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. 2007 Oct 31;2(10):e1085.
doi: 10.1371/journal.pone.0001085.

Nitric oxide destabilizes Pias3 and regulates sumoylation

Jing Qu  1 , Guang-Hui Liu, Kaiyuan Wu, Peiwei Han, Peng Wang, Jiangmei Li, Xu Zhang, Chang Chen
Affiliations

Nitric oxide destabilizes Pias3 and regulates sumoylation

Jing Qu et al. PLoS One. .

Abstract

Small ubiquitin-related protein modifiers (SUMO) modification is an important mechanism for posttranslational regulation of protein function. However, it is largely unknown how the sumoylation pathway is regulated. Here, we report that nitric oxide (NO) causes global hyposumoylation in mammalian cells. Both SUMO E2 conjugating enzyme Ubc9 and E3 ligase protein inhibitor of activated STAT3 (Pias3) were targets for S-nitrosation. S-nitrosation did not interfere with the SUMO conjugating activity of Ubc9, but promoted Pias3 degradation by facilitating its interaction with tripartite motif-containing 32 (Trim32), a ubiquitin E3 ligase. On the one hand, NO promoted Trim32-mediated Pias3 ubiquitination. On the other hand, NO enhanced the stimulatory effect of Pias3 on Trim32 autoubiquitination. The residue Cys459 of Pias3 was identified as a target site for S-nitrosation. Mutation of Cys459 abolished the stimulatory effect of NO on the Pias3-Trim32 interaction, indicating a requirement of S-nitrosation at Cys459 for positive regulation of the Pias3-Trim32 interplay. This study reveals a novel crosstalk between S-nitrosation, ubiquitination, and sumoylation, which may be crucial for NO-related physiological and pathological processes.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Nitric oxide caused loss of sumo conjugates.
(A) HEK293 cells transfected with HA-SUMO1, 2 or 3 were treated with 500 µM GSNO for 4 h, lysed in Laemmli buffer, and immunoblotted with αHA or α actin. Asterisk: free SUMO1/2/3. (B) Time-dependent effect of GSNO on SUMO1 conjugates in transfected HEK293 cells, treated as in (A). In a control experiment, cells were treated with H2O2 (1 mM) for 1h. (C) HeLa cells were treated with increasing concentrations of GSNO for 4 h, lysed in Laemmli buffer, and immunoblotted with αSUMO1, αubiquitin or αactin. (D) RAW264.7 cells pretreated with or without SMT were stimulated with LPS for 24 h, lysed in Laemmli buffer, and immunoblotted with αSUMO1, αiNOS or αactin.
Figure 2
Figure 2. S-nitrosation of Ubc9 did not compromise its SUMO E2 enzyme activity.
(A) S-nitrosation of HA-Ubc9 (wild-type or cysteine mutants) in HEK293 cells stimulated with 500 µM GSNO determined by biotin-switch assay. (B) HeLa cells were incubated with 500 µM H2O2 for 30 min or 500 µM GSNO for the indicated time, lysed in Laemmli buffer with (lower blot) or without (upper blot) DTT, and immunoblotted with αUbc9. (C) HEK293 cells were transfected with the indicated plasmids, treated with H2O2 (for 1h) or GSNO (for 4 h), lysed in Laemmli buffer, and immunoblotted with αp53 or αactin.
Figure 3
Figure 3. Effects of GSNO on the protein level of SUMO-related enzymes.
(A) HeLa cells were treated with increasing concentrations of GSNO for 4 h, lysed in Laemmli buffer and immunoblotted with the indicated antibodies. (B) Immunoblotting analysis of endogenous Pias3 and SUMO1 conjugates in HeLa cells transfected with non-specific, mismatched or Pias3-specific siRNAs.
Figure 4
Figure 4. GSNO promotes Pias3-Trim32 interplay.
(A) HeLa cells expressing His-Ub were stimulated with or without GSNO for 4 h and then subjected to His-Ub pull down. Both longer and shorter exposures are shown. Bracket: poly-ubiquitinated Pias3; Arrow: mono-ubiquitinated Pias3. (B) Effect of GSNO on Pias3-Trim32 interaction determined by GST-pull down assay. GST-Pias3 fusion proteins, immobilized on beads, were mixed with cell lysates with GFP-Trim32 expression in the absence or presence of 500 µM GSNO. Pull-downed and input GFP-Trim32 was analyzed by immunoblotting. (C) Confocal microscopic analysis of subcellular localization of Myc-Pias3 and GFP-Trim32 in cotransfected HEK293 cells. 4 h after transfection, cells were incubated with or without LMB for 16 h. (D) HeLa cells transfected with or without GFP-Trim32 were stimulated with or without GSNO for 4 h and then the denatured lysates were immunoprecipitated with αPias3, followed by detection for Ub. Bracket: poly-ubiquitinated Pias3; Arrow: mono-ubiquitinated Pias3; Asterisk: unmodified Pias3. (E) HEK293 cells transfected with the indicated plasmids were stimulated with or without GSNO for 1 h and then subjected to His-Ub pull down. Bracket: ubiquitinated GFP-Trim32. In panels (A), (C), (D) and (E), cells were incubated with MG132 to prevent potential protein degradation.
Figure 5
Figure 5. S-nitrosation of Pias3 and its effects on Pias3-Trim32 association.
(A) S-nitrosation of GFP-Pias3 in HEK293 cells stimulated with 500 µM GSNO determined by biotin-switch assay. (B) Recombinant Pias3 was exposed to 100 µM GSNO and blocked with MMTS. In-gel digested Pias3 was subjected to Nano-LC-MS/MS analysis. (C) Effect of GSNO on Pias3(C459S)-Trim32 interaction determined by GST-pull down assay.
Figure 6
Figure 6. Model demonstrating that oxidative and nitrosative stress block the sumoylation pathway through different mechanisms.
Top: In a normal redox environment, Ubc9 conjugates SUMO to the substrate with the help of E3 ligase. Middle: Oxidative stress leads to formation of a disulfide bond between the E1 subunit Uba2 and the E2 subunit Ubc9, resulting in inactivation of both E1 and E2 enzymes. Below: Under nitrosative stress, both Ubc9 and Pias3 are S-nitrosated. Whereas S-nitrosation of Ubc9 cannot interfere with its catalytic activity, S-nitrosation of Pias3 facilitates its degradation by promoting its interplay with Ub E3 ligase Trim32, thereby resulting in decrease of SUMO conjugating efficiency. Su, SUMO; S, substrate protein.
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