SUMOylation Is Required for Optimal TRAF3 Signaling Capacity

<div><p>TNF receptor–associated factors (TRAFs) are multifunctional adaptor proteins involved in temporal and spatial coordination of signals necessary for normal immune function. Here, we report that TRAF3, a TRAF family member with a key role in Toll-like and TNF family receptor signaling and suppressor of lymphomagenesis, is post-translationally modified by the small ubiquitin-related modifier (SUMO). Through yeast two-hybrid and co-immunoprecipitation assays we have identified Ubc9, the SUMO conjugating enzyme, as a novel TRAF3-interacting protein. We show that Ubc9-dependent SUMOylation of TRAF3 modulates optimal association with the CD40 receptor, thereby influencing TRAF3 degradation and non-canonical NF-κB activation upon CD40 triggering. Collectively, our findings describe a novel post-translational modification of a TRAF family member and reveal a link between SUMOylation and TRAF-mediated signal transduction.</p></div

TRAF3 is post-translationally modified by SUMO.

<p>(<b>A</b>) HeLa cells stably expressing His-tagged SUMO-1 or SUMO-2 and parental cells were lysed in a protein-denaturing buffer and lysates were subjected to enrichment of SUMOylated proteins on nickel-nitrilotriacetic acid (Ni-NTA) columns. Eluates were immunoblotted with a TRAF3 polyclonal antibody. (<b>B & C</b>) SUMO-1 modification of TRAF3. Protein lysates were obtained from EJ bladder carcinoma, BJAB lymphoma and HeLa cervical carcinoma cells stably transfected with SUMO-1 (B) or mouse splenocytes (C) in the presence or absence of iodocetamide (IDO) and immunoprecipitated (<i>I.P</i>) with anti-TRAF3 C20 antibody. The SUMO-1 conjugates were detected by anti-SUMO-1 specific antibody. (<b>D</b>) SUMO-2/3 modification of TRAF3. Protein lysates were obtained from EJ cells in the presence orabsence of IDA and TRAF3 immunoprecipitates (<i>I.P</i>) were immunoblotted (<i>I.B</i>) with an anti-SUMO-2/3 specific antibody.</p

TRAF3 interacts with Ubc9 in a yeast 2-hybrid assay.

<p>(<b>A</b>) <i>S. cerevisiae</i> strain PJ69-4A was transformed with plasmids expressing the GAL4 activation domain (AD) fused to Ubc9, the GAL4 binding domain (BD) fused to TRAF3 or with control empty vectors in all possible combinations. Cells were grown on standard medium (SD-leu-trp) in the presence or absence of X gal (SD-leu-trp+X gal), medium lacking adenine (SD-leu-trp-ade) to detect expression of the <i>GAL-ADE2</i> reporter gene or medium lacking histidine (SD-leu-trp-his + 5 mM 3-AT) to detect expression of the <i>GAL-HIS3</i> reporter gene. (<b>B</b>) Quantification of galactosidase reporter activity in yeast transformed with the vectors described in (A). Results shown represent the mean values of galactosidase activity (±SD) relative to controls from 4 independent experiments.</p

TRAF3 SUMOylation is Ubc9-dependent.

<p>(<b>A</b>) Over-expression of “dominant-negative” Ubc9^C93A which is unable to catalyze formation of SUMO conjugates reduces endogenous TRAF3 SUMOylation. HEK293 cells were transfected with Ubc9^C93A and lysates were obtained using an IDO-containing lysis buffer. Anti-TRAF3 immunoprecipitates (<i>I.P</i>) were immunoblotted (<i>I.B</i>) with either anti-SUMO-1 or anti-SUMO-2/3 antibodies, as indicated. Lysates were also immunoblotted with anti-MYC tag antibody to confirm expression of Ubc9^C93A or anti-TRAF3 to detect the levels of immunoprecipitated endogenous TRAF3. <i>CV</i>; control vector. (<b>B</b>) Knock-down of Ubc9 diminishes TRAF3 SUMOylation. HEK 293 cells were transfected with Ubc9 siRNA or an unrelated siRNA targeting <i>luciferase</i> (Luc) prior to lysis. Anti-TRAF3 immunoprecipitates were then immunoblotted with either anti-SUMO-1 or anti-SUMO-2 antibodies, as indicated. Results are representative of at least 4 independent experiments for (A) and (B).</p

TRAF3 SUMOylation affects its CD40-interacting capacity.

<p>(<b>A</b>) The binding of TRAF3 to GST-CD40CT increases when SUMO modification is maintained. HEK293 cells were lysed in the presence or absence of iodoacetamide (IDO) and lysates were incubated with bacterially produced GST-CD40 C-terminus (CT) or, as control GST-CD40CTA, carrying a T²⁵⁴→A mutation (GST-CD40CTA) which abolishes interaction with TRAF3, bound to glutathione sepharose beads. Interacting proteins were fractionated by SDS-PAGE and immunoblotted (<i>I.B</i>.) with anti-TRAF3 Ab. Whole cell lysates (<i>WCL</i>; 30 µg) were analyzed by immunoblot for TRAF3 expression levels. Lower right panel: Coomassie-stained gel showing GST-CD40CT and GST-CD40CTA produced in bacteria. (<b>B</b>) Over-expression of “dominant-negative” Ubc9^C93A reduces binding of TRAF3 to CD40. HEK293 cells were transfected with Ubc9^C93A or control vector (<i>CV</i>), lysates were obtained using an IDO-containing lysis buffer and incubated with GST-CD40CT or GST-CD40CTA bound to glutathione sepharose beads. Interacting proteins were fractionated by SDS-PAGE and immunoblotted (<i>I.B</i>.) with anti-TRAF3. Whole cell lysates (<i>WCL</i>; 30 µg) were analyzed for TRAF3 and Ubc9^C93A expression levels by immunoblotting using anti-TRAF3 and Myc tag Abs, respectively. Results in (A) & (B) are representative of 3 independent experiments.</p

TRAF3interacts with Ubc9 through its ring finger TRAF-N domain.

<p>(<b>A</b>) GST-Ubc9 produced in bacteria as fusion with GST interacts with FLAG-tagged TRAF3 expressed in HEK 293 cells. GST or GST-Ubc9 was incubated with protein lysates isolated from FLAG-TRAF3 transfected HEK293 cells. Pulled-down proteins were solubilized in SDS protein sample buffer, separated on 8% SDS polyacrylamide gels and immunoblotted for TRAF3. Coomassie blue staining was used to confirm equal utilization of GST and GST-Ubc9 proteins in the pull-down assays. (<b>B</b>) Ubc9 and TRAF3 interact <i>in vivo</i>. FLAG-tagged TRAF3 was co-expressed with myc-tagged Ubc9 in HEK293 cells. Ubc9 was detected in anti-FLAG immunoprecipitates. As positive control <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080470#pone.0080470-Lin1" target="_blank">[25]</a>, FLAG-tagged SMAD4 was used to monitor interactions with Ubc9. (<b>C &D</b>) Interaction of TRAF3 deletion mutants with Ubc9 in an <i>in vitro</i> GST pull-down assay. 293T cells were transiently transfected with the FLAG-tagged TRAF3 deletion mutants shown in <i>C</i>. Thirty six hours later, the extracts were incubated with purified GST-Ubc9 bound to glutathione sepharose beads. Interacting proteins were fractionated by SDS-PAGE and immunoblotted with anti-FLAG mAb (lanes 1-5; <i>D</i>). Whole cell lysates (30 µg) were analyzed in parallel to monitor the motility of the TRAF3 deletion mutant proteins (lanes 6-10). <i>I.P</i>.; immunoprecipitation. <i>I.B</i>.; immunoblot. <i>WCL</i>; Whole cell lysates</p

SUMOylation affects TRAF3 degradation and non-canonical NF-κB2 signaling in CD40-stimulated cells.

<p><b>(A)</b> Knock-down of Ubc9 diminishes CD40 ligand (CD154) induced TRAF3 degradation. EJ cells were transfected with Ubc9 siRNA or an unrelated siRNA targeting <i>luciferase</i> (Luc) prior to stimulation with 0,5 µg/ml recombinant CD154 and lysis. Lysates were immunoblotted with anti-TRAF3, anti-cIAP2 or anti-β-actin antibodies. Results are representative of at least 5 independent experiments. (<b>B</b>) Validation of siRNA targeting TRAF3. EJ bladder carcinoma cells were transfected with siRNAs against TRAF3, RIP1 or the unrelated <i>luciferase</i> and knock-down efficacy and specificity were determined by immunoblotting cell lysates with anti-TRAF3, RIP1 or β-actin antibodies. (<b>C</b>) Lysates from Ubc9 knocked-down cells were immunoblotted with anti-NF-κB2 mAb recognizing both the full-length p100 and processed p52 form of NF-κB2 or with β-actin as loading control. Results are representative of 3 independent experiments. (<b>D</b>) Nuclear and cytoplasmic protein extracts were prepared from control and Ubc9 siRNA-transfected EJ cells before and after stimulation with 0,5 µg/ml CD40L and analyzed for p65/RelA expression by immunoblot. The transcription factor Sp1 was used as a marker for the purity of the nuclear cell extract preparation. Results are representative of 3 independent experiments.</p