Identification of a new small ubiquitin-like modifier (SUMO)-interacting motif in the E3 ligase PIASy

Abstract

Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity.

Keywords: PARP1; PIASy; SIM; SUMO; SUMO-interacting motif (SIM); TopoIIa; UBC9; nuclear magnetic resonance (NMR); small ubiquitin-like modifier (SUMO); sumoylation.

Figure 1.

In vitro SUMO E3 ligase activity of C-terminal deletions of PIASy. A, schematic diagram showing the conserved domains of the Siz/PIAS family of E3 ligases. Solid lines indicate the PIASy truncation constructs tested in the SUMOylation assay. The time courses for in vitro SUMOylation of TopoIIα (B) and PARP1 (C) using WT PIASy and various truncation constructs of PIASy (N480, N454, and N414) as well as in the absence of PIASy (−) are shown. SUMOylated and unmodified forms of TopoIIα and PARP1 are indicated as are the various PIASy constructs used.

Figure 2.

Analysis of the interaction of SUMO-3 with PIASy lacking the original SIM. A, PIASy schematic as in Fig. 1A with the solid line indicating the fragment, residues 287–454, used in the NMR and FP experiments. B, 2D ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled SUMO-3 titrated with increasing molar ratios of PIASy(287–454). Expanded sections of representative SUMO-3 residues affected upon binding of PIASy(287–454) are shown on the right. C, plot of relative peak intensity for all assigned, non-overlapping SUMO-3 resonances in the ligand-bound versus -free form (I_1:1/I_1:0). Gray and red lines depict mean and one standard deviation from the mean (1σ), respectively. D, SUMO-3 residues displaying significant peak intensity reduction upon complex formation with PIASy(287–454) are highlighted red. E, fluorescence polarization binding assay of fluorescein maleimide-labeled SUMO-3 titrated with increasing concentrations of PIASy(287–454). ΔmP, change in millipolarization. Error bars represent S.D.

Figure 3.

NMR titrations of SUMO-3 and PIASy(287–501) containing both original and new SIMs. A, schematic of PIASy with the solid bar indicating the fragment 287–501 used in the experiment. B, 2D ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled SUMO-3 titrated with increasing amounts of PIASy(287–501). Expanded sections of selected SUMO-3 residues affected by PIASy(287–501) are shown on the right. C, relative peak intensity graph for SUMO-3 resonances in the ligand-bound versus -free state (I_1:0.25/I_1:0) where gray and red lines correspond to the mean and one standard deviation from the mean (1σ), respectively. D, the SUMO-3 residues affected upon binding of PIASy(287–501) are highlighted (red, significant peak intensity reduction; orange, significant change in the peak position). Error bars represent S.D.

Figure 4.

Characterization of the binding of SUMO-3 and the new SIM of PIASy. A, PIASy schematic as in Fig. 1A with new SIM shown in magenta. The solid bar corresponds to the PIASy fragment 409–454 (which contains the new SIM but lacks the original SIM) used in the experiment. The sequences of the WT and mut PIASy construct are shown. B, a section of the 2D ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled SUMO-3 titrated with increasing molar ratios of WT PIASy(409–454) demonstrates specific SUMO-SIM binding. C, expanded sections of selected SUMO-3 residues affected upon binding of WT PIASy(409–454). D and E, similar to B and C for SUMO-3 titrations with mut PIASy(409–454). F, plots of weighted CSD from titrations of SUMO-3 with WT PIASy(409–454) (top) and mut PIASy(409–454) (bottom). Gray and red lines correspond to the mean and one standard deviation (1σ) from the mean, respectively. G, SUMO-3 residues that are strongly affected by the binding of WT PIASy(409–454) are colored red.

Figure 5.

NMR titrations of SUMO-3 and PIASy(409–501) containing both original and new SIMs. A, schematic of PIASy indicating fragment 409–501 containing both the original SIM and the new SIM used in the experiment. B, 2D ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled SUMO-3 titrated with increasing amounts of PIASy(409–501). Expanded sections of representative SUMO-3 residues affected by the binding of PIASy(409–501) are on the right. C, plot of relative peak intensity for SUMO-3 resonances in the ligand-bound versus -free form (I_1:0.25/I_1:0). Gray and red lines depict mean and one standard deviation from the mean (1σ), respectively. D, the SUMO-3 residues affected upon complex formation with PIASy(409–501) are highlighted (red, residues displaying significant peak intensity reduction; orange, residues with significant change in the peak position). Error bars represent S.D.

Figure 6.

Functional role of the new SIM of PIASy. A, schematic of PIASy with solid bars illustrating the various SIM site-directed mutations examined in the SUMOylation assay. The time courses for SUMOylation of TopoIIα (B) and PARP1 (C) using the indicated PIASy SIM mutant in the in vitro SUMO conjugation assay are shown. SUMOylated and unmodified forms of TopoIIα and PARP1 along with the input PIASy are indicated. Quantification of the SUMOylation of TopoIIα (D) and PARP1 (E) at the final time point of the assay is shown. Assays were performed in triplicate. Error bars represent one standard deviation, and an asterisk indicates statistically significant differences from the WT activity.