title: 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.
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy. mirco.fanelli@uniurb.it
authors.forename: Mirco
authors.initials: M
authors.lastname: Fanelli
authors.forename: Anna
authors.initials: A
authors.lastname: Fantozzi
authors.forename: Pasquale
authors.initials: P
authors.lastname: De Luca
authors.forename: Sara
authors.initials: S
authors.lastname: Caprodossi
authors.forename: Shu-ichi
authors.initials: S
authors.lastname: Matsuzawa
authors.forename: Mitchell A
authors.initials: MA
authors.lastname: Lazar
authors.forename: Pier Giuseppe
authors.initials: PG
authors.lastname: Pelicci
authors.forename: Saverio
authors.initials: S
authors.lastname: Minucci
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004330:Drosophila; D005822:Genetic Vectors; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D009363:Neoplasm Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D000071156:Promyelocytic Leukemia Protein; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D025521:Tumor Suppressor Proteins; D020298:U937 Cells; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins
chemical_list: D018076:DNA, Complementary; D009097:Multienzyme Complexes; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex