Infection of Drosophila by Gram-negative bacteria triggers a signal transduction pathway (the IMD pathway) culminating in the expression of genes encoding antimicrobial peptides. A key component in this pathway is a Drosophila IkappaB kinase (DmIKK) complex, which stimulates the cleavage and activation of the NF-kappaB transcription factor Relish. Activation of the DmIKK complex requires the MAP3K dTAK1, but the mechanism of dTAK1 activation is not understood. In human cells, the activation of TAK1 and IKK requires the human ubiquitin-conjugating enzymes Ubc13 and UEV1a. This study demonstrates that the Drosophila homologs of Ubc13, (Bendless) and UEV1a, are similarly required for the activation of dTAK1 and the DmIKK complex. Surprisingly, the Drosophila caspase DREDD and its partner dFADD are required for the activation of DmIKK and JNK, in addition to their role in Relish cleavage. These studies reveal an evolutionarily conserved role of ubiquitination in IKK activation, and provide new insights into the hierarchy of signaling components in the Drosophila antibacterial immunity pathway (Zhou, 2005).
A cell culture system was established to study the IMD and Toll signaling pathways in S2 cells. The IMD pathway is activated by treating the cells with Gram-negative peptidoglycan (which is present in crude preparations of lipopolysaccharides), whereas activation of the Toll pathway is achieved by treating the cells with the Spätzle ligand. Active Spätzle is produced from a cell line stably transfected with a plasmid containing the copper-inducible metallothionein promoter driving the expression of active Spätzle C-106. When these cells are treated with copper, active SPZ is secreted into the medium, and this conditioned media can be used to activate naïve cells. Using the RNAi-mediated gene inactivation method, it was found that SPZ-induced Drosomycin gene activation in S2 cells requires the Drosophila Rel proteins Dif and Dorsal, as well as Toll, dMyD88, Tube, Pelle, and Slimb, as expected. In sharp contrast, these dsRNAs do not block the expression of antimicrobial peptide genes induced by peptidoglycan. Instead, RNAi studies demonstrate that peptidoglycan-induced gene expression requires all known components of the IMD pathway. Therefore this RNAi approach was used to determine the roles of candidate signaling components in the IMD and Toll signaling pathways (Zhou, 2005).
Previous studies have shown that activation of the mammalian IKK complex requires a ubiquitination step. In particular, TRAF6-mediated IKK activation was shown to require a dimeric ubiquitin-conjugating enzyme composed of the Ubc13 and UEV1a proteins. Bendless and dUEV1a are the Drosophila homologs of Ubc13 and UEV1a, respectively. Bendless and dUEV1a, like their mammalian counterparts, associate with each other in vivo. To investigate whether Bendless and dUEV1a are required for antibacterial gene expression in response to peptidoglycan, the RNAi-mediated gene inactivation method was used. S2 cells were transfected with various dsRNAs. After 48 h, cells were first treated with 20-hydroxy-ecdysone for 24 h to enhance their competence to induce antimicrobial genes in response to immune challenge, and then treated with peptidoglycan or SPZ to activate the IMD or Toll signaling pathways, respectively. Total RNA was isolated from these cells and subjected to Northern blotting analysis using cDNA fragments corresponding to Diptericin or Drosomycin as probes, to examine the activation of the IMD and Toll pathways, respectively. Both Bendless and dUEV1a are required for maximal levels of antibacterial peptide gene expression in response to peptidoglycan treatment. In fact, when both Bendless and dUEV1a are both targeted by RNAi, Diptericin induction is reduced to near background levels. (The partial effect of Bendless or dUEV1a RNAi alone is likely because of the fact that RNAi often does not generate a complete null phenotype.) By contrast, the induction of Drosomycin by Toll activation is unaffected by the RNAi-mediated knock-down of Bendless and dUEV1a. Note that the same cells were stimulated with either peptidoglycan or SPZ in. As a control, S2 cells treated with DmIKKgamma or Toll dsRNA showed significantly reduced peptidoglycan-induced Diptericin or SPZ-induced Drosomycin gene expression, respectively. As a control, mRNA and/or protein levels of targeted genes were examined by RT-PCR and/or Western blotting to confirm the effectiveness of RNAi (Zhou, 2005).
To provide further evidence that Bendless is involved in the IMD pathway, a dominant negative mutant Bendless was used to determine whether peptidoglycan-induced antibacterial gene activation can be blocked. Stable S2 cell lines were generated that express either wild-type or C87A Bendless under the control of the metallothionein promoter. The cysteine to alanine mutation at position 87 creates a dominant-negative mutant because this residue, located in the catalytic pocket in ubiquitin-conjugating enzymes, is crucial for the catalytic activity of E2s. These cells were then treated with various combinations of peptidoglycan and copper, and Northern blotting was employed to examine the expression of antibacterial genes including Attacin, Cecropin and Diptericin. Overexpression of wild-type Bendless has no effect on peptidoglycan-induced antibacterial gene activation, since similar levels of antibacterial peptide gene expression were detected in cells treated with or without copper. In contrast, overexpression of Bendless C87A leads to a significant reduction in peptidoglycan-activated expression of antibacterial peptide genes (Zhou, 2005). Bendless flies have been identified which carry a proline to serine substitution at position 97 within the strictly conserved active site region of E2s. In order to determine whether bendless flies are defective in response to Gram-negative bacterial infection, both wild-type and bendless flies were subjected to E. coli infection and Diptericin gene activation was examined by Northern blotting. bendless flies display significantly weaker Diptericin gene activation compared with wild-type flies. These results indicate that the Bendless-dUEV1a E2 complex is required for signaling by the IMD pathway (Zhou, 2005).
Experiments were carried out to determine whether Bendless and dUEV1a are required for peptidoglycan-induced activation of the DmIKK complex. Previous studies have shown that peptidoglycan treatment induces the kinase activity of the endogenous DmIKK complex in S2 cells. Cells were transfected with dsRNAs corresponding to various mRNAs. After 48 h, these cells were treated with peptidoglycan for 15 min, and the endogenous DmIKK complex was immunoprecipitated and subjected to in vitro kinase assays using recombinant Relish protein as substrate. Bendless or dUEV1a dsRNA treatment leads to a significant decrease in peptidoglycan-induced DmIKK kinase activity, suggesting that Bendless and dUEV1a are required for peptidoglycan-induced DmIKK activation. As a control, DmIKKgamma dsRNA treatment completely abolished peptidoglycan-induced DmIKK activation. It is concluded that the ubiquitin-conjugating enzymes Bendless and dUEV1a are specifically involved in the Drosophila IMD pathway, and they play a role upstream of the DmIKK complex (Zhou, 2005).
Overexpression of IMD in Drosophila results in the activation of antibacterial genes in the absence of bacterial infection. IMD overexpression, under control of the copper-inducible metallothionein promoter, can also strongly activate expression of the Diptericin gene in S2 cells. This stable cell line therefore provides a useful tool to perform an epistatic analysis to determine the position of Bendless/dUEV1a complex relative to IMD in the Drosophila antibacterial signaling pathway. The IMD stable cells were first transfected with dsRNAs derived from various genes and then stimulated with copper or peptidoglycan, and IMD- and peptidoglycan-induced Diptericin gene activation was examined. Overexpression of IMD, via the addition of copper, leads to strong activation of the Diptericin gene. In fact, copper-induced IMD expression is as potent as peptidoglycan in driving diptericin expression. Cells transfected with LacZ dsRNA show a similar Diptericin expression profile compared with cells that were mock-treated. Consistent with the observation that the DmIKK complex functions downstream of IMD, cells transfected with DmIKKgamma dsRNA are severely defective in both IMD- and peptidoglycan-induced Diptericin expression. Furthermore, cells treated with dsRNAs derived from Bendless and dUEV1a genes display a significant reduction in both peptidoglycan- and IMD-mediated Diptericin gene activation. These results indicate that the ubiquitin conjugating enzymes Bendless and dUEV1a function downstream of IMD in this signaling pathway (Zhou, 2005).
Thus, by differentially activating the IMD and Toll signaling pathways in Drosophila S2 cells, this study shows that Bendless (dUbc13) and dUEV1a are required for the Drosophila IMD signaling pathway. Using RNAi to target Bendless and/or dUEV1a significantly reduces the levels of peptidoglycan-induced antibacterial peptide gene expression and activation of the Drosophila IKK complex. This mechanism of IKK activation is highly conserved; in mammals Ubc13 and UEV1a are required for TNFalpha-, IL-1β-, and TCR-mediated IKK and NF-kappaB activation (Zhou, 2005).
This ubiquitin-dependent kinase activation does not involve proteasome-mediated degradation. Proteasome inhibitors do not block IKK activation, in flies or humans. Moreover, ubiquitination without degradation has been shown to activate the human IKK complex, and a similar Drosophila activity has been identified, The primary sequence of the mammalian Ubc13/UEV1a and those of the Drosophila Bendless/dUEV1a are highly conserved (90% similarity for Ubc13, 79% for UEV1a). The crystal structures of the yeast and human Ubc13/UEV1a (Mms2) have shown clearly that this E2 complex can make only K63-linked polyubiquitin chains. Thus, it is likely that the Drosophila Bendless/dUEV1a E2 catalyzes the formation of K63-linked ubiquitin chains (Zhou, 2005).
In a cell-free system, human TRAF6 was shown to be an E3 ligase that auto-ubiquitinates in conjunction with Ubc13/UEV1a. This results in the activation of TRAF6 and, in turn, the activation of TAK1. Activated TAK1 phosphorylates key serine residues in the activation loop of IKKβ, resulting in the activation of IKKβ. It is suspected that similar mechanisms are involved in the Drosophila IMD pathway. This study demonstrates that the Drosophila TAK1 homolog functions downstream of Bendless and dUEV1a. Furthermore, a Drosophila homolog of TAB2/TAB3 is also required for the IMD pathway. Interestingly, the C-terminal zinc finger domain of TAB2, which is conserved in the Drosophila protein, has recently been shown to bind specifically to K63-polyubiquitin chains. Strikingly, it has found that a galere/dTAB2 mutant, which is defective in the IMD pathway, carries a mutation in this zinc finger domain (Zhou, 2005).
The Drosophila E3 ligase, analogous to human TRAF2 or TRAF6, which functions with Bendless and dUEV1a in the activation of dTAK1 and DmIKK remains to be identified. In Drosophila, the dTRAF2 protein is the closest homolog of mammalian TRAF6, and it is the only Drosophila TRAF protein that contains the RING domain, typical of E3 ligases. However, RNAi knockdown and dominant-negative studies suggest that dTRAF2 is not involved in either the IMD or the Toll signaling pathways in S2 cells. In fact this gene is expressed at undetectably low levels in S2 cells. In one previous study dTRAF2 was reported to interact physically and functionally with Pelle, a key signaling component in the Toll signaling pathway that controls the antifungal immune response. However, these studies were based on overexpression experiments and in vitro binding assays, which might not reflect the physiological role of dTRAF2. In contrast, a recent publication demonstrated that dTRAF2 mutants are not fully able to induce antimicrobial peptide genes following E. coli infection. However, these studies did not clearly determine whether dTRAF2 is involved in the Toll or IMD pathways. The data suggest that dTRAF2 is not a critical component of the IMD pathway in S2 cells. Further studies, in cells and in flies, are necessary to elucidate the role of dTRAF2 in Drosophila immunity (Zhou, 2005).
The possibility is considered that other Drosophila RING-containing proteins might be involved in Drosophila immunity. However, RNAi knockdown studies with 10 different RING domain-encoding genes failed to block either the Toll or IMD pathways. Finally, since the structure of the RING domain of Rad5 has been successfully modeled to fit into the structure of the dimeric ubiquitin-conjugating enzyme complex Ubc13/UEV1a, it was reasoned that the potential ubiquitin ligase involved in the IMD pathway might physically interact with Bendless and dUEV1a. Therefore yeast two-hybrid screens were performed using Bendless and dUEV1a as baits in an effort to identify their protein interaction partners. A Drosophila RING protein, CG14435, was identified in such screens. CG14435 interacts robustly with both Bendless and dUEV1a in yeast two-hybrid assays. Furthermore, the CG14435-Bendless and CG14435-dUEV1a interaction was confirmed by co-immunoprecipitation of overexpressed proteins in S2 cells. However, RNAi-based studies suggest that CG14435 is not involved in the Drosophila innate immunity signaling pathways. It has been shown that bendless flies display defective synaptic connectivity and abnormal morphology within the visual system, suggesting Bendless functions in a variety of developmental processes. In addition, the Saccharomyces cerevisiae homologs of Bendless and dUEV1a have been implicated in DNA damage repair. Therefore it is possible that CG14435 is involved in some cellular processes other than immunity which require Bendless and dUEV1a. Further studies are necessary to elucidate the physiological role of CG14435 and to identify the ubiquitin ligase activity required for ubiquitination-dependent DmIKK activation (Zhou, 2005).
As in the TRAF6 pathway, dTRAF2 and/or other E3 ligases that function with Bendless and dUEV1a in the IMD pathway may be the target(s) of K63 polyubiquitination. Another possible target of Bendless/dUEV1a-mediated ubiquitination is the Drosophila IKKgamma subunit (also known as NEMO in mammals) of the IKK complex. In mammals, it has recently been shown that NEMO is K63 polyubiquitinated by the Ubc13/UEV1a complex in response to Bcl10 expression or T-cell activation. Other possible targets of ubiquitination by Bendless and dUEV1a in the IMD pathway include the Drosophila TAB2 homolog and IMD. Recently, it was shown that the two mammalian homologs of TAB2 and TAB3 were ubiquitinated or associated with other ubiquitinated proteins. In addition, the mammalian RIP1, which is homologous to IMD protein especially in its death domain, has recently been shown to be K63 polyubiquitinated and associated with TAB2 in a TNFalpha-dependent manner. In any case, K63 polyubiquitin chains likely function to recruit the Drosophila TAK1/TAB2 complex, via the TAB2 K63 polyubiquitin binding domain, to either (or both) the upstream activators, such as IMD, and/or the downstream target of dTAK1 kinase activity, the Drosophila IKK complex (Zhou, 2005).
As expected, the epistatic analyses presented in this study demonstrate that IMD functions upstream of all other components in the pathway except the receptor PGRP-LC, and is required for IKK activation. Moreover, Bendless and dUEV1a function downstream of IMD and upstream of dTAK1, as predicted from the model for Ubc13 and UEV1a in mammals. dTAK1 is required for activation of the Drosophila IKK complex and likely functions as the IKK kinase (Zhou, 2005).
Although it is established that dFADD and DREDD are required for the IMD pathway, previous experiments have suggested that they function downstream of the DmIKK complex. For example, DREDD overexpression in flies leads to Diptericin gene expression in the absence of Gram-negative bacterial infection, and DREDD-mediated Diptericin gene activation requires neither the DmIKK complex nor dFADD. Also, recent studies have shown that DREDD interacts with Relish, and that a caspase-cleavage site within Relish is required for peptidoglycan-induced Relish activation. Therefore, it was speculated that DREDD functions downstream of the DmIKK complex by directly cleaving DmIKK-phosphorylated Relish. This possibility is consistent with the observation that DREDD and dFADD are required for dTAK1Delta-mediated Diptericin gene activation. Surprisingly, dFADD and DREDD are required for peptidoglycan-induced DmIKK activation, arguing that DREDD and dFADD function upstream in the pathway. In addition, DREDD is also required for peptidoglycan-induced JNK activation, but neither DREDD nor dFADD are required for dTAK1- or dTAK1Delta-mediated DmIKK activation, suggesting that dFADD and DREDD act at a step upstream of dTAK1 in response to peptidoglycan. Based on these observations, it is proposed that dFADD and DREDD play dual roles in the Drosophila antibacterial signaling pathway. On the one hand, dFADD transduces signals from IMD to DREDD, resulting in DREDD activation and enabling Relish cleavage; in contrast, dFADD and DREDD contribute to peptidoglycan-induced DmIKK activation through a mechanism that remains to be elucidated. DREDD may function similarly to human Caspase-8, which is a DED-containing apical caspase similar to DREDD. Caspase-8 has recently been shown to be required for NF-kappaB activation in response TCR-signaling. This role of Caspase-8 requires the enzymatic activity of full-length protein Caspase-8 and is involved in recruiting the IKK complex to the upstream signaling complex of CARMA1, Bcl10, and MALT1. Interestingly MALT1 is also a caspase-like gene (sometimes referred to as a paracaspase), and it is thought to function as an E3-ligase accessory factor with Ubc13 and UEV1a in TCR-mediated NF-kappaB activation. DREDD may similarly function as E3-ligase accessory factors with Bendless and dUEV1a as the E2, in the IMD pathway (Zhou, 2005).
The following scheme is proposed for the Drosophila antibacterial signaling pathway. Peptidoglycan treatment or Gram-negative bacterial infection leads to the activation of the membrane-bound peptidoglycan-recognition protein, PGRP-LC. Activated PGRP-LC in turn transduces signal to IMD. IMD, in turn, interacts with dFADD and subsequently DREDD. It is proposed that IMD, dFADD and DREDD form a complex that contributes to dTAK1 activation, perhaps as part of an E3 ligase. This complex is likely to function in conjunction with Bendless/dUEV1a to activate dTAK1 and then DmIKK. Once activated, the Drosophila IKK complex phosphorylates Relish, which is subsequently cleaved. The N-terminal Relish cleavage product, an NF-kappaB transcription factor, then translocates to the nucleus where it activates antimicrobial peptide gene expression. In addition to their role in IKK activation, DREDD and dFADD are also proposed to function downstream in this pathway, in the signal-induced cleavage of phospho-Relish (Zhou, 2005).
Jun N-terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF-kappaB/Relish-mediated activation of antimicrobial peptide genes. However, this study demonstrates that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals is rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss-of-function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. The data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression (Delaney, 2006).
Innate immune responses are critical for a rapid host defense against pathogens. The signaling pathways that control these responses are present in all multicellular organisms, ranging from humans to flies, and are remarkably well conserved. Although the innate response lacks the antigen recognition capacity of vertebrate adaptive immunity, it is nevertheless complex and crucial for host survival. Drosophila is a proven genetic model organism for the study of innate immunity and has provided invaluable insights into the control of responses to infection (Delaney, 2006).
Toll and Imd are the founding members of two principal innate immune response signaling pathways in Drosophila. Toll signals through two NF-kappaB/Rel family transcription factors, Dif and Dorsal, and is required for responses to fungal and Gram+ bacterial infections. Imd signaling controls primarily Gram- bacteria-specific responses through the cleavage and activation of a third Rel family transcription factor, Relish, by the Drosophila caspase Dredd. Relish activation also requires an IkappaB kinase (IKK) complex that is itself activated by Imd signaling. The transcriptional targets of Dif and Relish are not entirely distinct. For example, cecropinA expression requires either Relish or Dif, or both, depending on the type and strain of infecting microorganism. More than 20 Drosophila genes have been implicated in these signaling pathways and nearly all of them have mammalian homologues with conserved immune functions (Delaney, 2006 and references therein).
Jun N-terminal kinase (JNK) signaling has been linked to stress responses, cell migration, apoptosis, and immune responses in both insects and mammals. JNK activity can be induced by infection, lipopolysaccharide, and inflammatory cytokines such as tumor necrosis factor (TNF) in flies and mammals. Null mutations in JNK signaling components are typically embryonic lethal in flies and thus unlikely to appear as targets of mutagenesis screens designed to detect immune response genes in living animals. An exception to this rule is dTAK1. Overexpression and dominant-negative studies indicated that dTAK1 can act as a JNK kinase kinase (Delaney, 2006 and references therein).
Previously characterized dTAK1 mutations, however, showed no apparent JNK-like phenotype, but failed to express Relish-dependent antimicrobial peptides, suggesting a role in the Imd pathway (Vidal, 2001). Previous epistasis analysis using the UAS/GAL4 overexpression system to ectopically express dTAK1 placed dTAK1 downstream of imd and upstream of the IKK complex in the Relish signaling pathway (Vidal, 2001). In vitro experiments implicated dTAK1 in the IKK-dependent phosphorylation of Relish in S2 cells (Delaney, 2006).
Evidence has been uncovered for a Relish-independent function of dTAK1 in the control of antimicrobial peptide gene expression. Several aspects of Relish activation appeared normal in infected dTAK1 mutant animals, including cleavage, nuclear localization, and promoter binding. Therefore whether JNK pathway components mediate dTAK1 function in the immune response was examined. Several lines of evidence are reported for dTAK1 acting through the JNK cascade in the innate immune response. First, overexpression of Hemipterous, a JNKK, rescued attacin and diptericin expression in dTAK1 mutant animals, whereas overexpression of the downstream Imd component Dredd did not. Second, it was found that expression of the Puckered (Puc) phosphatase, an inhibitor of JNK activity, suppressed the expression of antimicrobial peptide genes. To directly test for a JNK requirement in immune signaling, JNK mutant clones were induced in the fat body of larvae. Strikingly, diptericin, attacin, Metchnikowin, and Drosomycin expression was lost in the mutant tissue (Delaney, 2006).
It is concluded that the JNK pathway is required to mediate dTAK1 signaling during the Drosophila immune response. Furthermore, a model is proposed where the JNK and NF-kappaB signaling are both required to activate antimicrobial peptide gene expression during the immune response in the Drosophila fat body (Delaney, 2006).
The function of TAK1 in vertebrates has remained enigmatic. It was originally identified as a TGFβ-activated kinase, hence the name, in mammalian cell culture assays. However, follow-up work in multicellular contexts and in vivo analyses in vertebrates, C. elegans, and Drosophila have shown no clear link to TGFβ signaling, but rather suggest a role for TAK family kinases in JNK activation or as upstream activators of Nemo-like kinases. In mammalian systems, TAK1 is one of a number of kinases that can activate IKK complexes and, consequently, NF-kappaB signaling in vitro. In vitro studies of human cells have shown that targeting of TAK1 by RNAi reduces NF-kappaB activation by TNFalpha and IL-1 stimulation. Recent studies using fibroblasts derived from TAK1 mutant mouse embryos and mice with a B-cell-specific deletion of TAK1 showed that JNK activation was impaired in response to all stimuli tested in TAK1 mutant cells. Although NF-kappaB activation was impaired in response to stimulation by IL-1β, TNF, and TLR3 and TLR4 ligands, NF-kappaB activation by B-cell receptor or LT-β stimulation remained intact, suggesting a specific role for TAK1 upstream of IKKβ and JNK, but not IKKalpha. Interestingly, IKKalpha activation leads to the phosphorylation and processing of NF-kappaB2 from the p100 to the active p52 form, reminiscent of Relish activation in Drosophila (Delaney, 2006 and references therein).
Biochemical analyses in mammalian systems have demonstrated that TAK1 functions in multimeric protein complexes that can include TAB1, TAB2, and different TRAF proteins. The exact composition of these complexes seems to determine TAK1 responsiveness and downstream effects. In the fly, genetic studies found an interaction between dTRAF1 and dTAK1 in the activation of JNK signaling and apoptosis. Gain- and loss-of-function analyses indicate that dTRAF2, but not dTRAF1, is necessary for the activation of Relish-dependent gene expression; however, no interaction between dTRAF2 and dTAK1 in the activation of antimicrobial peptides has been reported (Delaney, 2006).
Genome-wide analyses that examined in vivo responses in Drosophila identified dJun and puc as genes potentially regulated by Toll and Imd signaling, suggesting a cross-regulation between these pathways and the JNK signaling pathway. A study recently reported that RNAi knockdown of kayak, msn, hep, or aop blocked E. coli-induced attacin and drosomycin expression in S2 cells. Furthermore, in related studies, it was also observed that, although dTAK1 RNAi-treated S2 cells failed to express an attacin reporter gene, Relish cleavage and nuclear localization remain intact in these cells. Other RNAi analyses in S2 cells have concluded that JNK signaling does not have a significant role in antimicrobial peptide gene expression. However, RNAi against hep or bsk seemed to partially block antimicrobial peptide induction, especially of attacin and cecropinA and, accordingly, attacinD levels were lower in microarrays when the JNK pathway was blocked. The current results confirm a positive role for JNK signaling in the antimicrobial peptide response in vivo (Delaney, 2006).
The placement of dTAK1 function upstream of JNK, rather than IKK, requires a remodeling of the signaling pathways that activate the antimicrobial peptide genes. Earlier models were based on studies that showed that dTAK1 mutations blocked the constitutive activation of diptericin by Imd overexpression (Vidal, 2001). In turn, IKK mutations blocked dTAK1-induced diptericin expression. One interpretation of these data places IKK directly downstream of dTAK1. However, if the activation of both JNK and IKK signaling pathways is required, then a disruption in either branch would be sufficient to suppress any upstream activation (Delaney, 2006).
Overexpression of dTAK1 is sufficient to induce antimicrobial peptide expression (Vidal, 2001). However, dTAK1 is an extremely potent activator of JNK signaling and apoptosis, and overexpression of dTAK1 could activate proteins that are not normal phosphorylation targets. Based on RNAi studies in S2 cells, dTAK1 is required for dIKK complex-dependent phosphorylation of Relish in vitro. This could reflect a stringent requirement for dTAK1 in blood cell-derived S2 cells that is different in fat body tissue (Delaney, 2006).
The new model would predict that overexpression of the Dredd caspase would be insufficient to activate fully the antimicrobial peptides in dTAK1 mutant animals and this is indeed the case. Overexpression of Dredd may be sufficient to induce antimicrobial peptide gene expression in a wild-type background because of inadvertent JNK pathway activation by ectopic caspase activity or by the heat-shock protocol itself. Alternatively, an additional role for Dredd has been proposed in the ubiquitin-mediated activation of dTAK1 and the dIKK complex (Zhou, 2005). The suppression by dTAK1 mutants of ectopic Dredd expression is consistent with this model as well, and does not distinguish between the two potential functions of Dredd. The current data are consistent with a model that places dTAK1 activity in a pathway parallel to the functions of IKK and Relish and in which both these pathways are required for the activation of antibacterial peptide genes such as diptericin and attacin (Delaney, 2006).
Promoter analyses of most antimicrobial peptide genes have not revealed any obvious binding sites for activator protein-1 (AP-1) complexes, the Jun/Fos heterodimer, and transcriptional mediator of JNK signaling. However, AP-1 binding sites can be quite diverse and are not always predictable directly from DNA sequence. Nevertheless, a recent study identified a functional AP-1 binding site in the attacinA promoter. These data suggest that AP-1 binding represses attacinA transcription by recruiting histone deacetylase 1 (dHDAC1) to the promoter. In contrast, in mammalian studies, c-Jun function is itself repressed by association with HDAC3. This repression is relieved upon JNK signaling. A similar mechanism may be employed in the Drosophila fat body. Accordingly, the sustained expression of attacin and other antimicrobial peptide genes in vivo would require an activation (or de-repression) of AP-1 function at the onset of the immune response. Such positive cooperation between AP-1 and NF-kappaB transcription factors was also seen in molecular studies of the human β-defensin-2 promoter (Delaney, 2006).
AP-1-dependent gene expression is normally rapid. Thus, if AP-1 activity is not directly required for diptericin expression, it could act indirectly through the activation of other genes. Alternatively, JNK could phosphorylate some targets other than the AP-1 complex proteins Jun and Fos. In mammalian studies, it has been shown that JNK can phosphorylate, and thereby inhibit, Insulin Receptor Substrate-1. However, the recent finding that RNAi against kayak/dFos can block antimicrobial peptide expression and the current dJun loss-of-function studies in vivo suggest that JNK does indeed signal through AP-1 to control expression of these genes (Delaney, 2006).
It is intriguing that overexpressed Puc not only blocks Relish-dependent antimicrobial peptide gene expression, but it also strongly blocks drosomycin expression, which is not true in dTAK1 mutants. This suggests that JNK or JNK-related proteins, for example, p38a, p38b, and MPK2, may also be important for other aspects of the immune response, for example, the Toll/Dif-dependent antimicrobial genes. The clonal analysis of JNK mutant tissue confirms that JNK is required not only for the expression of Gram--specific peptides diptericin and attacin, but also for Metchnikowin (Gram+/fungal specific) and drosomycin (fungal specific). Mutations in dTAK1 had less of an impact on Metchnikowin or drosomycin expression than on attacin, for example. Furthermore, reduced dJun activation occurred in dTAK1 mutant animals, indicating that other upstream kinases may be involved in the control of these genes. JNK is a member of a large family of mitogen-activated protein kinases (MAPKs). In the fly, there are at least five MAPKKKs, four MAPKKs, and five MAPKs, and so the potential redundancies are many. If these other proteins contribute to the immune response, how they do so has yet to be tested in genetic loss-of-function in vivo studies in the fat body (Delaney, 2006).
How JNK and NF-kappaB signals integrate to positively control gene expression is a critical question. This study has demonstrated that both are required for the expression of a particular set of immune responsive genes in vivo. Through the use of Drosophila genetics, it should be possible to identify novel immune response genes that are controlled cooperatively by JNK and NF-kappaB signaling. From promoter analysis of these genes, it may be possible to predict additional genes that are important for other biological processes. Both the JNK and NF-kappaB signaling pathways have been implicated many times in many different contexts. Continued analysis in Drosophila may lead to a general understanding of their roles in normal biological processes and developmental malignancies (Delaney, 2006).
CYLD encodes a tumor suppressor that is mutated in familial cylindromatosis. Despite biochemical and cell culture studies, the physiological functions of CYLD in animal development and tumorigenesis remain poorly understood. To address these questions, Drosophila CYLD (dCYLD) mutant and transgenic flies were generated expressing wild-type and mutant dCYLD proteins. dCYLD is essential for JNK-dependent oxidative stress resistance and normal lifespan. Furthermore, dCYLD regulates TNF-induced JNK activation and cell death through dTRAF2, which acts downstream of the TNF receptor Wengen and upstream of the JNKK kinase dTAK1. dCYLD encodes a deubiquitinating enzyme that deubiquitinates dTRAF2 and prevents dTRAF2 from ubiquitin-mediated proteolytic degradation. These data provide a molecular mechanism for the tumor suppressor function of this evolutionary conserved molecule by indicating that dCYLD plays a critical role in modulating TNF-JNK-mediated cell death (Xue, 2007).
Shortened animal lifespan may result from compromised oxidative stress tolerance. To examine the oxidative stress resistance, 3-day-old flies were challenged with paraquat for a prolonged period of time and their survival rates were measured. It was found that dCYLD mutants exhibited a significant reduction in survival rate as compared with wild-type or heterozygous dCYLD flies after 24 hr or 36 hr of exposure to paraquat, suggesting dCYLD plays a pivotal role in regulating oxidative stress resistance (Xue, 2007).
JNK signaling has been reported to play an important role in regulating oxidative stress resistance and lifespan in Drosophila (Wang, 2003). Ubiquitous expression of Bsk, the Drosophila JNK ortholog, under the control of tubulin promoter, rescues both lifespan and oxidative stress resistance defects in dCYLD mutants, suggesting that dCYLD regulates these two physiological effects through the JNK signaling pathway (Xue, 2007).
This study was extended to other stress conditions and it was found that dCYLD mutants are less resistant to dry starvation (no food and water), a phenotype that has been associated with reduced JNK activity (Wang, 2003). In contrast, dCYLD mutants do not affect animal survival at high and low temperature conditions (Xue, 2007).
To further examine the role of dCYLD in regulating JNK signaling in animal development, the genetic interactions between dCYLD and Eiger (Egr), the Drosophila ortholog of TNF that triggers the JNK pathway, was tested. Ectopic expression of Egr, under the control of the GMR promoter (GMR > Egr) and using the Gal4/UAS binary system, induces JNK activation and cell death in the developing eye that results in vastly reduced adult eye size. The Egr-induced JNK activation and small-eye phenotype was suppressed modestly by deleting one copy of dCYLD and suppressed soundly by removing both copies. The strong suppression of the Egr eye phenotype in homozygous dCYLD mutants was partially reverted by adding one copy of dCYLDRes. These results indicate that dCYLD is required for Egr-triggered JNK activation and cell death (Xue, 2007).
dCYLD encodes a protein of 640 amino acids, containing in its N terminal portion a cytoskeleton-associated protein (CAP) domain that is present in proteins associated with microtubules and the cytoskeletal network, two ubiquitin carboxyl-terminal hydrolases (UCH) domains that are commonly associated with deubiquitinating enzyme activity, and three CXXC zinc-finger (ZF) motifs with potential protein-protein interaction ability. To functionally characterize these motifs, UAS transgenes were generated expressing the wild-type or three mutant versions of dCYLD that delete the CAP domain, the two UCH domains, or the three ZF motifs. When expressed under the control of the GMR promoter, neither the full-length nor the dCYLD mutants displayed any detectable phenotype. When introduced into the GMR > Egr; dCYLD−/− background, wild-type dCYLD released the suppression of the Egr eye phenotype, confirming that the suppressive effect was due to the loss of dCYLD functions. In contrast, dCYLDΔUCH had no effect on the suppression of the Egr eye phenotype, and dCYLDΔCAP could only partially relieve this suppression, implying that the UCH domains are necessary for dCYLD functions and that the CAP domain is essential for dCYLD to execute its full activity in vivo. Interestingly, expression of dCYLDΔZF completely abolished the suppression effect, suggesting that the ZF motifs are dispensable in dCYLD regulation of Egr-induced cell death (Xue, 2007).
Ubiquitous expression of the full-length dCYLD, but not dCYLDΔUCH, rescues both shortened lifespan and hypersensitivity to paraquat in dCYLD mutants, suggesting that the deubiquitinating activity is indispensable for dCYLD to regulate JNK-dependent oxidative stress resistance and lifespan (Xue, 2007).
TNF receptor-associated factors (TRAFs) are important adaptor proteins that bind to TNF receptors and relay TNF signals to the JNK and NF-κB pathways in mammals. In Drosophila, Egr signal is mediated exclusively by the JNK pathway. However, the role of Drosophila TRAF proteins in Egr-JNK signaling remains unclear. The Drosophila genome encodes two TRAFs: dTRAF1, the TRAF2 ortholog; and dTRAF2, the TRAF6 ortholog. To determine the role of dTRAF1 and dTRAF2 in Egr-JNK signaling, the effects were examined of loss-of-function mutations and RNAi-mediated downregulation of dTRAF1 or dTRAF2 on the Egr eye phenotype. The Egr-induced small-eye phenotype was not suppressed by either deletion of one copy of the dTRAF1 gene or coexpression of a dTRAF1 RNAi. In contrast, the Egr eye phenotype was suppressed strongly by removing half of the dosage of dTRAF2 and suppressed completely by deleting the dTRAF2 gene. Consistently, coexpression of a dTRAF2 RNAi significantly suppressed the Egr eye phenotype. In agreement with genetic data, dTRAF2 exhibited a much stronger physical interaction with Wgn and dTAK1 than did dTRAF1 . Together, the above results point to dTRAF2, but not dTRAF1, as the adaptor protein that mediates Egr signaling in Drosophila (Xue, 2007).
To investigate the physiological functions of dCYLD and dTRAF2 in JNK activation, the expression pattern of puckered (puc) was checked in dCYLD or dTRAF2 mutants. puc encodes a JNK phosphatase whose expression is positively regulated by the JNK pathway, and thus, the puc-LacZ expression of the pucE69 enhancer-trap allele can be used as a readout of JNK activity in vivo. puc is weakly expressed in wild-type third-instar eye discs, and can be detected by prolonged staining. It has been previously shown that puc expression posterior to the morphogenetic furrow (MF) depends on endogenous Egr signaling. This study found that such expression patterns are reduced dramatically in dCYLD mutants and dTRAF2 RNAi animals. In contrast, puc expression in the disc margin, which is independent of Egr signaling, was not affected. GMR > Egr strongly activated puc transcription posterior to the MF. This ectopic Egr-induced puc expression was largely blocked by loss of dCYLD or by expression of dTRAF2 RNAi. Taken together, these observations indicate that both dCYLD and dTRAF2 are physiologically required by the endogenous JNK pathway (Xue, 2007).
The role of CYLD in modulating JNK signaling in mammalian cells has remained controversial. Consistent with the current observation, it was reported that JNK activity diminished in Cyld−/− thymocytes, which implies that CYLD is physiologically required for JNK activation. However, CYLD was also reported to negatively regulate JNK signaling in culture cells and macrophages. Thus, CYLD could positively or negatively regulate JNK signaling in a cell-type-specific manner (Xue, 2007).
To genetically map the epistasis of dCYLD and dTRAF2 in the Egr-JNK pathway, the genetic interaction between dTAK1 (JNKKK) and dCYLD or dTRAF2 was examined in the developing eyes. Expression of dTAK1 under the control of the GMR promoter resulted in pupa lethality, while dTAK1 expression under the control of the sevenless (sev) promoter (sev > dTAK1) induced extensive cell death in larval eye discs and gave rise to rough eyes with a reduced size. Loss of dCYLD or dTRAF2, or coexpression of a dTRAF2 RNAi, had no effect on the sev > dTAK1 phenotype, while removal of one copy of hep (JNKK) or bsk (JNK) partially suppressed the sev > dTAK1 phenotype, suggesting that dCYLD and dTRAF2 operate upstream of dTAK1 in the Egr-JNK pathway (Xue, 2007).
Ectopic Egr expression in the dorsal thorax driven by the potent pannier-GAL4 driver resulted in pupa lethality. However, when reared at 18°C, these animals survived to adulthood, presumably due to lessened Egr expression caused by reduced Gal4 activity, and produced a small-scutellum phenotype. This phenotype could be suppressed by RNAi inactivation of JNK signaling components, e.g., wgn, dTRAF2, dTAK1, hep, or bsk, suggesting that the phenotype is caused by activation of JNK signaling. Ectopic expression of dCYLD driven by pannier-GAL4 produced a similar but weaker phenotype, which could be fully suppressed by the coexpression of an RNAi of dTRAF2 and dTAK1, but not that of wgn. These results indicate that dCYLD functions downstream of Wgn, but upstream of dTRAF2 and dTAK1, in modulating JNK signaling (Xue, 2007).
RNAi-mediated downregulation of dTRAF2, but not dTRAF1, resulted in compromised oxidative stress resistance and shortened lifespan, suggesting that the role of dTRAF2 in dCYLD-JNK signaling has been conserved in different physiological contexts (Xue, 2007).
Previous studies have reported that the Shark tyrosine kinase and Src42A regulate JNK signaling in epidermal closure during embryogenesis and metamorphosis. However, null mutants for egr and dCYLD are fully viable and do not display the epidermal closure defect, implying that Shark and Src42A act in parallel to Egr and dCYLD in modulating JNK signaling. Consistent with this interpretation, loss of shark or src42A failed to suppress the GMR > Egr or pnr > dCYLD phenotype. In addition, it was found that loss of the transcription factor dFOXO could suppress both GMR > Egr and pnr > dCYLD phenotypes, suggesting that dFOXO acts downstream of dCYLD in JNK signaling. Consistent with this observation, dFOXO is required downstream of JNK in modulating cell death, oxidative stress resistance, and lifespan (Xue, 2007).
Overexpression of CYLD in mammalian tissue culture cells negatively regulates NF-κB signaling by deubiquitinating TRAF2/6. dCYLD, like its mammalian counterpart, contains two UCH deubiquitinating domains. Indeed, genetic analysis revealed that the UCH deubiquitinating domains are crucial for the in vivo functions of dCYLD. Furthermore, genetic epistasis data show that dCYLD acts upstream of dTRAF2 in the JNK pathway. Thus, it was hypothesized that dCYLD might act in the JNK pathway to deubiquitinate and subsequently stabilize dTRAF2 by preventing its ubiquitination-mediated proteolytic degradation. To examine this hypothesis in vivo, a FLAG-tagged dTRAF2 transgene (GMR > FLAG-dTRAF2) was introduced into dCYLD mutants and transgenic flies. Proteins were extracted from the heads of these flies for biochemical analyses. It was found that loss of dCYLD resulted in a significant reduction in dTRAF2 protein level, while the ubiquitination of dTRAF2 was markedly enhanced. Both changes were suppressed by dCYLDRes. Consistently, overexpression of dCYLD, but not dCYLDΔUCH, increased dTRAF2 protein level and decreased its ubiquitination. These results show that dCYLD functions as a deubiquitinating enzyme that deubiquitinates dTRAF2 and promotes dTRAF2 accumulation in vivo (Xue, 2007).
Polyubiquitination chains are usually formed on two lysine residues, K48 and K63. It is generally believed that the K48-linked polyubiquitination mediates proteasome-dependent protein degradation, while the K63-linked polyubiquitination mediates endocytosis and signal transduction. Previous work in mammalian culture cells has implicated that CYLD encodes a deubiquitinating enzyme that preferentially cleaves K63-linked polyubiquitin chain from its target proteins for NF-κB signaling. However, a recent in vivo study in Cyld−/− mice has reported that CYLD could regulate the stability of its target protein by effectively removing K48-linked polyubiquitin chain in thymocytes. Interestingly, JNK activity also diminished in Cyld−/− thymocytes. Thus, the role of CYLD in regulating protein stability and positively modulating JNK signaling could be conserved in mammals (Xue, 2007).
CYLD mutations in human patients cause dramatic skin tumors. However, the physiological function of CYLD and the mechanism underlying CYLD deficiency-induced tumorigenesis remain largely unknown. By generating the null dCYLD mutation and dCYLD transgenic animals and performing genetic analysis, this study has shown that dCYLD is a critical regulatory component of the JNK signaling pathway. Genetic epistasis and biochemical analysis further reveal that dCYLD modulates JNK signaling by deubiquitinating dTRAF2 and thus preventing dTRAF2 from ubiquitination-mediated proteolytic degradation. Loss of dCYLD results in augmented ubiquitination and degradation of dTRAF2, which renders cells resistant to apoptosis triggered by JNK signaling. Deregulation of apoptosis has been implicated as a major cause of tumorigenesis. Consistently, mice deficient for both jnk1 and jnk2 were resistant to apoptosis induced by UV irradiation, anisomycin, and MMS, and jnk1−/− mice exhibited enhanced skin tumor development, a phenotype that is pathogenically similar to cylindromatosis in CYLD human patients. Together these data argue that modulation of JNK signaling could be a conserved mechanism underlying familial cylindromatosis in CYLD patients (Xue, 2007).
Stimulation of the Drosophila immune response activates NF-kappaB and JNK signaling pathways. For example, infection by Gram-negative bacteria induces the Imd signaling pathway, leading to the activation of the NF-kappaB-like transcription factor Relish and the expression of a battery of genes encoding antimicrobial peptides. Bacterial infection also activates the JNK pathway, but the role of this pathway in the immune response has not yet been established. Genetic experiments suggest that the Drosophila homolog of the mammalian MAPK kinase kinase, TAK1 (transforming growth factor ß-activated kinase 1), activates both the JNK and NF-kappaB pathways following immune stimulation. Drosophila TAK1 functions as both the Drosophila IkappaB kinase-activating kinase and the JNK kinase-activating kinase. However, JNK signaling is not required for antimicrobial peptide gene expression but is required for the activation of other immune inducible genes, including Punch, Sulfated, and Malvolio (Mlv). Thus, JNK signaling appears to play an important role in the cellular immune response and the stress response (Silverman, 2003).
In the fly, the NF-kappaB homolog Relish is required for the induction of antimicrobial peptides in response to Gram negative bacterial infection, and relish mutants are hyper-susceptible to Gram-negative bacterial infections. Relish is a bipartite protein with an NF-kappaB (Rel homology) domain and an inhibitory IkappaB domain. In unstimulated cells, Relish is present in the cytoplasm as a full-length precursor protein and, upon infection, is endoproteolytically cleaved. The N-terminal Rel homology domain of Relish then translocates into the nucleus and activates antimicrobial gene expression, whereas the C-terminal IkappaB module remains in the cytoplasm. Signal-induced cleavage and activation of Relish requires a signaling pathway known as the Imd pathway, which includes the receptor PGRP-LC (peptidoglycan recognition protein LC), the intracellular signaling component Imd [a receptor interacting protein (RIP)-like death domain protein], TAK1 (a MAP3K), the Drosophila IKK complex (IKKß/Ird5 and IKKγ/Kenny), as well as the caspase Dredd and the adaptor known as the Fas-associated death domain (FADD). The detailed biochemical mechanisms required for this intracellular signaling pathway are not understood. Drosophila S2* cells have been used show that TAK1 is required for both LPS-induced JNK and IKK activation and for antimicrobial peptide gene induction. Surprisingly, the immune activation of the JNK pathway is not required for antimicrobial peptide gene induction. However, several other JNK-dependent, LPS-inducible genes were identified in microarray studies, suggesting a role for JNK signaling in the cellular immune response as well as protection from stress (Silverman, 2003).
To characterize the role of TAK1 in this pathway at a biochemical level, the Drosophila S2* cell line has been used. When these cells are treated with ecdysone, they differentiate and become responsive to LPS treatment, which leads to high levels of antimicrobial peptide gene expression. An additional advantage of this cell line is that RNAi can be used to target any gene of interest. Similar to the TAK1 mutant fly, targeting TAK1 with RNAi in S2* blocks LPS-mediated activation of antibacterial peptide gene expression, similar to that observed with IKKγ RNAi. TAK1 is unique among the Drosophila MAP3Ks tested because it is required for LPS signaling, whereas slpr and Drosophila MEKK4 are not (Silverman, 2003).
In mammals, TAK1 has been shown to play a critical role in IL-1-induced NF-kappaB activation. In vitro, TAK1 can directly phosphorylate and activate the human IKK complex. Thus, TAK1 has been proposed to function as the IKK activating kinase (IKK-K) in this signaling pathway. The immuno-compromised phenotype of TAK1 mutant flies and S2* cells suggest that TAK1 may function as the IKK-K in the insect immune response. To determine whether TAK1 function is required for IKK activation, an immunoprecipitation kinase assay was utilized. The endogenous Drosophila IKK complex was immunoprecipitated with anti-DmIKKgamma antisera from cell lysates prepared from LPS-treated or untreated cells. This immunoprecipitate was then tested for kinase activity in vitro using recombinant Relish as substrate. LPS treatment leads to a significant increase in Drosophila IKK activity. Treatment of cells with TAK1 RNAi inhibits the LPS-induced IKK activation as much as targeting DmIKKgamma itself. It is concluded that TAK1 is required for LPS-induced activation of the Drosophila IKK complex the subsequent expression of antimicrobial gene expression (Silverman, 2003).
The Drosophila TAK1 protein plays a critical role in the activation of the insect immune response. Genetic studies revealed that TAK1 mutant flies are unable to respond to Gram-negative infections and suggests that TAK1 functions upstream of the Drosophila IKK complex. Consistent with these results, TAK1 is shown to be required for activation of the LPS-induced immune signaling pathways in Drosophila cells in culture. In addition, TAK1 is required for activation of the Drosophila IKK complex in vitro. Thus, Drosophila TAK1 is likely to function as the IKK-K in the LPS signaling pathway, as has been proposed for human TAK1 (Silverman, 2003).
JNK signaling is also activated during the immune response in both flies and humans. However, the exact mechanism by which LPS leads to JNK activation in Drosophila is unclear, as is the role of JNK signaling during the immune response. Gene expression profiling has been used to infer that TAK1 is required for the activation of JNK signaling and that JNK signaling is important for wound healing. This study directly demonstrates that JNK activation requires TAK1. Thus, TAK1 appears to function both as a JNKK activating kinase and an IKK activating kinase, as proposed for mammalian TAK1. Furthermore, microarray results suggest that JNK signaling may have important functions in cellular immunity and the stress response (Silverman, 2003).
The gene expression profiling data presented in this study identifies a relatively small number of genes that specifically require the JNK signaling pathway for their LPS-induced expression. The expression of two genes (Punch and sulfated) identified in these experiments has been validated by real time RT-PCR. Punch is an immune inducible gene in cells in culture and in adult flies. However, although it has been shown that in adult flies the immune induction of Punch requires Relish (De Gregorio, 2001), the data presented in this study demonstrate that Punch induction in S2 cells requires JNK pathway components (hep, bsk, and TAK1) but not the Relish-activating kinase IKK. The experiments presented here were performed in an embryonic Drosophila cell line (that has macrophage-like qualities), whereas the data from De Gregorio (2001) was generated from entire adult flies. Thus, it is possible that the signaling pathways required for Punch induction vary depending on the developmental stage and cell type examined. In fact, Punch has at least two promoters that direct developmentally specific expression (Silverman, 2003).
Punch encodes the enzyme for GTP cyclohydrolase I, which is the first enzyme (and rate-determining step) in the formation of the cofactor tetrahydrobiopterin (BH4). This cofactor is required for the conversion of tyrosine to dopamine, which has at least two possible roles in immunity: (1) dopamine is one of two main Drosophila catecholamines, which are important for the stress response in both insects and mammals; (2) dopamine is the precursor of melanin, which is produced during wound healing and encapsulation processes in the fly. In fact, it has been proposed that increased Punch activity could lead to increased melanization (Silverman, 2003 and references therein).
The cofactor BH4 is also an essential cofactor for nitric oxide synthase (NOS). NO itself has at least two possible roles in the immune response: (1) NO is known to be a major microbicidal compound in mammalian phagocytic cells and is likely to function similarly in Drosophila macrophages; (2) NO has also been implicated in immune signaling in Drosophila. NO is required for transmitting a signal from the site of infection to the fat body, the major organ of immune responsive gene expression. Thus, Punch may contribute to the insect immune response in several ways, including protection against stress, melanization of wound sites, and activation of cellular and humoral immunity (Silverman, 2003 and references therein).
The potential role of sulfated in the immune response is less obvious. sulfated encodes an extracellular sulfatase that removes sulfate groups from heparin sulfate proteoglycans (HSPGs). In avian and Drosophila systems, it is thought that sulfated activity is crucial for the regulation of Wnt signaling, possibly by controlling the extracellular milieu in which the Wnt ligand travels (Silverman, 2003).
One of the most intriguing targets of both the JNK and IKK pathways is Mvl, the Drosophila NRAMP-1 homolog. Mvl mutants were first identified in the fly because they display gustatory behavioral defects caused by the inability to properly process sensory neuronal input. Mvl is expressed in both the nervous system and circulating hemocytes. In the mouse, NRAMP-1 is expressed in macrophages, and mutations in the NRAMP-1 gene are responsible for the sensitivity of some inbred mice strains to the Mycobacterium bovis bacille Calmette-Guérin (BCG) and other intracellular bacterial pathogens. NRAMP-1 is thought to control the levels of cations, possibly Fe2+ or Mn2+, in lysosomal compartments of mouse macrophages. A current model suggests that NRAMP-1 pumps cations out of the phagolysosome, thereby starving microbes of cations required by the enzymes (superoxide dismutase and catalase), which protect the bacteria from reactive oxygen intermediate (ROI)- and reactive nitrogen intermediate (RNI)-induced damage. In the fly, the role of Mvl in immunity is not yet characterized, but its induction during an immune response coupled with the activity of this protein in vertebrate macrophages suggests that it may play an important role in the cellular immune response (Silverman, 2003).
Microarray analysis has provided evidence that LPS-induced JNK activation is important for the stimulation of a gene expression program similar to that seen during dorsal closure. Thus, JNK may be important for wound healing. Expression of only a few of the JNK target genes (for example, Filamin) is reported in this study. The data argue that JNK signaling is required for the activation of cellular immunity and stress protection, whereas a connection to wound healing cannot be excluded by these data (Silverman, 2003).
Certain antimicrobial genes (e.g., diptericin) require a combination of transcription factors for their proper induction. It has been suggested, based on DNA footprinting and DNA sequence analysis, that Diptericin activation requires a kappaB binding site (now believed to be the site of Relish binding) as well as putative NF-IL6-like, and interferon regulatory factor (IRF)-like binding sites. However, of these only Relish is required for the immune inducible expression of diptericin. The data presented here show that the JNK signaling pathway and the AP-1-like factors activated by Drosophila JNK signaling are not involved in antimicrobial peptide gene induction in phagocytes. This would be quite different from immune activation of many mammalian cytokine genes, which require the coordination of several signaling pathways and the activity of several transcription factors for full immune induction. For example, IFN-ß induction requires the activation of three independent signaling cascades and the cooperative binding of three transcription factors, NF-kappaB, c-Jun/activating transcription factor 2 (ATF-2), and the interferon regulatory factor, to the enhancer region. Together, these transcription factors form a higher order complex known as the enhanceosome. Control of the insect antimicrobial genes may not require this complex enhancer architecture (Silverman, 2003).
These studies clearly demonstrate that activation of the innate immune response in Drosophila leads to the activation of JNK and NF-kappaB signaling pathways through a branched signal transduction cascade. The MAP3K TAK1 lies at the branch point of this cascade and likely functions as the JNKK activating kinase and the IKK activating kinase. These signaling pathways are highly conserved. TAK1 also serves similar functions in mammalian innate immune signaling. Furthermore, novel immune-induced targets of the JNK pathway have been identified, that may function in cellular immunity and stress protection (Silverman, 2003).
The molecular circuitry underlying innate immunity is constructed of multiple, evolutionarily conserved signaling modules with distinct regulatory targets. The MAP kinases and the IKK-NF-kappaB molecules play important roles in the initiation of immune effector responses. The Drosophila NF-kappaB protein Relish plays a crucial role in limiting the duration of JNK activation and output in response to Gram-negative infections. Relish activation is linked to proteasomal degradation of TAK1, the upstream MAP kinase kinase kinase required for JNK activation. Degradation of TAK1 leads to a rapid termination of JNK signaling, resulting in a transient JNK-dependent response that precedes the sustained induction of Relish-dependent innate immune loci. Because the IKK-NF-kappaB module also negatively regulates JNK activation in mammals, thereby controlling inflammation-induced apoptosis, the regulatory cross-talk between the JNK and NF-kappaB pathways appears to be broadly conserved (Park, 2004).
The JNK and IKK/Relish branches of the Imd pathway mediate distinct gene induction responses in Drosophila innate immunity. After diverging downstream from TAK1, these two signaling cascades regulate two separate groups of target genes that are distinct in their induction kinetics and function. The IKK/Relish targets have been extensively characterized and most encode products whose role in innate immunity is relatively well established. In contrast, the JNK-regulated, LPS-responsive genes represent a largely uncharacterized set of loci whose function in innate immunity is not clear. These genes exhibit transient induction kinetics, reaching a maximum ~1 h after induction. It was found that the transient kinetics of the JNK target genes is controlled by the transient kinetics of the JNK module of the Imd pathway and that the IKK/Relish branch plays an active role in turning off JNK activity. Hence, the two seemingly independent branches of the Imd pathway are wired in such a way as to coordinate the temporal order of individual responses (Park, 2004).
The evidence indicates that Relish-mediated JNK inhibition involves proteasomal degradation of TAK1, the MAPKKK responsible for JNK activation in response to LPS. Treatment with proteasomal inhibitors or RNAi against a component of the proteasome complex results in sustained JNK activation during the LPS response. Furthermore, in cells expressing constitutively active Relish, the stability of TAK1 is greatly decreased. Based on these findings, it is suggested that certain targets of Relish that are induced during immune responses facilitate destruction of TAK1 and switch off the JNK cascade. The fact that cycloheximide and actinomycin D also block the down-regulation of JNK activity indicates the involvement of targets of Relish rather than Relish itself. The Relish target involved in this cross-talk likely increases the susceptibility of TAK1 to proteasomal degradation by direct targeting of TAK1 or by antagonizing factors responsible for TAK1 stabilization (Park, 2004).
In considering this model, it should be noted that TAK1 is critically required for activating both IKK and JNK. Elimination of TAK1 during the LPS response thus turns off both of the downstream signaling cascades. Yet IKK/Relish target genes do not show a transient expression pattern, whereas JNK targets do. One possible explanation for this discrepancy lies in the nature of JNK and Relish activation. JNK is activated through its phosphorylation, a modification that is highly reversible. Thus, termination of the input that contributes to JNK phosphorylation is sufficient to result in its rapid inactivation, especially when one of the JNK targets encode a JNK phosphatase, Puckered. Relish, in contrast, is activated through proteolysis, an irreversible modification. Once activated Relish enters the nucleus, it may remain bound to its target genes for some time even after termination of the upstream signal (Park, 2004).
Interestingly, the antagonism between NF-kappaB and JNK signaling is evolutionarily conserved. In mice, inactivation of either IKKß or NF-kappaB RelA (p65) in cells leads to a sustained JNK activation in response to TNFalpha. The sustained JNK activation by TNFalpha has been associated with TNFalpha-induced apoptosis. Several independent studies have proposed different molecules as mediators for the NF-kappaB inhibition of JNK signaling in the TNFalpha pathway including XIAP, GADD45ß, and reactive oxygen species. Nevertheless, the underlying mechanism remains largely unknown. As the Drosophila Imd pathway and the signaling pathway downstream of mammalian TNF receptor share many conserved features, a similar mechanism may govern the antagonistic relationship between IKK/NF-kappaB and JNK in both flies and mammals. Thus, the mechanistic insights gained from studies in Drosophila should be relevant when elucidating the mechanism that connects NF-kappaB to JNK signaling in mammals (Park, 2004).
The Imd signaling cascade, similar to the mammalian TNF-receptor pathway, controls antimicrobial peptide expression in Drosophila. A large-scale RNAi screen was performed to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. Seven gene products required for the Attacin response in vitro were identified, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1 (TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. These results show that an RNAi-based approach is suitable to identify genes in conserved signaling cascades (Kleino, 2005).
Drosophila has developed a highly sophisticated immune defense, which is required for living in a natural environment that is rich in bacteria and fungi. In contrast to mammals, Drosophila has no adaptive, that is, antibody-mediated immunity, which makes it a good model for studying the pattern recognition receptors and signaling pathways of innate immunity. In Drosophila, there are two major pathways that respond to microbes: the Imd and the Toll pathways. Both of them are strikingly well conserved throughout evolution. Thus, novel findings from work on Drosophila immune response can fuel discoveries in the mammalian systems (Kleino, 2005).
In Drosophila, evolutionarily conserved peptidoglycan recognition proteins (PGRPs) are of paramount importance for microbial recognition. Several Drosophila PGRPs are necessary for normal resistance to bacteria . Secreted PGRP-SA is essential for induction of immune response genes via the Toll pathway in response to certain Gram-positive bacteria in vivo. In contrast, PGRP-LC is the first component of the Imd pathway (Choe, 2002; Gottar, 2002; Rämet, 2002). It is located on the cell membrane where it appears to act as a pattern recognition receptor for bacteria either alone or together with other PGRPs (Takehana, 2004). Recently, intracellular domain of PGRP-LC was shown to bind directly to the Imd, which is the next known component downstream of PGRP-LC (Choe, 2005). Imd contains a death domain with homology to the mammalian receptor-interacting protein 1. The signal is propagated via transforming growth factor-activated kinase 1 (TAK1) to Drosophila homologs for IKKgamma and IKKalpha/ß (Key and Ird5, respectively) (Rutschmann, 2000; Lu, 2001). Whether TAK1 phosphorylates the Drosophila IKKs directly is uncertain and the mechanism of TAK1 activation is elusive. TAK1 has also been shown to play a role in the regulation of the c-Jun N-terminal kinase (JNK) pathway (Park, 2004). Finally, the signal leads to the activation of the Drosophila NF-kappaB homolog Relish, involving its phosphorylation by the IKK complex (Silverman, 2000) and cleavage by a caspase currently believed to be Dredd (Leulier, 2000: Stöven, 2000; Stöven, 2003), which forms a complex with BG4, a homolog to mammalian Fas-associated death domain protein (FADD; Leulier, 2002). The phosphorylated and cleaved Relish is then translocated to the nucleus, where it binds to DNA leading to synthesis of antimicrobial peptides (Kleino, 2005 and references therein).
Normal response to most Gram-negative bacteria in Drosophila depends on the Imd pathway, which is very similar to the TNF receptor signaling pathway in mammals. In order to determine whether there are still unknown components in the Imd signaling pathway, a large-scale RNAi-based screen was carried out in Drosophila S2 cells using a luciferase-reporter-based quantitative assay. The activity of the pathway was assayed using Attacin-luciferase (Att-luc) reporter. Transfection efficiency and cell viability were monitored using Act5C-ß-gal reporter. The Imd signaling pathway was activated with heat-killed Escherichia coli. At first, tests were carried out to see if dsRNA targeting a known component of the pathway caused a decrease in Att-luc activity. Relish (Rel) RNAi blocked the Imd pathway activity in a dose-dependent manner. 10 ng of Rel dsRNA per 5.0 x 105 S2 cells in 500 microl of medium reduced the luciferase activity by >50% and more than 0.1 microg of Rel dsRNA blocked the luciferase activity almost completely. Therefore, RNAi very effectively silences the expression of the targeted gene in this assay, which thus can be used to identify essential components of the Imd pathway (Kleino, 2005).
6713 dsRNAs from an S2 cell-derived cDNA library were assayed for their effect on the Imd signaling pathway in S2 cells using Att-luc reporter as a read-out. Most dsRNA treatments had little or no effect. Seven genes decreased Att-luc activity by >80% without decreasing Act5C-ß-gal activity by more than 40%, indicating that viability and the translation machinery were unaffected. These genes included three (PGRP-LC, imd and Rel) out of eight known components of the Imd pathway. Rel was identified three times. Novel genes identified were kayak, longitudinals lacking (lola), inhibitor of apoptosis 2 (Iap2) and CG7417. The CG7417 protein is a homolog to the mammalian TAK1-binding proteins 2 and 3 (TAB2 and TAB3), hereafter called TAB. Interestingly, a dsRNA treatment silencing Rel, TAB, PGRP-LC, imd or lola also strongly decreased the Drosomycin reporter (Drs-luc) activity induced by the constitutively active form of Toll (Toll10b). Therefore, it appears that a low level of Rel activity is required also for normal Drs response via the Toll pathway in S2 cells (Kleino, 2005).
Kayak is a known component of the JNK signaling pathway; RNAi targeting kayak caused an 88 +/- 7% decrease in Att-luc activity. This is in accordance with recent results, which indicate that JNK is essential for normal antimicrobial peptide release in S2 cells (Kallio, 2005). RNAi targeting lola caused 87 +/- 5% decrease in Att-luc activity. Lola is a nuclear factor that is required for axon growth in the Drosophila embryo and normal phagocytosis of bacteria in S2 cells (Rämet, 2002). Lola has not been indicated to play a role in the synthesis of antimicrobial peptides. In the reporter assay, lola RNAi decreased Att-luc activity slightly less than known components of the Imd pathway. Of note, RNAi silencing of lola also decreased Drs-luc activity induced by Toll10b in S2 cells (Kleino, 2005).
In all, 35 dsRNA treatments representing 22 genes caused a greater than three-fold increase in the Att-luc activity in response to heat-killed E. coli after ecdysone treatment in S2 cells. These genes could be divided into the following categories based on the putative function of their encoding protein: (1) genes involved in microtubule organization or actin cytoskeleton regulation (par-1, Rab-protein 11, multiple ankyrin repeats single KH domain [mask], alpha-Tubulin at 84B, CG6509 and PDGF- and VEGF receptor related [Pvr]); (2) helicases and other genes involved in DNA replication (Helicase 89B, Rm62, kismet, mutagen-sensitive 209 and double parked); (3) signaling molecules (daughter of sevenless, CG32782 and Ecdysone-induced protein 75B); (4) transcription factors (E2F transcription factor and Zn-finger homeodomain 1) and (5) uncharacterized genes. Of note, kismet was identified eight times, Pvr six times and E2F transcription factor twice in this screen. The mechanisms for how these genes affect signaling through the Imd pathway remain to be studied. Of note, none of these dsRNA treatments notably induced the Imd pathway without E. coli (Kleino, 2005).
Two novel components of the Imd pathway, Iap2 and TAB, were identified that appear to be absolutely necessary for induction of Att-luc activity in S2 cells in response to heat-killed E. coli. dsRNA targeting either Iap2 or TAB causes a drastic, 98 +/- 1% decrease in Att-luc activity. Iap2 or TAB RNAi has no effect on cell growth as determined by cell counts, indicating that the result is not due to increased cell death. To verify that the observed phenotypes were caused by decreased expression of Iap2 and TAB, targeted RNAi with gene-specific primers was carried out. Specific dsRNA treatments targeting either Iap2 or TAB drastically decreases the Att-luc activity. TAB RNAi also decreases the Drs reporter activity via the Toll10b-induced Toll pathway. Whether the effect of Iap2 or TAB RNAi was ecdysone dependent was examined. If ecdysone was not used, Att-luc induction was clearly (35 +/- 2%, N=3) weaker, but also this induction was blocked by RNAi targeting either Iap2 or TAB, indicating an ecdysone-independent mechanism (Kleino, 2005).
To ascertain that the results were not due to an artifact related to the use of a reporter construct, the expression level of Cecropin A1 (CecA1), another well-characterized antimicrobial gene regulated by the Imd pathway, was analyzed by semiquantitative RT-PCR. A 6-h exposure to heat-killed E. coli increased the mRNA level of CecA1. This increase could be blocked entirely by RNAi targeting either Rel or TAB. In addition, induction was reduced by RNAi targeting Iap2. Corresponding results were obtained also for Att D and Diptericin (Dpt). dsRNA treatments targeting either Rel or TAB totally blocked the induction, while the effect of Iap2 RNAi was somewhat more moderate (Kleino, 2005).
To investigate whether these in vitro findings are of in vivo relevance, the inducible expression of Iap2 dsRNA was used in Drosophila in vivo. The UAS/GAL4 binary system to drive expression of dsRNA in a defined tissue has been previously used to block the expression of defined genes. To this end, transgenic flies were generated carrying the UAS-Iap2-IR. This construct has two 500 bp long inverted repeats (IR) of the gene, separated by an unrelated DNA sequence that acts as a spacer, to give a hairpin-loop-shaped RNA. These transgenic flies were crossed to flies carrying various GAL4 drivers in order to activate transcription of the hairpin-encoding transgene in the progeny. Iap2 has been shown to be required for the regulation of apoptosis in Drosophila (F. Leulier, personal communication to Kleino, 2005), and overexpression of UAS-Iap2-IR with the ubiquitous and strong daughterless-GAL4 (da-GAL4) driver leads to lethality at the pupal stage. To address the role of Iap2 in antimicrobial gene expression, the UAS-Iap2-IR transgene was expressed using the C564-GAL4 driver that expresses GAL4 in the adult fat body. Flies were kept at 25°C to avoid the induction of apoptosis in the fat body. Flies that express Iap2-IR ubiquitously through C564 showed no detectable defects. However, the expression of the antibacterial peptide gene Dpt was strongly reduced after infection with the Gram-negative bacteria Erwinia carotovora. This phenotype was similar, although weaker, than those generated by BG4-IR RNAi. Importantly, the expression of Drs remained inducible in Iap2-IR; C564 flies, indicating that Iap2 did not block the Toll pathway and that the fat body remained functional (Kleino, 2005).
To map the locations of Iap2 and TAB in the Imd signaling cascade, known components of the cascade were overexpressed including a constitutively active form of Relish (Rel DeltaS29-S45), wild-type Relish or wild-type Imd. All these caused an activation of Att expression in S2 cells. Att induction caused by expression of either Relish construct could not be blocked by RNAi targeting either imd, TAB or Iap2, indicating that both TAB and Iap2 are located upstream of Relish. In contrast, Att induction caused by overexpression of Imd is blocked by RNAi targeting either Rel, imd, TAB or Iap2, indicating that both TAB and Iap2 lie downstream of Imd in the hemocyte-like S2 cells (Kleino, 2005).
To assess whether Iap2 is located downstream of Imd in the fat body in vivo, the UAS-imd construct with a heat-shock-GAL4 (hs-GAL4) driver was overexpressed; this induced expression of the Dpt gene in the absence of infection. Although there is some constitutive Dpt expression in these flies, the level of Dpt increases after heat shock. Using these flies, Dpt expression was reduced by coexpression of UAS-Iap2-IR by 44 +/- 8% (N=2) in these flies. Total RNA was extracted from unchallenged adult flies, collected 6 or 16 h after a heat shock (37°C, 1 h) and RT−PCR analysis was used to monitor the expression level of Dpt. This indicates that Iap2 functions, genetically, downstream of Imd in the fat body in vivo (Kleino, 2005).
To map the exact location of Iap2 in the Imd signaling cascade, Iap2 was overexpressed in S2 cells, which resulted in a minimal but reproducible induction of Att expression. This induction was completely blocked by dsRNAs targeting the known components of the Imd pathway, except dsRNAs targeting either imd or TAK1. These results indicate that Iap2 lies downstream of TAK1 in the Imd signaling pathway. To ascertain efficacies of the dsRNA treatments used, effect of the dsRNA treatments on E. coli-induced Att response was simultaneously measured. All of the dsRNA treatments strongly decreased the Att response, suggesting that the expression of targeted genes was effectively silenced. Of note, it was not possible to stimulate the Imd pathway with the expression vector containing the full-length cDNA of TAB (Kleino, 2005).
Upon Imd pathway activation, the NF-kappaB homolog Relish becomes phosphorylated by the IKK complex and thereafter cleaved by a caspase putatively thought to be Dredd. Finally, Relish is translocated to the nucleus. To study the role of TAB and Iap2 on Relish cleavage, Drosophila hemocyte-like mbn-2 and S2 cells were stimulated with commercial lipopolysaccharide (LPS) known to contain a bacterial component that activates the Imd pathway, followed by Western blotting with Relish antibody (alpha-C; Stöven, 2000). In unstimulated, GFP dsRNA-treated mbn-2 cells, most of Relish is uncleaved (Relish-110), whereas upon LPS stimulus, Relish cleavage is induced. As expected, in Dredd and key dsRNA-treated cells Relish cleavage was blocked. Interestingly, TAB, Iap2 or TAK1 dsRNA did not affect Relish cleavage. Similar results were obtained also in S2 cells. This points to a novel mechanism of regulation of Relish activity. There was no Relish detected in Rel dsRNA-treated cells, indicating that the half-life of REL-49 (C-terminal Relish cleavage product) is less than the duration of the dsRNA treatment. Of note, REL-49 was observed also in all Dredd dsRNA-treated cells. It is possible that after RNAi knockdown, there is a small amount of Dredd left, sufficient to cleave REL-110 in unstimulated cells. Alternatively, there is some constitutively cleaved Relish in cell lines and this cleavage is Dredd independent (Kleino, 2005).
To investigate whether Iap2 or TAB play a role in the nuclear localization of the activated Relish protein, dsRNA-treated S2 cells were stained with alpha-RHD antibody (Stöven, 2000). In GFP dsRNA-treated cells, Relish is translocated into the nucleus upon LPS stimulus. As expected, there is no nuclear staining of Relish in Dredd or key dsRNA-treated, LPS-stimulated S2 cells. Importantly, the nuclear translocation of Relish appears to be affected in both Iap2 and TAB dsRNA-treated cells compared to GFP dsRNA-treated controls. This suggests that cleavage of Relish is not sufficient for translocation of Relish to the nucleus but another, yet to be characterized signal that is propagated via Iap2 and TAB is required. Alternatively, a different staining pattern could be due to decreased stability of nuclear Relish or slower kinetics. Of note, compared to key and Dredd dsRNA-treated cells, very faint nuclear staining can be seen in Iap2 dsRNA-treated cells. Surprisingly, Relish nuclear localization was normal in TAK1 dsRNA-treated cells. This implies a possibility that the role of TAK1 in the Imd pathway signaling is downstream of translocation of Relish into the nucleus. Altogether, these results show that the regulation of Relish activity is more complex than previously thought. The involvement of TAB and Iap2 in nuclear localization but not cleavage of Relish indicates a novel mode of regulation in the Imd pathway (Kleino, 2005).
Iap2 codes for a 498 amino-acid (aa) protein that has three N-terminal BIR (baculovirus IAP repeat) domains and a C-terminal RING-finger (Really Interesting New Gene) domain. Drosophila Iap2 is well conserved throughout phylogeny and has high sequence similarity with many mammalian Iap2s, such as human, rat and mouse (E values 9 x 10-66, 2 x 10-66 and 3 x 10-66, respectively). Interestingly, the CARD (caspase recruitment domain), identified in apoptotic signaling proteins, is present in the mammalian homologs but missing from Drosophila. It has been shown that RING domain containing proteins, including IAPs, bind E2 ubiquitin-conjugating enzymes catalyzing the transfer of ubiquitin from E2 to a substrate, therefore acting as E3 ligases. Ubiquitination can lead to either proteasomal degradation, or, in the case of non-K48-linked polyubiquitination, to multiple outcomes such as activation or relocalization of the substrate protein. Human c-Iap2 is expressed most strongly in immune tissues including spleen and thymus and has been proposed to associate with TRAFs through its BIR domains (Rothe, 1995). However, in a luciferase assay, neither TRAF1 nor TRAF2 dsRNA treatment reduced the Imd pathway activity, indicating that TRAFs are not essential for Imd pathway activity in Drosophila S2 cells (Kleino, 2005).
The Drosophila TAB codes for an 831 aa protein that has an N-terminal CUE domain (97-139 aa) and a C-terminal zinc-finger (ZnF) domain (765-789 aa). Only two other Drosophila genes code for a CUE domain: CG2701, and CG12024. Their function is unknown. TAB is the only Drosophila protein with both CUE and ZnF domains. These domains are homologous to the respective domains in mammalian TABs. The CUE domain carries a ubiquitin-binding motif, whereas the ZnF domain has an alpha-helical coiled-coil region. It has been shown that Drosophila TAK1 binds TAB (CG7417) in a two-hybrid protein interaction system (Giot, 2003). In humans, the C-terminal coiled-coil domain of TAB3 mediates the association with TAK1, and it is also required for stimulation of TAB3 ubiquitination by TRAF6 (Ishitani, 2003c). Apart from these two domains, there is very little sequence similarity, suggesting that these domains are functionally important. Indeed, it has been shown that the ZnF domain and the CUE domain, to a lesser extent, of human TAB2 and TAB3 are important to NF-kappaB activation (Kanayama, 2004). The exact molecular mechanism by which Iap2 and TAB modulate signaling via the Imd pathway in Drosophila remains to be studied (Kleino, 2005).
This study identified two novel components of the Imd signaling cascade: Iap2 and TAB. Both of these have mammalian homologs, further indicating high conservation of this signaling cascade. TAB is an 831 aa protein that has conserved CUE and ZnF domains. As in mammals, it is plausible that TAB regulates TAK1 activity also in Drosophila, since TAB was the only protein to bind TAK1 in a two-hybrid protein interaction system (Giot, 2003). Both Iap2 and TAB are located downstream of Imd. Interestingly, Relish is cleaved appropriately without Iap2 or TAB, but there appears to be an effect to the transportation of Relish to the nucleus. Therefore, it is speculated that there is another, previously unidentified level of regulation needed for Relish activation. Since the RING domain-containing Iap2 is a putative E3 ligase, it is hypothesized that this regulation could involve ubiquitination of Relish -- or another protein regulating the activity of Relish -- by Iap2. Possible interaction of Iap2 with the other Imd pathway components remains to be studied (Kleino, 2005).
Surprisingly, Relish nuclear localization is normal in TAK1 dsRNA-treated cells. This implies a possibility that the role of TAK1 in the Imd pathway signaling is downstream of translocation of Relish into the nucleus. These results are in line with recent results from Delaney (in preparation, reported by Kleino, 2005), which indicate that Relish activation is intact in TAK1 mutant flies. Therefore, TAK1 may control the activity of another transcription factor -- possibly via the JNK pathway -- required for normal antimicrobial peptide response in Drosophila. This is in line with identification of Kayak as an important factor for Att response in this study and with earlier results indicating that the JNK pathway is required for normal Att response in S2 cells (Kallio, 2005). Of note, the effect of dsRNA treatments targeting JNK pathway components is more modest compared to TAK1 RNAi in this experimental setting. This is in line with the earlier results of Silverman (2003), who showed that in S2* cells TAK1 RNAi totally blocks Dpt, Cecropin and Att response to LPS, whereas RNAi targeting JNK pathway components hemipterous and basket have a more moderate effect. Nevertheless, the regulatory interplay that has been detected between the Imd and the JNK pathway in the Drosophila innate immune response (Boutros, 2002: Park, 2004) is likely to attract more attention in the future (Kleino, 2005).
This present study underlines the convenience of RNAi-based screening in S2 cells. Importantly, two novel components of the Imd pathway have been identified. The exact roles Iap2 and TAB play in the activation of Relish remain to be solved. In addition, these findings will likely focus attention to investigate the importance of Iap2 in mammalian TNF receptor signaling. This methodology can be readily applied to study other conserved signaling cascades (Kleino, 2005).
Imd-mediated innate immunity is activated in response to infection by
Gram-negative bacteria and leads to the activation of Jun amino-terminal kinase
(JNK) and Relish, a NF-kappaB transcription factor responsible for the
expression of antimicrobial peptides. Plenty of SH3s (POSH) has been shown to
function as a scaffold protein for JNK activation, leading to apoptosis in
mammals. This study reports that POSH controls Imd-mediated immunity signalling in
Drosophila. In POSH-deficient flies, JNK activation and Relish induction were
delayed and sustained, which indicated that POSH is required for properly timed
activation and termination of the cascade. The RING finger of POSH, possessing
ubiquitin-ligase activity, is essential for termination of JNK activation.
POSH binds to and degrades TAK1, a crucial activator of both the JNK
and the Relish signalling pathways. These results establish a novel role for
POSH in the Drosophila immune system (Tsuda, 2005).
Among the components of the Imd pathway, TAK1 plays a crucial role as an
activator of both the JNK and the Relish pathways (Vidal 2001; Silverman, 2003; Park, 2004). Flies that are deficient in TAK1 do
not produce antibacterial peptides and are therefore highly susceptible to
Gram-negative bacterial infection (Vidal, 2001).
In Drosophila S2 cells, TAK1 has been shown to be required for the
peptidoglycan-induced activation of IkappaB
kinase (IKK) and JNK (Leulier, 2003; Silverman, 2003; Kaneko,
2004). TAK1 is also important for limiting the duration of JNK activation,
thereby resulting in a transient JNK-dependent response that precedes the
sustained induction of the Relish-dependent genes (Tsuda, 2005).
Although it has been shown that proteasomal degradation of
TAK1 leads to the rapid termination of JNK signalling (Park, 2004),
the degradation mechanism has yet to be entirely clarified,
and a ubiquitin ligase that promotes TAK1 degradation has not been reported.
This study identifies Plenty of SH3s (POSH) as a crucial component that controls the termination of immunity signalling in Drosophila. POSH, which was
initially isolated as a Rac1-interacting protein, consists of a RING-finger
domain and four SH3 domains, and has been shown to function as a scaffold
protein for JNK signalling components leading to apoptosis in mammals (Tapon, 1998; Xu, 2003). In Drosophila, overexpression of POSH in imaginal discs
activates JNK, which results in various defects in adult morphology (Seong, 2001). Based on flies that are deficient in
POSH, evidence was provided that POSH is required for the properly timed
activation and termination of Imd-mediated immune responses, and that the RING
finger of POSH, which shows ubiquitin-ligase activity, is essential for this
regulation (Tsuda, 2005).
To assess the role of POSH in vivo, a null allele of POSH
was created by the excision of a flanking P-element in EP(2)1026. A mutant was obtained
bearing a deletion that included the start codon and extended 1,282
nucleotide bases into the coding region of POSH. Northern blot analysis
indicated that POSH transcripts were not detectable in
POSH74 homozygous animals. Most
POSH74 homozygous individuals survived embryogenesis,
and the JNK cascade operating during dorsal closure remained intact. Furthermore,
POSH74 showed no genetic interaction with the JNK
pathway components in embryos. Taken together, these results
indicate that not every instance of JNK signalling requires POSH as a scaffold.
Although mutants showed no obvious developmental defects, adult
flies did not live as long as control flies, and older cultures often
contained dead larvae and pupae that were heavily melanized.
In addition, the viability of POSH-deficient flies
after injection with Escherichia coli was significantly lower than that
of wild type. A possible cause for such phenotypes is defective
immune function, and therefore a role for POSH in the immune
response was sought (Tsuda, 2005).
Whether the TAK1-mediated immune response was compromised by the
POSH74 mutation was tested. Infection with E. coli
activates TAK1, which consequently phosphorylates JNK and induces the expression
of Relish/NF-kappaB, resulting in the subsequent induction of a
battery of genes encoding antimicrobial peptides, such as Diptericin.
Northern blot analysis indicated that
the induction of relish and diptericin (dpt) messenger RNAs
after E. coli infection was significantly delayed in
POSH74 flies compared with that in controls.
Interestingly, it was also observed that expression of
relish was sustained for a longer period after infection in
POSH74 flies (Tsuda, 2005).
To investigate the effects of the POSH mutation more quantitatively,
real-time PCR analysis of immune response genes was performed.
Consistent with Northern blot analysis, the
induction of dpt was delayed, and relish induction was sustained,
with a maximum expression at 2 h after infection. The
expression pattern of attacin A (attA), which is dependent on
Relish, was also analyzed. Induction of attA was almost normal, but unlike
in the control flies, its expression level continued to increase up to 4 h after
infection. The maximum level of expression was twice that of control flies,
consistent with Relish activity being elevated in POSH mutant flies. The
expression profile of puckered (puc), a target
gene of the JNK pathway, was also analyzed. Induction of puc was slightly delayed, with the
highest level of expression at 1 h after infection, and expression was sustained
for a longer period in POSH74 flies compared with that
in controls. These results indicate that POSH is required for properly timed
activation and rapid termination of both JNK- and Relish-mediated immune
response pathways (Tsuda, 2005).
TAK1 has been shown to be required for the maintenance
of relish mRNA expression, and its
proteasomal degradation is responsible for the rapid termination of Relish and
JNK signalling (Park, 2004). POSH contains a
CH4C4-type RING-finger domain near its N terminus. It has been shown that the
RING-finger domain of mammalian POSH possesses E3 ubiquitin-ligase activity and
regulates the level of POSH protein through the proteasomal pathway (Xu, 2003).
Drosophila POSH also had E3 ubiquitin-ligase activity;
its auto-ubiquitination has been demonstrated. To assess the functional importance of the RING domain, POSHmR was constructed, bearing point mutations in the RING domain, and POSHDeltaR
lacking the RING-finger domain altogether. Neither of these
proteins was able to auto-ubiquitinate after expression in cultured
Drosophila cells (S2). It is conceivable, therefore, that POSH is
responsible for the feedback regulation of the immune response through TAK1
ubiquitination and its subsequent degradation. Observations indicate that
the RING-finger function of POSH is essential for the degradation of TAK1 (Tsuda, 2005).
To characterize further the role of
POSH in the regulation of JNK signalling, immunocompetent S2 cells were used in an
in vitro model system used to study the immune response triggered by
peptidoglycan in a commercial lipopolysaccharide preparation. Consistent with
puc expression results in POSH mutant flies, the reduction of POSH
by RNA interference (RNAi) significantly decreased the level of JNK activation,
but it was sustained for a longer period than in control cells.
In contrast, overexpression of POSH induced JNK
activation at a high level, but then inactivated it rapidly. When
POSHmR was transfected into S2 cells, phosphorylated JNK persisted
for a longer period than in control cells. The effects of POSH
overexpression and the role of the RING-finger function were examined
in vivo using
armadillo-GAL4 (arm-GAL4) as a ubiquitous expression driver.
Following bacterial infection, the expression level of
puc, a target of JNK signalling, rapidly declined in flies overexpressing
POSH (arm>POSH). In contrast, puc expression was sustained in
flies overexpressing POSHmR (arm>POSHmR),
which suggests that POSHmR has a dominant-negative effect on JNK
signalling. Together, these results indicate that POSH affects both activation
and inactivation of JNK signalling induced by microbial infection, and that the
RING-finger domain of POSH is essential for the negative feedback regulation of
JNK signalling. The profile of puc expression in
POSH74 flies is remarkably similar to that seen in
relish flies, which supports the idea that POSH mediates Relish-dependent inactivation of JNK in the immune system (Tsuda, 2005).
These results show that
POSH modulates the TAK1-mediated innate immune response. Following microbial
infection, POSH facilitates rapid JNK activation and the induction of
relish/NF-kappaB expression, both of which are mediated by TAK1
(Silverman, 2003). Conversely, POSH is also
required for the rapid termination of both JNK activation and relish
transcription through the E3 ubiquitin-ligase activity of the RING-finger
domain. The results indicate that POSH is involved in a mechanism of negative
feedback regulation of JNK and NF-kappaB
signalling. The Drosophila TAK1-mediated innate immune response is
similar to the tumour necrosis factor (TNF) signalling cascade in mammals.
Imd protein contains a death domain
with homology to that of mammalian TNF-receptor-interacting protein (RIP).
Most of the pathway
components (such as FADD, TAK1) and Drosophila homologues of
IKKgamma and IKKbeta are conserved in mammalian TNF signalling. The
activation of Relish involves its phosphorylation and cleavage by DREDD, the
Drosophila homologue of caspase 8. Eiger, a
Drosophila homologue of the TNF-alpha
superfamily ligand, has recently been identified. Overexpression of Eiger induces
cell death by activating the JNK signalling pathway, as in mammals. Although the
Eiger signalling pathway has not been fully elucidated in Drosophila, it
has been clearly shown that TAK1 is essential to transduce the cell-death
signal. It is possible that POSH is involved in Eiger signalling and modulates
TAK-mediated JNK signalling as in the immune system. It will be of interest to
determine whether POSH functions in mammalian TNF signalling (Tsuda, 2005).
TGF-ß activated kinase 1
The TAK1 plays a pivotal role in the innate immune response of Drosophila by controlling the activation of JNK and NF-kappaB. Activation of TAK1 in mammals is mediated by two TAK1-binding proteins, TAB1 and TAB2, but the role of the TAB proteins in the immune response of Drosophila has not yet been established. A TAB2-like protein has been identified in Drosophila called dTAB2. Similar to mammalian TAB2/3, dTAB2 contains a ubiquitin-binding domain (N-CUE) at its N-terminal, and a highly conserved Zinc Finger (ZnF) domain at its C-terminal, respectively. dTAB2 can interact with TAK1, and stimulate the activation of the JNK and NF-kappaB signaling pathway. Furthermore, silencing of dTAB2 expression by dsRNAi inhibits JNK activation by peptidoglycans (PGN), but not by NaCl or sorbitol. In addition, suppression of dTAB2 blocks PGN-induced expression of antibacterial peptide genes, a function normally mediated by the activation of NF-kappaB signaling pathway. No significant effect on p38 activation by dTAB2 was found. These results suggest that dTAB2 is specifically required for PGN-induced activation of JNK and NF-kappaB signaling pathways (Zhuang, 2006).
Studies in mammalian system have shown that activation of TAK1 is regulated by two TAK1 binding proteins, TAB1 and TAB2/3. The dTAB2 appears to associate with dTAK1, thus dTAB2 also acts as a dTAK1-associating protein in Drosophila. In mammalian cells, TAB2 not only binds with TAK1, but also associates with an upstream regulator TRAF6 to form a TAK1-TAB2-TRAF6 complex. TRAF6 contains an N-terminal RING domain, and functions as a ubiquitin ligase, which, in conjunction with a dimeric Ub-conjugating enzyme complex consisting of Ubc13 and Uev1A.Mms2, to catalyze the K63-linked ubiquitination of TRAF6. TAB2 binds to K63-linked polyubiquitinated TRAF6 though a highly conserved C-terminal zinc finger (ZnF) domain, thus facilitating the activation of TAK1. In Drosophila, TAB2 also contains a conserved ZnF domain at its C-terminal, and deletion of this region eliminated its ability to activate the NF-kappaB signaling pathway. In addition, two TRAF homologues, dTRAF1 and dTRAF2, have been identified in Drosophila. Only dTRAF2 contains a RING domain, and dTRAF2 mutant has a reduced level of Diptericin and Drosomycin induction after E. coli infection. However, whether dTRAF2 can be polyubiquitinated and whether dTAB2 binds with polyubiquitinated dTRAF2 to facilitate the activation of dTAK1 still requires further biochemical study (Zhuang, 2006).
In mammals, the physiological importance of TAB2 has been clarified by studies on TAB2-deficient mice and RNAi studies. The phenotype generated from the TAB2 deficiency is very similar to that of NF-kappaB-, IKK-beta-, and NEMO/IKK-deficient mice. However, IL-1 or TNF-induced activation of NF-kappaB and JNK signaling pathways appears to be normal in TAB2-deficient embryonic fibroblasts. Recently, another mammalian TAB2 homologue, TAB3 has been identified, suppression of both TAB2 and TAB3 by RNAi inhibit the activation of IKK and JNK by IL-1 and TNF. In Drosophila, the generation of dTAB2-deficient fly has not been reported so far. However, silencing of dTAB2 by using dsRNAi inhibits PGN-induced activation of JNK and the expression of antibacterial peptide diptericin and attacin gene, two typical peptides produced when the IMD signaling pathway is activated by Gram-negative bacterial pathogens. Since the dTAB2 is associated with dTAK1 and TAB2 is believed to be a key regulator of TAK1, these results are consistent with previous studies that have shown that the Drosophila TAK1 is essential for the activation of both NF-kappaB and JNK MAP kinase signaling after PGN stimulation. Considering the association of dTAB2 with dTAK1 and the functional role of its mammalian homologue TAB2 in the activation of TAK1, the physiological function of dTAB2 in the stimulation of PGN is most likely mediated through TAK1 (Zhuang, 2006).
Compared to the studies of TAB2 and TAK1 in the activation of IKK and JNK, the functional role of TAB2 and TAK1 in the activation of p38 MAP kinase is less clear. TAK1 has been shown to directly phosphorylate and activate the MKK6, a MAP2K of p38 in vitro. However, no significant defect of PGN-induced p38 activation in TAB2-silenced Drosophila cells was observed. In addition, PGN-induced p38 activation is normal in TAK1-silenced cells. These results suggest that TAK1 and its associating protein TAB2 may not be involved in p38 activation in PGN stimulation. The activation of p38 by TAK1 in previous reports may have been caused by TAB1 protein, another component of TAK1 activation complex (Zhuang, 2006).
In mammals, TAK1, a MAPKKK kinase, is implicated in multiple signaling processes, including the regulation of NF-kappaB activity via the IL1-R/TLR pathways. TAK1 function has been studied primarily in cultured cells, and its in vivo function is not fully understood. Null mutations have been isolated in the Drosophila Tak1 gene that encodes Tak1, a homolog of TAK1. Tak1 mutant flies are viable and fertile, but they do not produce antibacterial peptides and are highly susceptible to Gram-negative bacterial infection. This phenotype is similar to the phenotypes generated by mutations in components of the Drosophila Imd pathway. Genetic studies also indicate that Tak1 functions downstream of the Imd protein and upstream of the IKK complex in the Imd pathway that controls the Rel/NF-kappaB like transactivator Relish. In addition, epistatic analysis places the caspase, Dredd, downstream of the IKK complex, which supports the idea that Relish is processed and activated by a caspase activity. This genetic demonstration of Tak1's role in the regulation of Drosophila antimicrobial peptide gene expression suggests an evolutionary conserved role for TAK1 in the activation of Rel/NF-kappaB-mediated host defense reactions (Vidal, 2001).
The Toll signaling pathway, which was first identified as a regulator of embryonic dorsal-ventral patterning, is one regulator of antimicrobial peptide gene expression in Drosophila. Upon infection, the Spaetzle (Spz) protein is cleaved to generate a ligand for the Toll transmembrane receptor protein; Toll binding by Spz stimulates the degradation of the IkappaB homolog, Cactus, and the nuclear translocation of the Rel proteins Dorsal and Dorsal-like immunity factor (Dif). A second pathway regulating antimicrobial peptide gene expression in flies was initially identified by a mutation in the immune deficiency (imd) gene that results in susceptibility to Gram-negative bacterial infection and an impairment of antibacterial peptide gene expression (Lemaitre, 1995). imd encodes a homolog of the mammalian Receptor Interacting Protein (RIP) (P. Georgel, in preparation). The imd gene encodes a death domain-containing protein. In mammals, RIP appears to function in an adaptor complex associated with the tumor necrosis factor (TNF) receptor, and genetic placement of imd suggests that IMD has a conserved function in flies as part of a receptor-adaptor complex that responds to Gram-negative bacterial infection. Molecular studies have isolated four additional factors that appear to define the Imd pathway: Relish, a third Drosophila Rel protein; two members of a Drosophila IkappaB kinase (IKK) complex, that is, the kinase DmIKKß and a structural component DmIKKgamma (Kim, 2000; Rutschmann, 2000; Silverman, 2000; Lu, 2001) and Dredd, a caspase (Elrod-Erickson, 2000; Leulier, 2000). Relish is a homolog of the mammalian P100 and P105 compound Rel proteins that contain both Rel domains and inhibitory ankyrin domains. A receptor for the Imd pathway has not been identified, but infection triggers Relish cleavage and nuclear translocation of the Rel domain (Stöven et al. 2000). Relish cleavage requires DmIKKß activity, indicating that, like the mammalian IKK complex that functions in the IL1-R and TNF-R pathways, the Drosophila IKK complex regulates Rel protein activity. However, and in contrast to P100 and P105 processing, Relish cleavage is not blocked by proteasome inhibitors but does require a functional Dredd gene, suggesting that Dredd may cleave Relish directly after infection (Stöven, 2000). Like imd, mutations in DmIKKß, DmIKKgamma, Dredd, and Relish affect antibacterial peptide gene expression after infection and induce susceptibility to Gram-negative bacterial infections. However, mutations in these genes do not induce susceptibility to fungal infections, demonstrating that the immune responses regulated by the Imd pathway are required to resist Gram-negative bacterial but not fungal infections (Vidal, 2001 and references therein).
Some significant conclusions of recent studies on the regulation of Drosophila antimicrobial peptide gene expression are that the Toll and Imd pathways do not share any components and that each pathway regulates specific Rel proteins. The only evidence of interactions between the two pathways is the observation that both pathways are required to fully induce some of the antimicrobial peptide genes, suggesting that these genes respond to combinations of Rel proteins controlled by the two pathways. The influence of each pathway on the expression of each antimicrobial peptide gene is apparent in flies carrying mutations that affect either the Toll or the Imd pathway: Drosomycin is mainly controlled by the Toll pathway; Diptericin and Drosocin can be fully activated by the Imd pathway; and full Metchnikowin, Defensin, Cecropin A and Attacin activation requires both pathways (Vidal, 2001 and references therein).
None of the antimicrobial peptide genes are induced in imd;Toll double mutant flies, demonstrating that Imd and Toll are two essential pathways that regulate antimicrobial gene expression pathways. Despite an increased understanding of the regulation of antimicrobial peptide gene expression in flies, various intermediates in the Toll and Imd pathways remain uncharacterized: for example, neither the kinase that targets Cactus for degradation in the Toll pathway nor the receptor-adaptor complex that regulates the Imd pathway have been identified. Following the observations that null mutations affecting the Imd pathway are not required for viability, a search for additional members of the Imd pathway was initiated by screening for nonlethal mutations that induce susceptibility to Gram-negative bacterial infection in adult flies. Null mutations in the Drosophila transforming growth factor activated kinase 1 gene (Tak1) encoding the Drosophila homolog of the mammalian mitogen-activated protein kinase kinase kinase (MAPKKK) TAK1 (Takatsu, 2000) induce high susceptibility to Gram-negative bacterial infection and block antibacterial peptide gene expression. These results indicate that Drosophila Tak1 codes for a new component of the Imd pathway (Vidal, 2001).
To identify Drosophila genes that mediate defense reactions to bacterial infection, ~2500 lines carrying ethyl methanesulfonate (EMS)-induced mutations on the X chromosome were tested for susceptibility to bacterial infection: male adult flies were pricked with a needle dipped into a pellet of the Gram-negative bacterial species Erwinia carotovora carotovora 15 (E. carotovora 15) and screened for mutants that failed to survive infection. Using this assay, nine recessive, homozygous viable mutations were isolated that render flies highly susceptible to E. carotovora 15 infection: Less than 10% of the mutated flies survived 48 h postinfection, whereas more than 90% of the wild-type flies survived. These nine mutations fall into two complementation groups: B118, which represents five of the mutations, and D10, corresponding to Tak1, which represents the other four mutations. The B118 group corresponds to the caspase encoding gene Dredd (Vidal, 2001),
To compare the D10 phenotype with the phenotypes generated by mutations in other genes that regulate Drosophila immune responses, the susceptibility of D10 and other mutant lines to infection by four microorganisms was assayed: flies were pricked with the Gram-negative bacteria Escherichia coli (E. coli), the Gram-positive bacteria Micrococcus luteus (M. luteus), or the fungus Aspergillus fumigatus, and flies were naturally infected with the entomopathogenic fungus Beauveria bassiana (B. bassiana). The D10 phenotype is similar to the imd and Relish phenotypes; flies carrying the D10, imd, and Relish mutations are susceptible to Gram-negative bacterial infection and resistant to Gram-positive bacterial and fungal infections, although D10 flies, like imd flies, exhibit slightly lower susceptibility to Gram-negative bacterial infection compared to Relish mutants. In contrast, mutations in the spz gene render flies susceptible to fungal infections, and only flies carrying mutations in both spz and imd are susceptible to Gram-positive bacterial infection. This survival analysis demonstrates that the D10 gene product, like Imd and Relish, is required to resist Gram-negative bacterial infection (Vidal, 2001).
The Toll pathway is required for the full induction of the antifungal peptide genes and a subset of the antibacterial peptide genes. Mutations that block the Toll pathway reduce the expression of these genes; conversely, mutations that block the Imd pathway reduce the expression of genes with antibacterial activity. To determine how the D10 mutation affects antimicrobial peptide gene expression, the levels of Diptericin, Cecropin A, Defensin, and Attacin, which encode antibacterial peptides, Drosomycin, which encodes an antifungal peptide, and Metchnikowin, which encodes a peptide with both antibacterial and antifungal activity, were monitored in flies homozygous for two D10 alleles. In addition, the D10 phenotype was compared with all of the previously identified mutations affecting the Imd pathway and with a spz mutation that blocks the Toll pathway (Vidal, 2001).
Pricking adult flies with a mixture of Gram-positive and Gram-negative bacteria activates the expression of all the antimicrobial peptide genes; in the D10 mutants, however, mixed Gram-negative/Gram-positive infections induce significant levels of only Drosomycin and Metchnikowin. Quantitative measurements of three independent RNA blot experiments show that in D10 flies, Drosomycin is induced to wild-type levels; Metchnikowin is induced to 70% of wild-type levels; Cecropin A, Defensin, and Attacin are induced to <25% of wild-type levels, and Diptericin is induced to <5% of wild-type levels. This pattern of antimicrobial peptide gene expression in the D10 mutants is similar to the patterns displayed in mutants of the Imd pathway, although the D10 mutations, like imd, have slightly weaker effects on antimicrobial peptide gene expression compared to the Dredd, DmIKKß, DmIKKgamma, and Relish mutations. This weaker phenotype in D10 and imd flies correlates well with their lower susceptibility to E. coli infection (Vidal, 2001).
Because of its chromosomal location and the functions of its mammalian homologs, Tak1 was chosen as a candidate gene mutated in D10 flies. Several different experiments to determine whether the D10 alleles correspond to mutations in Tak1. Tested first was the ability of a 15 Kb genomic fragment that contains Tak1 to rescue the immune response deficiency in D10 flies. D10 adults carrying the Tak1 transgene (P[Tak1+]) both express Diptericin and resist Gram-negative bacterial infection at levels comparable to wild-type flies, demonstrating that this genomic fragment rescues the D10 phenotypes. Overexpression of the Tak1 cDNA using the UAS/GAL4 system partially rescues Diptericin expression in the D101 mutant. The Tak1 genomic coding sequence was tested. The four D10 alleles all contain mutations within the Tak1 kinase domain. This led to the renaming of the D10 alleles Tak11 to Tak14: Tak11 and Tak14 were generated by missense mutations in conserved residues, Tak12 was generated by a point mutation that creates a stop codon, and Tak13 contains a deletion of 31 base pairs that also results in a premature stop codon. All four Tak1 alleles inhibit Diptericin induction by Gram-negative bacterial infection to the same degree, and this inhibition is not enhanced in flies heterozygous for each allele and a deficiency spanning Tak1. In addition, flies homozygous for the four alleles are equally susceptible to Gram-negative bacterial infection. The apparent null phenotype manifested by the four Tak1 alleles indicates that the Tak1 kinase domain is essential for Tak1 function in the Imd pathway. This observation is supported by results from experiments with a kinase dead form of Tak1, Tak1-K46R, which acts as a dominant negative inhibitor of Tak1 (Takatsu, 2000). Tak1-K46R expression driven by the UAS/GAL4 system blocks Diptericin expression after mixed bacterial infection, confirming that Tak1 is required for the Drosophila antibacterial immune response. The results of the rescue experiments, sequencing data, and the dominant negative Tak1 mutant phenotypes together demonstrate that D10 encodes the MAPKKK Tak1 (Vidal, 2001).
The loss-of-function Tak1 mutations display immune response phenotypes that are very similar to the phenotypes generated by mutations in imd, DmIKKß, DmIKKgamma, Dredd, and Relish, suggesting that these genes function together in the Imd pathway. Previous studies indicated that DmIKKgamma and DmIKKß directly regulate Relish activity (Silverman, 2000); however, the positions of Imd, Tak1, and Dredd in the Imd pathway were not determined. To avail of a genetic approach for ordering the Imd pathway, both Dredd and Tak1 were overexpressed via the UAS/GAL4 system. In lines carrying a heat shock (hs)-GAL4 driver and either the Dredd or Tak1 cDNAs under the control of a UAS promoter, heat shock induces Diptericin expression to about 15%-20% of the level observed in adults 6 h after bacterial infection. This result indicates that the overexpression of these two genes is sufficient to activate the antibacterial pathway in the absence of infection. By using this UAS/GAL4 system to overexpress Tak1 and Dredd in various mutant backgrounds, the epistatic relationships were tested between Tak1 and Dredd, and the other genes of the Imd pathway. Mutations in DmIKKß and DmIKKgamma, but not imd, block Diptericin induction by Tak1 overexpression, indicating that Tak1 functions downstream of Imd and upstream of the IKK complex. However, the Diptericin expression induced by Dredd overexpression is not affected by mutations in imd or DmIKKgamma. For genetic reasons, Northern blot analysis could not be used to test the effect of mutations in Relish and DmIKKß, which are on the third chromosome, on the UAS-Dredd-induced Diptericin expression. Therefore, the UAS-Dredd transgene was overexpressed through a female, adult fat body driver (yolk-GAL4) and Diptericin expression was monitored with a Diptericin-LacZ reporter gene. LacZ titration assays have demonstrated that ß-galactosidase activity is induced in lines overexpressing UAS-Dredd in the absence of infection. The Dredd-mediated Diptericin-LacZ induction is strongly reduced in Relish but not in DmIKKß mutants, confirming the Northern blot results showing that Dredd functions downstream of the IKK complex and demonstrating that Dredd regulates Diptericin expression through Relish (Vidal, 2001).
Natural fungal infections highlight the ability of Drosophila to discriminate between pathogens and activate specific immune response pathways that lead to adapted immune responses. Although only the Imd pathway is required to resist Gram-negative bacterial infection, the Toll pathway is still activated to some degree in flies pricked with Gram-negative bacteria. This suggests that injury (and associated contamination) contributes to a nonspecific immune response. E. carotovora 15 naturally infects the Drosophila larval gut and triggers both the local and systemic expression of antimicrobial peptide genes. In contrast to infection by pricking, natural E. carotovora 15 infection induces Diptericin expression more strongly than Drosomycin expression. To compare the contribution of the Toll and Imd pathways to antimicrobial peptide induction after natural E. carotovora 15 infection, Diptericin and Drosomycin expression were quantified in larvae from various mutant lines after natural E. carotovora 15 infections. The Diptericin expression induced by E. carotovora 15 natural infection is entirely dependent on the genes of the Imd pathway, confirming that Diptericin is exclusively regulated by the Imd pathway. Interestingly, the level of Diptericin induction is higher in spz mutants than in wild-type larvae. In addition, Drosomycin induction after E. carotovora 15 natural infection of larvae is also dependent on the Imd pathway: mutations blocking the Imd pathway have stronger effects than the spz mutation on Drosomycin expression induced by natural infection. These data contrast somewhat with previous observations that the imd mutation does not block Drosomycin induction by E. carotovora 15 infection. However, the earlier analysis of Drosomycin induction by E. carotovora 15 was based on the qualitative analysis of a Drosomycin-lacZ reporter gene; it is thought that that the quantitative analysis of Drosomycin expression by Northern blot is a more accurate determination of Drosomycin expression patterns. In conclusion, the current results indicate that Drosomycin gene induction after natural Gram-negative bacterial infection is largely mediated by the Imd pathway (Vidal, 2001).
Natural infections by E. carotovora 15 also trigger the local expression of antimicrobial peptide genes in various epithelial tissues, and both Diptericin expression in the anterior midgut and Drosomycin expression in the trachea are dependent on the imd gene. By assaying the expression of Diptericin-lacZ and Drosomycin-GFP reporter genes in naturally infected Tak1 and Dredd mutant larvae, it has been shown that both genes are required for Diptericin and Drosomycin expression in epithelial tissues after natural E. carotovora. These data confirm the predominant role of the Imd pathway in antimicrobial peptide regulation after natural E. carotovora 15 infection and suggest that Gram-negative bacterial recognition in flies preferentially activates the Imd pathway (Vidal, 2001).
To study the in vivo function of TAK1, transgenic flies carrying the wild-type mouse TAK1 (UAS-mTAK1) or a truncated, constitutively active form of mTAK1 (UAS-mTAK1deltaN) were generated. Ectopic expression of the wild-type mTAK1 shows various defects in different adult tissues. For example, UAS-mTAK1/Dll-GAL4 flies lack the distal parts of legs and antenna, whereas UAS-mTAK1/elav-GAL4 flies show a rough eye phenotype. Ectopic expression of the activated form of mTAK1 (mTAK1delta1N) results in lethality in early developmental stages. To circumvent the early lethal