title: Identification of a non-covalent ternary complex formed by PIAS1, SUMO1, and UBC9 proteins involved in transcriptional regulation.
abstract: Post-translational modifications with ubiquitin-like proteins require three sequentially acting enzymes (E1, E2, and E3) that must unambiguously recognize each other in a coordinated fashion to achieve their functions. Although a single E2 (UBC9) and few RING-type E3s (PIAS) operate in the SUMOylation system, the molecular determinants regulating the interactions between UBC9 and the RING-type E3 enzymes are still not well defined. In this study we use biochemical and functional experiments to characterize the interactions between PIAS1 and UBC9. Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E2·E3 interaction as well as assembled into a previously unidentified non-covalent ternary complex with SUMO as evidenced by bioluminescence resonance energy transfer, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry studies. In this ternary complex, SUMO functions as a bridge by forming non-overlapping interfaces with UBC9 and PIAS1. Moreover, our data suggest that phosphorylation of serine residues adjacent to the PIAS1 SUMO-interacting motif favors formation of the non covalent PIAS1·SUMO·UBC9 ternary complex. Finally, our results also indicate that the non-covalent ternary complex is required for the known transcriptional repression activities mediated by UBC9 and SUMO1. Taken together, the data enhance our knowledge concerning the mode of interaction of enzymes of the SUMOylation machinery as well as their role in transcriptional regulation and establishes a framework for investigations of other ubiquitin-like protein systems.
authors.affiliation: From the Département de Biochimie, Université de Montréal, C. P. 6128 Succursale Centre-Ville, Montréal, Quebec H3C 3J7, Canada.
authors.forename: Xavier H
authors.initials: XH
authors.lastname: Mascle
authors.forename: Mathieu
authors.initials: M
authors.lastname: Lussier-Price
authors.forename: Laurent
authors.initials: L
authors.lastname: Cappadocia
authors.forename: Patricia
authors.initials: P
authors.lastname: Estephan
authors.forename: Luca
authors.initials: L
authors.lastname: Raiola
authors.forename: James G
authors.initials: JG
authors.lastname: Omichinski
authors.forename: Muriel
authors.initials: M
authors.lastname: Aubry
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D050828:Protein Inhibitors of Activated STAT / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization; D025842:SUMO-1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: C106529:PIAS1 protein, human; D050828:Protein Inhibitors of Activated STAT; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9
keywords: Bioluminescence Resonance Energy Transfer (BRET); Calorimetry; E2 SUMO Conjugating Enzyme; E3 SUMO Ligase; Gene Regulation; Nuclear Magnetic Resonance; Phosphorylation; Protein Complexes; SUMO Interacting Motif; Sumoylation