InteractiveFly: GeneBrief

immune response deficient 5 and kenny: Biological Overview | References

BIOLOGICAL OVERVIEW

The Drosophila genome encodes two IKK genes. DmIKKβ (or DLAK) is most similar to human IKKβ, and is involved in Relish activation (Stoven, 2003). That leaves the second Drosophila IKK, DmIKKepsilon (also known as IkappaB kinase-like 2 or Ik2), as a candidate for the Cactus kinase. However, a function of DmIKKepsilon as Cactus kinase has been ruled out. Instead, DmIKKepsilon modulates caspases for a non-apoptotic function and controls both actin and microtubule cytoskeletons (Bergmanm 2003).

Two roles for the Drosophila IKK complex in the activation of Relish and the induction of antimicrobial peptide genes

The Drosophila NF-kappaB transcription factor Relish is an essential regulator of antimicrobial peptide gene induction after gram-negative bacterial infection. Relish is a bipartite NF-kappaB precursor protein, with an N-terminal Rel homology domain and a C-terminal IkappaB-like domain, similar to mammalian p100 and p105. Unlike these mammalian homologs, Relish is endoproteolytically cleaved after infection, allowing the N-terminal NF-kappaB module to translocate to the nucleus. Signal-dependent activation of Relish, including cleavage, requires both the Drosophila IkappaB kinase [consisting of 2 subunits: a catalytic kinase subunit encoded by ird5 (IKKβ) and a regulatory subunit encoded by kenny (IKKγ)] and death-related ced-3/Nedd2-like protein (DREDD), the Drosophila caspase-8 like protease. This report shows that the IKK complex controls Relish by direct phosphorylation on serines 528 and 529. Surprisingly, these phosphorylation sites are not required for Relish cleavage, nuclear translocation, or DNA binding. Instead they are critical for recruitment of RNA polymerase II and antimicrobial peptide gene induction, whereas IKK functions noncatalytically to support Dredd-mediated cleavage of Relish (Ertürk-Hasdemir, 2009).

These data suggest a new model for the regulation of Relish activity. In this model, Relish is controlled by 2 distinct mechanisms, both of which signal downstream of the receptor Peptidoglycan recognition protein LC (PGRP-LC). One arm controls the cleavage of Relish and requires IMD, FADD, DREDD, and the IKK complex. The other arm controls Relish phosphorylation through TAK1 and the IKK complex. Robust induction of antimicrobial peptide expression requires that both mechanisms of control are fully active; Relish must be cleaved and phosphorylated (Ertürk-Hasdemir, 2009).

Phosphorylation of Relish is critical for signal-dependent transcriptional activation of target genes. By using mass spectrometry and in vitro kinase assays, serines 528 and 529 were identified as targets of IKKβ phosphorylation. These serines are phosphorylated rapidly, in cell lines and flies, after immune challenge. Mutation of these residues to alanine resulted in a protein that acted as dominant negative in cell culture, inhibiting the PGN-induced expression of antimicrobial peptide genes Diptericin and Attacin. A recent study reported that ectopic expression of REL-68 (the N-terminal portion of Relish) was not sufficient to drive the expression of the antimicrobial peptide genes Attacin and Cecropin (Wiklund, 2009). Because REL-68 is not expected to be phosphorylated, these results support the conclusion that phosphorylation is critical for Relish-mediated transcriptional activation of AMP genes. However, this article also reported that REL-68 was able to induce Diptericin, which appears to contradict the current findings. In these experiments, transgenic REL-68 is overexpressed, which may contribute to these confusing results (Ertürk-Hasdemir, 2009).

Further supporting the conclusion that Relish phosphorylation is critical, kinase-dead IKKβ transgenic rescue supported only very weak induction of AMP genes in flies. In these experiments, Relish is expressed at normal levels. Surprisingly, serines 528 and 529, and IKKβ catalytic activity itself, were not required for signal-dependent Relish cleavage. Serines 528 and 529 were also not essential for nuclear translocation or DNA binding. Instead, ChIP experiments show that these serines are required for the efficient recruitment of RNA Pol II to the Diptericin locus (Ertürk-Hasdemir, 2009).

These ChIP assays used the 8WG16 mAb, which preferentially recognizes unphosphorylated CTD repeats of the largest subunit of RNA Pol II. The unphosphorylated CTD is associated with the preinitiating RNA Pol II complex recruited to promoters. Thus, these results argue that phosphorylation of Relish on serines 528 and 529 is required for efficient recruitment of RNA Pol II to the Diptericin and Diptericin-B promoters. An alternate possibility, suggested by recent findings on gene regulation in Drosophila, is that phosphorylated Relish could stimulate elongation from paused RNA Pol II. However, a genome-wide analysis of promoters containing stalled RNA Pol II has found that many Drosophila antimicrobial peptide genes are not sites of paused RNA Pol II. The use of 8WG16 in the experiments presented in this study, further argues that phosphorylation of serines 528 and 529 does not modulate RNA Pol II pausing but instead regulates polymerase recruitment to the preinitiation complex at the Diptericin locus. The exact mechanism by which phosphorylation of serines 528 and 529 affect RNA Pol II recruitment remains to be elucidated. It may involve interaction with coactivators, such as components of the mediator complex, or it may involve the recently discovered IMD component Akirin (Goto, 2008), which is argued to function in the nucleus, downstream of Relish (Ertürk-Hasdemir, 2009).

This report also provides further supporting evidence that DREDD may be the caspase that directly cleaves Relish. This study shows that overexpression of DREDD is sufficient to cause Relish cleavage. Relish cleavage required catalytically active DREDD and expression of another apical caspase, the caspase-9 like DRONC, did not generate cleaved Relish. Interestingly, DREDD-mediated Relish cleavage did not lead to Relish phosphorylation and was not sufficient to drive Diptericin expression. Furthermore, immunopurified DREDD, but not drICE, cleaved Relish in vitro, albeit not very efficiently. The poor efficiency of Relish cleavage, in vitro, may be due to the highly oligomeric state of purified Relish and/or the low activity of DREDD, which has proven to be very difficult to produce in an active form. It was also found that a biotinylated peptide with the Relish cleavage site bound active DREDD; although strong evidence for a direct interaction, this assay is not particularly specific. Together, these data strongly suggest that DREDD directly cleaves Relish, but it cannot yet be concluded with certainty that other proteases, such as an effector caspase, are not involved (Ertürk-Hasdemir, 2009).

In addition to DREDD, Relish cleavage also requires both IKK subunits. However, Relish cleavage does not require catalytically active IKKβ. Delaney (2006) showed that TAK1 is not required for Relish cleavage. Because TAK1 is required for the immune-induced activation of the IKK kinase, this result is consistent with the data indicating that IKK catalytic activity is not involved in Relish cleavage. Instead, IKK complex may function as a scaffold or adaptor, but not as a kinase, in controlling the cleavage of Relish (Ertürk-Hasdemir, 2009).

Taken together, these data demonstrate that Relish is regulated by 2 distinct mechanisms. Relish is probably cleaved by DREDD and phosphorylated by the IKK complex. These 2 regulatory mechanisms appear to be independent, because phosphorylation can occur without cleavage, and vice versa, although they are both triggered by PGN stimulation of the receptor PGRP-LC. Surprisingly, the IKK complex also plays a role in the cleavage of Relish, but not through its kinase activity. Instead, IKK-mediated phosphorylation of Relish on serines 528 and 529, within its N-terminal transcription factor module, is necessary for transcriptional activation of target genes (Ertürk-Hasdemir, 2009).

A Drosophila IkappaB kinase complex required for Relish cleavage and antibacterial immunity

A Drosophila IkappaB kinase complex containing DmIKKß (Immune response deficient 5) and DmIKKγ (Kenny), homologs of the human IKKß and IKKγ proteins, has been identified. This complex is required for the signal-dependent cleavage of Relish, a member of the Rel family of transcriptional activator proteins, and for the activation of antibacterial immune response genes. In addition, the activated DmIKK complex, as well as recombinant DmIKKß, can phosphorylate Relish in vitro. Thus, it is proposed that the Drosophila IkappaB kinase complex functions, at least in part, by inducing the proteolytic cleavage of Relish. The N terminus of Relish then translocates to the nucleus and activates the transcription of antibacterial immune response genes. Remarkably, this Drosophila IkappaB kinase complex is not required for the activation of the Rel proteins Dif and Dorsal through the Toll signaling pathway, which is essential for antifungal immunity and dorsoventral patterning during early development. Thus, a yet to be identified IkappaB kinase complex must be required for Rel protein activation via the Toll signaling pathway (Silverman, 2000).

To identify the signaling components required for the Drosophila immune response a reverse genetic approach, taking advantage of the Drosophila Genome Project, was undertaken. A cDNA sequence with homology to the kinase domain of the human IKK genes was identified in the BDGP EST database. Examination of the amino acid sequence of the encoded protein, which has been designated DmIKKß (Drosophila melanogaster IKKß), displays significant similarity to the N-terminal region of hIKKalpha and hIKKß but is more similar to hIKKß. The amino acid sequence of the C terminus of DmIKKß is only weakly related to the corresponding regions of IKKalpha and IKKß. A predicted coiled-coil can be detected in a region corresponding to the predicted leucine zipper coiled-coil of hIKKalpha and hIKKß. The DmIKKß gene maps to chromosomal location 89B as determined with the BDGP P1 filter array (Silverman, 2000).

The mammalian IKKalpha and IKKß proteins are found in a high molecular weight IkappaB kinase complex that includes the structural component IKKγ or NEMO. To investigate the possibility that DmIKKß is also a component of a similar kinase complex, a yeast two-hybrid screen was performed using DmIKKß as bait. A Drosophila larval cDNA library was screened, and a total of 85 independent positive clones were analyzed. All of these clones were found to contain overlapping inserts from a cDNA that encodes a Drosophila protein with homology to hIKKγ primarily in its C terminus. This gene, referred to as DmIKKγ hereafter, maps to chromosomal location 60E as determined with the BDGP P1 filter array. Secondary structure predictions of the protein encoded by DmIKKγ suggest the existence of several coiled-coil regions. The overall amino acid sequence homology between DmIKKγ and hIKKγ suggests a putative structural and functional relationship between the two proteins, especially in their C-terminal halves. DmIKKß and DmIKKγ interact to form a complex both in vivo and in vitro (Silverman, 2000).

To determine whether DmIKKß and DmIKKγ are involved in the activation of antibacterial genes, an LPS-inducible Drosophila cell line was engineered to express different versions of these genes. When S2* cells are treated with LPS, the expression of antibacterial peptide genes, such as Diptericin, Cecropin, and Attacin, are induced. Thus, S2* cells were stably transfected with plasmids that express wild-type or potentially dominant negative versions of DmIKKß and DmIKKγ under the control of the copper-inducible metallothionein promoter. Four cell lines were generated that express one of the following proteins: DmIKKß wild type, DmIKKß K50A, DmIKKγ wild type, or DmIKKγ 201-387. DmIKKß K50A was chosen because similar mutations in hIKKalpha or hIKKß, which change a conserved lysine in the ATP binding domain, create dominant negative proteins, whereas the DmIKKγ_201-387 was designed because a similarly truncated hIKKγ acts as a dominant negative mutant in human cells. Full-length hIKKγ can also act as a dominant negative when expressed at high levels, presumably by titrating limiting components required for functional complex assembly. DmIKKß and DmIKKγ interact to form a complex both in vivo and in vitro (Silverman, 2000).

After the addition of copper, DmIKK proteins are rapidly produced at high levels as detected by immunoblotting. To determine whether these DmIKK proteins block LPS induction of antibacterial peptide expression in S2* cells, the stable cell lines were first treated with copper to induce expression of the DmIKKs and then stimulated with LPS. DmIKKß K50A blocks the LPS induction of the Diptericin, Cecropin, and Attacin genes. Similarly, either full-length or truncated versions of DmIKKγ are potent inhibitors of antibacterial peptide gene expression. These data clearly show that DmIKKß K50A, DmIKKγ full-length, or DmIKKγ_201-387 potently inhibit Diptericin, Cecropin, and Attacin gene induction. By contrast, the expression of wild-type DmIKKß slightly decreases the level of expression of these genes. It is concluded that DmIKKß and DmIKKγ are required for the activation of antibacterial gene expression in response to LPS (Silverman, 2000).

Genetic studies have shown that the Drosophila Relish gene is essential for the antibacterial immune response. Bacterial infection or LPS treatment activates the endoproteolytic cleavage of Relish. Once cleaved, the N-terminal RHD of Relish translocates into the nucleus, where it activates the transcription of antibacterial genes. Considering that the overexpression of dominant negative DmIKKß or DmIKKγ blocks the induction of antibacterial peptide genes, it is likely that DmIKKß (and the DmIKK complex) functions in the signaling pathway leading to the cleavage of Relish. In order to test this possibility, the DmIKK overexpressing cell lines were employed to follow the fate of Relish protein cleavage. The stable cell lines and the parental cells were first treated with copper and then induced with LPS for 15 min. LPS treatment of the parental cells, or any of the stable lines that were not treated with copper, results in Relish cleavage. Full-length Relish (~110 kD) is cleaved to generate fragments of ~68 kD and ~49 kD, corresponding to the N- and C-terminal fragments, respectively. Expression of DmIKKß K50A, DmIKKγ, or DmIKKγ_201-387 blocks the cleavage of Relish. Overexpression of wild-type DmIKKß causes a slight accumulation of full-length Relish (Silverman, 2000).

To obtain additional evidence that DmIKKß and DmIKKγ are required for LPS-induced antibacterial gene expression, the inhibitory effect of double-stranded RNA (dsRNA), also referred to as RNAi, was exploited. The presence of gene-specific dsRNA molecules in Drosophila or Caenorhabditis elegans embryos has been shown to destabilize the cognate mRNAs. dsRNA corresponding to the DmIKKγ or DmIKKß genes was synthesized in vitro and transfected into the LPS-inducible S2* cell line. Transfection of gene-specific dsRNA causes a significant reduction in the amount of the corresponding mRNA. For example, DmIKKγ dsRNA lowers DmIKKγ mRNA to nearly undetectable levels, while DmIKKß or LacZ dsRNA has no effect on DmIKKγ mRNA levels. DmIKKγ protein levels are also greatly reduced only in those cells transfected with DmIKKγ dsRNA; however, it is important to note that although DmIKKγ protein is reduced approximately 10-fold, it is still detectable after RNAi treatment. Importantly, dsRNA-mediated interference of either DmIKKß or DmIKKγ greatly inhibits the LPS-induced expression of antibacterial genes, such as Attacin, Cecropin, and Diptericin. The LPS-induced cleavage of Relish is also inhibited by either DmIKKß or DmIKKγ dsRNA. Full-length Relish protein persists after the induction with LPS only in those lanes transfected with DmIKK dsRNA. Although the inhibition of Relish cleavage seen with DmIKK RNAi is not as dramatic as that observed with the dominant negative DmIKKs, it is clear that full-length Relish is not as efficiently cleaved in those cells with reduced levels of DmIKKß or DmIKKγ. In both the dominant negative and the RNAi experiments, a stronger inhibition is always observed at the transcriptional level, as compared to that seen at the protein level. This suggests that small perturbations in the amount of the nuclear translocated Relish can have dramatic effects on the level of transcriptional activation. Small differences in the amounts of transcription factors have been shown to exhibit all-or-none effects on promoters that require the cooperative assembly of transcription enhancer complexes. dsRNA-mediated interference does not create a null allele. Thus, the reduced amount of Relish cleavage and antibacterial gene expression still observed in the RNAi-treated cells is most likely due to the remaining DmIKKs found in these cells. Both the dominant negative and RNAi experiments show that DmIKKß and DmIKKγ are required for LPS-induced Relish endoproteolytic cleavage and transcriptional activation of antibacterial peptide genes in vivo (Silverman, 2000).

The Toll signaling pathway, which leads to Cactus degradation and activation of Dif and Dorsal, is necessary for the antifungal immune response as well as early embryonic patterning. The DmIKKß/γ complex could be involved in the Toll signaling pathway in addition to its role in the antibacterial pathway. To test this possibility directly, the RNAi technique was used with a Drosophila cell line specifically engineered to assay the Toll signaling pathway. Schneider S2* cells were stably transfected with a torso-pelle fusion gene controlled by the metallothionein promoter, creating the S2*tpll cell line. Previous studies have demonstrated that fusing the transmembrane domain of torso to the pelle gene creates a constitutively active pelle kinase that can stimulate the Toll signaling pathway in fly embryos or in tissue culture cells. In the S2*tpll cell line, addition of copper leads to the activation of the Toll antifungal pathway. Drosomycin transcription is induced, but Diptericin transcription is unaffected. Transfection of dsRNA into the S2*tpll cells leads to specific loss of the corresponding mRNA and protein. DmIKKγ dsRNA causes a substantial reduction in the level of DmIKKγ protein. However, neither DmIKKß nor DmIKKγ dsRNA blocks the torso-pelle-mediated activation of Drosomycin transcription. These experiments strongly argue that DmIKKß and DmIKKγ are not required for the Toll-mediated antifungal pathway but are required for the LPS-induced antibacterial pathway (Silverman, 2000).

The finding that dominant negative DmIKKs or DmIKK RNAi can block Relish cleavage and Relish-dependent gene activation suggests that Relish may be a bona fide target of the DmIKK complex. To test this possibility, experiments were carried out to determine whether DmIKKß or the DmIKK complex can phosphorylate Relish protein in vitro. Flag-tagged Relish was immunoprecipitated with Flag antibodies from a Schneider cell line that expresses very high levels of the epitope-tagged protein. Immunoprecipitated Relish was then used as a substrate with recombinant DmIKKß in a kinase assay using [γ-³²P]ATP. A band the size of Relish was labeled with ³²P by the recombinant kinase. As a control, extracts from the parental Schneider cell line, which does not express Flag-Relish, were also used in a Flag immunoprecipitation. When these immunoprecipitates were used as substrates for DmIKKß, phosphorylation of Relish was not observed. To further demonstrate that the phosphorylated band is Relish, a similar experiment was performed using anti-Relish antibodies, instead of Flag antibodies, to precipitate Relish. Again, a band corresponding to Relish was phosphorylated in a DmIKKß-dependent manner. It is concluded that DmIKKß can directly phosphorylate Relish (Silverman, 2000).

To determine whether this DmIKKß phosphorylation of Relish is specific, the ability of other, related kinases to phosphorylate Relish was tested. For these experiments, the specificity of DmIKKß was compared to that of recombinant human IKKß and IKKepsilon. While DmIKKß readily phosphorylates Relish, phosphorylation was not observed with either hIKKß or hIKKepsilon. When these same recombinant human IKKs were used in a GST-IkappaBalpha kinase assay, they were both active. Thus, the ability to phosphorylate Relish is not shared between the Drosophila and human IkappaB kinases. Preliminary experiments have shown that the DmIKKß can phosphorylate the linker region between the RHD and the Ankyrin domain. Surprisingly, DmIKKß can also phosphorylate Cactus and IkappaBalpha, and this phosphorylation occurs within the N-terminal regulatory domain, which is required for signal-dependent degradation by the proteasome. Thus, the ability to specifically phosphorylate IkappaB proteins is shared by the Drosophila and human IkappaB kinases, whereas only the Drosophila kinase can phosphorylate Relish. The mammalian IkappaB kinase complex has been shown to phosphorylate p105; however, this phosphorylation leads to its degradation rather than its processing (Silverman, 2000).

To test the possibility that the activated DmIKK complex can phosphorylate Relish, an immunocomplex kinase assay was performed. First, the DmIKK complex was precipitated from an LPS-inducible Schneider cell line using an anti-DmIKKγ antibody. This antibody recognizes the endogenous DmIKKγ that is expressed in this cell line. Although the anti-DmIKKß antibodies are less sensitive (DmIKKß cannot be detected in crude extracts), the DmIKKß protein can be detected after immunoprecipitation with anti-DmIKKγ antibodies. Immunoprecipitation experiments were performed with extracts prepared from cells treated with LPS or left untreated; no differences were detected, with or without LPS, in the levels of DmIKKß or DmIKKγ expressed and precipitated. However, LPS causes a specific increase in the level of Relish kinase activity that was detected in the immunoprecipitated DmIKK complex. Thus, the DmIKK complex that is precipitated with anti-DmIKKγ antibodies contains an LPS-inducible Relish kinase activity. This observation supports the view that DmIKKß and DmIKKγ form (part of) an LPS-inducible kinase complex that phosphorylates Relish, activating its cleavage in response to LPS (Silverman, 2000).

Role of Drosophila IKKgamma in a Toll-independent antibacterial immune response

Loss-of-function mutants in the Drosophila homolog of the mammalian I-kappa B kinase (IKK) complex component IKK γ have been generated. Drosophila IKK γ is required for the Relish-dependent immune induction of the genes encoding antibacterial peptides and for resistance to infections by Escherichia coli. However, it is not required for the Toll-DIF-dependent antifungal host defense. The results indicate distinct control mechanisms of the Rel-like transactivators DIF and Relish in the Drosophila innate immune response and show that Drosophila Toll does not signal through a IKK γ-dependent signaling complex. Thus, in contrast to the vertebrate inflammatory response, IKK γ is required for the activation of only one immune signaling pathway in Drosophila (Rutschmann, 2000).

The antibacterial arm of the Drosophila innate immune response requires an IkappaB kinase

The ird5 gene was identified in a genetic screen for Drosophila immune response mutants. Mutations in ird5 prevent induction of six antibacterial peptide genes in response to infection but do not affect the induction of an antifungal peptide gene. Consistent with this finding, Escherichia coli survive 100 times better in ird5 adults than in wild-type animals. The ird5 gene encodes a Drosophila homolog of mammalian IkappaB kinases (IKKs) and is the catalytic subunit of the IKK complex that contains IKKγ (Kenny) as a regulatory subunit. The ird5 phenotype and sequence suggest that the gene is specifically required for the activation of Relish, a Drosophila NF-kappaB family member (Lu, 2001).

Mutations in ird5 prevent induction of a diptericin-lacZ reporter gene in response to infection and also prevent transcriptional induction of the endogenous diptericin gene. E. coli-induced expression of all seven classes of antimicrobial peptide genes in ird5 mutant larvae were examined by RNA blot hybridization, including the genes encoding antibacterial (Diptericin, Cecropin A, Defensin, Attacin, Drosocin, and Metchnikowin) and antifungal (Drosomycin) peptides. In wild-type larvae, all the antimicrobial genes are strongly induced after bacterial challenge. In contrast, in larvae homozygous for either ird5 allele, there is no detectable induction of the diptericin, cecropin A, defensin, drosocin, or metchnikowin genes. The attacin gene is induced in the mutants to ~30% of normal levels, while drosomycin is induced to normal levels. The same effects on the induction of antimicrobial peptide genes are seen in ird5¹/Df and ird5²/Df animals, suggesting that both alleles cause a complete loss of gene function (Lu, 2001).

Mutations in three other genes, imd, Relish, and Death related ced-3/Nedd2-like protein, have been shown to prevent normal induction of antibacterial peptide genes in adult Drosophila. The pattern of antimicrobial peptide gene induction in ird5 mutants was compared with that in imd and Relish mutants in both larvae and adults. Mutations in all three genes have very similar effects on antimicrobial gene induction in larvae: diptericin and cecropin A are not induced; attacin induction is reduced and drosomycin induction is normal. In adult animals, the antimicrobial gene expression phenotypes of ird5 and Relish mutants are very similar: diptericin induction is blocked, cecropin A and attacin induction is reduced, and drosomycin induction is normal. The antimicrobial gene expression phenotype of imd adults is slightly different, with some residual diptericin expression. Mutations in Dredd, a Drosophila caspase, prevent normal induction of diptericin and attacin and allow induction of drosomycin. These comparisons suggest that ird5, Dredd, Relish, and probably imd act in the same signaling pathway to control the induction of antibacterial peptide genes in response to infection (Lu, 2001).

To assess the importance of the ird5 gene in controlling the growth of invading bacteria, bacterial survival and growth were compared in wild-type, ird5, imd, and Relish animals. In wild-type larvae, most of the E. coli injected into the animal are killed by 6 h after infection. At this same time point, there are four to 15 times as many E. coli in ird5 mutant larvae as in wild-type animals. The effects of the ird5 mutations are more striking in experiments with adults: at 24 h after infection, there are 20-350 times as many bacteria per animal in ird5 mutants compared to wild type. The bacterial growth phenotype of ird5 mutants is similar to that seen in Relish mutant larvae and adults and somewhat stronger than that of imd mutants. This is consistent with the stronger effects of ird5 and Relish on the antimicrobial peptide genes: Mutations in either ird5 or Relish prevent normal induction of diptericin, cecropin, drosocin, attacin, and metchnikowin, while induction of metchnikowin is induced in imd mutants (Lu, 2001).

The mutations responsible for the failure to induce the diptericin-lacZ reporter gene for both ird5 alleles were mapped between the visible markers cu (86D1-4) and sr (90D2-F7) on the right arm of the third chromosome. The deficiency Df(3R)sbd⁴⁵(89B4-10) fails to complement the immune response defect of either ird5 allele. Further deficiency-complementation tests and male recombination mapping narrowed the ird5 interval to 89B4-9, between pannier and Stubble. Two molecular-defined genes in this interval were considered as candidates responsible for the ird5 phenotype, Akt and a gene defined by an EST that is related to mammalian IkappaB kinases (IKKs). Mutant alleles of Akt cause recessive lethality, and ird5/Akt[l(3)89Bq] heterozygous animals are viable and showed normal induction of the diptericin-lacZ reporter gene, indicating that the ird5 phenotypes are not caused by mutations in Akt (Lu, 2001).

Mammalian IKKbeta is required for activation of NF-kappaB in response to inflammatory signals such as TNF-alpha and IL-1; the IKK homolog was therefore considered as a candidate gene for ird5. A full-length cDNA was cloned for the IKK homolog, termed here DmIkkbeta. The same gene has also identified molecularly as encoding a kinase activated by LPS in a Drosophila cell line (Kim, 2000; Medzhitov, 2000; Silverman, 2000). Based on genomic DNA sequence, DmIkkbeta is located between pannier and mini-spindles. Two size classes of transcripts, 2.7 and 4.2 kb, were detected from the DmIkkbeta gene; the cDNA corresponds to the 2.7-kb transcript. Both transcripts are expressed at higher levels after infection. The complete open reading frame of DmIkkbeta was sequenced from the ird5¹ and ird5² chromosomes. A single C-to-T nucleotide substitution was found in ird5¹ that would change a glutamine codon (CAA) at amino acid 266 of the open reading frame to a stop codon (TAA) within the conserved kinase domain. No sequence changes were identified in the open reading frame in ird5²; however, neither DmIkkbeta transcript was detectable in ird5² homozygotes. This analysis indicates that both ird5 alleles are associated with mutations that should abolish DmIkkbeta activity (Lu, 2001).

The ird5 immune response phenotype shows striking specificity: all of the antibacterial peptide genes are strongly affected by the ird5 mutations, but the antifungal peptide gene drosomycin is induced normally in ird5 mutants. The specific immune response phenotype of ird5/DmIkkbeta in vivo contrasts with the global effects on antimicrobial peptide genes seen in cell lines when a dominant negative form of the same gene is expressed in cultured cells (Kim, 2000). The ird5/DmIkkbeta mutant phenotype implies that, in vivo, ird5 is not an essential component of the Toll pathway, which is required for the induction of drosomycin. The ird5/DmIkkbeta gene is therefore a component of an independent signaling pathway, which could be activated by another member of the Drosophila Toll-like receptor family (Lu, 2001).

Mammalian IKKalpha and IKKbeta phosphorylate serine residues in the N-terminal domain of IkappaB; these serine residues target IkappaB for degradation, thereby allowing the nuclear localization and activation of NF-kappaB. The ird5/DmIkkbeta sequence suggests that the protein encoded by this gene phosphorylates an IkappaB-like protein. There are two known Drosophila IkappaB-like proteins that could act as inhibitor proteins in the immune response: Cactus, and the C-terminal ankyrin repeat domain of Relish. In cactus mutants, drosomycin is expressed constitutively, but the antibacterial peptide genes are not, which indicates that Cactus is not involved in the pathways that regulate the antibacterial peptide genes. Furthermore, ird5/DmIkkbeta homozygous mutant females are fertile, demonstrating that this gene is not required for degradation of Cactus during dorsal-ventral patterning in the embryo (Lu, 2001).

The ird5/DmIkkbeta phenotype is similar to the phenotype of Relish mutants. For both genes, homozygous mutant flies are viable and fertile, indicating that the two genes are not essential for development. Mutations in either Relish or ird5/DmIkkbeta completely prevent induction of diptericin and cecropin but allow some induction of attacin and drosomycin. Mutations in either gene produce comparable effects on bacterial growth. These results argue that ird5/DmIkkbeta and Relish act in the same signaling pathway and suggest that Ird5/DmIkkbeta activates Relish-containing dimers. Relish activation requires proteolytic cleavage of Relish protein into an N-terminal Rel domain that translocates to the nucleus and a C-terminal ankyrin repeat domain that remains in the cytoplasm. Recent biochemical experiments have shown that DmIkkbeta can phosphorylate Relish protein (Silverman, 2000), which is consistent with the model that phosphorylation of Relish by DmIkkbeta leads to targeted proteolysis and activation of Relish (Lu, 2001).

Although ird5/DmIkkbeta is expressed maternally, ird5 mutant females are fertile, demonstrating that the gene is not required for embryonic dorsal-ventral patterning. However, a small fraction of embryos (~0.5%) produced by homozygous ird5¹ or ird5¹/ird5² females show a weakly dorsalized phenotype, suggesting that ird5/DmIkkbeta does have a minor role in the maternal pathway that activates Dorsal. It is suggested that there is another kinase in the early embryo that is primarily responsible for phosphorylation and degradation of Cactus. The normal induction of drosomycin in ird5/DmIkkbeta mutants suggests that there will also be another kinase activated by the Toll pathway in the immune response -- perhaps the same kinase that acts downstream of Toll to activate Dorsal in the embryo. The genome sequence indicates that there is one additional IkappaB kinase gene in Drosophila. Future experiments will test whether this gene plays a role in embryonic patterning and the antifungal immune response (Lu, 2001).

The data suggest that different Drosophila Rel dimers are activated by homologous but distinct signaling pathways. Given the similarities of innate immune response pathways in Drosophila and mammals, it is likely that similar pathway-specific signaling components will mediate the activities of the members of the mammalian Rel proteins (Lu, 2001).

Immune activation of NF-kappaB and JNK requires Drosophila TAK1

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 alpha-activated kinase 1), activates both the JNK and NF-kappaB pathways following immune stimulation. This report demonstrates that Drosophila TAK1 functions as both the Drosophila IkappaB kinase-activating kinase and the JNK kinase-activating kinase. However, this study found that 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. Thus, JNK signaling appears to play an important role in the cellular immune response and the stress response (Silverman, 2003).

Innate immune signaling pathways are highly conserved between insects and mammals. In both cases, pathogens are recognized through the detection of conserved molecules such as microbial cell wall components, which are shared by broad groups of microbes. For example, lipopolysaccharides (LPS) from Gram-negative bacteria are potent inducers of both insect and mammalian innate immunity. LPS and other microbial substances are recognized by germ line-encoded receptors such as Toll-like receptors (TLR) or peptidoglycan recognition proteins. Activation of these receptors stimulates conserved signaling pathways leading to the expression of antimicrobial proteins and immune stimulatory cytokines. For example, LPS treatment leads to the activation of NF-kappaB and c-Jun N-terminal kinase (JNK) signaling pathways in both insects and mammals. In mammals, the NF-kappaB pathway is required for the induction of cytokines in response to infection. Likewise, 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 (Silverman, 2003).

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 (IKKβ/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 (Silverman, 2003).

Another conserved immune signaling system is the JNK pathway. Activation of JNK (and other MAPK) signaling pathways in innate immune signaling is well documented in vertebrates. Three MAPK signaling pathways (JNK, ERK, and p38) are stimulated by LPS treatment. Pharmacological inhibition of the ERK and/or p38 pathways in monocyte cell lines inhibits the induction of cytokines such as IL-1, IL-8, and tumor necrosis factor α (TNFα). Also, in mouse embryonic fibroblasts JNK2 is required for induction of type I interferons in response to viral infection and for the induction of IL-12 and IL-6 in response to LPS. Although all the members of the JNK pathway are conserved in the fly, and Drosophila JNK signaling is activated in response to LPS stimulation, the role of this pathway in the insect immune responses has not been clearly delineated (Silverman, 2003).

By contrast, the role of the JNK signaling pathway in Drosophila development has been extensively studied. The best understood role of JNK signaling is in dorsal closure during embryonic development. The dorsal closure JNK pathway kinase cascade relies on the mixed lineage kinase (MLK)-type MAP3K slipper (slpr), which is thought to function as a JNKK kinase, activating the JNK kinase hemipterous (hep), which, in turn, activates the JNK basket (bsk). Bsk, in turn, phosphorylates and activates the transcription factor d-Jun (Silverman, 2003).

Dorsal closure is mechanistically similar to wound healing. Both processes involve pulling an epithelial sheet across an opening and sealing the hole. Recently, it has been proposed that JNK signaling is be required for wound healing in the fly. Consistent with this idea, the targets of JNK signaling during immune activation have been reported to include many cytoskeletal components similar to those reported to be JNK-inducible genes during dorsal closure. Although this is a reasonable hypothesis, it has not yet been demonstrated that inhibition of JNK signaling, for example by inactivating bsk or hep, prevents proper wound repair. In addition to its potential role in wound healing, Drosophila JNK signaling has also been implicated in the activation of stress-protective proteins. Also, earlier reports have suggested that AP-1-like transcription factors, which are the ultimate targets of the JNK signaling pathway, might regulate antimicrobial peptide gene expression. Thus, JNK may act at many different levels in the Drosophila immune response, but few mechanistic details have emerged (Silverman, 2003).

In mammals, the MAP3K TAK1 plays a central role in the IL-1R/Toll-like receptor signaling pathways. In vitro biochemical studies showed that TAK1 could be activated directly by tumor necrosis factor receptor-associated factor 6 (TRAF6), which is known to be a critical component of this signaling pathway. Furthermore, these in vitro studies demonstrated that, once activated, TAK1 can activate both the IKK complex (and NF-kappaB signaling) as well as the JNKK MKK6 (and JNK signaling) (Wang, 2001). Recent studies in mammalian cell culture have demonstrated that TAK1 is required for NF-kappaB activation following tumor necrosis factor alpha or IL-1 stimulation (Takaesu, 2003). Similarly, the Drosophila TAK1 is required for the Imd signaling pathway; Drosophila TAK1^-/- flies have a severely compromised immune response and do not express antimicrobial peptides upon infection with Gram-negative bacteria (Vital, 2001). Also, gene expression profiling experiments have implied that Drosophila TAK1 is responsible for activation of both JNK and NF-kappaB (Relish) signaling following immune stimulation (Butros, 2002). However, these studies provide no biochemical data supporting this hypothesis (Silverman, 2003).

This report used Drosophila S2* cells to 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).

This study has shown that 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 suggested that TAK1 functions upstream of the Drosophila IKK complex (Vidal, 2001). Consistent with these results, this study shows that TAK1 is 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. Boutros (2002) used gene expression profiling to infer that TAK1 is required for the activation of JNK signaling and that JNK signaling is important for wound healing. This study directly demonstrated 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 (Wang, 2001). 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, as shown in this stduy, and in adult flies as shown by De Gregorio (2001). However, that study found that, in adult flies, the immune induction of Punch requires Relish, whereas 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 in this study were performed in an embryonic Drosophila cell line (that has macrophage-like qualities), whereas the data from De Gregorio 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 (McLean, 1993) 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. First, dopamine is one of two main Drosophila catecholamines, which are important for the stress response in both insects and mammals. Second, 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).

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. First, NO is known to be a major microbicidal compound in mammalian phagocytic cells and is likely to function similarly in Drosophila macrophages. Second, NO has also been implicated in immune signaling in Drosophila. Foley (2003) recently reported that 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).

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 (Rodrigues, 1995). 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 (Nelson, 1999). 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).

A recent microarray study 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. Only a few JNK target genes have been reported in this study (for example, Filamin). Instead, this study identified a number of genes implicated in cell-cell interactions, stress responses, and macrophage activation. The differences between these studies could be the consequence of one or more of the following reasons. First, the experiments of Boutros (2002) targeted two MAP2Ks (Hep and dMKK4) by RNAi. As shown in the biochemical data presented in this study, Drosophila JNKK (hep) is responsible for LPS-induced phosphorylation of c-Jun. Thus, it is likely that in the experiments of Boutros additional MAPK pathways were inhibited. Second, ecdysone-treated S2* cells were used instead of undifferentiated S2 cells. Ecdysone treatment causes significant changes in the S2* cells, including altered morphology and adherence, cell cycle arrest, and, most importantly, a greater level of LPS-induced antimicrobial peptide gene expression. Also, in the current experiments printed cDNA arrays were used that include only ~40% of the predicted genes in the fly, whereas Boutros used oligo-based arrays that include features for all ~14,000 predicted genes. Therefore, 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 the current data (Silverman, 2003).

Earlier studies suggested that certain antimicrobial genes (e.g. diptericin) require a combination of transcription factors for their proper induction. It was 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, the last decade of research has identified only Relish as being required for the immune inducible expression of diptericin. The data presented in this study 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-gamma 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 were identified that may function in cellular immunity and stress protection (Silverman, 2003).

Caspase-mediated processing of the Drosophila NF-kappaB factor Relish

The NF-κB-like transcription factor Relish plays a central role in the innate immune response of Drosophila. Unlike other NF-κB proteins, Relish is activated by endoproteolytic cleavage to generate a DNA-binding Rel homology domain and a stable IκB-like fragment. This signal-induced endoproteolysis requires the activity of several gene products, including the IκB kinase complex and the caspase Dredd. This study used mutational analysis and protein microsequencing to demonstrate that a caspase target site, located in the linker region between the Rel and the IκB-like domain, is the site of signal-dependent cleavage. Physical interaction between Relish and Dredd is shown, suggesting that Dredd indeed is the Relish endoprotease. In addition to the caspase target site, the C-terminal 107 aa of Relish are required for endoproteolysis and signal-dependent phosphorylation by the Drosophila IκB kinase β. Finally, an N-terminal serine-rich region in Relish and the PEST domain were found to negatively regulate Relish activation (Stöven, 2003).

Innate immune responses rely on transcription factors of the Rel/NF-κB family. In unstimulated cells, Rel proteins reside in the cytoplasm complexed with an inhibitory IκB molecule. After an immune challenge, the inhibitor is phosphorylated by the IκB kinase (IKK) complex, ubiquitinated, and degraded by the 26S proteasome. The released Rel protein translocates to the nucleus where it activates target genes. The signaling cascades that activate Rel proteins are remarkably conserved between flies and human. Many proteins involved in the mammalian tumor necrosis factor receptor pathway have close homologs in the Drosophila immune deficiency (imd) pathway, which controls the immune-induced production of antimicrobial peptides. Recent genetic studies have established an order in which the participating genes may act in this signaling pathway. The central transcription factor in the imd signaling cascade is the NF-κB factor Relish. With its composite structure, comprising a Rel homology domain and an IκB-like domain, Relish is similar to the mammalian NF-κB precursors p100 and p105 (Stöven, 2003).

But in striking contrast to its mammalian counterparts, the activation of Relish does not require proteasome-dependent degradation of the IκB-like region. Instead, Relish is processed by rapid, signal-dependent endoproteolysis, generating two stable fragments: REL-68, which contains the Rel homology domain and translocates to the nucleus, and REL-49, which includes the IκB-like region and remains cytoplasmic. Unexpectedly, a role for a caspase in Relish activation was indicated by the fact that mutants in Dredd, a Drosophila caspase gene, are deficient in Relish processing and antimicrobial peptide production. But whether Dredd acts directly on Relish has been an open question (Stöven, 2003).

The Drosophila IKK complex also regulates Relish processing. The IKK complex is activated by immune stimulation and Drosophila IKKβ can directly phosphorylate Relish in vitro (Silverman, 2000). Moreover, mutants in ird5 (IKKβ) and kenny (IKKγ) have the same immune phenotype as Relish mutants (Lu, 2001; Rutschmann, 2000). It has not been clear though whether IKKβ-mediated phosphorylation of Relish occurs in response to an immune stimulus and whether it is required for Relish cleavage in vivo (Stöven, 2003).

This study further investigated the roles of Dredd and IKKβ in Relish cleavage and characterized those sequences in Relish that are required for its endoproteolysis. The actual cleavage site is reported, along with direct interactions between Dredd and Relish; this information together provide strong evidence that Relish endoproteolysis is indeed carried out by the caspase Dredd (Stöven, 2003).

The data presented here demonstrate that the signal-dependent cleavage of Relish occurs at a caspase target site. The residues immediately adjacent to the cleavage site fit the caspase consensus and the critical aspartate within this site, at position 545, is required for cleavage. These data strongly argue for a caspase as the Relish endoprotease. Although Dredd, by homology to the human caspases-8 and -10, is thought to be an initiator rather than an effector caspase, it is the prime candidate for the Relish endoprotease. Dredd mutants are unable to process Relish. This study demonstrated that Dredd and Relish interact physically. Furthermore, none of the other six known Drosophila caspases were found to be involved in Relish activation when RNA interference was used in cell culture. However, so far it has not been possible to reconstitute cleavage in vitro with purified Dredd and IKKβ-phosphorylated Relish (Stöven, 2003).

Dredd and Relish are bound to each other before an immune stimulus, suggesting the existence of a preassembled Dredd/Relish complex that is awaiting the incoming signal. This signal is most likely identical with phosphorylation by IKKβ. This set-up fits well with the speed of Relish processing, which occurs within seconds after LPS stimulation (Stöven, 2003).

This study identified additional regions in Relish that control its activation. The N-terminal serine-rich region and the PEST domain seem to negatively regulate Relish activation. One attractive model for Relish activation is that the precursor is held in a closed conformation, via an interaction between the serine-rich region and the PEST domain. This closed conformation would prevent nuclear translocation, inappropriate cleavage, and DNA binding by concealing the nuclear localization signal and the poorly structured linker with the caspase target site. Upon stimulation, Relish is phosphorylated in a reaction that requires IKKβ and the C-terminal 107 residues of Relish. This modification results in an open conformation in which the nuclear localization signal and the caspase target site would become accessible (Stöven, 2003).

The direct involvement of a caspase in Relish endoproteolysis represents a novel mechanism of NF-κB activation and caspase function. Interestingly, a similar mechanism may also exist in mammalian systems. For example, a caspase-8 loss-of-function mutation in human patients is connected with defective activation of lymphocytes, a process that is known to require NF-κB. This new function of caspase-8 is independent of death receptor signaling and apoptosis induction. Another parallel between Relish processing and NF-κB activation in mammals is given by the so-called noncanonical NF-κB pathway, which requires NF-κB inducing kinase and IKK for the signal-dependent processing of p100 (Stöven, 2003 and references therein).

The role of ubiquitination in Drosophila innate immunity: Signaling upstream of Relish

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. The IKK complex consists of the the Drosophila IkappaB kinase [consisting of 2 subunits: a catalytic kinase subunit encoded by ird5 (IKKβ) and a regulatory subunit encoded by kenny (IKKγ)]. 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 DmIKKγ (Kenny) 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, DmIKKγ 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 DmIKKγ 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 IKKγ 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).

Selective autophagic degradation of the IKK complex in Drosophila is mediated by Kenny/IKKgamma to control inflammation

Implication of autophagy in the downregulation of immune signaling pathways through the degradation of their components constitutes an emerging field of investigation. This work showed that the selective interaction of Drosophila protein Kenny/IKKgamma (CG16910) with the autophagic machinery is required for the degradation of the I-kappa B kinase complex. This regulatory mechanism is essential for the downregulation of the immune deficiency (IMD) pathway in response to commensal microbiota to prevent inflammation (Jacomin, 2020).

Search PubMed for articles about Drosophila Ird5 and Kenny

Bergmann, A. (2003). IKKepsilon signaling: not just NF-kappaB. Curr. Biol. 16(15): R588-R590. PubMed ID: 16890515

Boutros, M., Agaisse, H., and Perrimon, N. (2002). Sequential activation of signaling pathways during innate immune responses in Drosophila. Dev. Cell 3: 711-722. PubMed ID: 12431377

De Gregorio, E., Spellman, P. T., Rubin, G. M. and Lemaitre, B. (2001). Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays. Proc. Natl. Acad. Sci. 98: 12590-12595. PubMed ID: 11606746

Delaney, J. R., et al (2006). Cooperative control of Drosophila immune responses by the JNK and NF-kappaB signaling pathways. EMBO J. 25: 3068-3077. PubMed ID: 16763552

Ertürk-Hasdemir, D., et al. (2009). Two roles for the Drosophila IKK complex in the activation of Relish and the induction of antimicrobial peptide genes. Proc. Natl. Acad. Sci. 106(24): 9779-84. PubMed ID: 19497884

Foley, E., and O'Farrell, P. H. (2003). Nitric oxide contributes to induction of innate immune responses to gram-negative bacteria in Drosophila. Genes Dev. 17: 115-125. PubMed ID: 12514104

Goto, A., et al. (2008). Akirins are highly conserved nuclear proteins required for NF-kappaB-dependent gene expression in Drosophila and mice. Nat. Immunol. 9: 97-104. PubMed ID: 18066067

Jacomin, A. C. and Nezis, I. P. (2020). Selective autophagic degradation of the IKK complex in Drosophila is mediated by Kenny/IKKgamma to control inflammation. Mol Cell Oncol 7(1): 1682309. PubMed ID: 31993495

Lu, Y., Wu, L. P. and Anderson, K. V. (2001). The antibacterial arm of the Drosophila innate immune response requires an IkappaB kinase. Genes Dev 15: 104-110. 11156609

McLean, J. R., Krishnakumar, S. and O'Donnell, J. M. (1993). Multiple mRNAs from the Punch locus of Drosophila melanogaster encode isoforms of GTP cyclohydrolase I with distinct N-terminal domains. J. Biol. Chem. 268: 27191-27197. PubMed ID: 8262960

Medzhitov, R. and Janeway, C. (2000). Innate immune recognition: Mechanisms and pathways. Immun. Rev. 173: 89–97. PubMed ID: 10719670

Nelson, N. (1999). Metal ion transporters and homeostasis. EMBO J. 18: 4361-4371. PubMed ID: 10449402

Rodrigues, V., Cheah, P. Y., Ray, K. and Chia, W. (1995). malvolio, the Drosophila homologue of mouse NRAMP-1 (Bcg), is expressed in macrophages and in the nervous system and is required for normal taste behaviour. EMBO J. 14: 3007-3020. PubMed ID: 7621816

Rutschmann, S., Jung, A. C., Zhou, R., Silverman , N., Hoffmann, J. A. and Ferrandon, D. (2000). Role of Drosophila IKKgamma in a Toll-independent antibacterial immune response. Nat Immunol. 1: 342-347. 11017107

Silverman, N., Zhou, R., Stöven, S., Pandey, N., Hultmark, D. and Maniatis, T. (2000). A Drosophila IkappaB kinase complex required for Relish cleavage and antibacterial immunity. Genes Dev 14: 2461-2471. 11018014

Silverman, N., Zhou, R., Erlich, R. L., Hunter, M., Bernstein, E., Schneider, D. and Maniatis, T. (2003). Immune activation of NF-kappaB and JNK requires Drosophila TAK1. J. Biol. Chem. 278(49): 48928-34. PubMed ID: 14519762

Stöven, S., Silverman, N., Junell, A., Hedengren-Olcott, M., Erturk, D., Engström, Y., Maniatis, T. and Hultmark, D. (2003). Caspase-mediated processing of the Drosophila NF-kappaB factor Relish. Proc. Natl. Acad. Sci. USA 100: 5991-5996. 12732719

Takaesu, G., Surabhi, R. M., Park, K. J., Ninomiya-Tsuji, J., Matsumoto, K. and Gaynor, R. B. (2003). TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway. J. Mol. Biol. 326: 105-115. PubMed ID: 12547194

Vidal, S., Khush, R. S., Leulier, F., Tzou, P., Nakamura, M. and Lemaitre, B. (2001). Mutations in the Drosophila dTAK1 gene reveal a conserved function for MAPKKKs in the control of rel/NF-kappaB-dependent innate immune responses. Genes Dev. 15: 1900-1912. PubMed ID: 11485985

Wang, C., Deng, L., Hong, M., Akkaraju, G. R., Inoue, J. and Chen, Z. J. (2001). TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature 412: 346-351. PubMed ID: 11460167

Wiklund, M. L., et al. (2009). The N-terminal half of the Drosophila Rel/NF-kappaB factor Relish, REL-68, constitutively activates transcription of specific Relish target genes. Dev. Comp. Immunol. 33: 690-696. PubMed ID: 19135474

Zhou, R., Silverman, N., Hong, M., Liao, D. S., Chung, Y., Chen, Z. J. and Maniatis, T. (2005). The role of ubiquitination in Drosophila innate immunity. J. Biol. Chem. 280: 34048-34055. 16081424

date revised: 12 September 2022

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