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.
Highlights
Covalent coupling of SUMO by the E2 and E3 enzymes confers repression activity to transcriptional regulators
Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E21⁄7E3 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
HEK293T cells were co-transfected with a fixed amount of a DNA construct coding for either the wild-type PIAS1 or the PIAS1L337A mutant fused to Renilla Luciferase (RLuc-PIAS1or RLuc-PIAS1-L337A) along with increasing amounts of a DNA construct coding for UBC9 fused to the green fluorescent protein (UBC9-GFP) (Fig. 1B)
Summary
Covalent coupling of SUMO by the E2 and E3 enzymes confers repression activity to transcriptional regulators. Our results reveal that UBC9 and PIAS1 are engaged both in a canonical E21⁄7E3 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. Unlike what is observed with ubiquitination sites, a large proportion of SUMO-modified lysine residues are found within consensus motifs corresponding to the sequence KX(E/D) ( is a hydrophobic residue, and X corresponds to any residues) [29] This is consistent with the fact that a single E2-conjugating enzyme functions in the SUMOylation pathway and to the uniqueness of the catalytic cleft of UBC9 [30]. Our data support a model in which the PIAS11⁄7SUMO1⁄7UBC9 ternary complex functions in transcriptional repression and its formation is regulated by the phosphorylation state of the SIM module of PIAS1
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