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, 33 (5), 570-80

Protection From Isopeptidase-Mediated Deconjugation Regulates Paralog-Selective Sumoylation of RanGAP1

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Protection From Isopeptidase-Mediated Deconjugation Regulates Paralog-Selective Sumoylation of RanGAP1

Shanshan Zhu et al. Mol Cell.

Abstract

Vertebrates express three small ubiquitin-related modifiers (SUMO-1, SUMO-2, and SUMO-3) that are conjugated in part to unique subsets of proteins and, thereby, regulate distinct cellular processes. Mechanisms regulating paralog-selective sumoylation, however, remain poorly understood. Despite being equally well modified by SUMO-1 and SUMO-2 in vitro, RanGAP1 is selectively modified by SUMO-1 in vivo. We have found that this paralog-selective modification is determined at the level of deconjugation by isopeptidases. Our findings indicate that, relative to SUMO-2-modified RanGAP1, SUMO-1-modified RanGAP1 forms a more stable, higher affinity complex with the nucleoporin Nup358/RanBP2 that preferentially protects it from isopeptidases. By swapping residues in SUMO-1 and SUMO-2 responsible for Nup358/RanBP2 binding, or by manipulating isopeptidase expression levels, paralog-selective modification of RanGAP1 could be affected both in vitro and in vivo. Thus, protection from isopeptidases, through interactions with SUMO-binding proteins, represents an important mechanism defining paralog-selective sumoylation.

Figures

Figure 1
Figure 1
RanGAP1 is equally well modified by SUMO-1 and SUMO-2 in vitro and modification is not affected by Nup358 E3 ligase activity. RanGAP1 was expressed in vitro and incubated with recombinant E1 activating and E2 conjugating enzymes together with either SUMO-1 or SUMO-2 and in the presence or absence of Nup358-IR. A and B. Reactions were incubated for the indicated times (min), resolved by SDS-PAGE and visualized by autoradiography. C and D. Reactions were incubated with high concentrations of E1 and E2 enzymes (++) or low concentrations of E1 and E2 enzymes (+) together with increasing concentrations of Nup358-IR (0, 3, 10, 30, 100, 300 and 600 ng). Reactions were terminated after 10 min and analyzed by SDS-PAGE and autoradiography.
Figure 2
Figure 2
SUMO-1 modified RanGAP1 has a higher affinity for Nup358 relative to SUMO-2 modified RanGAP1. A. RanGAP1 was expressed in vitro and modified with either SUMO-1 or SUMO-2. Reaction products were incubated with glutathione beads containing GST-tagged Nup358-IR. Beads were washed with buffer containing the indicated MgCl2 concentrations (mM) and bound proteins were eluted and analyzed by SDS-PAGE and autoradiography. B. Biacore analysis was performed using sensor chips coated with GST-tagged Nup358-IR. SUMO-1 modified RanGAP1-NΔ419 was injected at multiple concentrations (8, 20, 50, 125 and 312 nm) together with Ubc9 and the resulting association and dissociation events were recorded. C. Biacore analysis of interactions between SUMO-2 modified RanGAP1-NΔ419 and Nup358-IR. SUMO-2 modified RanGAP1-NΔ419 was injected at multiple concentrations (10, 24, 61, 152 and 380 nm) together with Ubc9. RU=resonance units.
Figure 3
Figure 3
Nup358 binding differentially protects SUMO-1 and SUMO-2/3 modified RanGAP1 from isopeptidases. A. SUMO-1 modified His-RanGAP1-NΔ419 (input) was incubated with Ulp1 alone for 10 min (Ulp1) or incubated with Ulp1 for the indicated times (min) after pre-incubation with Nup358 and Ubc9 (Ulp1 + Nup358/Ubc9). Reactions were analyzed by immunoblotting with an anti-His antibody. B. Similar Ulp1 protection assays were performed with SUMO-2 modified His-RanGAP1-NΔ419. C. SUMO-1 or SUMO-2 modified His-RanGAP1-NΔ419 (input) were incubated with the catalytic domains of human SENPs alone (SENP1, SENP2, SENP) or after pre-incubation with Nup358 and Ubc9 (SENP1 + Nup358/Ubc9, SENP2 + Nup358/Ubc9, SENP5 + Nup358/Ubc9). Reactions were analyzed by immunoblotting with an anti-His antibody. SUMO modified and unmodified RanGAP1 are indicated. An asterisk indicates His-tagged SENP5.
Figure 4
Figure 4
Generation of a SUMO-1/2 chimera with Nup358-binding properties comparable to SUMO-2. A. A crystal structure illustrating interactions between the SIM of Nup358 and SUMO-1 (Reverter and Lima, 2005). A SUMO-1/2 chimera was produced by replacing the first α helix and second β strand of SUMO-1 with the same region from SUMO-2. B. In vitro sumoylation assays were performed using fluorescently-tagged GST-RanGAP1-NΔ419 and SUMO-1, SUMO-2 or SUMO-1/2 chimera. Reactions were terminated at different time points, resolved by SDS-PAGE and the fraction of RanGAP1-NΔ419 modified at each time point was quantified by fluorescence imaging and plotted. C. Biacore analysis was performed using sensor chips coated with GST-tagged Nup358-IR. RanGAP1-NΔ419 modified with the SUMO-1/2 chimera was injected at multiple concentrations (10, 25, 64, 159 and 397 nm) together with Ubc9 and the resulting association and dissociation events were recorded. RU=resonance units. D. SUMO-1/2 chimera modified His-RanGAP1-NΔ419 (input) was incubated with Ulp1 alone for 10 min (Ulp1) or incubated with Ulp1 for the indicted times (min) after pre-incubation with Nup358 and Ubc9 (Ulp1 + Nup358/Ubc9). Reactions were analyzed by immunoblotting with an anti-His antibody.
Figure 5
Figure 5
Stable SUMO modification of RanGAP1 in vivo correlates with high affinity Nup358 binding. A. Stable cell lines were induced to express HA-FKBP tagged SUMO-1, SUMO-1 chimera and SUMO-2. Whole cell lysates were resolved by SDS-PAGE and immunoblots were probed with anti-HA antibodies. Lysate from untransfected parental cells was included as a control. B. Cell lysates were probed with an antibody specific for RanGAP1. C. Stable cell lines expressing HA-FKBP tagged SUMO-1, SUMO-1 chimera and SUMO-2 were co-labeled with anti-HA and RanGAP1 specific antibodies and analyzed by immunofluorescence microscopy. D. Cells were transfected with Nup358-specific siRNAs for the indicated times (hrs) and whole cell lysates were prepared and resolved and SDS-PAGE. Immunoblots were probed with mAb 414 (recognizing Nup358 and Nup153 which serves as a loading control) and RanGAP1 specific antibodies.
Figure 6
Figure 6
Isopeptidases affect paralog selective RanGAP1 sumoylation in vivo. A. 293T cells were either mock transfected (Control) or co-transfected with SENP1 and SENP2 specific siRNAs (SENP1/2). 48 hr after transfection, whole cell lysates were prepared and analyzed by immunoblotting with SENP1 or tubulin specific antibodies (top panel). SENP2 knockdown was confirmed by co-transfecting FLAG-tagged SENP2 expressing cells (Control) with SENP2 specific siRNAs (SENP2) and immunoblotting with a SENP2 specific antibody (bottom panel). A non-specific cross-reacting band (*) served as a loading control. B. Whole cell lysates were prepared from mock-transfected cells (Control) or cells co-transfected with SENP1 and SENP2 specific siRNAs (SENP1/2). Lysates were analyzed by immunoblotting with SUMO-1 or SUMO-2/3 specific antibodies. C. Immunopurifications with SP2/0 control antibodies or SUMO-1 or SUMO-2/3 specific antibodies were performed using lysates from mock-transfected cells (Control) or cells co-transfected with SENP1 and SENP2 specific siRNAs (SENP1/2). Immunopurified SUMO conjugates were analyzed by immunoblotting with RanGAP1 or SUMO-2/3 specific antibodies. SUMO modified RanGAP1 and antibody heavy chains (h.c.) are indicated.
Figure 7
Figure 7
SUMO-1 and SUMO-2/3 modified RanGAP1 compete for Nup358 binding and protection from isopeptidases. A. Whole cell lysates were prepared from control 293T cells and 293T cells stably expressing shRNAs specific for SUMO-1. Lysates were analyzed by immunoblotting with antibodies specific for SUMO-1, SUMO-2/3, RanGAP1 and tubulin. B. Wild type and SUMO-1 deficient MEFs were analyzed by immunofluorescence microscopy. Cells were either co-labeled with SUMO-2/3 and SUMO-1 specific antibodies, or SUMO-2/3 and RanGAP1 specific antibodies. C. A model illustrating the roles of isopeptidases and Nup358 binding in determining SUMO-1 selective modification of RanGAP1. (1) SUMO-1 and SUMO-2/3 are equally well conjugated to RanGAP1. (2) SUMO-1 and SUMO-2/3 modified RanGAP1 are differentially recognized and deconjugated by isopeptidases. (3) SUMO-1 and SUMO-2/3 modified RanGAP1 compete for formation of a ternary complex with Nup358 and Ubc9 at NPCs. Due to a higher affinity for Nup358, SUMO-1 modified RanGAP1 competes more effectively for binding. Formation of the ternary complex protects both SUMO-1 and SUMO-2/3 modified RanGAP1 from isopeptidases. (4) SUMO-1 and SUMO-2/3 modified RanGAP1 dissociate from Nup358/Ubc9. Due to tighter association with Nup358, SUMO-1 modified RanGAP1 is more effectively protected from deconjugation.

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