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Genes Dev. 2000 Oct 1; 14(19): 2461–2471.
PMCID: PMC316979

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


Here we report the identification of a Drosophila IκB kinase complex containing DmIKKβ and DmIKKγ, homologs of the human IKKβ and IKKγ proteins. We show that this complex is required for the signal-dependent cleavage of Relish, a member of the Rel family of transcriptional activator proteins, and for the activation of antibacterial immune response genes. In addition, we find that the activated DmIKK complex, as well as recombinant DmIKKβ, can phosphorylate Relish in vitro. Thus, we propose that the Drosophila IκB kinase complex functions, at least in part, by inducing the proteolytic cleavage of Relish. The N terminus of Relish then translocates to the nucleus and activates the transcription of antibacterial immune response genes. Remarkably, this Drosophila IκB kinase complex is not required for the activation of the Rel proteins Dif and Dorsal through the Toll signaling pathway, which is essential for antifungal immunity and dorsoventral patterning during early development. Thus, a yet to be identified IκB kinase complex must be required for Rel protein activation via the Toll signaling pathway.

Keywords: insect immunity, NF-κB, IKK, Relish

NF-κB and other members of the Rel family of transcriptional activator proteins play essential roles in human innate immunity and in the Drosophila immune response (Hoffmann et al. 1999). In most cells Rel proteins are sequestered in the cytoplasm as a result of their association with an inhibitor protein, IκB. When human cells are infected by a microbial pathogen, signaling pathways are activated, culminating in the proteasome-dependent degradation of IκB and the nuclear translocation of NF-κB. Once in the nucleus, NF-κB activates the transcription of genes encoding antimicrobial proteins (Ghosh et al. 1998). The degradation of IκB is triggered by the activation of the IκB kinase (IKK) complex (Chen et al. 1996; Lee et al. 1997), containing IKKα and IKKβ, which are both catalytic subunits, and a structural component IKKγ (DiDonato et al. 1997; Mercurio et al. 1997; May and Ghosh 1998; Rothwarf et al. 1998; Yamaoka et al. 1998). The activated IκB kinase complex then phosphorylates IκB proteins, leading to their ubiquitination and subsequent degradation by the proteasome (Finco and Baldwin 1995).

Two distinct pathways for the activation of the Drosophila immune response have been identified. Infection by gram-negative bacterial pathogens leads to the production of antibacterial peptides, such as Attacin, Diptericin, and Cecropin, whereas fungal infection leads to the production of antifungals such as Drosomycin (Lemaitre et al. 1997). Three Drosophila Rel proteins are differentially required for the two immune response pathways. The antifungal response activates the Toll signaling pathway, causing the degradation of the IκB protein Cactus and the activation of two Drosophila Rel proteins, Dorsal and Dif (Lemaitre et al. 1996; Manfruelli et al. 1999; Meng et al. 1999; Rutschmann et al. 2000a). This pathway is triggered by fungal infection, which is believed to cause the activation of a serine protease cascade in the haemolymph. This protease cascade results in the proteolytic processing of Spätzle, the ligand for the transmembrane receptor Toll (Levashina et al. 1999). Activated Toll signals through two proteins, Tube and Pelle, leading to the degradation of Cactus (Belvin and Anderson 1996). Similar to mammalian IκBs, it is thought that Cactus degradation is triggered by phosphorylation of its N-terminal regulatory domain followed by ubiquitination, mediated by the Slimb-containing ubiquitin ligase complex and destruction by the 26S proteasome (Spencer et al. 1999). The identity of the Toll-activated Cactus kinase remains unknown. Degradation of Cactus leads to the nuclear translocation of Dif and Dorsal and the activation of transcription. Interestingly, in larvae either Dif or Dorsal is sufficient for antifungal immunity, whereas in adults Dif is required (Manfruelli et al. 1999; Meng et al. 1999; Rutschmann et al. 2000a). The Toll pathway also plays a critical role in early development, where it is required for dorsoventral patterning of the embryo. Dorsal, but not Dif, is required for the dorsoventral pathway (Belvin and Anderson 1996).

The antibacterial immune response requires the other Drosophila Rel protein, Relish (Hedengren et al. 1999). Relish is a homolog of the mammalian p105 precursor of NF-κB p50 protein. Like its mammalian counterpart, Relish consists of both an N-terminal Rel homology domain (RHD), and a C-terminal IκB-like Ankyrin-repeat domain that is believed to inhibit its own nuclear translocation (Dushay et al. 1996). However, the regulation of Relish appears to be quite different than that of p105. Recently S. Stöven and D. Hultmark found that Relish is activated by endoproteolytic cleavage in response to bacterial infection, leading to the production of the Relish N-terminal RHD, which translocates to the nucleus, and a stable C-terminal Ankyrin domain that remains in the cytoplasm. Furthermore, they have found that this cleavage is not mediated by the proteasome (Stöven et al. 2000). The activation of Relish appears to be the crucial event in the induction of the antibacterial immune response. However, the signaling pathway leading from bacterial infection to Relish cleavage is poorly understood. The cell surface receptors that recognize gram-negative bacteria or their cell wall component lipopolysaccharide (LPS) are unknown. In mammals, the Toll family of receptors, and TLR4 in particular, have been shown to be involved in LPS recognition and signaling, although none of the eight Drosophila Toll family members have yet been shown to be an LPS receptor (Medzhitov and Janeway 2000). Furthermore, the components and mechanisms used to transduce the signal from the cell surface receptors to Relish have not yet been identified. Here we identify and characterize a Drosophila IκB kinase complex that is activated by LPS and is in turn required for the activation of antibacterial immune response genes and for the LPS-dependent cleavage of Relish. Furthermore, we show that the activated kinase is capable of phosphorylating Relish in vitro. However, this Drosophila IKK complex is not required for the Toll signaling pathway, which is necessary for antifungal immunity and early embryonic dorsoventral patterning.


To identify the signaling components required for the Drosophila immune response we have undertaken a reverse genetic approach, taking advantage of the Drosophila Genome Project. A cDNA sequence with homology to the kinase domain of the human IKK genes was identified in the BDGP EST database (Berkeley Drosophila Genome Project/HHMI EST Project, unpubl.). Examination of the amino acid sequence of the encoded protein, which we designate DmIKKβ (Drosophila melanogaster IKKβ), displays significant similarity to the N-terminal region of hIKKα and hIKKβ but is more similar to hIKKβ. The amino acid sequence of the C terminus of DmIKKβ is only weakly related to the corresponding regions of IKKα and IKKβ. A predicted coiled-coil can be detected in a region corresponding to the predicted leucine zipper coiled-coil of hIKKα and hIKKβ (Fig. (Fig.1A).1A). The DmIKKβ gene maps to chromosomal location 89B as determined with the BDGP P1 filter array (Kimmerly et al. 1996). While this manuscript was in preparation, Kim et al. (2000) reported the initial characterization of the same gene, which they refer to as DLAK. They report that the autophosphorylation of DLAK is inducible by LPS, and they present evidence that Cactus may be a target of the kinase. In addition, they show that expression of a dominant negative mutant of DLAK inhibits the LPS-dependent activation of Rel proteins and a number of immune response genes (Kim et al. 2000).

Figure 1
DmIKKβ and DmIKKγ interact in vitro and in vivo. (A) Schematic representation of the Drosophila IKK homologs DmIKKβ and DmIKKγ. The N-terminal region of DmIKKβ displays significant homology to hIKKβ, and ...

DmIKKβ associates with DmIKKγ

As mentioned above, the mammalian IKKα and IKKβ proteins are found in a high molecular weight IκB kinase complex that includes the structural component IKKγ or NEMO (Rothwarf et al. 1998; Yamaoka et al., 1998). To investigate the possibility that DmIKKβ is also a component of a similar kinase complex we performed a yeast two-hybrid screen using DmIKKβ as bait. A Drosophila larval cDNA library was screened, and a total of 85 independent positive clones were analyzed. All of these clones were found to contain overlapping inserts from a cDNA that encodes a Drosophila protein with homology to hIKKγ primarily in its C terminus (Fig. (Fig.1A).1A). This gene, referred to as DmIKKγ hereafter, maps to chromosomal location 60E as determined with the BDGP P1 filter array (Kimmerly et al. 1996). Secondary structure predictions of the protein encoded by DmIKKγ suggest the existence of several coiled-coil regions (Fig. (Fig.1A).1A). The overall amino acid sequence homology between DmIKKγ and hIKKγ suggests a putative structural and functional relationship between the two proteins, especially in their C-terminal halves.

To confirm that DmIKKβ and DmIKKγ interact, coimmunoprecipitation experiments were performed. In vitro–translated full-length DmIKKβ and Flag-tagged DmIKKγ were both precipitated with anti-Flag beads (Fig. (Fig.1B,1B, lane 4). Only background levels of DmIKKβ associate with the beads in the absence of Flag-DmIKKγ (Fig. (Fig.1B,1B, lane 3). Similarly, DmIKKβ can be coimmunoprecipitated with Flag-DmIKKγ when the two proteins are coexpressed in Schneider cells by transfection (Fig. (Fig.1C).1C). We conclude that DmIKKβ and DmIKKγ interact to form a complex both in vivo and in vitro.

Dominant negative DmIKKs block LPS-stimulated induction of antibacterial peptide genes

To determine whether DmIKKβ and DmIKKγ are involved in the activation of antibacterial genes, an LPS-inducible Drosophila cell line was engineered to express different versions of these genes. When S2* cells are treated with LPS, the expression of antibacterial peptide genes, such as Diptericin, Cecropin, and Attacin, are induced (Samakovlis et al. 1992). Thus, S2* cells were stably transfected with plasmids that express wild-type or potentially dominant negative versions of DmIKKβ and DmIKKγ under the control of the copper-inducible metallothionein promoter. Four cell lines were generated that express one of the following proteins: DmIKKβ wild type, DmIKKβ K50A, DmIKKγ wild type, or DmIKKγ 201–387. DmIKKβ K50A was chosen because similar mutations in hIKKα or hIKKβ, which change a conserved lysine in the ATP binding domain, create dominant negative proteins (DiDonato et al. 1997; Mercurio et al. 1997; Regnier et al. 1997), whereas the DmIKKγ201–387 was designed because a similarly truncated hIKKγ acts as a dominant negative mutant in human cells (Mercurio et al. 1999). Full-length hIKKγ can also act as a dominant negative when expressed at high levels (Li et al. 1998), presumably by titrating limiting components required for functional complex assembly.

After the addition of copper, DmIKK proteins are rapidly produced at high levels as detected by immunoblotting (data not shown). To determine whether these DmIKK proteins block LPS induction of antibacterial peptide expression in S2* cells, the stable cell lines were first treated with copper to induce expression of the DmIKKs and then stimulated with LPS. As shown in the Northern blot in Figure Figure2,2, DmIKKβ K50A blocks the LPS induction of the Diptericin, Cecropin, and Attacin genes (Fig. (Fig.2,2, lane 12). Similarly, either full-length or truncated versions of DmIKKγ are potent inhibitors of antibacterial peptide gene expression (Fig. (Fig.2,2, lanes 16,20). As a control, the first panel in Figure Figure22 shows that copper treatment has no effect on LPS induction of Diptericin, Attacin, or Cecropin in the parental cell line (Fig. (Fig.2,2, lane 4). Furthermore, when the stable lines are induced with LPS but not pretreated with copper, the antibacterial genes are induced to high levels (lanes 10,14,18). The wild-type DmIKKβ has only a marginal effect on antibacterial induction (lane 8). These data clearly show that DmIKKβ K50A, DmIKKγ full-length, or DmIKKγ201–387 potently inhibit Diptericin, Cecropin, and Attacin gene induction. By contrast, the expression of wild-type DmIKKβ slightly decreases the level of expression of these genes. We conclude that DmIKKβ and DmIKKγ are required for the activation of antibacterial gene expression in response to LPS.

Figure 2
Dominant negative mutants of DmIKKβ or DmIKKγ block LPS induction of antibacterial peptide gene expression. S2* cell lines were generated that stably express various DmIKK genes under the control of a copper-inducible metallothionein promoter. ...

Dominant negative DmIKKs block LPS-induced Relish cleavage

As mentioned above, genetic studies have shown that the Drosophila Relish gene is essential for the antibacterial immune response (Hedengren et al. 1999). Bacterial infection or LPS treatment activates the endoproteolytic cleavage of Relish. Once cleaved, the N-terminal RHD of Relish translocates into the nucleus, where it activates the transcription of antibacterial genes (Stöven et al. 2000). Considering that the overexpression of dominant negative DmIKKβ or DmIKKγ blocks the induction of antibacterial peptide genes, it is likely that DmIKKβ (and the DmIKK complex) functions in the signaling pathway leading to the cleavage of Relish. In order to test this possibility, the DmIKK overexpressing cell lines were employed to follow the fate of Relish protein cleavage. The stable cell lines and the parental cells were first treated with copper and then induced with LPS for 15 min. Equal amounts of total protein were analyzed by immunoblotting with an anti-Relish antibody that recognizes the C-terminal Ankyrin domain. LPS treatment of the parental cells, or any of the stable lines that were not treated with copper, results in Relish cleavage. Full-length Relish (∼110 kD) is cleaved to generate fragments of ∼68 kD and ∼49 kD, corresponding to the N- and C-terminal fragments, respectively (e.g., see Fig. Fig.3,3, lanes 1,2; Stöven et al. 2000). Expression of DmIKKβ K50A, DmIKKγ, or DmIKKγ201–387 blocks the cleavage of Relish. Full-length Relish protein persists in those samples that were treated first with copper and then with LPS (Fig. (Fig.3,3, lanes 12,16,20, band labeled Rel 110), whereas it is completely cleaved in the control samples not pretreated with copper (lanes 10,14,18). Similar to the analysis of antibacterial mRNAs, overexpression of wild-type DmIKKβ causes a slight accumulation of full-length Relish.

Figure 3
Dominant negative mutants of DmIKKβ and DmIKKγ inhibit LPS-dependent cleavage of Relish. Extracts were prepared from cell lines expressing the copper-inducible DmIKK constructs and the control parental cell line (as labeled) 15 min after ...

Double-stranded DmIKK RNA blocks LPS-induced antibacterial gene activation and Relish cleavage but does not affect the Toll signaling pathway

To obtain additional evidence that DmIKKβ and DmIKKγ are required for LPS-induced antibacterial gene expression, we exploited the inhibitory effect of double-stranded RNA (dsRNA), also referred to as RNAi. The presence of gene-specific dsRNA molecules in Drosophila or Caenorhabditis elegans embryos has been shown to destabilize the cognate mRNAs (Kennerdell and Carthew 1998; Montgomery and Fire 1998; Fire 1999; Misquitta and Paterson 1999; Sharp 1999). The loss of specific mRNAs in Drosophila embryos leads to hypomorphic phenotypes, similar to mutations in the corresponding genes. Similarly, transfection of Drosophila cells in culture with specific dsRNAs can interfere with gene expression (Clemens et al. 2000; Hammond et al. 2000). Thus, dsRNA corresponding to the DmIKKγ or DmIKKβ genes was synthesized in vitro and transfected into the LPS-inducible S2* cell line. Transfection of gene-specific dsRNA caused a significant reduction in the amount of the corresponding mRNA. For example, DmIKKγ dsRNA lowers DmIKKγ mRNA to nearly undetectable levels (see Fig. Fig.4A),4A), while DmIKKβ or LacZ dsRNA has no effect on DmIKKγ mRNA levels. DmIKKγ protein levels were also greatly reduced only in those cells transfected with DmIKKγ dsRNA (Fig. (Fig.5B,5B, left); however, it is important to note that although DmIKKγ protein was reduced approximately 10-fold, it was still detectable after RNAi treatment. Importantly, dsRNA-mediated interference of either DmIKKβ or DmIKKγ greatly inhibits the LPS-induced expression of antibacterial genes, such as Attacin, Cecropin, and Diptericin (Fig. (Fig.4A,4A, top three panels). Double-stranded LacZ RNA has no effect on the expression of immune response genes, although LacZ dsRNA does inhibit the expression of a cotransfected LacZ reporter (data not shown). Furthermore, the LPS-induced cleavage of Relish is also inhibited by either DmIKKβ or DmIKKγ dsRNA, but not by LacZ dsRNA. As shown in Figure Figure4B,4B, full-length Relish protein (see band marked Rel 110) persisted after the induction with LPS only in those lanes transfected with DmIKK dsRNA. Although the inhibition of Relish cleavage seen with DmIKK RNAi is not as dramatic as that observed with the dominant negative DmIKKs (Fig. (Fig.3),3), it is clear that full-length Relish is not as efficiently cleaved in those cells with reduced levels of DmIKKβ or DmIKKγ. In both the dominant negative and the RNAi experiments, a stronger inhibition is always observed at the transcriptional level, as compared to that seen at the protein level. This suggests that small perturbations in the amount of the nuclear translocated Relish can have dramatic effects on the level of transcriptional activation. Small differences in the amounts of transcription factors have been shown to exhibit all-or-none effects on promoters that require the cooperative assembly of transcription enhancer complexes (Driever et al. 1989; Arnosti et al. 1996). As mentioned above and observed by others, dsRNA-mediated interference does not create a null allele. Thus, the reduced amount of Relish cleavage and antibacterial gene expression still observed in the RNAi-treated cells is most likely due to the remaining DmIKKs found in these cells. Both the dominant negative and RNAi experiments show that DmIKKβ and DmIKKγ are required for LPS-induced Relish endoproteolytic cleavage and transcriptional activation of antibacterial peptide genes in vivo.

Figure 4
DmIKK dsRNA specifically inhibits LPS-induced antibacterial gene expression and Relish cleavage. (A) RNA from cells transfected with various dsRNA—LacZ, DmIKKβ, or DmIKKγ as labeled at the top—was analyzed by Northern blotting ...
Figure 5
The Toll signaling pathway is not blocked by DmIKKβ or DmIKKγ RNAi. (A) RNA from the S2*tpll cell line was extracted and analyzed by Northern blot analysis. The two left-most lanes show that in response to torso–pelle expression, ...

As mentioned in the beginning, the Toll signaling pathway, which leads to Cactus degradation and activation of Dif and Dorsal, is necessary for the antifungal immune response as well as early embryonic patterning. The DmIKKβ/γ complex could be involved in the Toll signaling pathway in addition to its role in the antibacterial pathway. To test this possibility directly, we used the RNAi technique with a Drosophila cell line specifically engineered to assay the Toll signaling pathway. Schneider S2* cells were stably transfected with a torso–pelle fusion gene controlled by the metallothionein promoter, creating the S2*tpll cell line. Previous studies have demonstrated that fusing the transmembrane domain of torso to the pelle gene creates a constitutively active pelle kinase that can stimulate the Toll signaling pathway in fly embryos or in tissue culture cells (Grosshans et al. 1994; Galindo et al. 1995). In the S2*tpll cell line, addition of copper leads to the activation of the Toll antifungal pathway. Drosomycin transcription is induced (Fig. (Fig.5A),5A), but Diptericin transcription is unaffected (data not shown). Transfection of dsRNA into the S2*tpll cells leads to specific loss of the corresponding mRNA (data not shown) and protein. DmIKKγ dsRNA causes a substantial reduction in the level of DmIKKγ protein (Fig. (Fig.5B,5B, right). However, neither DmIKKβ nor DmIKKγ dsRNA blocks the torso–pelle-mediated activation of Drosomycin transcription (Fig. (Fig.5A).5A). Importantly, RNAi can be used to modify the torso–pelle-activated pathway. A combination of Dif and Dorsal dsRNA blocks torso–pelle-induced Drosomycin induction, and Cactus dsRNA causes the hyperactivation of Drosomycin in S2*tpll cell line, even without the addition of copper (Fig. (Fig.5A).5A). Additional experiments, which utilized transient transfection to express dominant negative versions of either DmIKKβ or DmIKKγ, failed to inhibit torso–pelle- or Toll-activated signaling (data not shown). These experiments strongly argue that DmIKKβ and DmIKKγ are not required for the Toll-mediated antifungal pathway but are required for the LPS-induced antibacterial pathway.

DmIKKβ phosphorylates Relish

The finding that dominant negative DmIKKs or DmIKK RNAi can block Relish cleavage and Relish-dependent gene activation suggests that Relish may be a bona fide target of the DmIKK complex. To test this possibility, we carried out experiments to determine whether DmIKKβ or the DmIKK complex can phosphorylate Relish protein in vitro. Flag-tagged Relish was immunoprecipitated with Flag antibodies from a Schneider cell line that expresses very high levels of the epitope-tagged protein (Han and Ip 1999). Immunoprecipitated Relish was then used as a substrate with recombinant DmIKKβ in a kinase assay using [γ-32P]ATP. As shown in Figure Figure6A,6A, a band the size of Relish was labeled with 32P by the recombinant kinase (see Fig. Fig.6,6, lanes 3,4). As a control, extracts from the parental Schneider cell line, which does not express Flag-Relish, were also used in a Flag immunoprecipitation. When these immunoprecipitates were used as substrates for DmIKKβ, phosphorylation of Relish was not observed (lanes 1,2). To further demonstrate that the phosphorylated band is Relish, a similar experiment was performed using anti-Relish antibodies, instead of Flag antibodies, to precipitate Relish. Again, a band corresponding to Relish was phosphorylated in a DmIKKβ-dependent manner (Fig. (Fig.6,6, lanes 5–8). We conclude that DmIKKβ can directly phosphorylate Relish.

Figure 6
LPS treatment activates the Relish kinase activity of the DmIKK complex. (A) Recombinant DmIKKβ phosphorylates Relish. Extracts from S2* cells (lanes 1,2,5,6) or from S2* cells that overexpress Flag-Relish (lanes 3,4,7,8) were immunoprecipitated ...

To determine whether this DmIKKβ phosphorylation of Relish is specific, the ability of other, related kinases to phosphorylate Relish was tested (Fig. (Fig.6B).6B). For these experiments, the specificity of DmIKKβ was compared to that of recombinant human IKKβ and IKKɛ. While DmIKKβ readily phosphorylates Relish (Fig. (Fig.6,6, lane 2), phosphorylation was not observed with either hIKKβ or hIKKɛ (lanes 3,4). When these same recombinant human IKKs were used in a GST-IκBα kinase assay, they were both active (Fig. (Fig.6,6, lanes 5,6; Peters et al., 2000). Thus, the ability to phosphorylate Relish is not shared between the Drosophila and human IκB kinases. Preliminary experiments have shown that the DmIKKβ can phosphorylate the linker region between the RHD and the Ankyrin domain. Surprisingly, DmIKKβ can also phosphorylate Cactus and IκBα, and we have shown that this phosphorylation occurs within the N-terminal regulatory domain, which is required for signal-dependent degradation by the proteasome (N. Silverman, N. Pandey, and T. Maniatis, unpubl.). Thus, the ability to specifically phosphorylate IκB proteins is shared by the Drosophila and human IκB kinases, whereas only the Drosophila kinase can phosphorylate Relish. Recently the mammalian IκB kinase complex has been shown to phosphorylate p105 (Heissmeyer et al. 1999); however, this phosphorylation leads to its degradation rather than its processing.

LPS treatment activates the DmIKK complex

To test the possibility that the activated DmIKK complex can phosphorylate Relish, an immunocomplex kinase assay was performed. First, the DmIKK complex was precipitated from an LPS-inducible Schneider cell line using an anti-DmIKKγ antibody. This antibody recognizes the endogenous DmIKKγ that is expressed in this cell line (Fig. (Fig.6C,6C, lanes 1,2). Although the anti-DmIKKβ antibodies are less sensitive (DmIKKβ cannot be detected in crude extracts), the DmIKKβ protein can be detected after immunoprecipitation with anti-DmIKKγ antibodies (lanes 3,4). Immunoprecipitation experiments were performed with extracts prepared from cells treated with LPS (lanes 2,4) or left untreated (lanes 1,3); no differences were detected, with or without LPS, in the levels of DmIKKβ or DmIKKγ expressed and precipitated. However, LPS caused a specific increase in the level of Relish kinase activity that was detected in the immunoprecipitated DmIKK complex (Fig. (Fig.6D,6D, lanes 3,4). Thus, the DmIKK complex that is precipitated with anti-DmIKKγ antibodies contains an LPS-inducible Relish kinase activity. This observation supports the view that DmIKKβ and DmIKKγ form (part of) an LPS-inducible kinase complex that phosphorylates Relish, activating its cleavage in response to LPS.


A number of genes required for Drosophila antibacterial immunity have been identified in genetic studies, including Relish (Hedengren et al. 1999), the gene encoding the Toll-like receptor (TLR) 18-wheeler (Williams et al. 1997), the immune deficient gene (imd; Lemaitre et al. 1995) and the immune response deficient genes 1–5 (ird1–5; Wu and Anderson 1998). In this article we report the characterization of DmIKKβ and DmIKKγ, Drosophila homologs of the essential components of the human IκB kinase complex. We show that the DmIKK complex is required for the signal-induced cleavage of Relish and the induction of antibacterial peptide gene expression. Thus, DmIKKβ and DmIKKγ play an essential role in the antibacterial immune response pathway. We also show that treatment of cells with LPS activates the DmIKK complex and that the activated kinase complex can then phosphorylate Relish. Consistent with this direct phosphorylation model, Relish was found to specifically interact with DmIKKβ in two-hybrid and coimmunoprecipitation assays (L. Burrack, R. Zhou, N. Silverman, T. Maniatis, unpubl.). Phosphorylated Relish may then be recognized by a yet to be identified protease that cleaves the protein between the RHD and the Ankyrin repeat domains. The N-terminal RHD of Relish then translocates into the nucleus, where it activates the expression of antibacterial genes. Additional studies will be required to identify the amino acid(s) phosphorylated in Relish and determine their role in Relish cleavage.

Using a tissue culture–based assay to specifically monitor the Toll signaling pathway, we have found that neither dominant negative DmIKKs nor DmIKK dsRNA interference inhibits the Toll pathway. Furthermore, other groups have identified mutations in both DmIKKβ and DmIKKγ, and their phenotypes in flies are consistent with our results. K. Anderson and colleagues (Y. Lu, L. Wu, and K. Anderson, pers. comm.) have found that ird5 encodes DmIKKβ, and ird5 mutants block the activation of the antibacterial gene Diptericin but have no effect on the activation of the antifungal gene Drosomycin. Likewise, J.A. Hoffmann and colleagues have isolated a mutant, kenny, which is specifically deficient in antibacterial immunity, and they have found that kenny alleles have mutations in the DmIKKγ gene (Rutschmann et al. 2000b). Similar to Relish mutants, mutations in ird5 and kenny inactivate the antibacterial immune response but have no effect on the dorsoventral pathway in early development and have little or no effect on the induction of antifungal peptides. On the basis of the work presented here and that from the K. Anderson and J.A. Hoffmann labs, it appears unlikely that the DmIKKβ/γ complex functions as the Cactus kinase required in the Toll antifungal and dorsoventral signaling pathway. Rather, our results strongly argue that the DmIKKβ/γ complex functions specifically in the LPS-induced Relish-dependent antibacterial pathway.

The data of Kim et al. (2000) showing that NF-κB binding activity and antimicrobial gene induction are blocked by dominant negative DmIKKβ (DLAK) are consistent with our conclusions. However, Kim et al. (2000) also showed that a dominant negative mutant of DmIKKβ blocks Cactus degradation in response to LPS treatment in cell culture. On the surface this observation would appear to contradict our results showing that the dominant negative DmIKKβ does not block Cactus degradation in the Toll signaling pathway. We note, however, that the Dif/Cactus complex can be specifically targeted in response to bacterial infection, and in this situation, the nuclear translocation of Dif is independent of the Toll signaling pathway (Wu and Anderson 1998). Taken together, the experiments of Kim et al. (2000) and Wu and Anderson (1998) suggest that, in addition to its role in Relish activation, the DmIKK complex may also phosphorylate Cactus in response to bacterial infection. However, this LPS-inducible pathway must be independent from the Toll signaling pathway, which is responsible for Cactus degradation during early embryonic patterning and the antifungal immune response. What then is the function of Cactus and Dif in the antibacterial pathway? Although Dif is not strictly required for the antibacterial pathway (Rutschmann et al. 2000a), perhaps it functions to augment the transcriptional activation potential of Relish. On the other hand, Dif could be necessary for the transcriptional activation of a certain subset of the antimicrobial peptide genes in response to gram-negative bacterial infection. As neither Dif nor cactus loss-of-function mutants have strong effects on the antibacterial pathway, more detailed investigation will be required to determine if they have a significant role in this pathway. Taken together, the results presented here and by Kim et al. (2000) are consistent with the conclusion that DmIKKβ functions specifically in the antibacterial signaling pathway and not the Toll antifungal pathway. Moreover, the LPS-induced DmIKK complex may phosphorylate both Relish and Cactus, causing the cleavage and activation of Relish and possibly the degradation of Cactus and activation of Dif.

In mammalian cells, it was thought that all known NF-κB-activating signals that lead to the degradation of IκB proteins activate a single IκB kinase complex containing IKKα, IKKβ, and IKKγ. On the basis of the data presented here, it seems likely that two IκB kinase activities are required in flies—one for the Toll pathway and another, DmIKKβ/γ, for antibacterial immunity. Similarly, a recent study in mammalian systems identified an alternate IKK complex, which lacks hIKK α, β, or γ and appears to be required for NF-κB activation in response to PMA or T-cell activation (Peters et al. 2000). This complex contains an unidentified IκB kinase capable of phosphorylating serines 32 and 36 of IκBα, as well as an IKK family member called IKKɛ (Peters et al. 2000) or IKKi (Shimada et al. 1999). Although recombinant IKKɛ/IKKi can phosphorylate serine 36 of IκBα, evidence suggests it may actually function as an IκB kinase kinase (IKK-kinase), activating an as yet unidentified S32/36-specific IκB kinase with which it is associated (Peters et al. 2000). A second hIKKɛ-like kinase known as NAK or TBK1 has also been characterized in mammals. This kinase also phosphorylates only S36 of IκBα but appears to function by activating the hIKKα/β/γ complex directly in response to certain stimuli (Pomerantz and Baltimore 1999; Tojima et al. 2000). Remarkably, the only additional IKK-like kinase in the Drosophila DNA database is a member of the human IKKɛ/IKKi/TBK/NAK subfamily. Thus, a Drosophila homolog of human IKKɛ may be a component of the Cactus kinase complex required for Toll signal transduction during early development and the antifungal immune response, or it could function upstream of the DmIKKβ/γ complex in the antibacterial pathway. Experiments are in progress to test these possibilities and to characterize the Toll-inducible Cactus kinase activity.

Materials and methods

Database analysis and two-hybrid selection

Sequence comparison was performed with BLAST, and the coiled-coil domains were predicted using the Paircoil program (Berger et al. 1995). Two-hybrid selection was performed as described (James et al. 1996) with modifications from T. Milne and B. Cali (pers. comm.). The third instar larval library (gift of S. Elledge, Baylor College of Medicine, Houston, TX) was in the λ ACT vector (Durfee et al. 1993).

Coimmunoprecipitation assays

In vitro translation was performed following the protocol of the manufacturer (Promega). Immunoprecipitations were carried out with anti-Flag M2 agarose beads (Sigma) in IP buffer (10% Glycerol, 20 mM Hepes at pH 7.6, 150 mM NaCl, 6 mM MgCl2, 1 mM EDTA, 0.1% NP-40, 0.05% BSA, 0.5 mM DTT). For immunoprecipitation from cells, Schneider S2* cells were first transfected by a calcium phosphate method with plasmids that express the DmIKK genes under the control of the Actin promoter (pPAC related plasmids). After 48 h, the cells were lysed, and immunoprecipitations were performed in lysis buffer (20 mM Tris at pH 7.6, 150 mM NaCl, 2 mM EDTA, 10% Glycerol, 1% Triton X-100, 1 mM DTT plus protease inhibitors) and analyzed by SDS-PAGE followed by immunoblot analysis with anti-DmIKKβ or anti-Flag antibodies. Antibodies were raised against purified recombinant protein DmIKKβ in chickens using standard protocols (Covance).

Stable cell lines and RNA blotting

The various DmIKK genes and the torso–pelle gene (a gift of S. Wasserman, UCSD) were cloned into the pRmHa-3 vector by standard methods to create constructs in which they were controlled by the metallothionein promoter. These constructs were then transfected into Schneider S2* cells in conjunction with pHS–neo, or pBmIEGlacZ–BmIEGhyg (Han and Ip 1999) for the torso–pelle line, at a ratio of 50:1; stable transfectants were then selected with G418 (800 μg/mL), or hygromycin (200 μg/mL) for the torso–pelle line. Northern blot analysis for the S2* was performed by treating the cells with copper sulfate (500 μM) for 6–8 h, then stimulating with LPS (Sigma, 055:B5, 10 μg/mL) for 3 h. The S2*tpll cell line was pretreated with 20-hydroxy-ecdysone (Sigma), 10−6 M for 24 h before copper treatment for 16 h. Then RNA was extracted using Trizol reagent (BRL). The Diptericin blot was performed on samples from cells first pretreated with 20-hydroxy-ecdysone (Sigma), 10−6 M for 24 h before copper and LPS treatment (Dimarcq et al. 1997). The Rp49 control for the Diptericin blot is not shown but is similar to that in Figure Figure2.2. Blots were probed using the ExpressHyb system (Clontech).

Protein analysis

Relish protein was examined in the stable cell lines that were treated with copper sulfate for 4 h and then stimulated with LPS for 15 min. Whole-cell extracts were made with lysis buffer, and then 50 μg of protein extract was loaded per lane for SDS-PAGE/immunoblot analysis. Monoclonal Relish antibodies were used for the immunoblot (Stöven et al. 2000)

RNAi experiments

The protocol of Hammond et al. (2000) was followed. Briefly, dsRNA was synthesized from a template amplified by PCR with T7 promoter sequences flanking a ∼500-bp fragment of the gene of interest (either DmIKKβ, DmIKKγ, or LacZ), using the Ribomax kit from Promega. dsRNA was purified by phenol/chloroform extraction and ethanol precipitation. dsRNA was transfected, along with a LacZ reporter plasmid, into S2* cells using a standard calcium phosphate method. The cells were split 24 h after transfection to a concentration of 0.5 × 106/mL and treated with 10−6 M hydroxy-ecdysone 24 h later. After an additional 24 h, cells were treated (or left untreated) with LPS (10 μg/mL for RNA extraction or 50 ng/mL for protein extraction) for 15 min (for protein extracts) or 3 h (for RNA isolation). The RNAi experiments in the S2*tpll cell line were performed similar to that described above except that the cells were split to 1.0 × 106/mL 24 h after transfection and treated with 10−6 M hydroxy-ecdysone. RNA and protein blots were performed as described above.

Kinase assays

His-tagged DmIKKβ was expressed in Sf9 cells using a Baculovirus expression system and purified on Ni-NTA (Graveley and Maniatis 1998). Relish was immunoprecipitated with anti-Flag antibodies or with a polyclonal Relish antibody, plus Protein G agarose, that recognizes the N-terminal domain (S. Stöven and D. Hultmark, unpubl.). Immunoprecipitated Relish was subjected to extensive washes before being used as a substrate. Kinase reactions were performed in kinase buffer (20 mM Hepes at pH 7.6, 20 mM β-Glycerolphosphate, 10 mM MgCl2, 50 mM NaCl, 1 mM DTT, 0.1 mM NaVO4, 200 μM ATP, and 5 μCi 32P-γ-ATP). hIKKα and hIKKɛ were also purified using a Baculovirus/Sf9 expression system; GST-IκBα was purified from Escherichia coli following standard procedures (gift of R. Peters, Harvard University, Boston, MA; Peters et al. 2000). For immunoprecipitation of the endogenous DmIKK complex, 100 μg of a detergent lysate from S2* cells, with or without LPS treatment (10 μg/mL for 15 min), was immunoprecipitated with 1 μL of anti-DmIKKγ antisera and Protein G beads. After washing in lysis buffer, the immunoprecipitates were equilibrated in kinase buffer and used with the substrates noted. A fraction of the immunoprecipitate was reserved for the immunoblot analysis shown in Figure Figure4C.4C. DmIKKγ antisera were raised in rabbits against recombinant DmIKKγ1–297 following standard protocols (Covance).


This work was supported by the grants from the NIH to T.M. (GM29379, GM59919), from the Helen Hay Whitney Foundation to N.S., and from the Göran Gustafsson Foundation for Scientific Research and the Swedish Natural Science and Medical Research Councils to D.H. The S2*tpll cell line was a gift of S. Liao. We thank T. Ip for sharing cell lines; S. Elledge for two-hybrid libraries; E. Bernstein and G. Hannon for assistance with RNAi; C. James, B. Cali, and T. Milne for two-hybrid reagents; S. Wasserman for the torso–pelle construct; and R. Peters for hIKK reagents.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.


E-MAIL ude.dravrah.phoib@sitainam; FAX (617) 495-3537.

Article and publication are at www.genesdev.org/cgi/doi/10.1101/gad.817800.


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