TGF-ß activated kinase 1


The role of ubiquitination in Drosophila innate immunity: The Drosophila homologs of Ubc13 are required in the Imd pathway for the activation of dTAK1 and the DmIKK complex

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).

Tumor suppressor CYLD regulates JNK-induced cell death in Drosophila

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).

Protein Interactions

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 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).

Targeting of TAK1 by the NF-kappaB protein Relish regulates the JNK-mediated immune response in Drosophila

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).

Inhibitor of apoptosis 2 and TAK1-binding protein are components of the Drosophila Imd pathway

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).

The RING-finger scaffold protein Plenty of SH3s targets TAK1 to control immunity signalling in Drosophila

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).

Drosophila TAB2, through interaction with Tak1, is required for the immune activation of JNK and NF-kappaB

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).

Domain specificity of MAP3K family members, MLK and Tak1, for JNK signaling in Drosophila

A highly diverse set of protein kinases function as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess fourteen MAPK kinase kinases (MAP3Ks) that activate Jun Kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, MAP3K signaling was assessed specificity in several JNK-dependent processes during development and stress response. The approach taken was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slipper and the TGF-beta activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure; then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. It was found that overexpression of the wildtype kinases stimulated JNK signaling in alternate contexts, so cells were capable to respond to both MAP3Ks, but with distinct outcomes. Relative to wildtype, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts including TNF-dependent cell death and innate immune signaling, however depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, these findings suggest that the selective deployment of a particular MAP3K can be attributed in part to their inherent sequence differences, cellular localization, and binding partner availability (Stronach, 2014).

Loss of Trabid, a new negative regulator of the Drosophila immune-deficiency pathway at the level of TAK1, reduces life span

A relatively unexplored nexus in Drosophila Immune deficiency (IMD) pathway is TGF-beta Activating Kinase 1 (TAK1), which triggers both immunity and apoptosis. In a cell culture screen, this study identified that Lysine at position 142 was a K63-linked Ubiquitin acceptor site for TAK1, required for signalling. Moreover, Lysine at position 156 functioned as a K48-linked Ubiquitin acceptor site, also necessary for TAK1 activity. The deubiquitinase Trabid interacted with TAK1, reducing immune signalling output and K63-linked ubiquitination. The three tandem Npl4 Zinc Fingers and the catalytic Cysteine at position 518 were required for Trabid activity. Flies deficient for Trabid had a reduced life span due to chronic activation of IMD both systemically as well as in their gut where homeostasis was disrupted. The TAK1-associated Binding Protein 2 (TAB2) was linked with the TAK1-Trabid interaction through its Zinc finger domain that pacified the TAK1 signal. These results indicate an elaborate and multi-tiered mechanism for regulating TAK1 activity and modulating its immune signal (Fernando, 2014).


Effects of Mutation and Ectopic Expression

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).

TAK1 participates in c-Jun N-terminal kinase signaling during Drosophila development

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 lethality, mTAK1 and mTAK1deltaN were ectopically expressed in the adult eye using an eye-specific expression vector, pGMR. pGMR contains multimerized Glass-binding sites and promotes gene expression in all cells in and posterior to the morphogenetic furrow in the larval eye disc. Eye-specific expression of the TAK1 transgenes induces specific defects in the visual system. pGMR-mTAK1 transgenic flies show relatively weak defects in the compound eyes. The phenotype varies among transgenic strains: 6 out of 11 transgenic strains reveal a rough eye phenotype and a reduction in size of the compound eye. The eye size is reduced to about 70% of the wild-type eye size. Many ommatidia, typically located in the medial region, are fused to each other. Electron micrographs of ommatidial cross sections reveal that some ommatidia are missing photoreceptors, while others show disrupted rows of pigment granules. In the pupal eye disc, a wild-type ommatidium contains four cone cells, two primary pigment cells (which are surrounded by six secondary pigment cells), three tertiary pigment cells, and three sensory bristles. In ommatidia from mTAK1 overexpression lines, only two to three cone cells are observed. The number of primary pigment cells is relatively normal, but occasionally one of the primary pigment cells is also missing. Secondary and tertiary pigment cells seem to be heavily affected by mTAK1 expression, since these cells are rarely observed in the mutant retina (Takatsu, 2000).

mTAK1deltaN was also expressed using the pGMR vector. In contrast to the pGMR-mTAK1 transgenic line, all pGMR-mTAK1deltaN transgenic files (eight independent transgenic lines) displayed severe eye phenotypes. In the weakest line, eye size was less than 30% of the wild type and ommatidial units were rarely identified. In addition, photoreceptor cells and pigment granules were difficult to recognize. In the mutant pupal eye disc, cone cells, primary pigment cells, and accessory cells exist in a disorganized array. The results suggest that the strength of signaling activity of mTAK1deltaN is stronger than that of intact mTAK1 in Drosophila (Takatsu, 2000).

To test whether Drosophila Tak1 is a true functional ortholog of mTAK1, Drosophila Tak1 was expressed under the control of the GMR-GAL4 driver. A small eye phenotype similar to that produced by pGMR-mTAK1deltaN (activated form) was obtained. The difference in the strength of Drosophila Tak1 enzymatic activity compared to the nonactivated mTAK1 might be explained by the difference in the NH2-terminal structures of these proteins. mTAK1 contains a serine-rich NH2-terminal domain that has been shown to down regulate mTAK1 kinase activity. Interestingly, Drosophila Tak1 lacks this domain, suggesting that Drosophila Tak1 may have a higher basal level of activity in the absence of an activator protein(s), such as TAB1 (Shibuya, 1996 and 1998), than does the vertebrate gene product (Takatsu, 2000).

The cause of the small eye phenotype induced by ectopic TAK1 signaling. Acridine orange staining, which identifies dying cells, was used to examine the extent of cell death in larval eye discs of Drosophila Tak1-overexpressing flies. This study reveals that dying cells increase dramatically in regions posterior to the morphogenetic furrow, suggesting that cell death occurred rapidly after Tak1 induction. The reduced eye phenotype of Tak1 overexpression can be rescued by coexpression of the apoptotic inhibitor protein p35. Also, the Drosophila inhibitor of apoptosis proteins 1 and 2 (DIAP1 and DIAP2) effectively rescues the phenotype. These data indicate that the cell death induced by Tak1 is apoptotic in nature; additional studies show that its induction is dependent on endogenous proapoptotic gene activity (Takatsu, 2000).

Sequential recruitment of the photoreceptor cells is disrupted in Tak1-overexpressing discs. The induction of early photoreceptor cells (R8 and occasionally R2 and R5) appears to occur normally. However, later photoreceptor induction is delayed or disrupted, and photoreceptor clustering shows an abnormal and diffuse pattern even in the presence of p35. This result indicates that ectopic TAK1 signaling may also directly or indirectly affect certain aspects of cell fate specification. Maybe this abnormal photoreceptor induction affects later cell development which includes cone cell and pigment cells. However, proper numbers of the cone cells and primary pigment cells are induced in the pupal eye discs from pGMR-mTAK1deltaN; pGMR-p35. It is concluded that TAK1 induces cell death after proper cell fate determination in at least some of the ommatidial cells, such as R8. However, in other cells, abnormal cell fate determination by the ectopic Tak1 signaling may also contribute to induction of cell death in the Tak1-expressing eye discs (Takatsu, 2000).

To address which signaling pathway(s) might be activated by Tak1 in vivo, genetic interactions between mutants in various signaling pathways and a Tak1-overexpressing line (either pGMR-mTAK1deltaN or GMR-GAL4/UAS-Tak1) were tested. Included in this screen were mutations that disrupt BMP signaling, the Raf-MAPK pathway, and the JNK pathway. Among the various mutants tested, only alleles of hep and bsk were found to interact strongly with lines overexpressing Tak1. In hemizygous hep or heterozygous bsk mutant backgrounds, the reduced size of compound eyes of GMR-GAL4/UAS-Tak1 flies is rescued to that of wild-type flies (Takatsu, 2000).

The Hep-Bsk signaling pathway has been shown to regulate the process of dorsal closure during the embryonic development. Two genes, puckered (puc) and decapentaplegic (dpp), are downstream targets of Hep-Bsk signaling and are induced in the leading-edge cells in the embryonic epidermis during dorsal closure. To further examine whether TAK1 signaling is mediated by Hep and Bsk, mTAK1 was misexpressed using the en-GAL4 driver, which promotes GAL4 expression in the posterior compartment of the embryonic ectoderm. Ectopic puc and dpp expression was observed in UAS-Tak1/en-GAL4 embryos in a striped pattern. This result indicates that exogenous mTAK1 activates the Hep-Bsk pathway in the embryo as well as during imaginal disc development. Whether Drosophila Tak1 expression leads to Bsk protein phosphorylation. Tak1 was transiently expressed in the third-instar larva by expressing UAS-Tak1 and hs-GAL4. Strong Bsk phosphorylation was observed only in the flies carrying UAS-Tak1 after heat shock. Phosphorylation of Drosophila p38 was not affected by Drosophila Tak1 expression. It is concluded that Drosophila Tak1 can activate the Hep-Bsk MAPK cascade in vivo (Takatsu, 2000).

A common feature exhibited by loss-of-function mutations in genes of the JNK signaling pathway is a failure of proper dorsal closure due to disruptions in the movement and the ability of cells to change shape in the leading edge of the lateral epidermis. Some of these mutants also exhibit defects in head structure and/or problems with germ band retraction. To test whether Tak1 also controls dorsal closure signals during normal development, a kinase-dead form (Tak1-K46R) was overexpressed in the embryonic epidermis, using a pnr-GAL4 driver. Various types of cuticle defects were observed. The most typical phenotype was a head structure defect in which mouth hooks were missing (36%). A certain fraction of these defective embryos also exhibited an anterior open phenotype similar to that exhibited by hep and bsk mutants. In the most extreme cases, almost the entire dorsal cuticle failed to close. Occasionally, embryos with a U-shaped phenotype were observed presumably due to insufficient germ band retraction. These phenotypes are consistent with the idea that Tak1 participates in the JNK signaling pathway (Takatsu, 2000).

Two different biological processes are known to be controlled by the JNK signaling pathway in Drosophila. One is the movement of leading-edge cells during the process of embryonic dorsal closure, and the other is in planar polarity determination of adult tissues. In neither case, however, is the role or identity of the putative MAPKKK molecule(s) that might be involved in these processes known. These studies show that Drosophila Tak1 could activate the JNK signaling cascade. Loss-of-function experiments, using dominant negative Tak1 and mTAK1, also revealed that TAK1 is likely to be required for the proper cell movement and/or shape changes in the embryo and visual system. Indeed, the dorsal open phenotype and head structure defects observed in the dominant negative Tak1-expressing embryos are highly reminiscent of the phenotype produced by JNK pathway loss-of-function mutants. Impaired control of cell shape was also noted in TAK1 gain-of-function phenotypes obtained by overexpression of Tak1 in the presence of p35 and also with dominant negative forms of TAK1. The function of JNK signaling in cell movement in the visual system has been studied only for ommatidial planar polarity determination. Although no defects in planar polarity were seen, abnormalities in the positioning and shape of interommatidial cells were seen in both dominant negative Tak1-expressing lines and hep mutants. This result suggests that the endogenous JNK signaling also participates in the positioning and shape of the interommatidial cells. Since a true loss-of-function hep allele also shows this phenotype, albeit more weakly, it is not likely that the phenotype caused by overexpression of dominant negative Tak1 is due to a novel neomorphic property of this protein. The weaker interommatidial phenotype of hemizygous hepr75 flies compared to that of pGMR-mTAK1-K63W (two copies) might indicate that there is some genetic redundancy for this class of kinase in Drosophila. Likewise, the lack of observable planar polarity defects in these animals may indicate either a nonrequirement for a Tak1 kinase in this process or a genetic redundancy for a Tak1-type kinase, or that this particular phenotype is less sensitive to Tak1 loss-of-function and so is not observable under the conditions used in this study (Takatsu, 2000).

Previous reports have shown that TAK1 is activated by TGF-ß/BMP stimuli and that a kinase-negative form of TAK1 prevents TGF-ß/BMP signaling in mammalian cells and in Xenopus. Surprisingly, no genetic interactions were observed between pGMR-mTAK1deltaN overexpression lines and mutations in the Dpp signaling pathway. Ectopic Tak1 signaling in the wing disc is unable to induce optomotor blind, a known downstream target of Dpp signaling and, furthermore, ectopic vein formation induced by a constitutively active Dpp receptor can not be suppressed by overexpression of Tak1-K46R expression. These results may indicate that Dpp signaling is not regulated by Tak1 in the visual system and wing or, alternatively, that the effects of Dpp-induced Tak1 signaling are mild compared to the Mad/Medea pathway of Dpp signaling and so are not observable with the genetic tests presently at hand (Takatsu, 2000).

The role of the Drosophila TAK homologue dTAK during development

Tak1 induces cell death in imaginal discs. Unlike the phenotype at 18°C, Tak1 overexpression at 25°C in imaginal discs is lethal with many GAL4 drivers and leads to a very severe eye phenotype with sev-GAL4. The adult eye is strongly reduced in size and the structure of the ommatidial clusters is completely disrupted with photoreceptor cells often lost or not differentiated. This phenocopies the eye ablation phenotypes by ectopic expression of inducers of cell death in the eye imaginal disc. One possibility to explain this small eye phenotype is an increased amount of cell death within the developing Drosophila retina. This would also be consistent with the JNK-mediated induction of apoptosis in imaginal disc tissue. This hypothesis was tested by monitoring cell death in third instar larval eye discs using Acridine Orange staining to mark dying cells. Tak1 overexpression dramatically increases cell death in the eye imaginal disc. Whereas wild-type eye disc tissue exhibits a very low background of Acridine Orange positive, apoptotic cells both in front and behind the morphogenetic furrow (MF), sev-GAL4/UAS-Tak1 discs exhibited massive cell death behind the MF, corresponding to the expression domain of the GAL4 driver (Mihaly, 2001).

Whether the Tak1-induced cell death is mediated by the normal apoptotic pathway and thus can be inhibited with the caspase inhibitor p35 was examined. Co-expression of the viral caspase inhibitor p35 with Tak1 results in a significant rescue of the phenotype caused by Tak1 overexpression alone. These data suggest that Tak1-induced cell death is largely caused by caspase-dependent apoptotic cell death (Mihaly, 2001).

Eiger, a TNF superfamily ligand that triggers the Drosophila JNK pathway

Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death, was identified in a large-scale gain-of-function screen. Eiger is a type II transmembrane protein with a C-terminal TNF homology domain. It is predominantly expressed in the nervous system. Genetic evidence shows that Eiger induces cell death by activating the Drosophila JNK pathway. Although this cell death process is blocked by Drosophila inhibitor-of-apoptosis protein 1 (DIAP1, Thread), it does not require caspase activity. Genetically, Eiger has been shown to be a physiological ligand for the Drosophila JNK pathway. These findings demonstrate that Eiger can initiate cell death through an IAP-sensitive cell death pathway via JNK signaling (Igaki, 2002).

Many mammalian TNF superfamily proteins activate both the NF-kappaB and the JNK pathway, and activation of the latter pathway facilitates cell death (Davis, 2000). To examine whether Eiger activates the JNK pathway, the genetic interactions of Eiger with the components of the Drosophila JNK cascade were examined. The reduced-eye phenotype induced by Eiger is strongly suppressed in basket (bsk), a heterozygous mutant of Drosophila JNK. In addition, overexpression of a dominant-negative form of Bsk almost completely suppresses the eye phenotype. Moreover, heterozygosity at the hemipterous (hep) locus, which encodes Drosophila JNKK, suppresses the reduced-eye phenotype much as does bsk, and its hemizygosity (null background) rescues the phenotype almost completely. Furthermore, the co-expression of a dominant-negative form of dTAK1 TGF-ß activated kinase 1; Drosophila JNKKK) also rescues the Eiger-induced phenotype completely. Misshapen (Msn) is a MAPKKKK that is genetically upstream of the JNK pathway in Drosophila. A half dosage of the msn gene strongly suppresses the Eiger-induced phenotype. Heterozygosity of Drosophila jun, a target of the JNK pathway, did not show any genetic interaction with Eiger, raising the possibility that the Eiger-stimulated death-inducing JNK pathway may not require new transcripts that are controlled by Jun (Igaki, 2002).

Puckered (Puc) is a dual-specificity phosphatase, the expression of which is induced by the Drosophila JNK pathway to inactivate Bsk, so that puc expression can be used to monitor the extent of activation of the JNK pathway. To confirm that the JNK pathway is actually activated by Eiger, puc expression level was assessed in the eye disc of GMR>regg1GS9830 flies using a puc-LacZ enhancer-trap allele. The strong induction of puc-LacZ was observed in the region posterior to the morphogenetic furrow of the eye disc compared with the control eye disc. Furthermore, Western blot analysis with an anti-phospho-JNK antibody has revealed that Bsk is phosphorylated by Eiger overexpression. These genetic and biochemical data led to a model in which Eiger activates Msn, thereby triggering the JNK signaling pathway, sequentially stimulating dTAK1, Hep and Bsk. Using RT-PCR analysis, whether Eiger could stimulate the NF-kappaB pathway was tested; however, no obvious upregulation of the antimicrobial peptide genes, the target genes of the Drosophila NF-kappaB pathway, was detected (Igaki, 2002).

Inducible expression of double-stranded RNA reveals a role for dFADD in the regulation of the antibacterial response in Drosophila adults

In Drosophila, the immune deficiency (Imd) pathway controls antibacterial peptide gene expression in the fat body in response to Gram-negative bacterial infection. The ultimate target of the Imd pathway is Relish, a transactivator related to mammalian P105 and P100 NF-kappaB precursor. Relish is processed in order to translocate to the nucleus, and this cleavage is dependent on both Dredd, an apical caspase related to caspase-8 of mammals, and the fly Ikappa-B kinase complex (dmIKK). dTAK1, a MAPKKK, functions upstream of the dmIKK complex and downstream of Imd, a protein with a death domain similar to that of mammalian receptor interacting protein (RIP). Finally, the peptidoglycan recognition protein-LC (PGRP-LC) acts upstream of Imd and probably functions as a receptor for the Imd pathway. Interference with dFADD (FlyBase designation: BG4) function by double-stranded RNA inhibition demonstrates that dFADD is a novel component of the Imd pathway. dFADD double-stranded RNA expression reduces the induction of antibacterial peptide-encoding genes after infection and renders the fly susceptible to Gram-negative bacterial infection. Epistatic studies indicate that dFADD acts between Imd and Dredd. These results reinforce the parallels between the Imd and the TNF-R1 pathways (Leulier, 2002).

dFADD is a gene encoding a death domain protein with an overall structure similar to that of mammalian Fas-associated death domain-containing protein (FADD), an adaptor that is believed to interact with the TNF-R1 complex through homophilic death domain interactions with the TNF-R-associated death domain-containing protein (TRADD). FADD then recruits pro-caspase-8 through homophilic death effector domain associations. Consequently, dFADD is an obvious candidate for linking the death domain protein Imd and the Dredd apical caspase in the Imd pathway. Inducible expression of dFADD double-stranded RNA has been used to determine if dFADD functions in the Imd pathway. This approach, which exploits the UAS/GAL4 binary system to drive expression of double-stranded RNA in a defined tissue is a form of RNA interference (RNAi) that has previously been shown to block the expression of defined genes (Leulier, 2002).

Transgenic flies carrying either UAS-dTAK1-IR or UAS-dFADD-IR have been generated. Both constructs consist of 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. Three GAL4 lines were used in this study: daughterless-GAL4 (da-GAL4), which expresses GAL4 strongly and ubiquitously; hs-GAL4, which directs expression of GAL4 ubiquitously after heat shocks; and yolk-GAL4, which expresses the yeast transactivator in the fat body of female adults (Leulier, 2002).

dTAK1-deficient flies do not express the antibacterial peptide-encoding gene Diptericin upon immune challenge and are highly susceptible to infection by Gram-negative bacteria. A similar phenotype is generated by mutations affecting the other components of the Imd pathway. Interestingly, the expression of UAS-dTAK1-IR induced by either the hs-GAL4 or the yolk-GAL4 drivers produces an immune deficiency phenotype similar to dTAK1 mutants: UAS-dTAK1-IR flies fail to express antibacterial-encoding genes after infection and are highly susceptible to Gram-negative bacterial infection. However, the UAS-dTAK1-IR expression phenotype is weaker than the dTAK1 null mutant phenotype, both in terms of survival and affect on anti-microbial peptide gene expression, suggesting that the inducible expression of RNAi mimics a partial loss-of-function mutation of the target gene. In agreement with what was observed in dTAK1 null mutants, expression of UAS-dTAK1-IR using the ubiquitous driver da-GAL4 does not lead to detectable developmental defects. This contrasts with the results obtained by expression of a dominant-negative construct of dTAK1, which leads to ectopic developmental defects. Taken together, these results demonstrate the suitability of the RNAi approach for functional studies of the antimicrobial response (Leulier, 2002).

To address dFADD's role in the regulation of antimicrobial gene expression, the UAS-dFADD-IR transgene was expressed using the three GAL4 insertions. Flies that express dFADD-IR ubiquitously through da-GAL4 show no detectable defects, suggesting that dFADD is not essential for development. These flies do, however, have phenotypes similar to those generated by mutations affecting the Imd pathway. The expression of antibacterial peptide genes Diptericin and Attacin are strongly reduced after septic injury, while the expression of the antifungal gene Drosomycin remains inducible. In addition, these flies exhibit a high susceptibility to Gram-negative bacterial infection but resistance to fungal infection. This phenotype is identical to that generated by the UAS-dTAK1-IR construct and is similar to (although slightly weaker than) those generated by null mutations in dTAK1, kenny, ird5, Dredd, Relish, and imd. These results demonstrate that, like the other components of the Imd pathway, dFADD is required for a full antibacterial response (Leulier, 2002).

Overexpression of the imd gene leads to constitutive transcription of antibacterial peptide genes, and this induction requires the Dredd caspase. Expression of both dTAK1-IR and dFADD-IR strongly reduces the Imd-mediated induction of antibacterial peptide-encoding genes, indicating that, genetically, dFADD and dTAK1 function downstream of Imd. This result was confirmed by demonstrating that the dTAK11 mutation also blocks the constitutive Diptericin expression induced by imd overexpression. Overexpressing Dredd via the UAS/GAL4 system also leads to Diptericin expression in the absence of infection, which can be monitored with a Diptericin-lacZ transgene. lacZ titration assays demonstrate that the Diptericin reporter gene expression induced by overexpressing the UAS-Dredd transgene is not affected by the coexpression of dFADD-IR. Consequently, these epistatic studies place dFADD function upstream of the Dredd caspase. This result is in agreement with cell culture experiments showing that dFADD binds to Dredd through its N-terminal prodomain and promotes the proteolytic processing of Dredd (Leulier, 2002).

Recent studies have shown that the Drosophila homolog of MyD88, dMyD88, is an essential component of the Toll pathway. In addition, dMyD88 has been shown to bind in vitro to dFADD, pointing to a possible interaction between dFADD and the Toll pathway. Expression of dFADD-IR does not, however, block the constitutive Drosomycin expression induced by the dominant, gain-of-function Toll10b mutation, and dFADD RNAi does not block Drosomycin induction by infection. This result indicates that, like the other components of the Imd pathway, dFADD is not required for Toll pathway function (Leulier, 2002).

Altogether, this analysis indicates that dFADD is a novel component of the Imd pathway that links Imd to Dredd. Biochemical studies show that dFADD contacts Dredd via homotypic dead effector domain interaction, and it is possible that dFADD interacts with Imd via its death domain. Consequently, dFADD, Dredd, and Imd may be components of a multiprotein adaptor complex functioning downstream of the receptor of the Imd pathway. Genetic studies suggest that the Imd pathway bifurcates downstream of Imd, with one branch leading to caspase activation via dFADD and the second branch leading to activation of the IKK complex via activation of dTAK1; both of these events are required for Relish processing (Leulier, 2002).

Studies using loss-of-function mutations in the genes encoding components of the Imd pathway did not provide clear evidence for a role of this cascade in developmentally regulated apoptosis. However, recently, it has been shown that the overexpression of imd with the da-GAL4 driver in flies induces an early larval lethality that can be partially rescued by coexpression of the viral caspase inhibitor P35, suggesting that Imd can also promote apoptosis. Interestingly, it was noted that the lethality induced by imd overexpression is totally suppressed in Dredd mutants but only marginally reduced in dTAK1 mutants, suggesting that this effect is mediated through the dFADD/Dredd arm but not the dTAK1-dmIKK arm of the Imd pathway (Leulier, 2002).

In conclusion, the implication of dFADD in the Drosophila Imd pathway strengthens the parallels between the Imd and TNF-R1 pathways: both cascades regulate NF-kappaB via RIP-MAPKKK-IKK intermediates and promote caspase activation through the FADD adapter. In Drosophila, these two processes are required to activate Relish, while, in mammals, current models suggest that the TNF-R1 pathway leads to either NF-kappaB activation or programmed cell death activation. Additional experiments are still required to demonstrate a clear role for the Imd pathway in the regulation of apoptosis. Finally, this study validates the use of the inducible expression of double-stranded RNA to address the in vivo function of genes that mediate the Drosophila antimicrobial response (Leulier, 2002).

Discrete functions of TRAF1 and TRAF2 in Drosophila melanogaster mediated by c-Jun N-terminal kinase and NF-kappaB-dependent signaling pathways

Two Drosophila tumor necrosis factor receptor-associated factors (TRAFs), Traf1 and Traf2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, as yet their in vivo functions and related signaling pathways are not fully understood. Traf1 is shown to be an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila. Ectopic expression of Traf1 in the developing eye induces apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses reveal that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by Traf1 are dependent on JNK and its upstream kinases, Hep and TGF-ß activated kinase 1. In support of these results, the Traf1-null mutant shows a remarkable reduction in JNK activity with an impaired development of imaginal discs< and a defective formation of photosensory neuron arrays. In contrast, Traf2 was demonstrated as an upstream activator of nuclear factor-kappaB (NF-kappaB). Ectopic expression of Traf2 induces nuclear translocation of two Drosophila NF-kappaBs, Dif and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and drosomycin. Consistently, the null mutant of Traf2 shows immune deficiencies in which NF-kappaB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, these findings demonstrate that Traf1 and Traf2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-kappaB-dependent signaling pathway, respectively (Cha, 2003).

To investigate the consequences of ectopic expression of Traf1 in the developing Drosophila eye, Traf1 was overexpressed by using an eye-specific gmr-GAL4 driver. The eyes of adults carrying one copy each of both gmr-GAL4 and Traf1 show a rough-eye surface with disorganized arrays of ommatidia, whereas the eyes of flies carrying either one copy of gmr-GAL4 or one copy of Traf1 alone appear normal. Examination of the retinal sections of adults carrying both gmr-GAL4 and Traf1 reveals the number of ommatidia to be reduced and the number and shape of the photoreceptor cells in each ommatidium also to be abnormal, compared to the controls, which carry only the gmr-GAL4 driver (Cha, 2003).

When two copies of Traf1 were overexpressed in the eye, it displayed a more severe phenotype and a reduced number of ommatidia, resulting in a size reduction of the compound eye, and some ommatidia were fused with each other. In contrast, ectopically expressed Traf2 had no effect on the eye development; ommatidial array, bristles, and compound eye size were all found to be normal (Cha, 2003).

Based upon the result that Traf1 is involved in JNK signaling, attempts were made to find out the signaling components between Traf1 and Hep by genetic interaction studies. Various kinases, such as Misshapen (msn), Slipper (slpr), and Drosophila Transforming growth factor ß-activated kinase 1 (Tak1), are known to be the upstream kinases for Hep in the eye development. Among these kinases, Tak1 synergistically increased the roughness of the compound eye surface and also reduced the eye size when coexpressed with Traf1. Moreover, Tak1-null mutation (Tak11), which has no effect on the eye morphology, is able to block the rough-eye phenotype caused by Traf1 overexpression. These data suggest that Traf1 activates the JNK signaling pathway via Tak1 and Hep (Cha, 2003).

Drosophila Sex-peptide stimulates female innate immune system after mating via the Toll and Imd pathways

Insect immune defense is mainly based on humoral factors like antimicrobial peptides (AMPs) that kill the pathogens directly or is based on cellular processes involving phagocytosis and encapsulation by hemocytes. In Drosophila, the Toll pathway (activated by fungi and gram-positive bacteria) and the Imd pathway (activated by gram-negative bacteria) leads to the synthesis of AMPs. But AMP genes are also regulated without pathogenic challenge, e.g., by aging, circadian rhythms, and mating. This study shows that AMP genes are differentially expressed in mated females. Metchnikowin (Mtk) expression is strongly stimulated in the first 6 hr after mating. Sex-peptide (SP), a male seminal peptide transferred during copulation, is the major agent eliciting transcription of Mtk and of other AMP genes. Both pathways are needed for Mtk induction by SP. Furthermore, SP induces additional AMP genes via the Toll (Drosomycin) and the Imd (Diptericin) pathways. SP affects the Toll pathway at or upstream of the gene spätzle, and the Imd pathway at or upstream of the gene imd. Mating may physically damage females and pathogens may be transferred. Thus, endogenous stimulation of AMP transcription by SP at mating might be considered as a preventive step to encounter putative immunogenic attacks (Peng, 2005).

The Toll and Imd signaling cascades are the major and best-characterized pathways involved in the activation of AMPs after pathogenic challenges. The effect of SP on AMP expression was studied by comparing the expression of Mtk, Drs, and Dipt in wt females or in females mutant in the Toll and Imd pathways, respectively, before and after mating with wt males. RNA was extracted from virgin and mated females and analyzed by quantitative PCR (Peng, 2005).

With the exception of dorsal (dl), all loss-of-function mutants of the Toll and Imd pathways abolish or strongly reduce Mtk expression after mating. Thus, Mtk expression induced by SP is dependent on both pathways. Furthermore, since spz and imd females fail to induce Mtk transcription after mating, SP must act on or upstream of spz and imd. dl and its functional homolog dif have been reported to be involved in AMP gene transcription under pathogenic challenge in the larval stage, but not functional in the adult immune defense. A partial response is observed in dl females, indicating that dl may be partially involved in the innate immune response elicited by SP in adult females (Peng, 2005).

Drs expression, controlled by the Toll pathway, is completely abolished in spz and Tl mutants. Correspondingly, Dipt expression, which is controlled by the Imd pathway, is completely abolished in the Imd pathway loss-of-function mutants imd, Tak1, and rel. It is concluded that SP can activate the Toll and the Imd pathways. The Toll pathway is essential for Drs expression, whereas the Imd pathway is essential for Dipt expression (Peng, 2005).

The SP-induced immune response activates the transcription of all three AMP genes studied. After pathogenic infections, Drs is induced by the Toll pathway and Dipt by the Imd pathway, whereas both pathways induce Mtk expression. The results obtained with the loss-of-function mutants follow this scheme. Whereas loss-of-function mutants of both pathways reduce or abolish Mtk expression after mating, induction of Drs expression is only abolished by loss-of-function mutants of the Toll pathway, whereas induction of Dipt expression is only lost in mutants of the Imd pathway. In sum, the classical pathways are activated to induce the transcription of AMP genes after mating as after microbial or fungal infections (Peng, 2005).

Detection of microorganisms and triggering the appropriate pathway is achieved by pattern recognition receptors (PRRs), immune proteins recognizing general microbial components. Two families of PRRs have been identified in Drosophila: the peptidoglycan recognition proteins (PGRPs) and the gram-negative binding proteins (GNBPs). Some of the 13 PGRPs encoded in the D. melanogaster genome have been implicated in the activation of specific immune responses. However, the signaling cascades between the PRRs and the Toll and the Imd pathways are not well characterized. Since in spz and imd null mutants AMP induction by SP is specifically abolished, the inducing signals must affect the signaling cascades at or upstream of those genes. At this stage, it cannot be determined whether SP enters the pathways at the PRR level or at an intermediate level between the PRRs and spz or imd, respectively. Furthermore, the induction of AMPs may occur systemically (e.g., in the fat body) or locally in the reproductive tract. Microarray analysis of AMP expression after mating of wt females with either wt or SP0 males, respectively, suggests that AMPs are mainly induced in the abdomen, but it does not discriminate between a systematic response in the abdomen and a specific response in the genital tract (Peng, 2005).

Drosophila females undergo dramatic physiological changes after mating, predominantly induced by SP. Mating may also physically damage females and may expose the female to pathogens transferred by the male as shown for the milkweed leaf beetle. Thus, the activation of the innate immune system to encounter putative immunogenic attacks during this sensitive phase of the life history of females makes biological sense. The signal is plausibly coupled to copulation in the form of SP transferred in the seminal fluid. Such a mechanism might allow the female to respond preventively to potential threats. In sum, these findings may describe the result of an optimal economical balance between spending costly energy for the innate immune response and preventive measures to fight a putative pathogenic attack (Peng, 2005).

Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila

Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Using a Drosophila genetic model for Ras-induced tumor progression, a molecular link has been shown between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/β-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic RasV12-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, since JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. The finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression (Igaki, 2006).

Most human cancers originate from epithelial tissues. These epithelial tumors, except for those derived from squamous epithelial cells, normally exhibit pronounced apicobasal polarity. However, these tumors commonly show defects in cell polarity as they progress toward malignancy. Although the integrity of cell polarity is essential for normal development, how cell polarity disruption contributes to the signaling mechanisms essential for tumor progression and metastasis is unknown. To address this, a recently established Drosophila model of Ras-induced tumor progression triggered by loss of cell polarity has been used. This fly tumor model exhibits many aspects of metastatic behaviors observed in human malignant cancers, such as basement membrane degradation, loss of E-cadherin expression, migration, invasion, and metastatic spread to other organ sites (Pagliarini, 2003). In the developing eye tissues of these animals, loss of apicobasal polarity is induced by disruption of evolutionarily conserved cell polarity genes such as scribble (scrib), lethal giant larvae (lgl), or discs large (dlg), three polarity genes that function together in a common genetic pathway, as well as other cell polarity genes such as bazooka, stardust, or cdc42. Oncogenic Ras (RasV12), a common alteration in human cancers, causes noninvasive benign overgrowths in these eye tissues (Pagliarini, 2003). Loss of any one of the cell polarity genes somehow strongly cooperates with the effect of RasV12 to promote excess tumor growth and metastatic behavior. However, on their own, clones of scrib mutant cells are eliminated during development in a JNK-dependent manner; expression of RasV12 in these mutant cells prevents this cell death (Igaki, 2006).

To better quantify the metastatic behavior of tumors in different mutant animals, the analysis focused on invasion of the ventral nerve cord (VNC), a process in which tumor cells leave the eye-antennal discs and optic lobes (the areas where they were born) and migrate to and invade a different organ, the VNC. It was further confirmed that the genotypes associated with the invasion of the VNC in this study also resulted in the presence of secondary tumor foci at distant locations, although the number and size of these foci were highly variable (Pagliarini, 2003; Igaki, 2006).

In analyzing the global expression profiles of noninvasive and invasive tumors induced in Drosophila developing eye discs, it was observed that expression of the JNK phosphatase puckered (puc) was strongly upregulated in the invasive tumors. Upregulation of puc represents activation of the JNK pathway in Drosophila. Therefore an enhancer-trap allele, puc-LacZ, was used to monitor the activation of JNK signaling in invasive tumor cells. Strong ectopic JNK activation was present in invasive tumors, while only a slight expression of puc was seen in restricted regions of RasV12-induced noninvasive overgrowth. Intriguingly, more intense JNK activation was seen in tumor cells located in the marginal region of the eye-antennal disc and tumor cells invading the VNC. Analysis of clones of cells with a cell polarity mutation alone revealed that JNK signaling was activated by mutation of cell polarity genes. Notably, JNK signaling was not activated in a strictly cell-autonomous fashion. JNK activation in these cells was further confirmed by anti-phospho-JNK antibody staining that detects activated JNK (Igaki, 2006).

To examine the contribution of JNK activation to metastatic behavior, the JNK pathway was blocked by overexpressing a dominant-negative form of Drosophila JNK (BskDN). As previously reported (Pagliarini, 2003), clones of cells mutant for scrib, lgl, or dlg do not proliferate as well as wild-type clones, while combination of these mutations with RasV12 expression resulted in massive and metastatic tumors. Strikingly, inhibition of JNK activation by BskDN completely blocked the invasion of the VNC, as well as secondary tumor foci formation. Drosophila has two homologs of TRAF proteins (DTRAF1 and DTRAF2), which mediate signals from cell surface receptors to the JNK kinase cascade in mammalian systems. It was found that RNAi-mediated inactivation of DTRAF2, but not DTRAF1, in the tumors strongly suppressed their metastatic behavior. Inactivation of dTAK1, a Drosophila JNK kinase kinase (JNKKK), or Hep, a JNKK, also suppressed metastatic behavior. Drosophila has two known cell surface receptors that act as triggers for the JNK pathway, Wengen (TNF receptor) and PVR (PDGF/VEGF receptor). Intriguingly, it was found that RNAi-mediated inactivation of Wengen partially suppressed tumor invasion. Inactivation of PVR, in contrast, did not show any suppressive effect on metastatic behavior. It was also found that the metastatic behavior of RasV12-expressing tumors that were also mutated for one of three other cell polarity genes, bazooka, stardust, or cdc42, was also blocked by BskDN. These data indicate that loss of cell polarity contributes to metastatic behavior by activating the evolutionarily conserved JNK pathway (Igaki, 2006).

Next, whether JNK activation is sufficient to trigger metastatic behavior in RasV12-induced benign tumors was examined. Two genetic alterations can be used to activate JNK in Drosophila. First, JNK signaling can be activated by overexpression of Eiger, a Drosophila TNF ligand. While mammalian TNF superfamily proteins activate both the JNK and NFκB pathways, Eiger has been shown to specifically activate the JNK pathway through dTAK1 and Hep. Indeed, the eye phenotype caused by Eiger overexpression could be reversed by blocking JNK through Bsk-IR (Bsk-RNAi). Second, overexpression of a constitutively activated form of Hep (HepCA) can also activate JNK signaling. However, the eye phenotype caused by HepCA overexpression was only slightly suppressed by Bsk-IR, suggesting that HepCA overexpression may have additional effects other than JNK activation. Therefore Eiger overexpression was used to activate JNK in RasV12-induced benign tumors, and it was found that the RasV12+Eiger-expressing tumor cells did not result in the invasion of the VNC. This indicates that loss of cell polarity must induce an additional downstream effect(s) essential for metastatic behavior. A strong candidate for the missing event is downregulation of the E-cadherin/catenin adhesion complex, since this complex is frequently downregulated in malignant human cancer cells and is also downregulated by loss of cell polarity genes in Drosophila invasive tumors (Pagliarini, 2003). In addition, it has been recently reported that higher motility of mammalian scrib knockdown cells can be partially rescued by overexpression of E-cadherin-catenin fusion protein, suggesting a role of E-cadherin in preventing polarity-dependent invasion. Furthermore, overexpression of E-cadherin blocks metastatic behavior of RasV12/scrib−/− tumors (Pagliarini, 2003), indicating that loss of E-cadherin is essential for inducing tumor invasion in this model. It was found that loss of the Drosophila E-cadherin homolog shotgun (shg), combined with the expression of both RasV12 and Eiger, induced the invasion of the VNC. Intriguingly, loss of shg in RasV12-expressing clones also showed a weak invasive phenotype at lower penetrance. In agreement with the essential role of JNK in tumor invasion, clones of shg−/− cells weakly upregulated puc expression. It was further found that JNK activation in dlg−/− clones is not blocked by overexpression of E-cadherin, suggesting that mechanism(s) other than loss of E-cadherin also exist for inducing JNK activation downstream of cell polarity disruption. The metastatic behavior of RasV12+Eiger/shg−/− tumors was completely blocked by coexpression of BskDN, indicating a cell-autonomous requirement of JNK activation for this process. Furthermore, it was found that loss of the β-catenin homolog armadillo also induced metastatic behavior in RasV12-induced benign tumors. In contrast, overexpression of HepCA in RasV12/shg−/− cells resulted in neither enhanced tumor growth nor metastatic behavior. Together, these results suggest that, although the RasV12+Eiger/shg−/− does not completely phenocopy the effect of RasV12/scrib−/−, activation of JNK signaling and inactivation of the E-cadherin/catenin complex are the downstream components of cell polarity disruption that trigger metastatic behavior in RasV12-induced benign tumors (Igaki, 2006).

Genetic analysis of Slipper/Mixed Lineage Kinase reveals requirements in multiple JNK-dependent morphogenetic events during Drosophila development

Mixed Lineage Kinases (MLKs) function as Jun-N-terminal kinase (JNK) kinase kinases to transduce extracellular signals during development and homeostasis in adults. slipper (slpr), which encodes the Drosophila homolog of mammalian MLKs is implicated in activation of the JNK pathway during embryonic dorsal epidermal closure. To further define the specific functions of Slpr, the phenotypic consequences of slpr loss- and gain-of function was studied throughout development, using a semi-viable maternal effect allele and wildtype or dominant negative transgenes. From these analyses it was confirmed that failure of dorsal closure is the null phenotype in slpr germline clones. In addition, there is a functional maternal contribution, which can suffice for embryogenesis in the zygotic null mutant, but rarely suffices for pupal metamorphosis, revealing later functions for slpr as the maternal contribution is depleted. Zygotic null mutants that eclose as adults display an array of morphological defects, many of which are shared by hep mutant animals, deficient in the JNK kinase (JNKK/MKK7) substrate for Slpr, suggesting that the defects observed in slpr mutants primarily reflect loss of hep-dependent JNK activation. Consistent with this, the maternal slpr contribution is sensitive to the dosage of positive and negative JNK pathway regulators, which attenuate or potentiate Slpr-dependent signaling in development. Though Slpr and TAK1, another JNKKK family member, are differentially used in dorsal closure and TNF/Eiger-stimulated apoptosis, respectively, a Tak1 mutant shows dominant genetic interactions with slpr suggesting potential redundant or combinatorial functions. Finally, it was demonstrated that SLPR overexpression can induce ectopic JNK signaling and that the Slpr protein is enriched at the epithelial cell cortex (Polaski, 2006).

Previous genetic studies have established a role for SLPR/MLK in JNK pathway activation during embryonic tissue closure. This study characterizes the phenotype of an allele affecting postembryonic development as well as protein products encoded by wildtype and mutant alleles. slprBS06 is a newly isolated null allele that encodes an early nonsense mutation and consequently, no protein product is detected in mutant tissue clones or by Western immunoblot. Phenotypic comparison between the null allele and existing alleles confirms the role for SLPR in dorsal closure, clarifies that slpr has a maternal contribution and that the prior two alleles encode dominant negative proteins, and uncovers additional roles for Slpr in metamorphosis of the adult (Polaski, 2006).

The severe dorsal open phenotype of slprBS06 germline clones, maternally and zygotically mutant, indicates that dorsal closure is the earliest requirement for Slpr in embryogenesis and that a failure of dorsal closure is the null phenotype, consistent with the phenotype of the previously characterized 921 and 3P5 slpr alleles. In contrast though, most slprBS06 zygotic mutants survive embryogenesis and adult mutant males are recovered at a low frequency. These males display several visible morphological phenotypes of variable penetrance, presumably as a consequence of the eventual depletion of functional maternal product. These observations indicate that the maternal slpr gene product is nearly sufficient for embryogenesis in the absence of zygotic product, but rarely provides enough function for metamorphosis, revealing additional roles for Slpr in postembryonic processes (Polaski, 2006).

Defects observed in mutant adults implicate Slpr function for proper metamorphosis of the genital discs, dorsal abdomen and notum, maxillary palps, and wing. Females show somatic defects during oogenesis demonstrating that Slpr is required for proper morphology of the chorionic dorsal appendages. Many of the defects, including those affecting the thorax, genitals and dorsal appendages, have been documented previously to result from loss of JNK signaling. Thus, the data reported here implicate Slpr as the upstream JNKKK family member required for JNK activation in these processes. The current study also suggests that slpr function is mediated primarily, if not entirely, via HEP/MKK7 and the JNK pathway, as evidenced by the fact that hep mutants share in common all of the defects observed in slpr mutants and that reducing the dosage of two known negative regulators of JNK signaling, puc and raw, suppresses the slpr phenotypes. In light of these results, it will be informative to systematically test whether, in vivo, the mammalian MLK proteins activate alternative substrates and pathways as has been suggested from tissue culture studies (Polaski, 2006).

Given that the slpr921 and slpr3P5 alleles are phenotypically more severe in zygotic mutants than the null allele indicates that the encoded products have dominant negative activity, which interferes with the functional pool of maternal slpr gene product. This is consistent with the molecular nature of the alleles, which predict that full length (921) or partial (3P5) protein product would be expressed in the mutants. Indeed, clonal analysis and immunofluoresence staining confirm the expression of mutant protein in slpr921 animals. Protein levels in slpr3P5 mutant tissue appear reduced relative to wildtype, but the encoded fragment retains the SH3 domain and most of the kinase domain, each of which, if folded properly, could engage in protein-protein interactions. Similarly, the catalytically inactive, full length protein encoded by slpr921 retains several functional protein interaction motifs, which could account for the antimorphic properties of the protein (Polaski, 2006).

What candidates are known to interact with the various regions of the Slpr protein to account for dominant negative activity? By analogy with the mammalian MLK proteins, at least three recognized domains have potential protein binding activity. The leucine zipper mediates homodimerization, which is requisite for autophosphorylation and substrate activation. Mutant Slpr protein in slpr921 cells might trap wildtype protein in unproductive dimers. The CRIB domain binds to the activated form of the small GTPases, Cdc42 and Rac1, both implicated in dorsal closure. Titration of these GTPases by non-catalytic Slpr921 protein could also contribute to dominant interference of the wildtype Slpr protein. Finally, the N-terminal SH3 domain, retained in the proteins encoded by both slpr3P5 and slpr921, has the potential to engage in both intra- and intermolecular interactions. The SH3 domain of mammalian MLK3 can bind to a region between the LZ and CRIB domains through a critical proline residue that is conserved in Drosophila Slpr. The postulated intramolecular binding is thought to negatively regulate MLK activation by locking the protein in a closed conformation (Polaski, 2006).

This type of autoinhibition has been demonstrated for other modular kinases, such as Src tyrosine kinase. Also, the SH3 domain may serve as a docking site for upstream activating kinases of the Ste20 family, for which titration by an SH3-containing protein fragment could impair signal relay to the JNK pathway. Therefore, the modular domain organization of the Slpr protein with the potential for multiple regulatory protein interactions is likely to explain why residual mutant protein is more detrimental than complete loss of protein in the null mutant (Polaski, 2006).

Why then does overexpression of an engineered kinase dead Slpr transgene that is functionally equivalent to the protein encoded by slpr921 have such mild consequences in the embryo? Evidence suggests that it is due to a substantial functional maternal component, in addition to the zygotic contribution, because reducing the maternal pool in embryos derived from slpr-/+ heterozygous mothers exacerbates the effect of dominant negative Slpr transgene expression. Further support for the function of the maternal gene product is demonstrated by the sensitivity of the maternal contribution to the dosage of additional positive and negative JNK pathway regulators, which has been monitored as the extent of recovery of slprBS06 mutant adult males (Polaski, 2006).

Given that the maternal product is nearly sufficient for mutant males to survive to adulthood, it is curious that immunodetection of endogenous Slpr protein in the embryo has been difficult relative to the ease with which transgenic protein can be detected. This may suggest that the maternal pool of slpr gene product is largely mRNA rather than protein, that an active mechanism exists to maintain low levels of embryonic protein, or that protein complexes mask the ability to detect the epitope on the Slpr protein, either of which could be overcome by the abundant expression of exogenous transgenic protein. Though the mechanism is not clear, the genetic loss-of-function and overexpression data together indicate that certain cell types or developmental contexts are sensitive to the levels of Slpr protein in modulating JNK signaling. For example, while exogenous Slpr can induce JNK signaling in embryonic dorsal ectoderm cells, normally limited for JNK activity, not all cells are equally inducible, suggesting there may be other limiting components or brakes that modulate the precise levels of JNK activity in cells. Inferring function from overexpression experiments in the absence of loss-of-function data can be misleading however, because wildtype transgene expression can stimulate JNK signaling promiscuously, or at least where the endogenous protein appears not to be required. For example, transgenic expression of either Slpr or TAK1 can induce JNK signaling ectopically in the embryonic dorsal ectoderm under the control of pnr-GAL4, even though endogenous levels of TAK1 cannot provide enough JNK signaling activity in slpr null embryos to rescue dorsal closure. Moreover, Tak1 mutants are viable providing corroborating evidence that Tak1 is not required for dorsal closure. In sum, JNK signaling activity may be at a threshold level in most cells, easily overactivated by expression of many different upstream regulators, but whose selective use in physiological circumstances is only revealed through analysis of loss-of-function (Polaski, 2006).

The combined gain- and loss- of function analysis for Slpr described here supports two proposed mechanisms of signaling specificity among JNKKK proteins; first, that individual family members are used selectively in particular contexts and second, that potential combinatorial or redundant functions may exist among members with common substrates. With respect to TNF/Eiger signaling, both loss-of-function analysis and dominant negative constructs consistently implicate TAK1 rather than Slpr in this JNK-dependent response. In addition, JNK-dependent developmental morphogenetic events, in particular dorsal closure in the embryo, selectively require Slpr. It was consistently observed that the slprBS06, Tak1 double mutant is more severe than either single mutant alone, suggesting that there are likely to be additional, perhaps redundant, functions of Slpr and TAK1 that are only revealed in the double mutant. These functions have yet to be investigated in detail (Polaski, 2006).

At face value, genetic analysis has allowed the assignment of Slpr to mediate many JNK-dependent morphogenetic events and TAK1 to mediate JNK-dependent homeostatic responses including apoptosis and immunity. However, it is still unclear whether the selective functions reflect differential transcriptional responses or whether cell and developmental context shapes what appear to be quite different cellular behaviors. In other words, though the developmental defects that arise as a consequence of loss of Slpr function may suggest a common failure in cell shape change or cytoskeletal functions, similar to the defects that underlie the failure of embryonic dorsal closure in the mutants, that assumption may be too simplistic. It is a formal possibility that Slpr could regulate additional or alternative JNK-dependent cell responses in distinct contexts. For example, though Slpr appears not to mediate TNF-induced apoptosis in the Drosophila eye under conditions where Eiger is overexpressed, the male genital misrotation phenotype observed in slpr mutants may be linked to JNK-dependent developmental programmed cell death. Defective genital rotation is observed in certain viable alleles of hid, encoding a protein with proapoptotic function. The basis of the rotation defect may be due to an excess of genital disc cells, similar to the embryonic defects in head involution, the namesake phenotype of hid (Polaski, 2006).

Thus, JNK signaling and HID function are both required for proper genital rotation and interestingly, there is precedent for hid being a transcriptional target of the JNK pathway downstream of Eiger. Thus, it will be important to determine specifically whether Slpr mediates JNK-dependent HID expression or even apoptosis in imaginal discs, or whether the requirement for Slpr in male genital rotation is unrelated to apoptosis. More generally, a full understanding of Slpr function will require systematic definition of the molecular and cellular mechanisms that underlie the morphological defects. If Slpr functions to regulate different outputs in different contexts, determining what regulates a selective response will be of considerable interest for future studies (Polaski, 2006).

Molecular interface of neuronal innate immunity, synaptic vesicle stabilization, and presynaptic homeostatic plasticity

This study defines homeostatic function for innate immune signaling within neurons. A genetic analysis of the innate immune signaling genes IMD, IKKβ, Tak1, and Relish demonstrates that each is essential for presynaptic homeostatic plasticity (PHP). Subsequent analyses define how the rapid induction of PHP (occurring in seconds) can be coordinated with the life-long maintenance of PHP, a time course that is conserved from invertebrates to mammals. This study defines a novel bifurcation of presynaptic innate immune signaling. Tak1 (Map3K) acts locally and is selective for rapid PHP induction. IMD, IKKbeta, and Relish are essential for long-term PHP maintenance. How Tak1 controls vesicle release is defined. Tak1 stabilizes the docked vesicle state, which is essential for the homeostatic expansion of the readily releasable vesicle pool. This represents a mechanism for the control of vesicle release, and an interface of innate immune signaling with the vesicle fusion apparatus and homeostatic plasticity (Harris, 2018).

This study has tested the core signaling components of an innate immune signaling pathway (IMD signaling) for a role during presynaptic homeostatic plasticity. The data support a model in which IMD signaling bifurcates downstream of the presynaptic innate immune receptor PGRP-LC to achieve immediate, local modulation of the presynaptic release apparatus via Tak1, and prolonged maintenance of the homeostatic response via the transcription factor Relish. This model allows the innate immune signaling system to rapidly alter presynaptic release (seconds to minutes) and simultaneously initiate a Rel-dependent consolidation of PHP. It is noteworthy that the consolidation of PHP can persist for months in insects and decades in humans. It has become clear that the molecular mechanisms responsible for the rapid induction and sustained expression are genetically separable. These findings provide an explanation for how the rapid induction and sustained expression of PHP are mechanistically coordinated (Harris, 2018).

The canonical function of innate immunity is to recognize invading pathogens or non-normal molecular patterns and induce a rapid, inflammatory reaction that is sustained for as long as the invasion persists. During an innate immune response, Tak1-dependent signaling turns on more rapidly, and turns off more rapidly, than Rel-mediated transcription. As such, Tak1 signaling can be considered as a feedforward activator of the cellular immune response. Based on the data, it is proposed that innate immune signaling in the nervous system is transfigured to detect non-normal neurophysiology, although the molecular event that reports altered neural (synaptic) function and is subsequently detected by pre-synaptic PGRP-LC remains a mystery. In the current study, Tak1 is placed at the presynaptic release site where homeostatic plasticity is rapidly induced. Thus, just as in canonical innate immunity, Tak1 is ideally situated to act as a feedforward potentiometer that controls vesicle fusion, thereby achieving a rapid compensatory change in presynaptic release following postsynaptic glutamate receptor inhibition. Subsequent activation of Rel- mediated transcription provides a sustained response that can be maintained for the duration of the perturbation (Harris, 2018).

Evidence is provided that Tak1 has a potent, constitutive function to stabilize the docked vesicle pool, defined both electrophysiologically and at the EM level. This can explain the dramatic defect in spontaneous miniature release frequency, which occurs without a change in the total number of presynaptic release sites. Fewer docked vesicles can explain the defect in EPSC amplitude despite normal action-potential-induced calcium influx. It was also demonstrated that vesicles can be mobilized to the release site during a stimulus train, temporarily achieving WT release rates before declining back to a reduced baseline. This effect reflects a stimulus-dependent re-population of the docked vesicle pool, as demonstrated by EM analysis directly following a train of APs. The stimulus, and presumably calcium-dependent, re-population of the docked vesicle pool explains why there is pronounced short-term synaptic facilitation in the Tak1 mutants compared to short-term depression in WT. Essentially, the number of docked vesicles is increased in a stimulus-dependent manner, something that does not occur in WT, based on EM analysis. Finally, analysis of recovery from vesicle depletion pinpoints the time at which Tak1 activity becomes essential. The recovery of synaptic transmission is completely normal in Tak1 for the first 500 ms following a stimulus train. It is only after this time point that recovery becomes defective (Harris, 2018).

As a working model, it is proposed that Tak1 functions to inhibit the rate of vesicle undocking and, thereby, persistently stabilizes the docked/primed vesicle pool. Immediately following a stimulus train, calcium- dependent mechanisms actively mobilize vesicles to the active zone and drive calcium-dependent vesicle docking and priming. Thus, calcium-dependent potentiation of the forward rate of docking/priming transiently overcomes the enhanced un-docking rate caused by loss of Tak1 and the number of docked vesicles is restored to WT levels. As intra-terminal calcium levels drop following the stimulus train, the rates of docking and un-docking re-equilibrate in the Tak1 mutant, reaching a steady state with many fewer docked vesicles compared to WT (Harris, 2018).

This model can also explain the failure of PHP in a Tak1 mutant. The rapid induction of PHP occurs following inhibition of postsynaptic glutamate receptors and is expressed in response to a single AP. This requires an expansion of the resting pool of docked/primed vesicles and this cannot be achieved in the absence of Tak1. Accordingly, enhanced Tak1 activity, following activation of the innate immune receptor PGRP, would inhibit the rate of vesicle un-docking and potentiate the pool of docked/primed vesicles, necessary for the rapid induction of PHP. Thus, Tak1 functions as a molecular potentiometer controlling the docked/primed vesicle pool during PHP. A question for future experimentation concerns how baseline Tak1 activity is established to achieve highly reproducible, baseline synaptic transmission in WT (Harris, 2018).

Most of the knowledge regarding the organization of innate immune signaling pathways is based on assays that detect the nuclear translocation of Rel/NF-κB, or quantify Rel/NF-κB-mediated transcription. Thus, the logic and spatial organization of innate immune signaling in neurons has yet to be clearly defined. The data argue for several novel features of signaling organization in a neuron, beyond the local synaptic action of Tak1. In canonical innate immune signaling, receptor activation catalyzes the assembly of an intracellular complex that includes Imd, Tak1, and IKKβ. Based on the evidence in Drosophila motoneurons, and consistent with published models of IMD signaling, it is proposed that activation of the PGRP receptor drives assembly of the IMD signaling complex that includes a poly-ubiquitin chain as well as the three proteins IMD, Tak1, and IKKβ. Then, it is suggested that IKKβ is necessary for this signaling complex to either assemble correctly or signal efficiently. Thus, IKKβ is necessary for both the short- and long-term forms of PHP, just like the PGRP receptor. Finally, once this complex is assembled and activated, two independent signal transduction cascades are initiated. One requires Tak1 and acts locally within the presynaptic terminal to achieve the rapid induction of PHP. The second pathway requires the function of IMD and signals via Rel-mediated transcription, necessary for the long-term maintenance of PHP. It is noted, however, that other models for IKKβ activation should also be considered. Finally, the data raise an important question regarding how signaling is conveyed from synaptic IKKβ to nuclear Rel, a topic of future studies (Harris, 2018).


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TGF-ß activated kinase 1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation

date revised: 23 August 2017

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