A Drosophila IkappaB kinase complex required for Relish cleavage and antibacterial immunity.
Journal: 2000/November - Genes and Development
ISSN: 0890-9369
PUBMED: 11018014
Abstract:
Here we report the identification of a Drosophila IkappaB kinase complex containing DmIKKbeta and DmIKKgamma, homologs of the human IKKbeta and IKKgamma 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 DmIKKbeta, can phosphorylate Relish in vitro. Thus, we propose that the Drosophila IkappaB kinase complex functions, at least in part, by inducing the proteolytic cleavage of Relish. The N terminus of Relish then translocates to the nucleus and activates the transcription of antibacterial immune response genes. Remarkably, this Drosophila IkappaB kinase complex is not required for the activation of the Rel proteins Dif and Dorsal through the Toll signaling pathway, which is essential for antifungal immunity and dorsoventral patterning during early development. Thus, a yet to be identified IkappaB kinase complex must be required for Rel protein activation via the Toll signaling pathway.
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Genes Dev 14(19): 2461-2471

A <em>Drosophila</em> IκB kinase complex required for Relish cleavage and antibacterial immunity

Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA; Umeå Center for Molecular Pathogenesis, Umeå University, S-901 87 Umeå, Sweden
Present address: R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ 08869, USA.
These authors contributed equally to this work.
Corresponding author.
Received 2000 Jun 30; Accepted 2000 Aug 7.

Abstract

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
Abstract

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.

Acknowledgments

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.

Acknowledgments

Footnotes

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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