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 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 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 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

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

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

Rapamycin decreases neoblast proliferation and increases the survival rate in <i>Smed-smg-1</i> RNAi animals.

<p>A. Number of H3P positive cells per mm² of un-cut planarians or 6 hR planarians that were previously treated with daily injections of DMSO, 20 nM rapamycin, 30 nM rapamycin or 40 nM rapamycin during 2 weeks. Error bars are s.d from the mean, the asterisk indicate P<0.05 while ns indicates “not significant” (P>0.05) using two-tailed Student's test with equal sample variance relative to the corresponding DMSO treated planarians. 6 hR shows an increased mitotic peak respect to the pre-cut condition despite the treatments (P<0.01). No significant differences (P>0.05) are observed either between the 30 nM and 40 nM rapamycin treatment in the pre-cut planarians or between the different rapamycin treatments in the 6 hR planarians. B. Number of H3P positive cells per mm² in control RNAi and <i>Smed-smg-1</i> RNAi planarians at 20 hR that were treated with daily injections of DMSO, 20 nM rapamycin, 30 nM rapamycin or 40 nM rapamycin. Outside the bars two asterisks (p<0.01) show significant differences while ns (p>0.05) shows no significant differences relative to the control RNAi animals. The asterisks inside the blue bars show significance relative to the 20 hR control RNAi worms DMSO treated whereas inside the red bars the significance is shown relative to the 20 hR <i>Smed-smg-1</i> RNAi worms DMSO treated. Error bars are s.d from the mean and asterisks indicate P<0.05 (one asterisk) or P<0.01 (two asterisks) or P>0.05 (ns) using two-tailed Student's test with equal sample variance. ≥8 planarians per time point. C. Kaplan-Meier curves demonstrate increased survival of <i>Smed-smg-1</i> RNAi animals when treated with 20 nM, 30 nM or 40 nM rapamycin. Four asterisks indicate p<0.0001 with log-rank analysis of survival curves. Representative images of surviving animals after <i>Smed-smg-1</i> RNAi and treated with 40 nM rapamycin are shown at 45 dR. Scale bars indicate 300 µm.</p

<i>Smed-smg-1</i> is required to restrict blastema growth during regeneration.

<p>A. The cartoon shows the levels of transverse amputation performed in the experiment (arrows) and highlights the trunk piece (dotted red circle), which was kept to follow anterior and posterior regeneration in all the experiments. Panels show 20–25 dR trunks. Dotted red lines define the blastemas. ph indicates the pharynx. <i>Smed-smg-1(RNAi)</i> animals show varying size of abnormal unpigmented blastemas with several degrees of eye differentiation displayed by the eye pigmentary cup in the live images (black arrows) and the VC1 staining. Asterisk shows a thickened optic chiasm, the white arrow shows an extra eye as an accumulation of unilateral photoreceptor clusters, always more anteriorly positioned than the original eyes, the yellow arrow shows abnormal axonal projections, and the white arrowhead shows an aberrant antero-dorsal photoreceptor projection. The black arrowhead shows epidermal hyperplasia. B. Table summarising the phenotypes observed in trunks at 25 dR; hyperplasia refers to epidermal hyperplasia observed in live animals. C, D. Mitotic numbers during different time points of regeneration and volume of the different neoblast compartments at 20 dR, respectively. Error bars are s.d from the mean and asterisks indicate P<0.05 (one asterisk) or P<0.01 (two asterisks) using two-tailed Student's test with equal sample variance. C. n≥5 planarians per time point in at least two independent experiments; D. n≥4. E. Panels show maximum confocal projections for the dorsal-most sections (1, 7, 4 and 10), the medial sections (2, 8, 5 and 11) and the ventral-most sections (3, 9, 6 and 12) of 20 d anterior regenerating trunks. Asterisks indicate the position of the eyes. Panels show the distribution of neoblasts (<i>cat-1</i>), neoblast early progeny (<i>cat-2</i>) and neoblast late progeny (<i>cat-3</i>) markers in controls versus <i>Smed-smg-1(RNAi)</i> animals. The inset in 8 and 11 show the presence of <i>cat-1</i> and H3P⁺ cells in the <i>cat-2</i> and <i>cat-3</i> compartment in front of the eyes in <i>Smed-smg-1(RNAi)</i> animals. Strips represent confocal XZ or YZ projections (D is dorsal and V is ventral) demonstrating a greater volume of neoblasts and progeny, an accumulation of <i>cat-1</i> cells dorsal to the brain ganglia (X2 and Y1) and a higher accumulation of <i>cat-3</i> at the anterior tip of the planarian (X1, Y1, Y2) in <i>Smed-smg-1(RNAi)</i> compared to controls. n≥4. Lower panels show H3P⁺ cells (panels 13 and 16, blue arrow) in front of the eyes (maximum confocal projection; n = 4/8), a very undeveloped brain labelled by anti-SYNAPSIN staining (panels 14 and 17; dorsal maximum confocal projection), an anterior gut branch (seen in BCAT-2 staining; maximum projection) aberrantly located with respect to the position of the eyes (n = 4/6, the white arrow indicates the most anterior level reached by the anterior gut branch) and the epithelium of protonephridial tubules stained by anti-BCAT-2 not reaching the tip of the planarian (panels 15 and 18; white arrow head; n = 6/6) in <i>Smed-smg-1(RNAi)</i> animals. F. Panels show maximum confocal projections of 20 d anterior regenerating trunks. The tip of the blastema shows minimal differentiation of muscle cells (stained with anti-TMUS13) in <i>Smed-smg-1(RNAi)</i> planarians. n = 3/3. G. Cartoons summarising the phenotype observed during anterior regeneration of 25 dR trunks. Scale bars represent 300 µm. Scale bars in (E) high magnification images represent 50 µm.</p

mTORC1 is necessary for <i>Smed-smg-1</i> RNAi phenotype.

<p>A. Table summarising the phenotypes observed after combinatorial RNAi experiments of <i>Smed-smg-1</i> with the members of TORC1. B. Panels show that double RNAi experiments of <i>Smed-smg-1</i> with either <i>Smed-tor</i> or <i>Smed-raptor</i> lead to lack of blastema formation, the same phenotype that is obtained after RNAi of either <i>Smed-tor</i> or <i>Smed-raptor</i> with <i>gfp</i>. Red dotted lines define the blastema. Arrows indicate outgrowths. C. Relative expression of <i>Smed-smg-1</i>, <i>Smed-tor</i> and <i>Smed-raptor</i> at 7 days of regeneration after single or double RNAi experiments. Expression levels are relative to <i>gfp</i> RNAi injected animals (dotted line). Similar levels of down regulation for the different genes are observed in single or double RNAi experiments (P>0.05). Error bars are s.d from the mean and asterisks indicate P<0.05 (one asterisk) or P<0.01 (two asterisks) or P<0.001 (three asterisks) using two-tailed Student's test with equal sample variance and relative to expression in control animals. Scale bars indicate 300 µm.</p