TRIM13 regulates ubiquitination and turnover of NEMO to suppress TNF induced NF-κB activation

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Cellular Signalling xxx (2014) xxx–xxx

CLS-08254; No of Pages 8

Contents lists available at ScienceDirect

Cellular Signalling

j ourna l homepage: www.e lsev ie r .com/ locate /ce l l s ig

TRIM13 regulates ubiquitination and turnover of NEMO to suppress TNFinduced NF-κB activation

FDhanendra Tomar, Rajesh Singh ⁎Department of Cell Biology, School of Biological Sciences and Biotechnology, Indian Institute of Advanced Research, Gandhinagar, India
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⁎ Corresponding author at: Department of BiochemMaharaja Sayajirao University of Baroda, Vadodara, Gu9377155303.

E-mail address: [email protected] (R. Singh).

http://dx.doi.org/10.1016/j.cellsig.2014.08.0080898-6568/© 2014 Published by Elsevier Inc.

Please cite this article as: D. Tomar, R. Singh,Cellular Signalling (2014), http://dx.doi.org/

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Article history:Received 7 July 2014Accepted 17 August 2014Available online xxxx

Keywords:TRIM13TNFNF-κBNEMOTumor suppressorClonogenic ability
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The NF-κB family of transcription factors is activated in response to various intracellular or extracellular stimuliand its dysregulation leads to pathological conditions like infection, cancer, neurodegenerative disorders. Thepost-translational modification by ubiquitination regulates various steps of NF-κB pathway. In the currentstudy, we have described the role of TRIM13, an endoplasmic reticulum (ER) membrane anchored E3 ligase inregulation of NF-κB. The expression of TRIM13 represses TNF induced NF-κB while the knockdown has theopposite effect. The E3 ligase activity and ER localization is essential for NF-κB suppression whereas TRIM13regulated autophagy is not essential. TRIM13 interacts with NEMO and modulates its ubiquitination andturnover, hence may regulate IKK complex activity. TRIM13 mediated NF-κB repression is essential for negativeregulation of clonogenic ability of the cells. This study for the first time demonstrated the role of TRIM13, ERresident RING E3 ligase as a novel regulator of NEMO ubiquitination, negative regulator of NF-κB signaling andits role as a tumor suppressor.

© 2014 Published by Elsevier Inc.

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The NF-κB family of transcription factor is activated by variousphysiological and pathological stimuli. It had been implicated in severalcellular processes like cell proliferation, death, inflammation and innateimmunity by regulating the expression of several inflammatorycytokine [1]. The dysregulation of NF-κB leads to several pathologicalconditions like cancer, neurodegeneration, metabolic and aging relateddiseases [2,3]. It is now well understood that different cytokines likeTNF, IL-1β, IL-8 activate NF-κB pathway leading to distinct outcomeslike proliferation, death or inflammation. Similarly, different intracellu-lar stresses like proteotoxic, oxidative, genotoxic and endoplasmicreticulum stress activate NF-κB, ultimately deciding the fate of cells[4–6]. The regulatory mechanisms of NF-κB activation by differentphysiological stimuli leading to unique cellular response is of immenseimportance however it is still not well understood.

The ubiquitination of different proteins involved in NF-κB pathwayis an additional level of regulation in different physiological and patho-logical conditions [7,8]. In quiescent cells, NF-κB inhibiting molecule,IκBα retains the NF-κB1/RelA dimer in the cytoplasm [2]. In stimulatedcells, the phosphorylation of IκBα, by the IκBα kinase (IKK) complex,primes it for K-48-linked poly-ubiquitination and subsequent degrada-tion by the proteasome [9]. The free NF-κB dimer, translocates to the

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istry, Faculty of Science, Thejarat 390002, India. Tel.: +91

TRIM13 regulates ubiquitinat10.1016/j.cellsig.2014.08.008

nucleus and binds to specific sequences (kappa B elements) in promoterregions of responsible genes [3]. The subunits of IKK complex alsoundergo different types of ubiquitination. The K-63 and linearpoly-ubiquitination of NF-κB essential modulator (NEMO) subunit isknown to regulate the assembly of IKK complex and its kinaseactivity [10,11]. The mono-ubiquitination of IKKβ involved in itsautophagic degradation [12]. The process of ubiquitination also playsessential role in the initiation of NF-κB pathway after the binding ofcytokines to their cognate receptor [13]. The binding of TNF to TNF-Rleads to the recruitment of specific ubiquitin ligases at the differenttime point leading to distinct pattern of ubiquitination and discreteoutcome. TRAF proteins (RING E3 Ligase) are recruited to TNF-R1,which may self-ubiquitinate or target other proteins to regulate NF-κBpathways [13]. Similarly, emerging evidences suggest thatubiquitination acts at different levels of NF-κB pathway and may eitherinhibit or activate it [9].

The process of ubiquitination involves sequential action of threeenzymes: E1, E2 and E3, for transferring the ubiquitin to the targetprotein [14]. The terminal enzymeE3, transfersUb fromE2 to a lysine res-idue on a substrate protein, resulting in an iso-peptide bond formationbetween the substrate lysine and the C-terminus glycine of Ub [15]. E3ligases provide specificity to the pathway as they recognize the substrate,interact with definite E2 and determine the topology of ubiquitination[15,16]. Several ubiquitin ligases have been identified that criticallydetermine the pattern of ubiquitination leading to either degradation ofproteins through UPS or regulation of their activity [17,18].

TRIM family proteins (more than 70 proteins) are members of RINGtype ubiquitin E3 ligases and characterized by the presence of N-terminal

ion and turnover of NEMO to suppress TNF induced NF-κB activation,

http://dx.doi.org/10.1016/j.cellsig.2014.08.008

mailto:[email protected]

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RING, B-Box, Coiled Coil (CC) domain and variable C-terminal domain[19]. The role of TRIM family proteins is emerging in several cellularprocesses like innate immune response, cell survival, miRNA biogenesisand stem cellmaintenance [20–22]. TRIM13 is an ER anchored ubiquitinE3 ligase, involved in degradation of ERAD substrates via proteasomeand autophagy pathway [23,24]. TNF is one of the pleotropic cytokinethat regulates NF-κB and caspase-8 in distinct cellular conditions lead-ing to either cell survival or cell death [25]. We previously reportedthat TRIM13 regulates caspase-8 ubiquitination and induces cell deathduring ER stress [26], however role of TRIM13 in regulation of TNFinduced NF-κB and its physiological relevance had not yet beenexplored. Here we described the role of TRIM13 in regulation of TNFinduced NF-κB pathway. TRIM13 modulates the IKK complex byregulating ubiquitination and turnover of NEMO in the presence ofTNF and decreases the clonogenic ability of the cells.

2. Materials and methods

2.1. Cell culture and reagents

The human cell lines HEK293, HeLa, A549 and MCF7 cells weregrown using procedures as described previously [27]. Full lengthTRIM13, GST-TRIM13, Flag-TRIM13, deleted constructs of TRIM13(TRIM13-ΔRING, GST-TRIM13-ΔCC and GST-TRIM13-ΔTM) andTRIM13-shRNA has been described previously [24]. HA-NEMO wasfrom Dr. Gilles Courtois (INSERM, Paris, France). The shRNA for ATG5,Beclin1 and control have been described earlier [26]. The primaryantibodies used were: Anti-HA-peroxidase (Roche, Germany), rabbitpolyclonal against β-Actin and ubiquitin (Abcam, USA), anti-IκBα,IKKα, IKKβ, IKKγ, ATG5 and Beclin1 (Cell Signaling Technology, Inc.,USA), anti-Flag-HRP (Sigma, USA). HRP-conjugated secondary antibod-ies: anti-rabbit and anti-mouse were purchased from Jackson ImmunoResearch Laboratories, Inc., USA. EZview™ Red ANTI-FLAG®M2 AffinityGel, trypan blue, acridine orange, propidium iodide, tunicamycin,wortmannin, pyrrolidine dithiocarbamate (PDTC) and TNFα werepurchased from Sigma-Aldrich, USA, zVAD.fmk from BioVision, USA,and G418 from Gibco, Invitrogen, USA.

2.2. RT-PCR analysis

To check the expression of TRIM13 in knockdown condition and indifferent cell lines RT-PCR was performed. Total RNA was isolatedfrom the cells using TRIzol® Plus RNA purification Kit (Life Technolo-gies, USA) according to the manufacturer's protocol. The first strandcDNA was synthesized using PrimeScript First Strand cDNA SynthesisKit (Clontech, Takara, Japan). The specific primers for TRIM13: forward,5′-CCGGAATTCAACTTCAGCTACTGGAATT-3′ and reverse, 5′-CGCGGATCCTTATAATAGTTTATATTT-3′; and β-Actin: forward, 5′-TCGTGCGTGACATTAAGGGG-3′ and reverse, 5′-GTACTTGCGCTCAGGAGGAG-3′ wasused for PCR reactions. The cycling conditions were 95 °C for 10 min,followed by 30 cycles at 95 °C for 30 s, at 48 °C for 30 s, and at 72 °Cfor 90 s and a final extension at 72 °C for 7 min. The PCR productswere analyzed by 1% agarose gel electrophoresis and imaged by CN-08Infinity gel imaging system (Vilber Lourmat, France).

2.3. NF-κB luciferase assay

Activation of NF-κB pathway was analyzed by dual luciferase assayas described previously [27]. Briefly, HEK293 were plated at a densityof 1.5 × 105 cells per well in a 24well plate. After overnight incubation,the cells were co-transfected with NF-κB firefly luciferase reporterconstruct and indicated expression clone using calcium phosphatetransfection method. Renilla luciferase expressing construct was usedfor normalizing transfection efficiencies. After 24 hours of transfection,the cells were treated with indicated chemical inhibitor or stimulator.The luciferase activity was assessed using Dual-Glo® luciferase assay

Please cite this article as: D. Tomar, R. Singh, TRIM13 regulates ubiquitinatCellular Signalling (2014), http://dx.doi.org/10.1016/j.cellsig.2014.08.008

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systemas per themanufacturer's instructions (Promega, USA) andmea-sured with a Centro LB 960 Luminometer (Berthold Technologies,Germany). All the assays were performed in triplicate and repeatedminimum three times.

2.4. Western blotting and immunoprecipitation

To monitor the protein turnover, HEK293 or MCF7 cells were platedat density of 5 × 105 cells per well in a six well plate and transfectedwith indicated constructs using calciumphosphatemethod as describedpreviously [24]. After 24 h of transfection cells were treated withindicated chemical and after incubation cells were collected in ice coldPBS. The cells were lysed with RIPA buffer (100 mM NaCl, 50 mMTris–HCl, 1% NP40 containing 1 mM PMSF) and protein content ineach sample was quantified using Bradford assay. The equal protein ofeach sample was loaded to SDS-PAGE and resolved proteins weredetected by specific antibodies using western blotting as describedpreviously [26].

To detect the protein–protein interaction, co-immunoprecipitationexperiments were performed. Briefly, HEK293 cells were seeded at adensity of 2 × 106 per 100-mm2-diameter dish and co transfectedwith Flag-TRIM13, HA-NEMO with vector using calcium phosphatetransfection method. After 36 h of transfection, cells were treated withindicated chemical and incubated for 10 h. Cells were harvested,washed with ice cold PBS (Gibco, Invitrogen) and lysed in immunopre-cipitation buffer (100 mMNaCl, 50 mM Tris–HCl, 0.1% NP40 containingcomplete protease inhibitor cocktail (Roche, Germany)). Cell lysateswere incubated with M2 FLAG-Affinity Gel (Sigma, USA) on rollershaker overnight at 4 °C. The gel beads were washed four times withIP buffer, re-suspended in 2× SDS-PAGE sample buffers and separatedon 12% SDS-PAGE. The resolved proteins were analyzed by westernblotting using specific antibodies as described earlier.

2.5. Colony forming assay

The colony forming assay was performed to monitor the clonogenicability of the cells as described previously [24,27]. Briefly, the cells weretransfected with indicated constructs in a 24 well plate by standardCaPO4 transfectionmethod. After 24 h of transfection, cells were count-ed and 3000 cells per 60mm2 dishwere seeded. The cells were culturedfor seven days in standard conditions. After incubation, the cells werefixed with cold methanol, washed with PBS and stained with 0.2%crystal violet. The plates were imaged for colony formation usingCN-08 Infinity gel imaging system (Vilber Lourmat, France). Thenumber of colonies was manually counted and clonogenic capability ispresented as the percentage of plating efficiency. The plating efficiencyis the ratio of the number of colonies to the number of cells seeded andwas calculated as described earlier [24].

2.6. Statistical analysis

The statistical significance between the different test groupswas de-termined by Student's t-test. All the experiments were repeated at leastthree times independently and *P b 0.05, **P b 0.01 and ***P b 0.001were considered as significant for ±SEM.

3. Results

3.1. TRIM13 suppresses TNF induced NF-κB

TNF is one of the important regulators of inflammation and celldeath [28]. RING E3 Ligases mediated ubiquitination plays importantrole in regulation of TNF induced NF-kB activation. The role of TRIM13,RING E3 Ligase, in regulation of TNF induced NF-κB activation is notknown. To elucidate the role of TRIM13 in regulation of NF-κB pathway,HEK293 cells were transfected with TRIM13 and vector, treated with



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TNF andmonitored for NF-κB activation. TheNF-κB activity significantlyincreased in vector transfected cells, whereas the expression of TRIM13significantly repressed the same in the presence of TNF (Fig. 1A). Tofurther confirm, TRIM13 was knocked down using shRNA andmonitored NF-κB activation. Interestingly, knockdown of TRIM13significantly increased TNF induced NF-κB activation (Fig. 1B). DuringNF-κB activation, IκBα protein is phosphorylated and degraded byproteasome leading to nuclear translocation of hetero-dimer of p50and p65. IκBα phoshporylation and protein turnover is also an indicatorof NF-κB activation, hence wemonitored the IκBα turnover by westernblotting after TNF treatment in TRIM13 overexpression and knockdownconditions. The level of 39 kDa protein band corresponding to IκBαdecreased in vector transfected cells in the presence of TNF as comparedto untreated cells (Fig. 1C). The expression of TRIM13 showed stabiliza-tion of IκBα in the presence of TNF as compared to vector transfectedcells (Fig. 1C). Similarly, TRIM13 knockdown showed low level of IκBαas compared to control shRNA transfected cells suggesting high

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Fig. 1. TRIM13 negatively regulates TNF induced NF-κB pathway. (A) TRIM13 suppresses TNF itreated with TNF (20 ng/ml) for 10 h, NF-κB activity was measured by Dual Glo luciferaseHEK293 transfected with shRNA of TRIM13 and control treated with TNF and NF-κB activity moverexpression rescues TNF induced IκBα degradation. TRIM13 was transfected in HEK293 cellantibodies. (D) TRIM13 knockdown enhanced IκBα turnover in response to TNF. HEK293 transdetecting IκBα turnover using western blotting. (E) TRIM13 overexpression suppresses TNF indfirefly luciferase reporter and Renilla luciferase control, treatedwith TNF and NF-κB activitymonsion of TNF induced IκBα degradation inMCF7 cells. MCF7 cellswere transfectedwith TRIM13 awestern blotting. Asterisk (*) indicates that p value b 0.05, for SEM of minimum three indepen


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turnover of IκBα (Fig. 1D). We also validated these observations inother cell lines. TRIM13 overexpression significantly decreased TNFinduced NF-κB activity in HeLa cells (Fig. 1E). The expression ofTRIM13 suppresses IκBα turnover in MCF7 cell line (Fig. 1F). Theseevidences clearly suggests that TRIM13 negatively regulates TNFinduced NF-κB activation.

3.2. The E3 ligase activity and ER localization of TRIM13 is essential forNF-κB regulation whereas autophagy is not essential

TRIM13 is a multi-domain protein (Fig. 2A) and each domain mayhave distinct functions to regulate the assembly of different signalingcomplexes in various cellular processes [24]. To define the domain ofTRIM13 essential for negative regulation of NF-κB, we have transfecteddifferent deletion constructs of TRIM13 in HEK293 cells and monitoredNF-κB activation in the presence of TNF. The deletion or mutation ofRING domain negates TRIM13 mediated suppression of TNF induced

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nduced NF-κB luciferase activity. HEK293 cells were transfected with vector and TRIM13,assay system. (B) TRIM13 knockdown enhances TNF induced NF-κB luciferase activity.onitored as described earlier. TRIM13 knockdown was confirmed by RT-PCR. (C) TRIM13s, treated with TNF and IκBα turnover was monitored by western blotting using indicatedfected with control and TRIM13 shRNA, treated with TNF and NF-κB activity monitored byuced NF-κB in HeLa cells. HeLa cells were transfectedwith TRIM13 and vector with NF-κBitored by Dual Glo Luciferase assay system. (F) TRIM13 overexpression results in suppres-nd vector, treatedwith TNF andNF-κB activitymonitored by detecting IκBα turnover usingdent experiments.


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Fig. 2. E3 Ligase activity and ER localization of TRIM13 is essential for NF-κB suppression. (A) Domain architecture of TRIM13 showing different deletion andmutant constructs of TRIM13used in present study. (B) RING domain of TRIM13 is essential for NF-κB suppression. HEK293 cells were transfectedwith deletion construct of TRIM13, stimulatedwith TNF for 10 h andNF-κB activity was monitored by luciferase assay. (C) Autophagy suppression does not have any effect on TRIM13 mediated negative regulation of NF-κB. TRIM13 and NF-κB luciferasereporter clones were transfected in HEK293 cells, treated with TNF with our without wortmannin and luciferase activity was monitored. (D) Knockdown of autophagy essential genesATG5 and Beclin1 does not have any effect on TRIM13 mediated suppression of NF-κB pathway. HEK293 cells were transfected with ATG5 and Beclin1 shRNA in TRIM13 overexpressioncondition. The cells were treated with TNF and monitored the NF-κB activation using luciferase assay. D′ and D″ shows the knockdown of ATG5 and Beclin1. Asterisk (*) indicates that pvalue b 0.05, for SEM of minimum three independent experiments.


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tion of NF-κB (Fig. 2B). The deletion of TM domain also results in theabolishment of TRIM13 mediated negative regulation of NF-κB(Fig. 2B). This suggests that localization of TRIM13 at ER is essentialfor negative regulation of NF-κB. Interestingly, deletion of CC domainshowed no effect on TRIM13 mediated NF-κB repression (Fig. 2B).

Wehave previously reported that TRIM13 induces autophagy duringER stress, and CC domain is essential for this activity [24]. This stronglysuggests that TRIM13 induced autophagymaynot be essential for NF-κBregulation. To further validate this hypothesis, we examined the role ofautophagy in regulation of TRIM13 mediated NF-κB suppression. Theexpression of TRIM13 negatively regulates TNF induced NF-κB activa-tion even in the presence of wortmannin (inhibitor of autophagy)(Fig. 2C). The effect of autophagy inhibitor wortmannin on IκBαturnover was also monitored by western blotting, which also showsthe same results (data not shown). The knockdown of autophagyessential genes, ATG5 and Beclin-1 do not recover TRIM13 mediatedsuppression of TNF induced NF-κB (Fig. 2D). The ATG5 and Beclin-1knockdown was confirmed by western blotting using their specificantibodies (Fig. 2D′, D″). These results convincingly demonstrate thatE3 ligase activity of TRIM13 is essential for TNF induced NF-κBregulation whereas autophagy is not essential.

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3.3. TRIM13 suppresses NF-κΒ pathway by acting upon IKK complex

To identify the regulatory step of NF-κB pathway targeted byTRIM13, HEK-293 cells were co-transfected with TRIM13 and NF-κB


regulatory genes and its activation was monitored. TRIM13 suppressedcIAP1, RIP1 and IKKβ induced NF-κB activation (Fig. 3A, B, and C)suggesting that TRIM13 acts downstream of these proteins in NF-κBpathway. TRIM13 was not able to suppress p65 mediated NF-κBactivation suggesting that TRIM13 may act upstream of p65 (Fig. 3D).The transfection of TRIM13 and treatment of PDTC, an inhibitor ofIκBα phosphorylation [29], in the presence of TNF, showed furthersuppression of TRIM13mediated NF-κB suppression (Fig. 3E) indicatingthat TRIM13 may act upstream of IκBα.

3.4. TRIM13 modulates IKK complex by regulating NEMO ubiquitination

The results of the previous sections clearly indicate that TRIM13mayact on the IKK complex to negatively regulate NF-κB signaling. TRIM13is an E3 ligase, therefore we hypothesized that TRIM13 may regulatethe ubiquitination of IKK complex, which can further affect the turnoveror assembly of the IKK complex. To validate this hypothesis, firstly thelevels of specific proteins of IKK complex were analyzed in TRIM13overexpression and knockdown conditions by western blotting. Thelevels of 48 kDa band corresponding to NEMO increased in the presenceof MG132 in vector transfected cells. The expression of TRIM13 furtherdecreased the levels of 48 kDa band corresponding to NEMO whichremained low even in the presence of MG132 as compared to vectortransfected cells. This strongly suggests the TRIM13 regulates turnoverof NEMO (Fig. 4A). There was no change in the levels of other proteinof IKK complex (IKKα and IKKβ). The knockdown of TRIM13 showedno affect on the turnover of NEMO (Fig. 4B).


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Fig. 3. TRIM13 suppresses NF-κΒ downstream of IKK complex and upstream of p65. HEK293 cells were co-transfectedwith TRIM13, vector and (A) cIAP1, (B) RIP1, (C) IKKβ, and (D) p65.NF-κB activity was monitored by luciferase assay as described earlier. Data represent mean SEM (error bars) and asterisk (*) indicates that p value b 0.05, and ns shows data is not sig-nificant for SEM of minimum three independent experiments. (E) PDTC further suppress TRIM13 mediated NF-κB suppression. HEK293 cells were transfected with TRIM13 treatedwith TNFwith orwithout PDTC (100 μM) for NF-κB activity monitored by luciferase assay. Asterisk (*) indicates that p value b 0.05, for SEM of minimum three independent experiments.


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NEMO is regulatory as well as essential subunit of IKK complex thatregulates the assembly and kinase activity of the complex. The previousstudies suggest that NEMO is ubiquitinated by different Ub E3 ligasesthrough discrete lysine linkages leading to different fates [10]. Tofurther elucidate the molecular mechanism of TRIM13 mediatedNEMO turnover, TRIM13 and NEMO interaction and its ubiquitinationwas monitored. HEK293 cells were co-transfected with Flag-TRIM13and HA-NEMO, treated with TNF to activate NF-κB in the presence ofNH4Cl and MG132 to block the autophagic and proteasomal proteindegradation. The pull down showed that the band of 48 kDacorresponding to NEMO in TRIM13 transfected cells and was notdetected in vector transfected cells. The levels of 48 kDa protein bandcorresponding to NEMO increased in the presence of TNF and furtherincreased when both autophagy and proteasomal protein degradationpathwayswere blocked (Fig. 4C). This evidence suggests the interactionof TRIM13 and NEMO in the presence of TNF and its increased turnoverin the presence of TNF.

To further characterize the domain of TRIM13 mediating theinteraction with NEMO, co-immunoprecipitation was performed usingdifferent domain deletion constructs of TRIM13. The deletion of RINGdomain increased the interaction of TRIM13 and NEMO as comparedto full length TRIM13, as the level of 48 kDa protein band correspondingto NEMO was increased in immunoprecipitation of TRIM13-ΔRING(Fig. 4D). The deletion of both RING and B-Box abolished the interactionof TRIM13 and NEMO as no band at 48 kDa was observed in this pulldown assay (Fig. 4D). This strongly suggests that B Boxmay be essentialfor interaction with NEMO and IKK complex.

The E3 ligase activity of TRIM13 may be essential for NEMOubiquitination for repression of NF-κB in the presence of TNF. Toelucidate the role of TRIM13 in ubiquitination of NEMO, the immuno-precipitation of NEMO in overexpression/knockdown conditions ofTRIM13 was performed and ubiquitination was monitored by westernblotting. TRIM13 overexpression increased ubiquitination of NEMO(Fig. 4E), whereas the knockdown showed the opposite effect


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(Fig. 4F). These results clearly indicate that TRIM13 interacts andubiquitinates NEMO to regulate NF-κB pathway.

3.5. TRIM13 mediated negative regulation of NF-κB involved in regulationclonogenic ability of the cells

The evidences in the current study showed that TRIM13 acts as anegative regulator of NF-κB pathway. We have previously reportedthat TRIM13 acts as a novel tumor suppressor by regulating autophagydynamics, caspase-8 activation and cell death during ER stress [26].The activation of NF-κB is one of the major pathways activated inmany tumors that provide survival advantage to the tumor cells.Hence, we analyzed the role of TRIM13 mediated repression of NF-κBin regulation of clonogenic ability of the cells. As observed above theRING domain is essential for negative regulation of NF-κB activity,therefore, we checked the effect of deletion on clonogenic ability ofthe cells. The transfection of FL-TRIM13 in MCF-7 decreased theclonogenic ability of the cells as observed earlier [24]. The deletion ofRING domain significantly increased the clonogenic ability of the cellsand plating efficiency, compared to full length TRIM13 and vector(Fig. 5A, B). As we observed above, RING domain is essential for NF-κBregulation, hence it may have regulatory role in clonogenic ability ofthe cells. To further confirm this hypothesis, we co-transfected fulllength TRIM13 with p65-GFP and clonogenic ability was monitored.The overexpression of p65 significantly increased the plating efficiencyof TRIM13 transfected cells as compared to control (Fig. 5C). Theseobservations clearly suggest that TRIM13 mediated NF-κB suppressionis essential for suppression of clonogenic ability of the tumor cells.

4. Discussion

TNF is one of the pleotropic cytokine that activates NF-κB leading tosecretion ofmany trophic factors involved in different physiological andpathological conditions. The regulation of TNF induced NF-κB pathway


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Fig. 4. TRIM13 interacts with NEMO and regulates its ubiquitination. (A) TRIM13 overexpression increases NEMO turnover. HEK293 cells were transfected with TRIM13, treated withMG132 to inhibit proteasomal degradation and IKK component proteins turnover was monitored using western blotting. (B) TRIM13 knockdown showed no effect on NEMO turnover.HEK293 cells was transfected with control-shRNA and TRIM13-shRNA, treated with MG132 and IKK complex proteins turnover wasmonitored using western blotting. (C) TRIM13 inter-acts with NEMO. HEK-293 cells were co-transfected with HA-NEMO and Flag-TRIM13, TRIM13 immunoprecipitation was performed using Flag antibody, analyzed by western blottingusing specific antibodies. (D) B-Box domain of TRIM13 is essential for its interaction with NEMO. The Flag-tagged different deletion constructs of TRIM13 were co-transfected with HA-NEMO in HEK293 cells and immunoprecipitation and western blotting was performed as described above. (E) TRIM13 overexpression increased ubiquitination of NEMO. HEK-293cells were transfectedwith HA-NEMO and TRIM13. HA-immunoprecipitationwas performed and analyzed using specific antibody bywestern blotting. (F) TRIM13 knockdown decreasedNEMO ubiquitination. Immunoprecipitation and western blotting was performed as described above after co-transfection of HA-NEMO and TRIM13-shRNA in HEK293 cells.


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generating different outcomes in different conditions is one of themajorfocus of recent studies. The post-translational modification of targetproteins by E3 ubiquitin ligases plays an important role in regulationof NF-κB pathway. Here we provided several evidences that TRIM13,RING E3 ligase, negatively regulates TNF induced NF-κB pathway bymodulating NEMO, subunit of IKK complex through ubiquitinationnegatively regulates clonogenic ability of the cells.

The emerging evidences suggest that ER and mitochondria providenovel platform for assembly of signaling complexes to regulate cellularprocesses including inflammation and cell death. Assembly of MAVSsignaling complex and its interaction with upstream regulators on themitochondria associated membrane is an interesting observation [30].This suggests the dynamic interaction of sub-cellular organelles includ-ing mitochondria, ER, lysosome and plasma membrane [30]. Similarly,MITA, ER localized protein also develops signaling complex on ERduring viral infection to mount innate immune response [31]. In thecurrent study we provided several evidences that TRIM13, ER resident


ubiquitin ligase negatively regulates TNF induced NF-κB pathway. Theobservation is not specific and seems to be a general phenomenon inseveral human cell lines. TRIM13 is a multi domain protein and theevidences in the current study again showed that each domain mayhave discrete function in regulation of NF-κB pathway. The evidenceshere suggest that RING domain having ubiquitin ligase activity, isessential for NF-κB activation. The CC domain of TRIM family proteinsare known to form higher order molecular structures [32], regulateautophagy induction and flux [24]. This domain is not essential forNF-κB activation suggesting that TRIM13 induced autophagy wasinvolved in regulation of NF-κB. This hypothesis was further validatedby using ATG5 and Beclin-1 knockdown studies as well as the use ofautophagy inhibitor. TRIM13 is anchored to ERmembrane by its TMdo-main and its deletion results in abolishment of TRIM13mediated NF-κBsuppression. This suggests that ER may also provide a surface to assem-ble novel signalosomes that may regulate the NF-κB pathway furthersupporting the emerging hypothesis that sub-cellular organelles


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Fig. 5. TRIM13 mediated NF-κB suppression regulates clonogenic ability of the cells.(A) RING and CC domain is essential for clonogenic ability suppression. MCF7 cells weretransfected with full length, ΔRING, ΔCC constructs of TRIM13. After 24 h of transfection,3000 cells were plated in 60mm2 dishes. Cells were incubated for 7 days and stainedwith0.2% crystal violet. (B) RING andCCdomain is essential for suppression for plating efficien-cy. Graphical representation for plating efficiency of Fig. 5A. (C) p65 overexpressionabolished TRIM13 mediated suppression of plating efficiency. Graphical representationfor plating efficiency of MCF7 cells co-transfected with p65 and TRIM13. Asterisk (*)indicates that p value b 0.05, for SEM of minimum three independent experiments.


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specifically ER andmitochondria are novel platform for dynamic proteincomplexes. These evidences collectively suggest that TRIM13 at ERmayassemble signalosome in the presence of TNF which may sequestercomponents of NF-κB pathway at ER may regulate its turnover via itsE3 ligase activity to suppress NF-κB.

The evidences using different luciferase assay and western blottingfor IκBα in this study strongly suggest that TRIM13mediated repressionmay act at the level of IKK complex. It is now established that IKKcomplex (~700 kDa complex) contains two catalytic proteins called asIKKα and IKKβ and a structural subunit called asNEMO [33]. The severalstudies showed that NEMO is absolutely essential for the assembly ofthe IKK complex and its further catalytic activity [33–35]. Theubiquitination of NEMO by different ubiquitin ligases leads to differentpattern of ubiquitination and different fate of the complex [10,11,36,37].

The deletion of RING domain results in stabilization of TRIM13 andNEMO interaction. This strongly suggests that in normal conditionsthe turnover of NEMO is regulated through TRIM13 mediatedubiquitination and degradation through proteasome. This is furtherconfirmed as the deletion of the B-Box also leads to further stabilizationof the interaction. B-Box has been shown to be essential for the higherorder structure and complex formation [38,39], hence may be essentialfor the interaction. Interestingly, it had been reported that B-Box of


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TRIM family proteins folds in RING domain like structure andco-ordinates to Zn atoms. Interestingly, it had been observed inTRIM16 and other TRIM proteins may act as E3 Ligase domain indepen-dent of RING domain [38]. The results here clearly suggest that B-Boxand RING domain may act synergistically and regulate the levels ofNEMO and possible hitherto unknown proteins of NF-κB pathway. Itwill be interesting to study E3 ligase activity of the role of B-Box ofTRIM13 and its possibility to ubiquitinate target proteins in the inflam-matory conditions. It is possible that TRIM13 interaction with NEMO,sequestration at ER and its turnover may compromise the IKK kinaseassembly and activity thus negatively regulates NF-κB activity. This isinteresting hypothesis and needs experimental validation.

The negative regulation of NF-κB by TRIM13 in the presence of TNF,suggest that TRIM13 may sensitize to TNF/ER stress induced cell deathpathways. NF-κB is known to regulate the expression of severalanti-apoptotic proteins [1,2] as well as act synergistically with othertranscription factor to regulate the process of apoptosis [40]. Theprevious report on TRIM13 mediated regulation of cell death [26]during stress conditions further corroborates this hypothesis. Thecolony forming assay clearly suggests that, TRIM13 overexpressionleads to suppression of colony forming ability [24]. This suppression ofcolony forming abilitymay be a cumulative effect of NF-κB suppression,autophagy and cell death induction. These observations clearly suggestthat TRIM13 acts tumor suppressor by regulating multiple cellularsignaling events. These observations should be further validated indifferent animal models for tumorigenesis to assign tumor suppressoractivity to TRIM13. The autophagy, NF-κB and cell death also playcritical role in the pathogenesis of number of diseases. The role ofTRIM13 in the other pathological conditions like neurodegeneration,infection and inflammation, aging, metabolic disorders needs to befurther studied.

5. Conclusion

RING E3 ligases play an essential role in regulation of NF-κB pathwayat different steps. The evidences in the current study shows that ERanchored RING E3 ligase TRIM13, negatively regulates NF-κB activation.TRIM13 acts at the IKK complex by regulating NEMO ubiquitination andturnover of NEMO. TRIM13 mediated suppression of NF-κB regulateclonogenic ability of the cancer cell, hence may act as potential tumorsuppressor.

Conflicts of interest

The authors declare there is no conflict of interest.

Acknowledgment

The current research work was financially supported by the Depart-ment of Biotechnology, Government of India (grant number BT/PR13924/BRB/10/794/2010 to Rajesh Singh). This work constitutes thePh.D. thesis of Dhanendra Tomar. Authors acknowledge the researchfellowships from Council of Scientific and Industrial Research (CSIR),Government of India to Dhanendra Tomar.

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TRIM13 regulates ubiquitination and turnover of NEMO to suppress TNF induced NF-κB activation