its cellular localization, but reduced its interaction with c-Src. This led to an inhibition of hnRNP K-dependent c-Src activation and a reduced hnRNP K phosphorylation. Plasmids—pET16b-hnRNP K and pET16b-hnRNP E1 have been described in Ref. 46Ostareck D.H. Ostareck-Lederer A. Wilm M. Thiele B.J. Mann M. Hentze M.W. Cell. 1997; 89: 597-606Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar and pSG5 His-hnRNP K and pSGT c-Src in Ref. 48Ostareck-Lederer A. Ostareck D.H. Cans C. Neubauer G. Bomsztyk K. Superti-Furga G. Hentze M.W. Mol. Cell. Biol. 2002; 22: 4535-4543Crossref PubMed Scopus (195) Google Scholar. Arginine to glycine mutants (hnRNP K 5RG) were generated by site-directed mutagenesis (Stratagene). GFP-hnRNP K or -hnRNP K 5RG were generated using the plasmid pEGFP-C1 (Clontech). His6-PABPN1 and His6-PABPN1ΔC49 are described in Ref. 55Kühn U. Nemeth A. Meyer S. Wahle E. J. Biol. Chem. 2003; 278: 16916-16925Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar. The plasmids coding for GST-PRMT1 and pCDNA-PRMT1 (38Côté J. Boisvert F.M. Boulanger M.C. Bedford M.T. Richard S. Mol. Biol. Cell. 2003; 14: 274-287Crossref PubMed Scopus (212) Google Scholar) were a kind gift from Stéphane Richard, McGill University, Montreal, Canada. The plasmid pET28b-PRMT1 (56Zhang X. Cheng X. Structure. 2003; 11: 509-520Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) was a kind gift from X. Cheng, Emory University, Atlanta, GA. Plasmids encoding GST-GAR (2Gary J.D. Clarke S. Prog. Nucleic Acids Res. 1998; 61: 65-133Crossref PubMed Google Scholar) and GST-PRMT2 were a kind gift from Mark T. Bedford, University of Texas, Smithville, TX. Primers used to PCR-amplify the human PRMT3 cDNA, cloned into SalI/NotI of pGEX 5X1 (Amersham Biosciences) are listed in the supplemental materials. The full-length human cDNAs encoding PRMT4, PRMT5, and PRMT6 were PCR-amplified (primers in the supplement) and inserted into pGEX-6P-1 (Amersham Biosciences). pET28a-PRMT3 was generated by PCR and subcloned into pET28a. The leukemia inhibitory factor (LIF) was expressed as GST-LIF from the plasmid pGEX-2T-LIF (57Mereau A. Grey L. Piquet-Pellorce C. Heath J.K. J. Cell Biol. 1993; 122: 713-719Crossref PubMed Scopus (55) Google Scholar), a kind gift from John K. Heath, University of Birmingham, UK. Recombinant Proteins—His-hnRNP K, His-hnRNP K 5RG, and His-hnRNP E1 were prepared as described (46Ostareck D.H. Ostareck-Lederer A. Wilm M. Thiele B.J. Mann M. Hentze M.W. Cell. 1997; 89: 597-606Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar) except that the proteins were dialyzed against 100 mm potassium acetate, 20 mm Hepes, pH 7.4, 5% glycerol, 1 mm EDTA, 1 mm dithiothreitol, 7 mm β-mercaptoethanol. Expression and purification of recombinant His-PABPN1 and His-PABPN1ΔC49 was described in Ref. 55Kühn U. Nemeth A. Meyer S. Wahle E. J. Biol. Chem. 2003; 278: 16916-16925Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar. His-PABPN1 was further purified by Mono-S fast protein liquid chromatography (Amersham Biosciences). His-PRMT1 and His-PRMT3 were prepared as described for His-hnRNP E1 (46Ostareck D.H. Ostareck-Lederer A. Wilm M. Thiele B.J. Mann M. Hentze M.W. Cell. 1997; 89: 597-606Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). GST fusion proteins were expressed and purified as described for the pGEX system (Amersham Biosciences). GST-GAR and GST-PRMT1, His-PRMT1 and His-PRMT3 used in the in vitro methylation assay were dialyzed against 150 mm potassium acetate, 20 mm Hepes, pH 7.4, 5% glycerol, 1 mm EDTA, 1 mm dithiothreitol. Protein concentrations were determined by the Bradford assay, SDS-PAGE, and Coomassie staining using bovine serum albumin as a standard. GST-LIF was expressed as described in a previous study (57Mereau A. Grey L. Piquet-Pellorce C. Heath J.K. J. Cell Biol. 1993; 122: 713-719Crossref PubMed Scopus (55) Google Scholar). SWISSPROT accession numbers for the expressed PRMTs are: PRMT1: Q63009; PRMT2: P55345; PRMT3: O60678; PRMT4: Q9NR22; and PRMT6: Q96LA8. Antibodies—Anti-PRMT1 antibody was a kind gift from Stéphane Richard, McGill University, Montreal, Canada. PRMT2, PRMT4, and PRMT6 antibodies were raised against GST-tagged proteins. PRMT3 and PRMT5 antibodies were generated against GST-tagged proteins and affinity-purified using the respective His-tagged proteins immobilized on CnBr-activated Sepharose beads (Amersham Biosciences). Immunopurification and Identification of hnRNP K—HnRNP K was precipitated from 1.7 mg of cytosolic HeLa cell extract prepared according to a previous study (58Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) (a kind gift from Reinhard Lührmann, Max Planck Institute, Göttingen, Germany) with 50 μg of a polyclonal hnRNP K antibody (Matritech R20475) using 200 μl of protein-A Sepharose CL-4B (Amersham Biosciences). Precipitated protein was eluted from affinity beads with a small volume of formic acid at 4 °C. The solution was diluted to 5% formic acid, and hnRNP K was purified by HPLC on a Nucleosil 500-5 C3-PPN column (150 × 2 mm, Macherey-Nagel) equilibrated with 0.09% trifluoroacetic acid and eluted by a 30–60% solvent B (0.08% TFA in acetonitrile) gradient over 28 min with a flow rate of 0.2 ml/min at 40 °C. HnRNP K was identified in column fractions by SDS-PAGE and Western blot analysis as well as peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) following digestion with proteinase Lys-C. An aliquot was submitted to N-terminal Edman degradation. Purification of hnRNP K from HeLa Cell Extract—Cytosolic HeLa cell extract (58Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) was centrifuged for 30 min at 20,000 × g, and the supernatant was loaded onto a DEAE-Sepharose column (10 mg of protein per ml column volume) equilibrated in 50 mm Tris, pH 7.4, 50 mm KCl, 1 mm EDTA, 0.5 mm dithiothreitol, 10% glycerol (buffer A). Proteins were eluted by a gradient of 50 mm to 500 mm KCl in buffer A (10 column volumes). Fractions were assayed for PRMT activity as described below with 500 ng of recombinant hnRNP K as a substrate. Fractions containing hnRNP K were pooled, dialyzed against buffer B (50 mm Hepes, pH 7.4, 50 mm KCl, 1 mm EDTA, 0.5 mm dithiothreitol, 10% glycerol), and applied to a Macroprep-S column (7 mg of protein per ml column volume). The column was washed with one volume of buffer B, and proteins were eluted by a gradient of 50 mm to 500 mm KCl in buffer B (10 column volumes). Fractions containing hnRNP K were pooled, dialyzed against buffer B, and applied to a Heparin-Sepharose column (2 mg of protein per ml column volume). The column was washed with 2.5 volumes of buffer B, and proteins were eluted by a gradient of 50 mm to 1 m KCl in buffer B (10 column volumes). Fractions containing hnRNP K were further purified by reversed-phase HPLC as above. The protein in HPLC fractions was dried under a nitrogen stream, alkylated with vinylpyridine as described (59Lee T.D. Shively J.E. Methods Enzymol. 1990; 193: 361-374Crossref PubMed Scopus (61) Google Scholar), and desalted by HPLC. An aliquot was used for MALDI-TOF MS and electrospray ionization (ESI) mass spectrometry. In Vitro Methylation Assay—Reaction mixture (25μl volume): 1 nmol of [S-14C]adenosylmethionine (60 mCi/mmol, Amersham Biosciences), 50 mm Hepes, pH 8.0, 40 mm potassium acetate, 0.2 mg/ml bovine serum albumin, 0.01% Nonidet P-40, 10% glycerol, 1 mm EDTA, 0.5 mm dithiothreitol, and protein as indicated, incubated at 30 °C for 2 h. After incubation the reaction was split: 11 μl were added to 500 μl of bovine serum albumin (50 μg/ml), followed by the addition of 500 μl of 20% trichloroacetic acid. The mixture was incubated for 30 min on ice and filtered through Glass Microfibre Filters GF/C (Whatman). Filters were washed with 4 ml of ice-cold trichloroacetic acid (10%) and 4 ml of ice-cold ethanol (96%), air dried, and added to 3 ml of scintillation mix (Lumasafe Plus). Precipitated radioactivity was measured in a Liquid Scintillation Analyzer (Tri-Carb 2100PR, Packard). The remaining volume was subjected to SDS-PAGE and autoradiography. In Vitro Transcription and Northwestern Analysis—In vitro transcription and Northwestern blot assays were carried out as described previously (48Ostareck-Lederer A. Ostareck D.H. Cans C. Neubauer G. Bomsztyk K. Superti-Furga G. Hentze M.W. Mol. Cell. Biol. 2002; 22: 4535-4543Crossref PubMed Scopus (195) Google Scholar). ES Cell Culture—ES(+/+) and ES(-/-) cells (17Pawlak M.R. Scherer C.A. Chen J. Roshon M.J. Ruley H.E. Mol. Cell. Biol. 2000; 20: 4859-4869Crossref PubMed Scopus (279) Google Scholar) obtained from Earl H. Ruley, Vanderbilt University, Nashville, TN, were cultured on 0.2% gelatinized cell culture plates in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum, l-glutamine, nonessential amino acids, penicillin/streptomycin (all Invitrogen), and 0.5 ng/ml GST-LIF. For the in vitro methylation assay, 1 × 106 cells were lysed in 0.5 ml of lysis buffer (300 mm NaCl, 1% Triton, 50 mm Tris, pH 7.4, 10 μg/ml leupeptin, and 0.1 mm phenylmethylsulfonyl fluoride) and centrifuged for 5 min at 20,800 × g to obtain a cytoplasmic extract. Metabolic labeling of ES(+/+) and ES(-/-) cells was performed in methionine-free Dulbecco's modified Eagle's medium supplemented with 10 μCi l-[methyl-3H]methionine (70–85 Ci/mmol, Amersham Biosciences) per ml for 3 h in the presence of cycloheximide and chloramphenicol as described previously (53Liu Q. Dreyfuss G. Mol. Cell. Biol. 1995; 15: 2800-2808Crossref PubMed Scopus (270) Google Scholar). Cells were lysed as above. For immunoprecipitation, anti-hnRNP K antibody (Matritech R20475) was used. Precipitated 3H-labeled proteins were subjected to SDS-PAGE and fluorography. HeLa Cell Transfection and Analysis—HeLa cells were transiently transfected by the calcium phosphate method (60Graham F.L. Van der Eb A.J. Virology. 1973; 52: 456-464Crossref PubMed Scopus (6495) Google Scholar). For the experiment shown in Fig. 7B, 5 μg of GFP-hnRNP K or GFP-hnRNP K 5RG was cotransfected with 5 μg of pcDNA or PRMT1. For the experiments shown in Fig. 8, 5 μg of cDNAs coding for pSG5 His-hnRNP K or His-hnRNP K 5RG was cotransfected with 5 μg of pSGT-cSrc or pcDNA PRMT1 or empty vector (pSGT or pcDNA, respectively) as indicated. HeLa cell lysate preparation and immunoprecipitation was performed as described (48Ostareck-Lederer A. Ostareck D.H. Cans C. Neubauer G. Bomsztyk K. Superti-Furga G. Hentze M.W. Mol. Cell. Biol. 2002; 22: 4535-4543Crossref PubMed Scopus (195) Google Scholar) with an anti-His antibody (H5, Qiagen). Antibodies against His and p-Tyr (Santa Cruz Biotechnology), c-Src (Abcam), and PRMT1 were used (Fig. 8).FIGURE 8Arginine methylation of hnRNP K strongly reduces its interaction with c-Src. HeLa cells were transiently transfected with His-tagged hnRNP K (A) or His-tagged hnRNP K 5RG (B) and PRMT1, c-Src, or empty vector (pSGT or pcDNA, respectively) as indicated. HeLa cell total lysates (A and B, upper panel) were resolved on a 12% SDS-PAGE and analyzed by Western blot assays with either anti-His, anti-PRMT1, anti-Src, anti-phosphotyrosine (p-Tyr) or anti-glyceraldehyde-3-phosphate dehydrogenase antibodies. His-tagged hnRNP K or hnRNP K 5RG was immunoprecipitated with an anti-His antibody (A and B, lower panel), resolved on a 12% SDS-PAGE and analyzed by Western blot assays with antibodies against His, to assess the amounts of precipitated hnRNP K or hnRNP K 5RG, c-Src, and PRMT1 to visualize coprecipitation and p-Tyr to analyze the tyrosine phosphorylation status of hnRNP K or hnRNP K 5RG.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Immunofluorescence—For single or double immunofluorescence, cells were essentially processed as described (61Huettelmaier S. Illenberger S. Grosheva I. Rüdiger M. Singer R.H. Jockusch B.M. J. Cell Biol. 2001; 155: 775-786Crossref PubMed Scopus (93) Google Scholar). An E600 (Nikon) microscope equipped with a digital camera (Hamamatsu) was used for conventional fluorescence microscopy. Images were acquired by Lucia software (Nikon) and compiled with Adobe Photoshop. Enzymatic Digestion of hnRNP K—Sequencing grade proteases were obtained from Roche Applied Science. Lys-C digestion of alkylated hnRNP K was performed in 50 μl of 25 mm Tris-HCl, pH 8.5, with a Lys-C to hnRNP K ratio of 1:200 (w/w) overnight at 37 °C. The digest was separated by HPLC as above with a 0–40% solvent B gradient over 60 min followed by a 40–60% solvent B gradient over 20 min and a flow rate of 0.2 ml/min at 40 °C. Fractions were dried under a nitrogen
Год издания: 2006
Авторы: Antje Ostareck‐Lederer, Dirk H. Ostareck, Karl Peter Rücknagel, Angelika Schierhorn, Bodo Moritz, Stefan Hüttelmaier, Nadine Flach, Lusy Handoko, Elmar Wahle
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Cancer-related gene regulation, Epigenetics and DNA Methylation, RNA modifications and cancer
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