TRAF6 is a novel regulator of Notch signaling in Drosophila melanogaster

Cellular Signalling 26 (2014) 3016–3026

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TRAF6 is a novel regulator of Notch signaling in Drosophila melanogaster Abhinava K. Mishra, Nalani Sachan 1, Mousumi Mutsuddi, Ashim Mukherjee ⁎ Department of Molecular and Human Genetics, Banaras Hindu University, Varanasi 221 005, India

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Article history: Received 18 August 2014 Received in revised form 25 September 2014 Accepted 25 September 2014 Available online 30 September 2014 Keywords: Drosophila TRAF6 Notch Deltex Kurtz

a b s t r a c t Notch signaling pathway unravels a fundamental cellular communication system that plays an elemental role in development. It is evident from different studies that the outcome of Notch signaling depends on signal strength, timing, cell type, and cellular context. Since Notch signaling affects a spectrum of cellular activity at various developmental stages by reorganizing itself in more than one way to produce different intensities in the signaling output, it is important to understand the context dependent complexity of Notch signaling and different routes of its regulation. We identified, TRAF6 (Drosophila homolog of mammalian TRAF6) as an interacting partner of Notch intracellular domain (Notch-ICD). TRAF6 genetically interacts with Notch pathway components in trans-heterozygous combinations. Immunocytochemical analysis shows that TRAF6 co-localizes with Notch in Drosophila third instar larval tissues. Our genetic interaction data suggests that the loss-of-function of TRAF6 leads to the rescue of previously identified Kurtz–Deltex mediated wing notching phenotype and enhances Notch protein survival. Co-expression of TRAF6 and Deltex results in depletion of Notch in the larval wing discs and down-regulates Notch targets, Wingless and Cut. Taken together, our results suggest that TRAF6 may function as a negative regulator of Notch signaling. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Notch pathway is an evolutionarily conserved signaling system that plays a major role in modulating cell fate decisions throughout metazoan development. Notch function is highly pleiotropic in nature that includes its involvement in differentiation, proliferation, apoptosis and self-renewal processes of different tissues [1–5]. Pleiotropic actions of Notch in different cell types come from its multiple levels of regulation. Since a large number of cellular processes are dependent on Notch signaling, it is not surprising that aberrant Notch signaling is associated with several human diseases including carcinogenesis. Notch is synthesized as a 300 kDa precursor protein which is first cleaved by furin-like convertases in the trans-Golgi compartment resulting in an N-terminal extracellular subunit and a C-terminal trans-membrane intracellular subunit [6]. This heterodimeric receptor is then transferred to the cell membrane where it is activated by ligands of the DSL family (Delta and Serrate/Jagged in Drosophila and mammals and LAG-2 in Caenorhabditis elegans). Binding of ligands to extracellular domain initiates proteolytic cleavages by metalloproteases and γ-secretase resulting in the release of Notch-ICD [7–10]. The Notch-ICD binds to Importin-α3

Abbreviations: TRAF6, Tumor necrosis factor Receptor Associated Factor 6; ICD, Intracellular domain; Dx, Deltex; Krz, Kurtz; en, Engrailed; Sgs3, Salivary gland secretion 3; GMR, Glass Multimer Reporter. ⁎ Corresponding author. Tel.: +91 542 6702490; fax: +91 542 2368457. E-mail address: [email protected] (A. Mukherjee). 1 Present address: The Stowers Institute for Medical Research, Kansas City, Missouri 64110, USA.

http://dx.doi.org/10.1016/j.cellsig.2014.09.016 0898-6568/© 2014 Elsevier Inc. All rights reserved.

and translocates to the nucleus using the canonical Importin-α3/ Importin-β transport pathway [11]. After translocation to the nucleus, the Notch-ICD directly binds to CSL transcription factor (mammalian CBF1/Drosophila Suppressor of Hairless/C. elegans LAG-1) and recruits transcriptional co-activators like mastermind (Mam) leading to the activation of Notch targets such as the Enhancer of Split (E(spl)) complex genes. E(spl) complex genes encode basic helix–loop–helix (bHLH) transcription factors that in turn repress achaete–scute complex (As-C) proneural genes [12,13]. The tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adaptor protein, which transduces the signal from TNFRs and Toll-like receptor/interleukin-1 receptor (TLR/IL-1R) superfamily to induce a wide spectrum of cellular responses [14]. In humans and mice, six members of TRAF family proteins, TRAF1 to TRAF6, are known whereas in Drosophila there are only two well-characterized TRAF proteins, TRAF4 and TRAF6 along with a relatively less characterized TRAF-like/TRAF3 [15–17]. TRAF proteins have a conserved TRAF domain at their C-termini and most TRAF proteins contain several zinc-finger motifs and a RING finger domain at the N-terminus [15]. Among these Drosophila homologs of mammalian TRAFs, the TRAF domain of TRAF6 is most closely related to that of mammalian TRAF6 [15]. Drosophila TRAF6 interacts with ECSIT and induces an antimicrobial response [18]. There have been several reports delineating TRAF6 contribution to dorsal activation through its physical association with Pelle [19] and immune responses by activating NF-κB and its downstream target genes diptericin, dptlp and drosomycin in vivo [20]. It has also been reported in some studies that TRAF6, but not TRAF4, acts as the adaptor protein that mediates Eiger/JNK signaling in

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Drosophila and TRAF6, together with dCYLD, is required for endogenous JNK signaling [21]. Several lines of evidences prove that mammalian TRAF6 interacts with known components of Notch pathway such as Presinilin1 [22, 23], RUNX1 [24], β-arrestin [25] and NUMBL [26]. These observations suggest that it can serve as a potent modulator in Notch signaling pathway. Since no study till date explains precise involvement of TRAF6 in Notch signaling, we have carried out experiments to propose a novel mechanism of regulation of Notch signaling involving Drosophila TRAF6. Using molecular and genetic analyses, we found that TRAF6, together with Deltex (Dx), acts as a negative regulator of Notch signaling. 2. Materials and methods 2.1. Yeast two-hybrid A 393 bp Drosophila Notch cDNA (accession number M11664) fragment, which encodes amino acids 1765–1895, was amplified by polymerase chain reaction (PCR). It was further cloned in frame with the sequence encoding LexA DNA-binding domain of bait vector. This construct was used as bait to screen oligo(dT)-primed Drosophila melanogaster 0–24 h embryo cDNA libraries cloned in pGAD prey vectors that contained GAL4 activation domains. A yeast two-hybrid screen was carried out as described previously [27]. All positive pGAD plasmids from His+ colonies were isolated and sequenced to identify interactors. 2.2. GST-pull down, co-immunoprecipitation and immunoblotting Full length (1–1428 bp), amino-terminus (1–441 bp) and carboxyterminus (604–1428 bp) Drosophila TRAF6 cDNA (CG10961 and GenBank accession number AE119793) were amplified by PCR and cloned into pGEX-4T-1 vector (Amersham) using EcoRI and NotI restriction sites. Sequence verified constructs were then transformed into E. coli BL21 (GE Healthcare). Single colonies from all three sets of transformants were freshly inoculated in 5 ml Luria Broth (HiMedia Laboratories) with 100 μg/ml ampicillin (Sigma) at 37 °C in a shaking incubator. For large-scale induction of GST-fusion proteins, 1–2% primary inoculum was added to 100 ml Luria Broth (HiMedia Laboratories) with 100 μg/ml ampicillin (Sigma) at 37 °C in a shaking incubator and 2 mM IPTG (Fermentas) was added when the O.D.600 of the culture reached 1.00. Lysate fraction was collected after 3 h of induction in a buffer containing CelLytic Express tablets (Sigma) and 1× complete Protease Inhibitor (Roche). To prepare crude lysate containing Notch-ICD protein, UAS-NotchICD flies were crossed with P{Sgs3-GAL4.PD}TP1 line and third instar larval salivary glands were lysed in a buffer containing 50 mM Tris, 1 mM PMSF, 200 mM NaCl, 2 mM MgCl2, 200 μg/ml lysozyme, 10% Glycerol and 0.1% Triton-X-100. Glutathione Sepharose (GE Healthcare) beads were washed in cold phosphate buffered saline (PBS), three times 30 min each and 50% slurry was made in PBS. Bacterial lysate containing GST alone and GST-fusion proteins of Drosophila TRAF6 (Full length, N-terminus and C-terminus) and the crude lysate containing Notch-ICD protein were mixed in 50% slurry of Glutathione-Sepharose 4B Matrix with end-over-end rotation for 3 h at 4 °C. Beads were collected after washing three times with Tri-PBS (1×-PBS and 1% Triton-X-100) and boiled with 2×-SDS sample buffer for 5 min before separating on 12% denaturing SDS polyacrylamide gel. Spectra multicolor broad range protein ladder (Fermentas) was used as a marker. Gel was transferred onto Immun-Blot PVDF membranes (Bio-Rad). After blocking in 4% skimmed milk in TBST (50 mM Tris, pH 7.5, 150 mM Nacl, 0.1% Tween-20) for 30 min, blots were then probed with mouse anti-Notch antibody (C17.9C6, Developmental Studies Hybridoma Bank) in 1:3000 dilution for 1 h followed by three washes in TBST buffer and another blocking for 30 min. Goat antimouse IgG-AP conjugate in 1:2000 dilution (Molecular Probes) in

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blocking solution was added then for 90 min. After washing in TBST thrice, color was detected by Sigma FAST BCIP/NBT (Sigma). For co-immunoprecipitation of Notch-ICD and TRAF6, w; UAS-HATRAF6/CyO-GFP; UAS-Notch-ICD flies were crossed with P{Sgs3GAL4.PD}TP1 line and third instar larval salivary glands were lysed in a buffer containing 50 mM NaCl, 50 mM KCl, 25 mM Tris, 25 mM Sucrose, 10 mM EDTA, 10 mM EGTA, 1 mM DTT, 1 mM PMSF, 10% Glycerol, 0.5% NP-40 and 1× Protease Inhibitor to prepare crude lysate of HA-tagged TRAF6 with Notch-ICD proteins. The lysate containing 1 mg of total protein was added to EZview Red Anti-HA Affinity Gel (Sigma) in an end-over-end rotator for 3 h at 4 °C. Beads were collected after washing three times with immunoprecipitation buffer, boiled with 2×-SDS sample buffer for 5 min, separated on 12% denaturing SDS polyacrylamide gel and transferred onto Immun-Blot PVDF membranes (Bio-Rad). After blocking for 30 min, blots were probed with mouse anti-Notch antibody (C17.9C6, 1:3000 dilution, Developmental Studies Hybridoma Bank), Rabbit monoclonal anti-HA antibody (1:2000, Sigma) or betatubulin (E7, 1:500, Developmental Studies Hybridoma Bank). Again after washing three times in TBST and another blocking for 30 min, goat anti-mouse IgG-AP conjugate or anti-rabbit IgG-AP conjugate in 1:2000 dilution (Molecular Probes) in blocking solution was added for 90 min followed by three washings in TBST. Color was detected by Sigma FAST BCIP/NBT (Sigma). 2.3. Drosophila genetics All stocks were maintained on standard cornmeal/yeast/ molasses/ agar medium at 25 °C. UAS-FLAG-Dx, C-96GAL4UAS-HA-Krz, UASNotch-ICD, UAS-Notch-Full Length and Notch pathway alleles N1/FM7, N54l9/FM7c, dx were kindly provided by Spyros Artavanis Tsakonas. UAS-FLAG-TRAF6, UAS-TRAF6-IR and TRAF2ex1/FM7 lines were gifted by Tian Xu and hsp70-flp; Act FRT y + FRT GAL4, UAS-GFP/CyO; H2YFP/ TM2 flies were obtained by Enrique Martín-Blanco [28]. GAL4 lines enGAL4, C96-GAL4, Sgs3-GAL4, A9-GAL4 and GMR-GAL4 were from Bloomington Stock Centre. For generation of the P(UAS-HA-TRAF6) strain, a full-length TRAF6 cDNA with HA tag at the amino-terminus was cloned in the pUAST vector in KpnI and XbaI restriction sites. Intact reading frames for all constructs were verified by DNA sequence analysis. This construct was introduced into w1118 embryos by germline transformation according to the standard procedures. After microinjection, multiple independent insertions were obtained for each chromosome. Transgenic lines were individually balanced and checked for the ability to overexpress HATRAF6 by crossing these lines to en-GAL4 flies followed by anti-HA staining. To co-express TRAF6 with Notch-ICD, TRAF6 with Notch-Full length and TRAF6 with Deltex respectively, w; UAS-HA-TRAF6/CyO-GFP; UAS-Notch-ICD, another line UAS-HA-TRAF6; UAS-Notch-Full length and UAS-HA-TRAF6; UAS-FLAG-Dx stocks were generated by appropriate genetic crosses. For clonal overexpression of UAS construct, we used a combination of the FLP/FRT system and the GAL4/UAS system [29] by using an yw hsp70-flp; Act FRT y + FRT-GAL4 UAS-GFP strain and crossed it with UAS-HA-TRAF6 line. Heat shock was given 30 h after egg laying at 37 °C for 60 min. 2.4. Immunostaining of imaginal discs Imaginal discs were dissected from third instar larvae in phosphatebuffered saline (PBS) and kept on ice until fixation. A 1:1 mixture of 3% paraformaldehyde in PBS and heptane was added at room temperature. After 1 min, the paraformaldehyde and heptane mixture was replaced by 3% paraformaldehyde and 5% DMSO and incubated for 20 min. Discs were then washed four times in Tri-PBS (a mixture of 1×-PBS and 0.2% Triton-X-100) containing 0.1% bovine serum albumin (BSA) for 10 min each, followed by blocking for 30 min in Tri-PBS with 0.1% BSA and 8% normal goat serum. Primary antibodies, mouse anti-Notch

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(C17.9C6) at 1:300, mouse anti-Wg (4D4) and mouse anti-Cut (2B10) at 1:100 (all three from Developmental Studies Hybridoma Bank), rabbit anti-HA at 1:200 (Sigma), mouse anti-HA at 1:200 (Sigma) or rabbit anti-FLAG at 1:100 (Sigma) were diluted in blocking solution, added to the discs and incubated overnight at 4 °C. After four washes in Tri-PBS with 0.1% BSA and 30 min blocking, discs were incubated with goat anti-rabbit IgG antibodies conjugated to FITC at 1:100 dilution (Jackson ImmunoResearch Laboratories) or goat anti-mouse IgG conjugated to alexa fluor 555 at a dilution of 1:200 (Molecular Probes) for 90 min at room temperature, followed by four washings in Tri-PBS containing 0.1% BSA. Discs were then mounted in Vectashield mounting media with DAPI (Vector Lab) and images were obtained with a Carl Zeiss LSM510 confocal microscope. A detergent free staining was performed to monitor the status of extracellular domain of Notch using mouse anti-Notch extracellular domain specific antibody (C458.2H, at 1:100 dilution, Developmental Studies Hybridoma Bank) with the above mentioned protocol except using Triton-X-100. 2.5. RT-PCR Total RNA was isolated from imaginal discs and other tissues (brain, eye-antennal, wing, leg discs, fat body, salivary glands and gut) of the third instar larvae using RNAquous-4 PCR kit (Ambion Inc.) as per the manufacturer's instructions and quantified spectrophotometrically. Equal amount of RNA samples (1 μg) was incubated with TURBO DNA-free DNase (Ambion Inc.) for 30 min at 37 °C to remove any residual DNA. First-strand cDNA was synthesized using ProtoScript M-MuLV Taq RT-PCR Kit (New England Biolabs) as per manufacturer's protocol. One-tenth volume of reaction mixture was subjected to PCR using primers specific for TRAF6 (forward primer — 5′-ATGCAGCGAGTCCA GGCC-3′ and reverse primer — 5′-CGATGCAAATGGCGCACT-3′). rps17 primers (forward primer — 5′-AAGCGCATCTGCGAGGAG-3′ and reverse primer 5′-CCTCCTCCTGCAACTTGATG-3′) were used as internal controls. PCR products were electrophoresed on 1.5% agarose gel. 2.6. Fluorescence in situ hybridization For generation of riboprobes, a unique fragment of TRAF6 (1-325 bp) was cloned in pGEM-3Z vector (Promega) and sense and anti-sense probes were generated utilizing the SP6 and T7 RNA Polymerases respectively. For in situ hybridization, third instar larval tissues were dissected in cold PBS and fixed with 4% paraformaldehyde for 20 min on ice followed by another fixation with 4% paraformaldehyde and 0.6% Triton-X-100 for 20 min at room temperature. Tissues were washed in PBT (1×-PBS with 0.1% Tween-20) twice for 5 min and once with PBT containing 0.1% active DEPC (Di Ethyl Pyro Carbonate) for 1 min. After another wash in PBT for 5 min, digestion was done with 10 μg/ml Proteinase K (prepared in PBT) for 2–3 min at room temperature. Tissues were again washed twice in chilled Glycine (2 mg/ml in PBT), thrice in PBT for 5 min and conditioned with 1:1 solution of PBT and Hyb B (50% de-ionized formamide and 5× saline-sodium citrate buffer, pH 5.0) for 10 min at room temperature before pre-hybridization in Hyb A (5× saline-sodium citrate buffer-pH 5.0, 10 μg/ml yeast tRNA, 100 μg/ml sheared salmon sperm DNA, 50 μg/ml heparin, 0.1% Tween20 in DEPC water) for 12 h at 62 °C. Hybridization with riboprobe in Hyb A was carried out at the same temperature for 24 h. After hybridization, four times washing, 15 min each, in Hyb B was done at the same hybridization temperature prior to three washings, 10 min each at room temperature, in a 4:1, 1:1, 1:4 solutions of Hyb B and PBT respectively. Again after five washes for 5 min in PBT, tissues were incubated with rhodamine labeled anti-dig antibody (1:500) for 3 h at room temperature. Further after five washes in PBT and twice in PBS, tissues were dissected and mounted in DABCO. Images were obtained with a Carl Zeiss LSM510 confocal microscope.

3. Results 3.1. Identification of TRAF6 as a Notch interactor We identified TRAF6 as an interacting partner of Notch receptor using yeast two-hybrid screen. In the same screen, multiple positive clones of an established binding partner of Notch-ICD, Suppressor of Hairless, were also identified which validates our approach. The yeast two-hybrid screen of 6 × 106 cDNAs from a Drosophila 0–24 h embryonic library was carried out using amino terminus of Notch intracellular domain (amino acids 1765–1895) as bait. Eight positive clones (His+) were isolated and found to encode overlapping TRAF6 cDNAs. Sequence analysis revealed that carboxy-terminal part of TRAF6 (amino acids 224 to 475) was sufficient for binding Notch (Fig. 1A). GST-pull down and co-immunoprecipitation experiments confirmed the interaction between TRAF6 and Notch. Different GSTTRAF6 fusion proteins (full-length 1–475, amino terminus 1–147 and carboxy terminus 202–475) were expressed in bacteria and incubated in Glutathione Sepharose beads along with extracts from third instar larval salivary glands in which Notch-ICD was overexpressed using Sgs3-GAL4 driver. Deletion analysis of TRAF6 showed that TRAF-C/ MATH domain (carboxy-terminus receptor binding domain) was required to interact with Notch-ICD (Fig. 1B). Furthermore, coimmunoprecipitation experiment with HA-tagged TRAF6 revealed that HA-TRAF6 pulled down Notch-ICD when co-expressed in larval salivary glands (Fig. 1C). We also investigated the sub-cellular localization of these proteins when UAS-HA-TRAF6 and UAS-Notch-FL (Notch full length) were coexpressed in larval wing discs and eye-antennal discs using C96-GAL4 and GMR-GAL4 drivers respectively. Immunocytochemical analysis revealed that TRAF6 and Notch proteins indeed co-localized in the same sub-cellular compartment (Fig. 1D1–G4). Moreover, to study the gain-of-function effect of TRAF6 on Notch, we generated gain-offunction clones of TRAF6 to overexpress TRAF6 clonally in an otherwise wild type background, using the flip-out technique, in third instar larval wing discs and carried out a detergent free staining to monitor extracellular domain of Notch. We noticed that localization of extracellular domain of Notch was altered in the clonal area in sub-apical region as compared to the cells with endogenous level of TRAF6. This suggests that TRAF6 may revamp localization of Notch, however, whether it also has a role in Notch cleavage remains to be seen (Fig. 1H1–H4). 3.2. Expression of TRAF6 transcripts in Drosophila third instar larval tissues To determine the expression of TRAF6 transcripts specifically, we investigated spatial localization of TRAF6 transcripts in third instar larval tissues using in situ hybridization and RT-PCR. Fluorescence in situ hybridization (FISH) demonstrated distinctive expression pattern of TRAF6 transcripts in larval tissues. TRAF6 transcripts expressed in all third instar larval discs and tissues and its localization was preferentially cytoplasmic. In wing discs, it had a moderately high expression in notum region (Fig. 2A4). Eye-antennal discs showed the expression of transcripts in photoreceptor neurons and TRAF6 expression was slightly higher in antennal region (Fig. 2B4). Semi-quantitative RT-PCR corroborated FISH expression data (Fig. 2C). 3.3. TRAF6 genetically interacts with Notch pathway components To address the functional implications of physical association between the Notch and TRAF6 proteins, we examined whether mutations in TRAF6 and Notch pathway components display genetic interactions in trans-heterozygous combinations. We used a homozygous viable, lossof-function TRAF6 allele, TRAF2ex1. A trans-heterozygous combination of Notch null allele, N1 or N54l9 and loss-of-function allele of TRAF6 (TRAF2ex1) resulted in rescue of wing nicking phenotype, indicating an increase of Notch function (n = 317/414 and 241/367 for N1 and N54l9

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Fig. 2. Expression of TRAF6 transcripts in Drosophila third instar larval tissues. (A1–B5) Fluorescence in situ hybridization (FISH) unveiled distinct expression pattern of TRAF6 transcripts in larval tissues. SG: salivary gland, OL: optic lobe, FB: fat body, VG: ventral ganglion, Eye-ant.: eye antennal disc. Scale bars, 50 μM, salivary gland (high magnification B1), 10 μM. (C) RT-PCR augments the FISH data. rps17 served as an internal control.

respectively, Fig. 3A2, B2). Single copy deletion of deltex (dx), which is a cytoplasmic modulator of Notch activity, resulted in a normal wing whereas we noticed that dx, in trans-heterozygous combination with TRAF6 allele, showed a wing phenotype that mimicked exactly the phenotype of a dx null mutant (n = 224/286, Fig. 3C2). Larval wing discs from this trans-heterozygous combination also displayed reduced Cut expression (a downstream target of Notch) at the dorso-ventral (DV) wing margin as reported earlier in wing discs from dx null mutant [30] (Fig. 3E4). These genetic interactions with TRAF6 at multiple levels in Notch pathway establish a functional relationship between TRAF6 and Notch consistent with their molecular interaction. 3.4. TRAF6 interacts genetically with Krz-Dx-Notch trimeric complex and alters Notch degradation Earlier Kurtz (Krz), the single Drosophila homolog of mammalian non-visual β-arrestins, was identified as an interacting partner of Dx, and the existence of a trimeric Notch–Dx–Krz protein complex was demonstrated [27]. This complex mediates the degradation of the Notch receptor through a ubiquitination-dependent pathway [27]. In an independent study, it was shown that mammalian TRAF6 physically

associates with β-arrestins 1 and 2 and this interaction negatively regulates the Toll-like Receptor (TLR)-interleukin-1 receptor (IL-1R) signaling and subsequent activation of NF-kB signaling [25]. We have also observed a distinct co-localization of FLAG-TRAF6 and HA-Krz when they were co-expressed at the wing margin in the third instar larval wing disc (Fig. 4G1–G3). Since we observed a genetic interaction between TRAF6 and dx, we asked whether the gain-of-function and loss-of-function of TRAF6 have a modifying effect on Krz-Dx mediated wing-nicking phenotype under the control of the C96-GAL4 driver in adult fly as well as on the vesicular status of Notch. Ectopic expression of TRAF6 in cells of the wing margin did not alter the Krz-Dx mediated nicking phenotype in the adult wing margin (Fig. 4A2), however some of the Notch vesicles now appeared to be slightly enlarged and colocalized to Deltex vesicles (Fig. 4D1–D3). Reducing the dose of TRAF6 by one copy in the HA-Krz and FLAG-Dx co-expression background resulted in a rescued wing and even loss of L4 vein material, which suggested an increase in Notch function (Fig. 4A3). When the status of Notch was examined in the larval wing discs of the above-mentioned combinations, Notch survival was found to be enhanced in the discs having a reduced dose of TRAF6 in HA-Krz and FLAG-Dx co-expression background as compared to the discs with endogenous or ectopically

Fig. 1. Drosophila Notch binds TRAF6. (A) Schematic representation of TRAF6 protein and its conserved domain. A region of TRAF6 (amino acids 224–475) that was sufficient for binding to Notch, based on yeast two-hybrid analysis, is shown below the full-length protein. RING — Really Interesting New Gene, CC — Coiled Coil, TRAF-C-TRAF- carboxy terminus. (B) GST-pull down assay was performed with salivary gland lysate over-expressing Notch-ICD (using Sgs3-GAL4) and recombinant GST-TRAF6 full-length (1–475 amino acids), amino-terminus (1– 147 amino acids), carboxy-terminus (202–475 amino acids) and other controls as indicated. GST-pulled down proteins were detected by western blot with anti-Notch (C17.9C6) antibody. (C) Co-immunoprecipitation was carried out with salivary gland lysate over-expressing HA-TRAF6 and Notch-ICD using Sgs3GAL4. + symbol indicates the presence of lysate and – symbol shows the absence of lysate. HA-TRAF6 immunoprecipitated Notch-ICD that was detected by anti-Notch antibody (C17.9C6). Input, self-immunoprecipitation and unbound fractions are shown. Beta-tubulin served as a loading control. (D1–G4) HA-TRAF6 and Notch-ICD were expressed under the control of C96-GAL4 and GMR-GAL4 drivers. Merged images of panels E4 and G4 show that HA (TRAF6) and Notch co-localized in Notch positive vesicles (arrowheads). Images in D4, E4, F4 and G4 are merges of those in D1–D3, E1–E3, F1–F3 and G1–G3, respectively. Scale bars, 100 μM (D1–D4, F1–F4) and 5 μM (E1–E4, G1–G4). (H1–H4) Gain-of-function clones of TRAF6 were generated to overexpress TRAF6 clonally in a wild type background using the flip-out technique in third instar larval wing discs. Clonal cells are marked by GFP expression. Detergent free staining revealed pronounced alteration in the extracellular epitope of Notch in the sub-apical region in the clonal area as compared to the cells with endogenous level of TRAF6. Scale bar, 5 μM.

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Fig. 3. Genetic interactions of TRAF6 with Notch pathway components. (A1 to C2) Representative wings from individuals with genotypes as indicated. Wings from N1 (A1) and N54l9 (B1) heterozygote showed wing-notching phenotype, which were completely rescued in trans-heterozygous combination with loss-of function allele of TRAF6 (TRAF2ex1) (A2 and B2). dx heterozygote (C1) wings displayed no phenotypic abnormality however in trans-heterozygous combination with TRAF6 loss-of- function mutant (TRAF2ex1), it showed wing vein thickening at distal ends of L2 and L3 and forked vein tip at L4 and L5 veins (C2), which exactly mimicked the dx null wing phenotype. Cut expression in w1118 and TRAF6 wing discs was detected in a narrow stripe along the DV boundary by antiCut antibody (D1, E1 and D2, E2 in low and higher magnification respectively). Cut expression in dx homozygous (D3, E3) and TRAF6/dx trans-heterozygous (D4, E4) third instar larval wing discs displayed an interruption at the intersection of antero-posterior and dorso-ventral boundaries, which was similar to the dx null wing discs [30]. Scale bars, 300 μM (A1–C2), 20 μM (D1–D4) and 5 μM, (E1–E4).

expressed TRAF6 (Fig. 4E1). Altogether, these results suggest that TRAF6 may play an important role in Krz-Dx mediated regulation of Notch receptor. 3.5. TRAF6 and Dx modulate Notch protein levels and signaling activity in vivo We strove to substantiate the strong genetic interaction between dx and TRAF6 loss-of-function with the gain-of-function analysis and observed the combinatorial effect of FLAG-Dx and HA-TRAF6 overexpression

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on Notch status and signaling activity. To this end, we overexpressed UASFLAG-Dx and UAS-HA-TRAF6 in Drosophila wing using C96-GAL4 and A9GAL4, that drove the expression of transgene in cells of dorso-ventral boundary at the wing margin and wing blade, respectively. Expression of HA-TRAF6 alone with the C96-GAL4 driver did not cause any abnormality at the wing margin (Fig. 5A1) while expression of only FLAGDx with the same driver resulted in mild irregularities of bristle pattern along the wing margin (Fig. 5A2). When FLAG-Dx and HA-TRAF6 were co-expressed at the wing margin, it ensued notching around the entire wing margin that was a manifestation of Notch loss-offunction phenotype (Fig. 5A3). Similar results were obtained using A9-GAL4 driver where expression of HA-TRAF6 alone did not result in any abnormality in wing (Fig. 5B1) while expression of FLAG-Dx followed the loss of vein material at the distal end of L4 and L5 veins with moderate loss in bristle pattern along the wing margin (Fig. 5B2). Co-expression of HA-TRAF6 and FLAG-Dx together with A9-GAL4 brought about a deformed wing with severe abnormalities throughout the wing (Fig. 5B3). In parallel, with the gain-of-function phenotypic analysis in the wing, we examined the levels of endogenous Notch protein in wing imaginal discs in a background of HA-TRAF6 and FLAG-Dx expression driven by en-GAL4 driver that drove the expression of transgene in posterior compartment of the wing discs. Expression of HA-TRAF6 alone did not affect the levels or localization of the Notch protein (Fig. 5C1D4) while FLAG-Dx, when expressed alone, resulted in re-localization of Notch protein in intracellular vesicles in the posterior compartment of wing discs and Dx also co-localized with Notch (Fig. 5E1–F4). Coexpression of HA-TRAF6, together with FLAG-Dx, prompted a significant depletion of Notch from Deltex positive vesicles and reduction in Notch protein level (Fig. 5G1–H4). Moreover, co-expression of HA-TRAF6 and FLAG-Dx also displayed distinct loss of Notch targets, Cut in posterior compartment of wing disc (Fig. 6C1–C3) and loss of Wingless at the dorso-ventral boundary region of the posterior compartment (Fig. 6 F1–F4) which further validated that TRAF6 and Dx together impaired Notch signaling activity. Therefore, the combinatorial effects of coexpression of TRAF6 and Dx, which mimic Notch loss-of-function phenotypes, appear to be caused by the decrease in Notch protein levels. DIC images revealed that the morphology of the posterior compartment of the wing discs expressing FLAG-Dx and HA-TRAF6 together, was also more irregular with a shift in the antero-posterior boundary more towards the posterior side (Fig. 5G3). It will be interesting to observe whether this was likely a consequence of depletion of Notch function in the posterior compartment. From the above data we concluded that overexpression of Dx, together with TRAF6, caused reduction in Notch protein levels and resulted in the down-regulation of Notch signaling. 4. Discussion Notch signaling presents a very distinct mechanism that sways a myriad of cell fate choices including differentiation, proliferation, and apoptotic programs. It is therefore ineluctable to fine-tune this signal right from the early developmental stages. Several mechanisms have been studied till date that explains how controlling mechanism operate at the level of receptor biosynthesis, processing, post-translational modifications, receptor trafficking, ligand–receptor interaction and post-signaling events that control the doses of Notch receptor and signal

Fig. 4. TRAF6 modifies the wing phenotype of Krz-Dx co-expression and altars the Notch protein levels. Co-expression of UAS-HA-Krz and UAS-FLAG-Dx transgenes under the control of the C96-GAL4 driver resulted in wing notching that resembled defects associated with reduced Notch signaling (A1). Overexpression of UAS-HA-TRAF6 transgene in this background caused no distinguishable phenotypic alteration (A2) however deleting one copy of TRAF6 (A3) or knocking down the levels of TRAF6 using UAS-TRAF6-IR (RNAi line) (B1) resulted in a rescue of the wing phenotype. B2 and B3 represent w1118 and TRAF2ex1 wings, respectively. TRAF2ex1 homozygous adult wings show mild irregularities and forked tips at the distal end of veins. Co-expression of HA-Krz and FLAG-Dx resulted in a marked depletion of Notch from Dx-positive vesicles (C1–C3) [27]. Expression of HA-TRAF6 in this background did not result in alteration in endogenous Notch levels (D1–D3). Reducing the dose of TRAF6 displayed marked enhancement of Notch however it did not co-localize to Dx vesicles (E1–E3). C3, D3 and E3 are merges of those in C1–C2, D1–D2 and E1–E2. Bar graph in F represents the fold change in Notch intensity and is representative of three independent experiments. Scale bars, 200 μM (A1–B3) and 5 μM (C1–E3). UAS-HA-Krz and UAS-FLAG-TRAF6 were co-expressed using C96-GAL4 and larval wing discs were stained with anti-HA and anti-FLAG antibodies. An evident co-localization was observed between the two proteins (G1–G3). Scale bar, 50 μM.

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longevity. Although many recent advances have been made to reveal different aspects of the Notch signaling mechanism and its intricate regulation, there are still many unanswered questions as to how

Notch signaling pathway operates to influence an astonishing array of cell fate decisions in different developmental contexts. In this study, we bring to light, a yet unknown mechanism of regulation of Notch

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signaling by Drosophila TRAF6 that, together with Dx, modulates Notch signaling. Drosophila TRAF6 is a cytoplasmic adapter protein that mediates signals through NF-kB [20] and JNK pathways [21]. Mammalian TRAF6 is involved in both the TNF receptor superfamily and the Interleukin-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily signal transduction pathways [14]. TRAF-C domain contributes to TRAF6 oligomerization and mediates the interaction of TRAF6 with upstream signaling molecules, however RING-domain of TRAF6 can function as a ubiquitin ligase that generates non-degradative K63-linked ubiquitin chains and mediates self-polyubiquitination [31]. Autoubiquitylation of TRAF6 activates TAK1-p38/JNK pathway in HEK293 cells [32] and overexpression of TRAF6 activates NF-kB pathway [33]. However, polyubiquitination of TRAF6 impedes Eiger/JNK signaling in Drosophila [21]. An array of emerging dataset interestingly suggests that TRAF6 and Notch may act antagonistically. One of the recent reports suggests that inhibition of TRAF6 improves adult myofiber regeneration upon injury through up-regulation of Notch signaling [34]. However, ours is the first report that Drosophila TRAF6 binds Notch intracellular domain and together with Dx, down-regulates Notch signaling. Drosophila dx gene encodes a cytoplasmic protein that positively regulates Notch signaling [35]. Drosophila Dx protein contains a RINGH2 and two WWE domains [36] and an E3 ubiquitin ligase activity has been shown to exist for mammalian homologs of Drosophila Dx [37]. It has also been studied that dx is involved in Su(H)-independent Notch signaling in Drosophila [30] however, further studies have provided a clear understanding that Dx signaling is Su(H) dependent [38]. In recent years, studies have suggested a new role of Drosophila dx in Notch signaling. It has been demonstrated that together with Kurtz (Drosophila non-visual β-arrestin), Dx negatively regulates Notch [27]. More recently, Shrub, a core component of the ESCRT­III complex, was identified as a key modulator of molecular synergy between Kurtz and Dx [39]. We present evidences that TRAF6 physically interacts with NotchICD and co-localizes with Notch in the same sub-cellular compartment. Overexpression of full-length Notch results in its accumulation at apical junctions and throughout internal vesicles where Notch extracellular and intracellular domains co-localize to each other. Co-expression of Notch with Su(dx)ΔHECT (Suppressor of Deltex deprived of HECT domain) prompts strong Notch accumulation in these vesicles however their shape is strikingly irregular and enlarged. Both the extracellular and intracellular epitopes of Notch co-localize with Su(dx) in these vesicles which suggests that accumulated Notch is predominantly fulllength and endocytosed prior to ligand-dependent cleavage and Notch activation [40]. We observed co-localization of Notch-full length and TRAF6 in vesicular structures and sections in sub-apical region in TRAF6 mis-expression clones also reveal altered status of Notch extracellular epitope, however, it remains to be seen whether TRAF6 expression can facilitate endosomal/lysosomal entry of Notch. Coexpression of Krz-Dx leads to degradation of Notch in a ubiquitination dependent manner however the identity of Krz-Dx vesicles has not been established [27]. Loss of TRAF6 in Krz-Dx co-expression background hinders with the degradation of Notch. Co-expression of TRAF6-Krz-Dx does not seem to interfere with the Notch levels as evident from the quantification data, nevertheless, the morphology of Notch positive vesicles appears to be changed as some of the vesicles are larger and irregular in shape than those in Krz-Dx co-expression only. Therefore, our data puts TRAF6 as an important mediator that

can affect the vesicular trafficking during Krz-Dx co-expression and subsequent ubiquitination dependent degradation of Notch. Earlier work has elucidated divers Dx function in vesicular trafficking of Notch where Dx promotes internalization of full-length Notch, which is subsequently targeted to the late endosomal limiting membrane, possibly through interactions with the AP-3 (Adaptor Protein- 3) complex. HOPS (Homotypic fusion and vacuole protein sorting) complex mediates maturation of Notch in late endosomes that, in turn, promotes fusion with the lysosome. Notch in internal vesicles is degraded, while Notch present at the limiting membrane may serve as a substrate for Presenilin-mediated cleavage and release of Notch-ICD [41]. In another study, it was observed that Dx function is required at two steps in Notch signaling. It promotes inclusion of Notch into endocytic compartments from the plasma membrane and is required in the transport of Notch from the early endosomes to lysosomes, where it is degraded [42]. The fact that loss-of-function mutants of dx display reduced Notch signaling [43–45], it is hypothesized that at least one of these two processes positively regulates the activation of Notch signaling [42]. In a more recent study, Dx function is discerned to induce Notch endocytosis through GPI (Glycophosphatidylinositol) protein-negative endosomes, which leads to Notch signaling, by the lysosomal activation mechanism. Alternatively, Notch entry to a GPI-protein-positive endocytic route leads to Notch activation independent of late endosomal trafficking but is sensitive to the reduction of early endosomal trafficking components. Su(dx) pits itself against Dx to divert more Notch into the GPI-proteinpositive vesicles and acts to limit endosomal Notch activation by facilitating the transfer of Notch into the multivesicular body [46]. Data from our initial experiments unambiguously present that, TRAF6 together with Dx, ensues loss of Notch and its signaling activity. Since both TRAF6 and Dx possess E3 ubiquitin ligase activity, this negative regulation is expected to be a consequence of a ubiquitination dependent phenomenon. Studies have unveiled that assembly of a chain of at least four ubiquitins linked together via their Lys48 residue targets cellular proteins for degradation by the 26S proteasome. However, monoubiquitination or polyubiquitination with chains linked together via Lys63 serve as non-proteolytic signals in intracellular trafficking, DNA repair, and signal transduction pathways [47]. The E3 ubiquitin ligases involved in Notch pathway have been proposed to cause dominant-negative effects on Notch signaling by their E3-deleted forms [48]. Deltex lacking the proline-rich motif, also behaves as a dominant-negative form of Deltex and inhibits Notch signaling during wing margin development however, this requires the Deltex domain essential for binding to the intracellular domain of Notch [44]. It will be further interesting to explore the possibility whether carboxy-terminal of TRAF6 that binds to Notch-ICD can act as a dominant negative form and affect the Notch-TRAF6 interaction at a functional level. The precise role of endogenous TRAF6 in Notch ubiquitination and vesicular trafficking and the context dependent involvement of TRAF6 in Dx dependent and Krz-Dx dependent Notch signaling are not known at the moment and is currently under investigation. A clear understanding towards this end will not only vindicate regulation of Notch pathway but will also be able to corroborate our understanding as to how TRAF6 may play an important role in receptor-trafficking and non-canonical pathways including cross-talk of Notch pathway with JNK and Rel/NF-κB pathways that involve TRAF6 as one of the principle cytoplasmic adaptor/scaffold protein.

Fig. 5. Co-expression of TRAF6 and Dx results in alteration of Deltex induced wing phenotype and reduction in Notch protein levels. Over-expression of UAS-HA-TRAF6 under the control of the C96-GAL4 driver did not cause any visible defect (A1), over-expression of UAS-FLAG–Dx alone caused mild irregularities of bristle pattern along the wing margin (A2) and co-expression of UAS-HA-TRAF6 and UAS-FLAG–Dx resulted in severe nicking along the wing vein margin (A3). Likewise, over-expression of UAS-HA-TRAF6 alone, with A9-GAL4, did not lead to phenotypic defects (B1), over-expression of UAS-FLAG-Dx resulted in irregularities in bristle pattern along the wing margin (B2) and co-expression of UAS-HA-TRAF6 and UAS-FLAG-Dx showed severely deformed wing (B3). Expression of HA-TRAF6 alone did not alter endogenous Notch protein levels in third instar larval wing discs (C1–C4 and D1–D4). Expression of FLAG-Dx caused the re-localization of Notch in Dx-positive vesicles (E1–E4 and F1–F4), however, co-expression of HA-TRAF6 and FLAG-Dx showed depletion of Notch protein (G1–G4 and H1–H4). Co-expression of HA-TRAF6 and FLAG-Dx also displayed folded invaginations and shift in posterior compartment boundary of the wing disc (G3). C4, D4, E4, F4, G4 and H4 are merged images of C1–C3, D1–D3, E1–E3, F1–F3, G1–G3, and H1–H3. Bar graph in I represents the fold change in Notch intensity and is representative of three independent experiments. Scale bars, 200 μM (A1–B3), 100 μM (C1–C4, E1–E4 and G1–G4), and 10 μM (D1–D4, F1–F4, H1–H4).

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Fig. 6. Co-expression of TRAF6 and Dx results in down-regulation of Cut and Wingless proteins. Expression of HA-TRAF6 alone did not result in any alteration in endogenous Cut level in third instar larval wing discs (A1–A3). Expression of FLAG-Dx alone showed a mis-regulated Cut pattern in the posterior compartment of the wing discs (B1–B3) and co-expression of HA-TRAF6 and FLAG-Dx resulted in the loss of Cut expression in the posterior compartment of the wing discs (arrowheads) (C1–C3). Insets in A1, B1 and C1 are Cut expression at the dorso-ventral boundary. Expression of HA-TRAF6 alone did not alter endogenous Wingless (Wg) pattern at the dorso-ventral margin in third instar larval wing discs (D1–D3). Expression of FLAG-Dx showed the Wg pattern in the wing discs (E1–E3) and co-expression of HA-TRAF6 and FLAG-Dx resulted in the loss of Wg expression at the dorso-ventral boundary in posterior compartment of the wing disc (arrowhead) and showed a broadening in Wg expansion in wing pouch (arrows) (F1–F3). D4, E4 and F4 are magnified images for better visibility. Scale bars, 100 μM (A1–F3 except D4, E4 and F4).

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5. Conclusion Our analyses add towards developing new insights into the regulation of Notch signaling by a new player TRAF6. A physical and functional interaction between TRAF6 and Notch presents cues to fortify our current understanding about Notch signaling. It not only adds a novel modulator in Notch interactome, but also helps in developing better sagacity to undertake unanswered questions related to upstream events in Notch signaling, ubiquitination, vesicular trafficking and its cross-talk with other signaling pathways. Acknowledgments The authors extend sincere thanks to Spyros Artavanis-Tsakonas, Tian Xu, Enrique Martín-Blanco and the Bloomington Stock Centre for fly stocks. Some of the antibodies used in this work were obtained from the DSHB. A.K.M. and N.S. were supported by fellowships from CSIR and ICMR, India respectively. This work was supported by grants from DST, India and DBT, India to A.M. and M.M. We also acknowledge the help from DST-Confocal facility in Banaras Hindu University for confocal microscopy. References [1] [2] [3] [4] [5] [6] [7] [8]

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