The Coiled-coil Domain Is the Structural Determinant for Mammalian Homologues of Drosophila Sina-mediated Degradation of Promyelocytic Leukemia Protein and Other Tripartite Motif Proteins by the Proteasome

Abstract

Mammalian homologues of Drosophila Seven in Absentia (SIAHs) target for proteasome-mediated degradation several factors involved in cell growth and tumorigenesis. Here we show that SIAH-1/2 binds and targets for proteasome-mediated degradation the putative tumor suppressor and tripartite motif (TRIM) family member PML, leading to the loss of its transcriptional co-activating properties and a reduction in the number of endogenous PML nuclear bodies. Association with PML requires the substrate-binding domain (SBD) of SIAH-1/2 through an interacting surface apparently distinct from those predicted by the structural studies, or shown experimentally to mediate binding to SIAH-associated factors. Within PML, the coiled-coil domain is required for Siah- and proteasome-mediated degradation, and deletions of regions critical for the integrity of this region impair the ability of Siah to trigger PML-RAR degradation. Fusion of the coiled-coil domain to heterologous proteins resulted in the capacity of mSiah-2 to target their degradation. All of the TRIM proteins tested were degraded upon mSiah-2 overexpression. Finally, we show that the fusion protein PML-RAR (that retains the coiled-coil domain), which causes acute promyelocytic leukemias, is also a potential substrate of mSiah-2. As a result of mSiah-2 overexpression and subsequent degradation of the fusion protein, the arrest in hematopoietic differentiation because of expression of PML-RAR is partially rescued. These results identify PML and other TRIMs as new factors post-translationally regulated by SIAH and involve the coiled-coil region of PML and of other SIAH substrates as a novel structural determinant for targeted degradation. Mammalian homologues of Drosophila Seven in Absentia (SIAHs) target for proteasome-mediated degradation several factors involved in cell growth and tumorigenesis. Here we show that SIAH-1/2 binds and targets for proteasome-mediated degradation the putative tumor suppressor and tripartite motif (TRIM) family member PML, leading to the loss of its transcriptional co-activating properties and a reduction in the number of endogenous PML nuclear bodies. Association with PML requires the substrate-binding domain (SBD) of SIAH-1/2 through an interacting surface apparently distinct from those predicted by the structural studies, or shown experimentally to mediate binding to SIAH-associated factors. Within PML, the coiled-coil domain is required for Siah- and proteasome-mediated degradation, and deletions of regions critical for the integrity of this region impair the ability of Siah to trigger PML-RAR degradation. Fusion of the coiled-coil domain to heterologous proteins resulted in the capacity of mSiah-2 to target their degradation. All of the TRIM proteins tested were degraded upon mSiah-2 overexpression. Finally, we show that the fusion protein PML-RAR (that retains the coiled-coil domain), which causes acute promyelocytic leukemias, is also a potential substrate of mSiah-2. As a result of mSiah-2 overexpression and subsequent degradation of the fusion protein, the arrest in hematopoietic differentiation because of expression of PML-RAR is partially rescued. These results identify PML and other TRIMs as new factors post-translationally regulated by SIAH and involve the coiled-coil region of PML and of other SIAH substrates as a novel structural determinant for targeted degradation. SIAH 1The abbreviations used are: SIAHmammalian Sina homologueSinaDrosophila Seven in AbsentiaSiahmammalian Sina homologue genesPMLpromyelocytic leukemia proteinTRIMtripartite motifRAretinoic acidRARretinoic acid receptorCCcoiled-coilLLnLN-acetyl-Leu-Leu-norleucinalGFPgreen fluorescent proteinTGF-βtransforming growth factor-βPBSphosphate-buffered salineMBPmaltose-binding proteinMBP-PMLmaltose-binding protein-PML fusionNBiesnuclear bodiesGAL4Gal4 DNA-binding domainGal4Gal4 coding sequenceTKthymidine kinase.1The abbreviations used are: SIAHmammalian Sina homologueSinaDrosophila Seven in AbsentiaSiahmammalian Sina homologue genesPMLpromyelocytic leukemia proteinTRIMtripartite motifRAretinoic acidRARretinoic acid receptorCCcoiled-coilLLnLN-acetyl-Leu-Leu-norleucinalGFPgreen fluorescent proteinTGF-βtransforming growth factor-βPBSphosphate-buffered salineMBPmaltose-binding proteinMBP-PMLmaltose-binding protein-PML fusionNBiesnuclear bodiesGAL4Gal4 DNA-binding domainGal4Gal4 coding sequenceTKthymidine kinase. proteins are the mammalian homologues of Drosophila Seven in Absentia (Sina) (Ref. 1Amson R.B. Nemani M. Roperch J.P. Israeli D. Bougueleret L. Le Gall I. Medhioub M. Linares-Cruz G. Lethrosne F. Pasturaud P. Piouffre L. Prieur S. Susini L. Alvaro V. Millasseau P. Guidicelli C. Bui H. Massart C. Cazes L. Dufour F. Bruzzoni-Giovanelli H. Owadi H. Hennion C. Charpak G. Telerman A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3953-3957Crossref PubMed Scopus (141) Google Scholar). Sina specifies eye cell fate by induction of proteasome-mediated degradation of the transcription factor tramtrack (2Li S. Li Y. Carthew R.W. Lai L.C. Cell. 1997; 90: 469-478Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Likewise, mammalian SIAHs behave as the RING domain-containing components of E3 ubiquitin ligase complexes and target for proteasome-mediated degradation several factors involved in transcriptional regulation, cell growth, and tumorigenesis (3Hu G. Zhang S. Vidal M. Baer J.L. Xu T. Fearon E.R. Genes Dev. 1997; 11: 2701-2714Crossref PubMed Scopus (178) Google Scholar, 4Zhang J. Guenther M.G. Carthew R.W. Lazar M.A. Genes Dev. 1998; 12: 1775-1780Crossref PubMed Scopus (190) Google Scholar, 5Germani A. Bruzzoni-Giovanelli H. Fellous A. Gisselbrecht S. Varin-Blank N. Calvo F. Oncogene. 2000; 19: 5997-6006Crossref PubMed Scopus (77) Google Scholar, 6Liu J. Stevens J. Rote C.A. Yost H.J. Hu Y. Neufeld K.L. White R.L. Matsunami N. Mol. Cell. 2001; 5: 927-936Abstract Full Text Full Text PDF Scopus (375) Google Scholar, 7Matsuzawa S.I. Reed J.C. Mol. Cell. 2001; 5: 915-926Abstract Full Text Full Text PDF Scopus (529) Google Scholar). mammalian Sina homologue Drosophila Seven in Absentia mammalian Sina homologue genes promyelocytic leukemia protein tripartite motif retinoic acid retinoic acid receptor coiled-coil N-acetyl-Leu-Leu-norleucinal green fluorescent protein transforming growth factor-β phosphate-buffered saline maltose-binding protein maltose-binding protein-PML fusion nuclear bodies Gal4 DNA-binding domain Gal4 coding sequence thymidine kinase. mammalian Sina homologue Drosophila Seven in Absentia mammalian Sina homologue genes promyelocytic leukemia protein tripartite motif retinoic acid retinoic acid receptor coiled-coil N-acetyl-Leu-Leu-norleucinal green fluorescent protein transforming growth factor-β phosphate-buffered saline maltose-binding protein maltose-binding protein-PML fusion nuclear bodies Gal4 DNA-binding domain Gal4 coding sequence thymidine kinase. SIAH-1 is a transcriptional target of p53 in human cells in vitro, suggesting that targeted degradation of SIAH substrates may be relevant to modulate p53 response to stress (growth arrest and/or apoptosis). The p53-SIAH connection remains to be fully clarified, because recent results suggest that in vivo (at least, in mouse) SIAH proteins are not induced by p53 and do not play a significant role in the p53-mediated stress response (8Matsuzawa S. Takayama S. Froesch B.A. Zapata J.M. Reed J.C. EMBO J. 1998; 17: 2736-2747Crossref PubMed Scopus (185) Google Scholar, 9Frew I.J. Dickins R.A. Cuddihy A.R. Del Rosario M. Reinhard C. O'Connell M.J. Bowtell D.D.L. Methods Cell Biol. 2002; 22: 8155-8164Google Scholar). Among p53 coregulators, promyelocytic leukemia protein (PML) belongs to the so-called “tripartite motif” (TRIM) gene family (10Reymond A. Meroni G. Fantozzi A. Merla G. Cairo S. Luzi L. Riganelli D. Zanaria E. Messali S. Cainarca S. Guffanti A. Minucci S. Pelicci P.G. Ballabio A. EMBO J. 2001; 20: 2140-2151Crossref PubMed Scopus (1046) Google Scholar). The TRIM is composed of a RING domain followed by a cysteine/histidine-rich region (B1/B2 boxes) and by a coiled-coil region mediating self-association (10Reymond A. Meroni G. Fantozzi A. Merla G. Cairo S. Luzi L. Riganelli D. Zanaria E. Messali S. Cainarca S. Guffanti A. Minucci S. Pelicci P.G. Ballabio A. EMBO J. 2001; 20: 2140-2151Crossref PubMed Scopus (1046) Google Scholar, 48Minucci S. Maccarana M. Cioce M. De Luca P. Gelmetti V. Segalla S. Di Croce L. Giavara S. Matteucci C. Gobbi A. Bianchini A. Colombo E. Schiavoni I. Badaracco G. Hu X. Lazar M.A. Landsberger N. Nervi C. Pelicci P.G. Mol. Cell. 2000; 5: 811-820Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). PML is a nuclear protein and a structural component of the nuclear bodies (NBies) (11Lamond A.I. Earnshaw W.C. Science. 1998; 280: 547-553Crossref PubMed Scopus (775) Google Scholar, 12Hodges M. Tissot C. Howe K. Grimwade D. Freemont P.S. Am. J. Hum. Genet. 1998; 63: 297-304Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). PML is induced upon stress and facilitates post-translational modifications of p53 required for its full activity (13Pearson M. Carbone R. Sebastiani C. Cioce M. Fagioli M. Saito S. Higashimoto Y. Appella E. Minucci S. Pandolfi P.P. Pelicci P.G. Nature. 2000; 406: 207-210Crossref PubMed Scopus (1130) Google Scholar, 14Ferbeyre G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar). PML-null cells are more resistant to several kinds of stresses, suggesting an important role for PML in the induction of growth arrest/apoptosis (15Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (99) Google Scholar). Mechanistically, PML can act as a transcriptional coregulator of both transcriptional activators and repressors (16Vallian S. Gaken J.A. Trayner I.D. Gingold E.B. Kouzarides T. Chang K.S. Farzaneh F. Exp. Cell Res. 1997; 237: 371-382Crossref PubMed Scopus (41) Google Scholar, 17Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 18Wu W.S. Vallian S. Seto E. Yang W.M. Edmondson D. Roth S. Chang K.S. Mol. Cell. Biol. 2001; 21: 2259-2268Crossref PubMed Scopus (129) Google Scholar, 19Tsuzuki S. Towatari M. Saito H. Enver T. Mol. Cell. Biol. 2000; 20: 6276-6286Crossref PubMed Scopus (50) Google Scholar). In acute promyelocytic leukemia, PML is fused to the retinoic acid receptor (RAR)α, to yield the PML-RAR fusion protein (20Warrell Jr., R.P. de The H. Wang Z.Y. Degos L. N. Engl. J. Med. 1993; 329: 177-189Crossref PubMed Scopus (832) Google Scholar, 21Grignani F. Fagioli M. Alcalay M. Longo L. Pandolfi P.P. Donti E. Biondi A. Lo Coco F. Grignani F. Pelicci P.G. Blood. 1994; 83: 10-25Crossref PubMed Google Scholar). PML-RAR inhibits hematopoietic differentiation by aberrant transcriptional repression of RAR target genes, associating with the N-CoR/histone deacetylase complex. Pharmacological doses of retinoic acid (RA) release PML-RAR from N-CoR/histone deacetylase, induce differentiation of acute promyelocytic leukemia blasts, and target PML-RAR for proteasome degradation (22Chomienne C. Fenaux P. Degos L. FASEB J. 1996; 10: 1025-1030Crossref PubMed Scopus (121) Google Scholar, 23Raelson J.V. Nervi C. Rosenauer A. Benedetti L. Monczak Y. Pearson M. Pelicci P.G. Miller Jr., W.H. Blood. 1996; 88: 2826-2832Crossref PubMed Google Scholar, 24Fanelli M. Minucci S. Gelmetti V. Nervi C. Gambacorti-Passerini C. Pelicci P.G. Blood. 1999; 93: 1477-1481Crossref PubMed Google Scholar). In this report, we investigate the functional relationships among TRIMs, PML-RAR, and SIAHs. Our results unravel a novel structural determinant for SIAH-targeted degradation, and suggest a potential role for SIAH proteins in the regulation of normal PML function. Plasmids—Full-length hSiah-1 cDNAs (wild-type, mutant A, mutant D, and deleted of ΔRING) were cloned in the vector pcDNA3-myc as described (25Matsuzawa S. Li C. Ni C.-Z. Takayama S. Reed J.C. Ely K.R. J. Biol. Chem. 2003; 278: 1837-1840Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Wild-type PML224RAR-B cDNA and deletion mutants for the individual heptads of the coiled-coil region (PML273RAR-B, PML224Δ(274–300)RAR-B, PML224Δ(301–326)RAR-B, and PML-224Δ(327–360)RAR-B) were cloned in pTL2 as described (26Perez A. Kastner P. Sethi S. Lutz Y. Reibel C. Chambon P. EMBO J. 1993; 12: 3171-3182Crossref PubMed Scopus (286) Google Scholar). Coiled-coil (CC)-RAR cDNA cloned in as described F. Gelmetti V. M. D. De S. D. Grignani F. Nervi C. Pelicci P.G. Oncogene. 1999; PubMed Scopus Google Scholar). mSiah-2 cDNA fused in to the green fluorescent protein and cloned in the vector F. T. A. M. Riganelli D. Grignani F. L. C. Pelicci P.G. Res. 1998; Google Scholar). and described J. Guenther M.G. Carthew R.W. Lazar M.A. Genes Dev. 1998; 12: 1775-1780Crossref PubMed Scopus (190) Google Scholar). Cell and cells were in with N-acetyl-Leu-Leu-norleucinal from The and described J. Guenther M.G. Carthew R.W. Lazar M.A. Genes Dev. 1998; 12: 1775-1780Crossref PubMed Scopus (190) Google Scholar, L. Fagioli M. L. S. M. R. Grignani F. T. Pelicci P.G. Blood. PubMed Google Scholar). of cells were as described V. Zhang J. M. Minucci S. Pelicci P.G. Lazar M.A. Mol. Cell. Biol. 1998; 12: Scopus Google Scholar). cells were by with the The from the that the and used to the of target cells F. T. A. M. Riganelli D. Grignani F. L. C. Pelicci P.G. Res. 1998; Google Scholar). cells were or in the of to induce of differentiation as described V. Zhang J. M. Minucci S. Pelicci P.G. Lazar M.A. Mol. Cell. Biol. 1998; 12: Scopus Google Scholar). and cells were by as the cells were and for by of in the cells were in and in of proteins with the for followed by an of with protein The were in in and for as described F. U. D. P. A. D. Gelmetti V. Fagioli M. Alcalay M. J. Grignani F. I. C. Pelicci P.G. EMBO J. 1996; PubMed Scopus Google Scholar). cells were with in for and in for The used for the were in and for in the cells were for with the protein and maltose-binding protein-PML fusion were in and by of and proteins to were with in mSiah-2 or PML in for were in the in and by followed by of PML and the of both PML and PML-RAR are regulated through that are not fully M. Minucci S. Gelmetti V. Nervi C. Gambacorti-Passerini C. Pelicci P.G. Blood. 1999; 93: 1477-1481Crossref PubMed Google Scholar, F. De F. M. J. de H. Nat. Genet. 1998; 20: PubMed Scopus Google Scholar, C. M. M. Gelmetti V. Gambacorti-Passerini C. D. Grignani F. Pelicci P.G. R. Blood. 1998; Google Scholar, H. K. K. S. T. T. R. T. Res. 1996; Google Scholar). SIAH proteins are involved in their degradation, we in used to degradation of several other proteins J. Guenther M.G. Carthew R.W. Lazar M.A. Genes Dev. 1998; 12: 1775-1780Crossref PubMed Scopus (190) Google Scholar, Reed J.C. Mol. Cell. 2001; 7: 915-926Abstract Full Text Full Text PDF PubMed Scopus Google Scholar, J. Stevens J. Rote C.A. Yost H.J. Hu Y. Neufeld K.L. White R.L. Matsunami N. Mol. Cell. 2001; 7: 927-936Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). hSiah-1 or mSiah-2 were with PML, or the fusion protein of protein from cells of PML and and PML-RAR, not of RAR of cells to with the proteasome PML and PML-RAR suggesting the of proteasome-mediated degradation mSiah-2 induced degradation of the of PML in the PML-RAR fusion protein that SIAH-targeted degradation of PML-RAR PML and not As J. Guenther M.G. Carthew R.W. Lazar M.A. Genes Dev. 1998; 12: 1775-1780Crossref PubMed Scopus (190) Google Scholar, G. Fearon E.R. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google the RING domain of SIAH-1/2 is for proteasome-mediated degradation of target or to induce PML degradation and of the substrate-binding domain of the substrate-binding are not required for PML degradation. cells were with PML and or mutant A, mutant D, mutant deletion of RING domain (25Matsuzawa S. Li C. Ni C.-Z. Takayama S. Reed J.C. Ely K.R. J. Biol. Chem. 2003; 278: 1837-1840Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). were by and/or the of endogenous PML The of an to in to cells were with a the mSiah-2 cDNA fused in with the or as and with a to the endogenous PML As shown in the number of is in cells with to we an of in cells in cells not or to induce in results were by of mSiah-2 fused to or in cells not PML inhibits transcription to through fusion to the DNA-binding domain (16Vallian S. Gaken J.A. Trayner I.D. Gingold E.B. Kouzarides T. Chang K.S. Farzaneh F. Exp. Cell Res. 1997; 237: 371-382Crossref PubMed Scopus (41) Google Scholar). used the fusion protein to transcription by the in in of mSiah-2 expression vector not the transcription and repression of transcription and results suggest that SIAH proteins induce proteasome-mediated degradation of PML and PML-RAR in a in a loss of PML co-activating PML with SIAH investigate PML a with SIAH we upon of mSiah-2 mSiah-2 ΔRING) and PML in As shown in we in we not in we protein, by in suggesting that the of a by from the reduction in PML upon not mSiah-2 overexpression. investigate PML and we in binding a to and in As shown in not the protein, results suggest that with PML through its region and requires the RING domain to target PML for proteasome-mediated degradation. The region for binding of not to PML to the so-called substrate-binding domain of the of this domain G. N. J.P. F. Bowtell D.D.L. Nat. Biol. 2002; PubMed Scopus Google Scholar). of the and other (25Matsuzawa S. Li C. Ni C.-Z. Takayama S. Reed J.C. Ely K.R. J. Biol. Chem. 2003; 278: 1837-1840Abstract Full Text Full Text PDF PubMed Scopus (38) Google that Siah may to Siah substrates through distinct to impair the with other to trigger PML degradation. hSiah-1 mutant for and which is in a surface required for binding to the SIAH (25Matsuzawa S. Li C. Ni C.-Z. Takayama S. Reed J.C. Ely K.R. J. Biol. Chem. 2003; 278: 1837-1840Abstract Full Text Full Text PDF PubMed Scopus (38) Google to trigger PML degradation Likewise, in a region mutant D, for and as a of G. N. J.P. F. Bowtell D.D.L. Nat. Biol. 2002; PubMed Scopus Google and for the Drosophila homologue to mediate with substrates as S. Xu C. Carthew R.W. Mol. Cell. Biol. 2002; 22: PubMed Scopus (50) Google not impair the capacity of Siah to PML protein, not Siah through suggesting that are the substrate-binding domain (25Matsuzawa S. Li C. Ni C.-Z. Takayama S. Reed J.C. Ely K.R. J. Biol. Chem. 2003; 278: 1837-1840Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). is not for degradation by Siah (25Matsuzawa S. Li C. Ni C.-Z. Takayama S. Reed J.C. Ely K.R. J. Biol. Chem. 2003; 278: 1837-1840Abstract Full Text Full Text PDF PubMed Scopus (38) Google PML, apparently a new of Siah which are targeted to the proteasome do not the described SIAH required for with The Coiled-coil of PML and other TRIM the for in cells to which of PML is required for degradation. the shown in we that the region required for binding and degradation in the of PML in the fusion region the tripartite we PML of the or the coiled-coil and their to degradation. the coiled-coil not degraded by and not with mSiah-2 the boxes) expression were in an the coiled-coil region the required for degradation, we used a the domain is fused to RAR (that not behave as an substrate as shown in the by in a RAR were not or by These results show that the coiled-coil region of PML is and for and proteasome-mediated degradation. The coiled-coil region of PML distinct heptads the of the of the fusion protein shown that heptads to the of the coiled-coil domain (26Perez A. Kastner P. Sethi S. Lutz Y. Reibel C. Chambon P. EMBO J. 1993; 12: 3171-3182Crossref PubMed Scopus (286) Google Scholar, F. U. D. P. A. D. Gelmetti V. Fagioli M. Alcalay M. J. Grignani F. I. C. Pelicci P.G. EMBO J. 1996; PubMed Scopus Google Scholar, F. Gelmetti V. M. D. De S. D. Grignani F. Nervi C. Pelicci P.G. Oncogene. 1999; PubMed Scopus Google Scholar, F. Lo M. M. 2003; PubMed Scopus Google Scholar). deletion of individual to of properties of and with PML, binding to a (26Perez A. Kastner P. Sethi S. Lutz Y. Reibel C. Chambon P. EMBO J. 1993; 12: 3171-3182Crossref PubMed Scopus (286) Google Scholar, F. U. D. P. A. D. Gelmetti V. Fagioli M. Alcalay M. J. Grignani F. I. C. Pelicci P.G. EMBO J. 1996; PubMed Scopus Google Scholar, F. Gelmetti V. M. D. De S. D. Grignani F. Nervi C. Pelicci P.G. Oncogene. 1999; PubMed Scopus Google Scholar, F. Lo M. M. 2003; PubMed Scopus Google critical for the integrity of the coiled-coil because of the properties are in the deletion of this region (26Perez A. Kastner P. Sethi S. Lutz Y. Reibel C. Chambon P. EMBO J. 1993; 12: 3171-3182Crossref PubMed Scopus (286) Google Scholar, F. U. D. P. A. D. Gelmetti V. Fagioli M. Alcalay M. J. Grignani F. I. C. Pelicci P.G. EMBO J. 1996; PubMed Scopus Google Scholar, F. Gelmetti V. M. D. De S. D. Grignani F. Nervi C. Pelicci P.G. Oncogene. 1999; PubMed Scopus Google Scholar, F. Lo M. M. 2003; PubMed Scopus Google Scholar). the of PML-RAR and deletion in individual to degradation of or or and of proteins to be degraded by In deletion of degradation by These results suggest that the integrity of the coiled-coil domain is critical for the to PML belongs to a family of the tripartite the of a coiled-coil region (10Reymond A. Meroni G. Fantozzi A. Merla G. Cairo S. Luzi L. Riganelli D. Zanaria E. Messali S. Cainarca S. Guffanti A. Minucci S. Pelicci P.G. Ballabio A. EMBO J. 2001; 20: 2140-2151Crossref PubMed Scopus (1046) Google Scholar). other TRIMs may behave as of mSiah-2 with several TRIM family in to of protein that TRIM proteins are potential substrates for degradation by mSiah-2 of the of is the capacity to the differentiation of hematopoietic cells F. U. G. Fagioli M. Alcalay M. A. Grignani F. C. I. Pelicci P.G. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). PML-RAR degradation we can with As a we used hematopoietic PML-RAR a F. De S. Nervi C. L. Gelmetti V. Cioce M. M. M. I. C. Grignani F. Lazar M.A. Minucci S. Pelicci P.G. Nature. 1998; PubMed Scopus Google Scholar). In induction of PML-RAR expression to the of differentiation induced by F. De S. Nervi C. L. Gelmetti V. Cioce M. M. M. I. C. Grignani F. Lazar M.A. Minucci S. Pelicci P.G. Nature. 1998; PubMed Scopus Google Scholar). cells were with or the expression by mSiah-2 induce cells were with of surface differentiation that PML-RAR differentiation in cells to mSiah-2 partially the differentiation induced by cells were in the of the fusion protein and of that is a of PML-RAR we SIAH-1/2 as a novel for PML and suggest a potential role for SIAHs in the regulation of PML structural in PML and in of the other TRIM family coiled-coil the critical for degradation. region self-association and the of in (10Reymond A. Meroni G. Fantozzi A. Merla G. Cairo S. Luzi L. Riganelli D. Zanaria E. Messali S. Cainarca S. Guffanti A. Minucci S. Pelicci P.G. Ballabio A. EMBO J. 2001; 20: 2140-2151Crossref PubMed Scopus (1046) Google Scholar). by E. Kim P.S. Sci. 1997; PubMed Scopus Google of the of several mSiah-2 substrates in proteins with a to a suggesting that this may be a more structural for degradation. a potential SIAH motif described for SIAH substrates I.J. G. N. E. Bowtell D.D.L. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). to in the coiled-coil region of the TRIM proteins a motif of this suggesting that proteins to a distinct of SIAH is by the of SIAH mutants to with of the that of the the capacity of Siah to target PML for degradation a novel of are in to the complex. SIAH expression to both PML-RAR and target critical The differentiation by the fusion protein, partially rescued. are several for this of may be to the or by SIAH of substrates is required for may both to the we the that PML-RAR may induce of the of its target genes and other modifications PML-RAR expression to a of gene expression through cell the differentiation in the of PML-RAR or Croce L. M. F. M. M. F. Lo Coco F. Kouzarides T. Nervi C. Minucci S. Pelicci P.G. Science. 2002; PubMed Scopus Google Scholar). SIAH-1 is induced by p53 in human cell and apparently a role in the p53 stress response (8Matsuzawa S. Takayama S. Froesch B.A. Zapata J.M. Reed J.C. EMBO J. 1998; 17: 2736-2747Crossref PubMed Scopus (185) Google Scholar). results from suggest that this regulation in cells or not play a role in the the of the I.J. Dickins R.A. Cuddihy A.R. Del Rosario M. Reinhard C. O'Connell M.J. Bowtell D.D.L. Methods Cell Biol. 2002; 22: 8155-8164Google Scholar). the that SIAH may to the of PML is a p53 G. de Stanchina E. Querido E. Baptiste N. Prives C. Lowe S.W. Genes Dev. 2000; 14: 2015-2027Crossref PubMed Google Scholar, M. Carbone R. Sebastiani C. Cioce M. Fagioli M. S. H. Appella E. Minucci S. Pandolfi P.P. Pelicci P.G. Nature. 2000; 207-210Crossref Scopus Google Scholar, P. Pandolfi P.P. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google a to p53 the described is induced by and p53 degradation through the proteasome C. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The transcriptional p53 SIAH and trigger degradation of critical components of the p53 and its of The among and SIAH in the p53 is and may the to stress and C. and for and their to