Covalent Modification of PML by the Sentrin Family of Ubiquitin-like Proteins

• DOI: 10.1074/jbc.273.6.3117
• Publication Date: 2002-07-26T14:56:15Z
• Authors: Tetsu Kamitani; Hung Phi Nguyen; Katsumi Kito; Taeko Fukuda-Kami...; Edward T.H. Yeh
• Keywords: Promyelocytic leukemia protein; NEDD8

PML, a RING finger protein with tumor suppressor activity, has been implicated in the pathogenesis of acute promyelocytic leukemia that arises following reciprocal chromosomal translocation fuses PML gene retinoic acid receptor α (RARα) gene. Immunocytochemical analysis demonstrated is co-localized novel ubiquitin-like nuclear bodies, which could be disrupted by PML-RARα fusion protein. The physical nature this co-localization unknown. Using COS cell expression system, we show covalently modified all three members sentrin family proteins. Covalent modification requires conserved Gly residue near C termini Sentrinization highly specific because neither NEDD8 nor ubiquitin modify PML. Similar specificity also observed for covalent RanGAP1 member These observations highlight fine substrate sentrinization pathway. In leukemia, two forms proteins have reported. Remarkably, both not sentrinized. Thus differential and play an important role regulating biological function leukemia. Chromosomal t(15;17), detected majority patients generates composed portions 1The abbreviations used are: RARα, α; HA, hemagglutinin; HA-PML, HA epitope-tagged PML; PML-RH, RH-tagged called (1Larson R.A. Kondo K. Vardiman J.W. Butler A.E. Golomb H.M. Rowley J.D. Am. J. Med. 1984; 76: 827-841Abstract Full Text PDF PubMed Scopus (354) Google Scholar, 2de H. Lavau C. Marchio A. Chomienne Degos L. Dejean Cell. 1991; 66: 675-684Abstract (1188) 3Kakizuka Miller Jr., W.H. Umesono Warrell R.P. Frankel S.R. Murty V.V.V.S. Dmitrovsky E. Evans R.M. 663-674Abstract (1282) 4Kastner P. Perez Lutz Y. Rochette-Egly Gaub M.P. Durand B. Lanotte M. Berger R. Chambon EMBO 1992; 11: 629-642Crossref (416) Scholar). normal myeloid cells, localized to multiprotein complex bodies or oncogenic domains (5Weis Rambaud S. Jansen Carvalho T. Carmo-Fonseca Lamond 1994; 345-356Abstract (620) 6Dyck Maul G. W. Chen Kakizuka 333-343Abstract (722) lines derived from are into microparticulate pattern, reversible treatment shown suppress transformation NIH3T3 cells activated neu oncogene (7Liu J.H. Mu Z.M. Chang K.S. Exp. 1995; 181: 1965-1972Crossref (99) full-length as bait yeast two-hybrid interaction screening, Boddy et al. isolated PIC1 interacted specifically assay (8Boddy M.N. Howe Etkin L.D. Solomon Freemont P.S. Oncogene. 1996; 13: 971-982PubMed They further was bodies. NB4 patient there no significant However, treatment, relocalization observed. suggest association may Our laboratory recently reported cloning molecule, sentrin, interacts death Fas necrosis factor 1 (9Okura Gong Kamitani Wada Okura I. Wei C.K. Yeh E.T.H. Immunol. 157: 4277-4281PubMed Overexpression protects against anti-Fas factor-induced death. We form conjugates other process analogous ubiquitination (10Kamitani Nguyen H.P. Biol. Chem. 1997; 272: 14001-14004Abstract (145) Furthermore, sentrinized nucleus. amino sequences identical. Thus, it tempting speculate (PIC1). Scholar) were unable demonstrate vitro between (sentrin) possibly due technical problems their co-precipitation assay. system 11Kamitani Katsumi 28557-28562Abstract (350) Scholar), This participate reaction. 16B12 (BAbCo, Richmond, CA) mouse monoclonal antibody peptide sequence YPYDVPDYA influenza hemagglutinin (HA). Mouse anti-RH (specific sequence, RGSHHHH) purchased Qiagen. anti-PML Santa Cruz Biotechnology. Rabbit polyclonal antiserum kindly provided Dr. Chang, (M. D. Anderson Cancer Center). cDNAs Rad51, ubiquitin, NEDD8, sentrins, amplified polymerase chain reaction using appropriate primers human testis cDNA library (Life Technologies, Inc.). sentrins ligated pcDNA3/HA-N N-terminal tagging epitope. RARα inserted pcDNA3/RH-N (11Kamitani pCDNA3/RH-C respectively. prepared plasmid pMAMneoPML (12Mu Chin K.V. Liu Lozano Mol. 14: 6858-6867Crossref (294) pcDNA3/RH-C express HA-PML PML-RH. PML-RARα-A PML-RARα-B (4Kastner generated pcDNA3/RH-C. Plasmids transfected LipofectAMINE harvested Western blotting nickel precipitation described previously expressed total lysates analyzed Three different antibodies employed analysis. As Fig. A, 70-kDa (arrowhead) 90-kDa band (lane 2). A rabbit 70- bands 4). There weak higher molecular mass lysate several lower bands, most likely degradation products sensitive anti-HA than 70 kDa 6). Because predicted only kDa, presence these suggests either migrate aberrantly molecules, such ubiquitin. Next, tagged RH epitope (RGSHHHHHH) at terminus Transfected lysed 6 m guanidine HCl denature prevent any proteolysis desentrinization. PML-RH then precipitated nickel-charged beads immunoblotted anti-sentrin B, distinct identified lanes (lanes 2 control, 3). experiments multiple sites. do favor possibility single site multimers does contain Lys48 equivalent required multimer formation (13Hershko Ciechanover Annu. Rev. Biochem. 61: 761-807Crossref (1190) (Fig. notion supported observation molecule (SUMO-1) 14Mannen Tseng Cho C.L. Li S.S.L. Biophy. Res. Commun. 222: 178-180Crossref (50) 15Lapenta V. Chiurazzi van der Spek Pizzuti Hanaoka F. Brahe Genomics. 40: 266-362Crossref (102) Sentrin belongs homologues Arabidopsis thaliana Homo sapiens, suggesting evolutionary perform unique functions cellular metabolism. Further data base revealed additional homologous (14Mannen interest one homologue nonmammalian genera species. renamed original sentrin-1 sentrin-2 sentrin-3. It known whether sentrin-3 determine modifiers. For purpose, tag placed N sentrin-1, sentrin-2, positive control Scholar,16Mahajan Delphin Guan Gerace Melchior 88: 97-107Abstract (999) 17Matunis M.J. Coutavas Blobel Cell 135: 1457-1470Crossref (948) known, however, HA-tagged co-expressed RH-RanGAP1 cells. Total incubated precipitate its derivatives. 3 4, 5, Rad51 1), 2), 3) RanGAP1. purified derivatives utilizing 7–12). shown, unmodified samples. should noted p90 7–9 native doublet HA-sentrin-1, -2, -3 sample 10–12). upper bandof HA-sentrin, thelower expressing 4–6), molecule. provide first evidence members. linkage similar approach applied 4–6). appears more efficient modifying sentrin-1. relative abundance (see below). Again, Ub stable conjugate 8–12). samples inability insufficient 8 9 conjugation present To confirm constructed mutants involved (marked aclosed triangle 2) adjacent C-terminal acids deleted expected, molecules lacking residues (data shown). predicated approximately kDa. his colleagues migrates about 90 during SDS-polyacrylamide gel electrophoresis, probably acidic contributed proline-rich domain 18Le X.F. Yang 271: 130-135Abstract (123) view results presented here, reports sentrin. abundant desentrinizing activity contribute difficulty demonstrating clearly after RanGAP1, second target via residue. difference pathway recent finding preferential Ubc9 supports distinction pathways (19Gong Fujise Caskey 28198-28201Abstract (165) postulated dominant negative mutants. RH-PML-RARα-A -B blotting. confirmed immunoblotting 7 8). currently know why lack sites binding E3 wrong compartment. Elucidation issues will bring insights pathobiochemistry thank providing us critical reading manuscript.