Decreased ubiquitination in the dTAK1^K156R mutant.

<p>(A) Mutant dTAK1^K156R showed decreased polyubiquitination when compared with dTAK1. Expression vectors encoding dTAK1-V5 or dTAK1^K156R -V5 were transfected along with dTAB2-HA and cMyc-Ub in combinations shown in S2 cells. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. (B) K48-linked polyubiquitination was decreased in the dTAK1^K156R mutant while K63-linked polyubiquitination was somewhat reduced. Expression vectors encoding dTAK1-V5 or dTAK1^K156R-V5 were transfected along with dTAB2-HA and cMyc-Ub, cMyc-UbK63 (left panel) or cMyc-UbK48 (right panel) in combinations shown in S2 cells. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. Protein size marker is depicted adjacent to the top panels with values in kDa.</p

<i>Drosophila</i> TAB2 ZnF domain regulates IMD signalling.

<p>(A) Sequence alignment of the C-terminal ZnF domain of hTAB2, hTAB3 and dTAB2. Conserved Cys residues are in red and indicated by arrows. (B) IMD signalling is significantly increased in dTAB2^ZnFDel mutant. S2 cells were co-transfected with expression vectors in combinations shown and <i>diptericin</i> expression was assayed 48 hrs post-transfection by qPCR. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004117#s2" target="_blank">Results</a> are mean values from 3 independent experiments and the error bars represent the Standard Error. Single, and triple asterisk indicates significance value of the result when compared to EGFP as determined by Student's t-Test (t = 17.8435, p<0.0025; t = 6.2838, p<0.025 respectively). However, dTAK1+dTAB2 was significantly lower than dTAK1+dTAB2^{ZnFDe<b>l</b>} when compared. (C) Mutation of dTAB2 ZnF stabilises dTAK1 and increases its K63-linked ubiquitination. <i>Drosophila</i> TAK1-HA was co-transfected with cMyc-Ub^K63 and either dTAB2-V5 (wt) or dTAB2^ZnFDel–V5 into <i>Drosophila</i> S2 cells. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-HA antibody, resolved on 10% SDS PAGE and immunoblotted with anti-cMyc antibody. Observe that TAK1 is stabilised in the cell lysate in the presence of dTAB2^ZnFDel (dTAK1 panel left lane). Protein size markers (NEB) are depicted with values in kDa. (D) <i>Drosophila</i> Trabid binds strongly to dTAB2^ZnFDel. S2 cells were transfected with dTrabid-HA alone or with either dTAB2^ZnFDel –V5 or dTAB2-V5. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE and immunoblotted with anti-HA antibody. Trabid bound very weakly to wild type TAB2 (middle lane). Observe the increase in binding of Trabid to dTAB2^ZnFDel (right lane) in comparison to wild type dTAB2.</p

<i>Drosophila</i> Trabid binds to dTAK1 and negatively regulates IMD signalling.

<p>(A) <i>Drosophila</i> Trabid interacted with dTAK1. <i>Drosophila</i> TAK1-HA was co-transfected with or without dTrabid-V5 in S2 cells, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE and immunoblotted with anti-HA antibody. (B) AMP gene expression was highly elevated in <i>trabid</i> mutants in the absence of infection. Expression levels of various AMPs (<i>attacinA; drosocin; diptericin; cecropinA1</i>) were checked in whole flies (3–6 days old) using qPCR compared with <i>yw</i> as control for the genetic background (wild type) of <i>pirk</i> and <i>trbd </i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004117#pgen.1004117-Tran1" target="_blank">[30]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004117#pgen.1004117-Bellen1" target="_blank">[31]</a>. We also used the <i>w¹¹⁸</i> strain as an additional control (to check the range between two independent controls). Error bars represent the Standard Error of 3 separate experiments. *p<0.05, **p<0.01, ***p<0.001 indicate significant value when <i>trbd</i> mutants are compared with the <i>yw</i> control. Expression of <i>diptericin</i> (C) and <i>drosocin</i> (D) was not significantly different in <i>yw</i>, <i>pirk</i>, <i>trabid</i> or <i>pirk; trbd</i> mutants in response to systemic infection. <i>yw</i> (wild type), <i>trbd, pirk and pirk; trbd</i> flies, were injected with two doses of <i>Ec1106</i> (low dose ≈400 cells, high dose ≈4000 cells) and AMP levels assayed after 6 and 24 hrs using qPCR. Error bars represent the Standard Error of 3 separate experiments. Expression in <i>yw</i> was set as 100%. Calculations using paired t-test showed that the difference between mutants and <i>yw</i> was not significant (E) Drosophila Trabid negatively regulated IMD signalling in a dose-dependent manner during gut infection. <i>yw</i> (wild type), <i>dredd, trbd/+, trbd, pirk; trbd and dredd; pirk; trbd flies</i>, were fed with Ecc15 and <i>diptericin</i> levels assayed after 24 hrs using qPCR. Error bars represent the Standard Error of 3 separate experiments. The Dpt/Rp49 ratio was normalized with the level obtained in <i>yw</i> guts. All the mutants showed statistically significant increase of diptericin levels (p<0.05). Of note that, any up-regulation over and above the wild type levels was suppressed in a <i>dredd</i> background in agreement with the hypothesis that <i>trbd</i> was negatively regulating the Imd pathway (p***<0.001 when <i>pirk; trbd</i> and <i>dredd; prik; trbd</i> were compared as determined by Student's t-Test). *p<0.05 indicates significance value when <i>trbd/+</i> and <i>trbd</i> were compared as determined by Student's t-test.</p

Loss of Trabid, a New Negative Regulator of the <i>Drosophila</i> Immune-Deficiency Pathway at the Level of TAK1, Reduces Life Span

<div><p>A relatively unexplored nexus in <i>Drosophila</i> Immune deficiency (IMD) pathway is TGF-beta Activating Kinase 1 (TAK1), which triggers both immunity and apoptosis. In a cell culture screen, we identified that Lysine at position 142 was a K63-linked Ubiquitin acceptor site for TAK1, required for signalling. Moreover, Lysine at position 156 functioned as a K48-linked Ubiquitin acceptor site, also necessary for TAK1 activity. The deubiquitinase Trabid interacted with TAK1, reducing immune signalling output and K63-linked ubiquitination. The three tandem Npl4 Zinc Fingers and the catalytic Cysteine at position 518 were required for Trabid activity. Flies deficient for Trabid had a reduced life span due to chronic activation of IMD both systemically as well as in their gut where homeostasis was disrupted. The TAK1-associated Binding Protein 2 (TAB2) was linked with the TAK1-Trabid interaction through its Zinc finger domain that pacified the TAK1 signal. These results indicate an elaborate and multi-tiered mechanism for regulating TAK1 activity and modulating its immune signal.</p></div

Ubiquitination profile is altered in the dTAK1^K142R mutant.

<p>(A) Mutant dTAK1^K142R showed enhanced ubiquitination. Expression vectors encoding dTAK1-V5 or dTAK1^K142R-V5 were transfected in S2 cells along with dTAB2-HA and cMyc-Ub in combinations as shown. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc. Observe the enhanced degradation of dTAK1^K142R-V5 in the cell lysate (right lane). (B) Mutant dTAK1^K142R showed enhanced K48-linked ubiquitination and very little K63-linked ubiquitination. Expression vectors encoding dTAK1-V5 or dTAK1^K142R-V5 were transfected in S2 cells along with dTAB2-HA and cMyc-UbK63 (left panel) or cMyc-UbK48 (right panel) in combinations as shown. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE, and immunoblotted with anti-cMyc antibody. Protein size markers (NEB) are depicted adjacent to the top panels with values given in kDa.</p

<i>Drosophila</i> Trabid significantly decreases K63-linked polyubiquitination in dTAK1 through its catalytic C518 and NZF domains.

<p>(A) Schematic representation of dTrabid and sequence alignment of human and <i>Drosophila</i> Trabid in the OTU domain. The putative catalytic residue is in red & is indicated by an arrow. (B) Point mutations in C518 and the 3 NZF domains significantly affect dTrabid function in Imd signalling. Expression vectors containing dTrabidC518S, dTrabidC518S+3xNZFDel, dTAK1 and dTAB2 were co-transfected in S2 cells in the combinations shown and <i>diptericin</i> and <i>puckered</i> expression was assayed by qPCR 48 hrs post-transfection. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004117#s2" target="_blank">Results</a> represent mean values of 3 independent experiments and the error bars SEM. Asterisk (*) indicates significance value of the results when compared to dTAK1+dTAB2 as determined by Student's t-Test (t = 9.8004, <i>p</i><0.025). (C) <i>Drosophila</i> Trabid reduces K63-linked ubiquitination in dTAK1. Expression vectors containing C-terminally HA-tagged dTrabid, dTrabidC518S or dTrabidC518S+3xNZFDel, along with dTAK1-V5, dTAB2-HA and cMyc-UbK63 were co-transfected in S2 cells in the combinations shown. Cells were lysed 48 hrs post-transfection, immunoprecipitated with anti-V5 antibody, resolved on 10% SDS PAGE and immunoblotted with anti-cMyc peroxidise. Protein size markers (NEB) are depicted adjacent to top panels with values given in kDa.</p

Loss of Trabid reduces life span.

<p>(A) Lifespan analysis of conventionally reared <i>trbd</i> mutans. Using the Log-Rank and Wilcoxon tests, life span analysis of flies revealed a statistically significant decrease in the survival rate of <i>trbd</i> and <i>trbd; pirk</i> mutant flies in comparison to <i>yw</i> flies. (p<0.0001 for both tests). Consistent with the assumption that decreased lifespan was a consequence of an excessive activation of Imd signalling <i>dredd; pirk; trbd</i> flies were significantly longer lived than the <i>pirk; trbd</i> double mutants (p<0.0001 for both tests). Each survival curve was a result of 12 independent experiments (360 flies in total per genotype; see experimental procedures) (B) Lifespan analysis of <i>trbd</i> mutants reared in germ-free conditions. Growing <i>trbd</i> or <i>pirk; trbd</i> flies in germ free conditions did not ameliorate life span as they were statistically inseparable to their normally grown siblings (p = 0.6206/0.6872 and p = 0.0004/0.0048 respectively). Each survival curve was a result of 12 independent experiments (360 flies in total per genotype; see experimental procedures). (C) Statistical analysis of life span results in A and B. Experimental and control populations are compared using Log-Rank and Wilcoxon tests (ChiSquare and p-values). All the analysis was performed using GraphPad Prism statistical software. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004117#s4" target="_blank">materials and methods</a> for protocol used for germ free flies.</p

Lys 142 and Lys 156 of dTAK1 are essential for immune signalling.

<p>(A) Sequence alignment of 1 to 230 amino acids of human TAK1 (hTAK1) and <i>Drosophila</i> TAK1 (dTAK1). Lys 142 (dark grey) and 156 (light grey) are indicated (arrows) and the kinase activation loop is underlined. (B) <i>Drosophila</i> TAK1^K142R and TAK1^K156R mutants failed to activate <i>diptericin</i>. <i>Drosophila</i> TAK1 or dTAK1^K142R or dTAK1^K156R mutant was expressed along with or without dTAB2, in S2 cells in combinations shown. <i>Diptericin</i> expression was assayed 48 hrs post-transfection. Error bars represent Standard Error of 3 separate experiments. *p<0.05, **p<0.01 indicate significant value when dTAK1 wild type or dTAK1 (+dTAB2) were compared with the empty vector.</p