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Toll Signaling PathwayDrosophilaThe Susanna Valanne, Jing-Huan Wang and Mika Rmet

http://www.jimmunol.org/content/186/2/649doi: 10.4049/jimmunol.1002302

2011; 186:649-656; ;J Immunol

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The Drosophila Toll Signaling PathwaySusanna Valanne,* Jing-Huan Wang,* and Mika Ramet*,

The identification of the Drosophila melanogaster Tollpathway cascade and the subsequent characterization ofTLRs have reshaped our understanding of the immunesystem. Ever since, Drosophila NF-kB signaling hasbeen actively studied. In flies, the Toll receptors areessential for embryonic development and immunity. Intotal, nine Toll receptors are encoded in the Drosophilagenome, including the Toll pathway receptor Toll. Theinduction of the Toll pathway by Gram-positive bacteriaor fungi leads to the activation of cellular immunity aswell as the systemic production of certain antimicro-bial peptides. The Toll receptor is activated when theproteolytically cleaved ligand Spatzle binds to the recep-tor, eventually leading to the activation of the NF-kBfactors Dorsal-related immunity factor or Dorsal. Inthis study, we review the current literature on the Tollpathway and compare the Drosophila and mammalianNF-kB pathways. The Journal of Immunology, 2011,186: 649656.

The Toll pathway was initially identified in a series ofgenetic screens for genes involved in early Drosophilaembryonic development. These screens were based on

the revolutionary saturation mutagenesis screen developed byC. Nusslein-Volhard and E.F. Wieschaus, who identified 15genes that control embryonic segmentation (1). This approachearned them, together with E.B. Lewis, the Nobel Prize inMedicine in 1995 (http://nobelprize.org/nobel_prizes/medicine/laureates/1995/). Subsequent genetic screens led to the discoveryof genes important in the dorsal-ventral (DV) patterning of theembryo (i.e., the dorsal group of genes, including Toll, tube,pelle, cactus, the NF-kB homolog dorsal, and seven genes up-stream of Toll) (2).Because NF-kB was implied to be involved in mammalian

immunity, and because the moth Hyalophora cecropia expressesan NF-kBlike immunoresponsive factor (3), it graduallybecame evident that parallels between the signaling pathwaysin Drosophila embryonic development and activation of the

immune system exist (4). Hultmark and colleagues (5) firstidentified Toll (Toll-1) as an activator of the immune responsein a Drosophila cell line in 1995. Around the same time, a hu-man homolog of Toll was identified and mapped to chromo-some 4p14 (6). Soon after this, a compelling in vivo study inDrosophila demonstrated that the DV regulatory gene cassettesignaling from the Toll ligand spatzle to cactus is involved in theantifungal response in Drosophila (7). The first mammalianTLR was described 1 y later in 1997 (8). This was shortlyfollowed by the characterization of five human TLRs (9) estab-lishing the role of the Drosophila Toll pathway as an evolution-arily conserved signaling cascade. However, mammalian TLRsare believed to have no role in development (10), whereas theDrosophila Toll pathway is involved both in immunity (7) anddevelopmental processes (2, 11, 12).

The Toll pathway in the immune response

The Drosophila immune system is composed of humoraland cellular components. A Gram-positive or fungal infectiontriggers the activation of the Toll pathway, which leads to thesystemic production of antimicrobial peptides (AMPs) (13,14). The antifungal peptide Drosomycin appears to be theprincipal target of the Toll humoral response. The Toll path-way also plays a role in the cellular immune response, whichincludes the phagocytosis of microbes, and the encapsulationand killing of parasites (15). Infecting Drosophila with theparasitic wasp Leptopilina boulardi activates a cellular immuneresponse (16), which is manifested by increased production ofcirculating plasmatocytes, and the differentiation of a groupof plasmatocytes into another specialized class of hemocyte,the lamellocyte. Lamellocytes participate in the encapsulationand killing of the parasite. Mutations in the gene cactus,a gain-of-function mutation in the Toll receptor gene, orthe constitutive expression of dorsal can induce lamellocytedifferentiation and cause the formation of melanotic tumorphenotypes (12, 17). Moreover, the Toll signaling pathwaytogether with other pathways has been found to control he-mocyte proliferation and hemocyte density (16, 18). In Dro-sophila larvae, Toll signaling is required for melanization (19).In gain-of-function Toll mutant flies, or cactus mutant flies

*Laboratory of Experimental Immunology, Institute of Medical Technology, Universityof Tampere, 33014 Tampere, Finland; and Department of Pediatrics, Tampere Uni-versity Hospital, 33014 Tampere, Finland

Received for publication July 28, 2010. Accepted for publication November 2, 2010.

This work was supported by grants from the Academy of Finland, the Foundation forPediatric Research, the Sigrid Juselius Foundation, and the Emil Aaltonen Foundation(to M.R.), the Foundation of the Finnish Anti-Tuberculosis Association (to S.V.), theTampere Tuberculosis Foundation, Competitive Research Funding of the PirkanmaaHospital District, and Biocenter Finland (to M.R. and S.V.).

Address correspondence and reprint requests to Prof. Mika Ramet, Laboratory of Ex-perimental Immunology, Institute of Medical Technology, University of Tampere,33014 Tampere, Finland. E-mail address: mika.ramet@uta.fi

Abbreviations used in this article: AMP, antimicrobial peptide; DAP, diaminopimelicacid; DD, death domain; DEAF-1, deformed epidermal autoregulatory factor-1; Dif,dorsal-related immunity factor; DREDD, death-related Ced-3/Nedd2-like protein; DV,dorsal-ventral; GNBP, Gram-negative binding protein; Gprk2, G protein-coupled re-ceptor kinase 2; Grass, Gram-positivespecific serine protease; IKK, IkB kinase; IRAK,IL-1Rassociated kinase; ModSP, modular serine protease; PGN, peptidoglycan; PGRP,peptidoglycan recognition protein; RNAi, RNA interference; SPE, Spatzle-processingenzyme; Spz, Spa(e)tzle; TAB, TGF-bactivated kinase 1 binding protein; TAK1, TGF-bactivated kinase 1; TIR, Toll/IL-1R; TRAF, TNFR-associated factor.

Copyright 2011 by TheAmerican Association of Immunologists, Inc. 0022-1767/11/$16.00

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that exhibit melanotic tumor phenotypes, the Toll-responsiveNF-kB factor Dorsal is constitutively nuclear (17). However,this melanotic tumor phenotype is independent of Dorsal,suggesting a redundant role for Dorsal and the Dorsal-relatedimmunity factor (Dif) in this context (17).The cellular response can also affect the activation of the Toll

pathway. Under normal conditions, Spn77Ba, a protease in-hibitor of the serpin family, inhibits a phenol oxidase proteasecascade. It was reported that tracheal melanization resultingfrom Spn77Ba disruption induces the systemic expression ofthe antifungal Drosomycin via the Toll pathway (20). Suchsignaling between local and systemic immune responses maybe an alarm mechanism that prepares the host in case a path-ogen breaches the epithelial barrier (20).

Drosophila Toll receptors

To date, nine genes encoding Toll-related receptors have beenidentified in the Drosophila genome. Toll, or Toll-1, was thefirst Toll identified and is responsible for AMP induction viathe Toll pathway. All Drosophila Toll receptors share a similarmolecular structure, with an ectodomain mainly composed ofleucine-rich repeat and cystein-rich flanking motifs. Phyloge-netically, Toll-5 is the closest relative to Toll (21). In contrastto other Tolls, Toll-9 has only one cystein-rich motif betweenthe transmembrane domain and leucine-rich repeats, a struc-ture very similar to mammalian TLRs (22). Drosophila Tollsand the IL-1Rs in mammals share a cytosolic homology do-main called Toll/IL-1R (TIR) domain, which interacts withadaptor molecules, thereby activating downstream events(22).As all mammalian TLRs are involved in the immune re-

sponse, it is tempting to speculate the involvement of otherDrosophila Tolls in immunity. Some Tolls could well playroles in immunological events; for example, Toll-5 may in-duce Drosomycin and Metchnikowin expression (21, 23, 24).In addition, Toll-5 has been shown to interact with the intra-cytoplasmic domains of Toll and Pelle, leading to the acti-vation of Dorsal-dependent transcription in a synergisticmanner with Toll (24). Also, Toll-9 has been reported toactivate the constitutive expression of Drosomycin (25), andfor this, Toll-9 may take advantage of the Toll signaling path-way components (26).

Spatzle activation

To activate the Drosophila Toll pathway either in develop-ment or in immunity, extracellular recognition factors initiateprotease cascades leading to the activation of the Toll receptorligand Spatzle ( or Spaetzle, Spatzle [Spz]) (27, 28). In non-signaling conditions, the prodomain of Spz masks a pre-dominantly hydrophobic C-terminal Spz region. Activationinduces proteolysis, which causes a conformational changeexposing determinants that are critical for binding of the Tollreceptor (29). Interestingly, the prodomain remains associatedwith the C terminus and is only released when the Toll ex-tracellular domain binds to the complex (30). Two models forthe binding of Spz to Toll have been suggested, the first ofwhich implies that one Spz dimer binds to two Toll receptors(31). In a newer model, two Spz dimers, each binding to theN terminus of one of the two Toll receptors, trigger a confor-mational change in the Tolls to activate downstream signaling(32) (Fig. 1).

Spz is synthesized and secreted as an inactive precursorconsisting of a prodomain and a C-terminal region (C-106)(33). In DV patterning, Spz is processed into its activeC-106 form by a serine protease cascade including Nudel,Gastrulation Defective, Snake, and Easter (34, 35). In addition,sulfotransferase Pipe is required independently of the proteasecascade to activate Easter (36). In microbe recognition, Spz-processing enzyme (SPE) is responsible for Spz cleavage (37).The current model for activation of SPE contains three up-stream cascades depending on the activating microorganism(Fig. 1). Two protease cascades leading to the activation ofGram-positivespecific serine protease (Grass) are initiatedby cell wall components of both fungi (b-glucan) and Gram-positive bacteria (Lysine-type peptidoglycan) (38). Grasswas originally identified to be specifically involved in therecognition of Gram-positive bacteria (39), but was latershown to be important also for the recognition of fungalcomponents (38). In addition, four other serine proteases,namely spirit, spheroide, and sphinx1/2, were identified inresponse to both fungi and Gram-positive bacteria (39).Upstream of Grass, a modular serine protease (ModSP),conserved in insect immune reactions, plays an essential rolein integrating signals from the recognition molecules Gram-negative binding protein (GNBP) 3 and PGN recognitionprotein (PGRP)-SA to the Grass-SPE-Spatzle cascade (40).A third protease cascade leading to the activation of SPE ismediated by the protease Persephone, which is proteolyti-cally matured by the secreted fungal virulence factor PR1(41) and Gram-positive bacterial virulence factors (38).Similar detection mechanisms have been suggested to occurin mammals, in which TLRs or Nod-like receptors directlydetect virulence factors or endogenous proteins released bydamaged cells (42, 43).The recognition of the Gram-positive bacterial lysine-type

peptidoglycan and/or the b-glucan from fungal cell walls ismediated by extracellular recognition factors. GNBP3 is re-sponsible for yeast recognition (41). The other identified fac-tors, namely GNBP1, PGRP-SA, and PGRP-SD, appear tomainly recognize Gram-positive bacteria. Upon Gram-positivebacterial recognition, PGRP-SA and GNBP1 physically inter-act and form a complex (4446). Thereafter, activated GNBP1hydrolyzes the Lys-type PGN and produces new glycan reduc-ing ends, which are presented to PGRP-SA (47). In contrast,Buchon et al. (40) showed that full-length GNBP1 had noenzymatic activity. They suggested a role for GNBP1 as a linkerbetween PGRP-SA and ModSP. PGRP-SD functions as a re-ceptor for Gram-positive bacteria with partial redundancy tothe PGRP-SAGNBP1 complex (48). It appears that PGRP-SD can also recognize diaminopimelic acid (DAP)-type PGNsfrom Gram-negative bacteria, thereby activating the Tollpathway (49).

The core Toll signaling pathway

After binding the processed Spz, the activated Toll receptorbinds to the adaptor protein MyD88 via intracellular TIRdomains (5052). Upon this interaction, MyD88, an adaptorprotein, Tube, and the kinase Pelle are recruited to forma MyD88-Tube-Pelle heterotrimeric complex through deathdomain (DD)-mediated interactions (5254). MyD88 andPelle do not come into contact with each other; instead,two distinct DD surfaces in the adaptor protein Tube

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separately bind MyD88 and Pelle (52). Recently, a highlyconserved Pelle/IL-1Rassociated kinase (IRAK) interactingprotein Pellino was shown to act as a positive regulator ofToll signaling (55). Drosophila Pellino mutants have impairedDrosomycin expression and reduced survival against Gram-positive bacteria (55). As all Pellinos contain a RING domain,it is tempting to speculate that Drosophila Pellino may ubiq-uitinate Pelle in a similar fashion as mammalian Pellinospolyubiquitinate IRAK1 (56).From the oligomeric MyD88-Tube-Pelle complex, the sig-

nal proceeds to the phosphorylation and degradation of theDrosophila IkB factor Cactus. In nonsignaling conditions,Cactus is bound to the NF-kB transcription factor(s) Dorsaland/or Dif in a context-dependent manner, inhibiting theiractivity and nuclear localization. So, the nuclear translocationof both Dorsal and Dif requires Cactus degradation (57). Tobe degraded, Cactus needs to be phosphorylated, and al-though it has not been directly shown, it is possible that thisis achieved by Pelle, because its kinase activity is required for

Cactus phosphorylation (58). Also, in a recent screening (59)in which 476 dsRNA were targeted against all the known andpredicted Drosophila kinases, Pelle was found to be the onlykinase implicated in Cactus phosphorylation. Cactus needsto be phosphorylated in two distinct N-terminal motifs (60)that resemble IkB kinase (IKK) targets, yet the DrosophilaIKK-b (Ird5) or IKK-g (Kenny) are not involved in theToll/Cactus pathway (61, 62). After phosphorylation, nu-clear translocation of Dorsal/Dif leads to activation of thetranscription of several sets of target genes. The Drosophilacore Toll signaling pathway is shown in Fig. 2.The Drosophila Dorsal is a Rel protein originally identified

as an important morphogen in DV polarization. In larvae andadult Drosophila, Dorsal is expressed in the fatbody, and bothits expression level (63) and nuclear localization (17) are en-hanced upon microbial challenge. Dorsal interacts with Pelle,Tube, and Cactus (6466), and, upon pathway activation,Dorsal translocates to the nucleus and binds to the kB-relatedsequence of AMP genes (63). Dorsal can activate the dip-

FIGURE 1. Extracellular cleavage of Spz leading

to Toll pathway activation. In early embryogenesis,

the protease cascade Gastrulation Defective-Snake

activates the protease Easter, which cleaves full-

length Spz. In the immune response, three protease

cascades lead to the activation of SPE to cleave full-

length Spz; the Persephone (PSH) cascade senses

virulence factors and is activated by live Gram-

positive bacteria and fungi. The other two cascades

are activated by pattern recognition receptors bind-

ing cell wall components from Gram-positive bac-

teria and fungi, respectively. All cascades converge

at ModSP-Grass for downstream activation of SPE.

Upon proteolytical processing, the Spz prodomain

is cleaved, exposing the C-terminal Spz parts crit-

ical for binding of Toll. Spz binding to the Toll

receptor initiates intracellular signaling.

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tericin promoter in vitro (67), and, moreover, bacterial cul-ture supernatants can stimulate nuclear translocation of Dor-sal in vivo in dissected fatbodies in a hemolymph-dependentmanner (19). Also, Dorsal activity is required to restrict theinfectivity of Pseudomonas aeruginosa in adult Drosophila, pro-viding evidence for Dorsal function in resistance against mic-roorganisms (68).Dif was identified in Drosophila as a dorsal-related immune

responsive gene that does not participate in DV patterning.Instead, it mediates an immune response in Drosophila larvae(69) and interacts with Cactus in vitro (70). Dif (71), but

not Dorsal (7), mediates Toll-dependent induction of the an-tifungal peptide gene Drosomycin in Drosophila adults, whereasDorsal and Dif seem to be redundant in larvae (71, 72). Fur-thermore, Dif and Dorsal can form heterodimers in vitro (67),and in a Drosophila macrophage-like S2 cell line, Dorsal seemsto play a more important role in Drosomycin promoter activa-tion than does Dif (73).

RNA interference screening for new components of the Toll pathway

Drosophila cells are ideal for large-scale in vitro RNA inter-ference (RNAi) screens (74, 75). Long dsRNA fragments up

FIGURE 2. Comparison of Drosophila Imd, Toll, and mammalian TLR signaling pathways. Homologies between signaling components are depicted by similarshape. The Imd pathway is activated by DAP-type PGN binding of the PGRP-LC dimer. Other PGRP family members play either negative or positive roles.

IMD is connected to the caspase DREDD via the adaptor protein Fas-associated DD protein (FADD). DREDD proteolytically cleaves IMD and Relish. Cleaved

IMD associates with the E3-ligase IAP2, E2-ubiquitin-conjugating enzymes UEV1a, Bendless (Ubc13), and Effete (Ubc5) and is K63 polyubiquitinated. This

activates the downstream kinase cascade leading to the phosphorylation and activation of Relish and AP-1, which activate the transcription of AMP and stress

genes, respectively. Akirin is required for Imd pathway function at the level of Relish (105). Pirk (106), Caspar (107), and Dnr1 (108) are negative regulators of

the Imd pathway. The Toll pathway is activated by Spz. One Spz dimer is depicted to bind the N terminus of Toll and to induce a conformational change leading

to the formation of a 4Spz:2Toll complex. Intracellular signaling leads to the phosphorylation and degradation of Cactus, which releases Dif and/or Dorsal to

translocate to the nucleus and activate transcription. Gprk2 associates with Cactus in a kinase domain (KD)-dependent manner. DEAF-1 is required to induce

Toll pathway target genes at or downstream of Dif/Dorsal. Mammalian TLRs are activated by bacterial-, viral-, and self-derived products. Depicted are MyD88-

dependent signal transduction events. TLR1, -2, -4, -5, and -6 signal through the plasma membrane, whereas TLR7, -8, and -9 function in the endosome. TLR1,

-2, -4, and -6 use the adaptors TIR domain-containing adaptor protein (TIRAP)/MyD88 adaptor-like (Mal) and MyD88, whereas TLR5, -7, -8, and -9 use

MyD88 only. MyD88 recruits IRAKs and TRAF6, which activates the TAK1/TAB complex via K63-linked ubiquitination. The activated TAK1 complex

stimulates the IKK complex and the MAPK pathway, thereby activating NF-kB and AP-1, respectively. Activated NF-kB translocates to the nucleus to activate

transcription. The signal from the endosome activates a complex containing TRAF3 in addition to MyD88, TRAF6, IRAKs, and IKK-a. The activated complex

phosphorylates and activates IFN regulatory factor 7 (IRF7) for its nuclear translocation and subsequent transcriptional activation of target genes.

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to several kilobases are readily internalized and processed byDrosophila S2 cells, which makes RNAi in S2 cells a veryfeasible tool for identifying genes involved in various processes(76, 77). In general, robust degradation of target RNA isobtained without a need for any transfection reagents. RNAiscreening strategies have revealed several new important find-ings related to Drosophila Toll signaling. Recently, deformedepidermal autoregulatory factor-1 (DEAF-1), which was firstidentified as a transcription factor that binds to Metchnikowinand Drosomycin promoters (78), was confirmed to be requiredfor full Drosomycin expression as well as for defending againstfungal infections (79). Moreover, endocytic machinery compo-nents, including Myopic, were indicated to play a role in theendocytosis of the Toll receptor upon pathway activation (59).In a recent genome-wide RNAi screen in S2 cells, G Protein-

coupled receptor kinase 2 (Gprk2) was identified as a regulatorof the Toll pathway (73). Gprk2 was found to be importantagainst a Gram-positive bacterial infection as well as in Tollpathway-mediated hemocyte activation in Drosophila in vivo.Gprk2 interacts with Cactus in S2 cells, but is not involved inCactus degradation, adding a new level of complexity to Dro-sophila Toll/Cactus regulation (73). Other genes identified inthe screen include a Friend of GATA factor U-shaped andToll activation mediating protein (TAMP; CG15737), witha previously unknown function. RNAi knockdown of ush orTAMP was shown to reduce the activity of the Drosomycinreporter in S2 cells in vitro as well as Drosomycin expression invivo in infected flies. However, the molecular mechanisms ofthe effect of these components on the Toll pathway remain tobe investigated (73).

Synergistic activation of the Drosophila immune-responsive pathways

It is clear that the Drosophila Toll pathway plays a key role inGram-positive bacterial and fungal infections (80). In turn,Imd signaling is initiated by the PGRP-LCmediatedrecognition of mainly a DAP-type PGN from Gram-nega-tive bacteria (74, 81). Imd pathway activation ultimately leadsto the activation of the NF-kB factor Relish (8284), itstranslocation to the nucleus, and the transcriptional activationof a group of target genes including AMPs (13, 14) (Fig. 2).Although the Drosophila immunity pathways get selectivelyactivated to a certain degree (85), synergistic interactionsbetween the Toll and the Imd pathways have graduallybecome evident (73, 8688). For example, although thebacterial branch of the Toll pathway is mainly activatedby a Lys-type PGN, the crystal structure of the Toll pathwaymediator PGRP-SD suggests binding to a DAP-type PGNrather than a Lys-type one (49). Moreover, in a Drosophilacell line, Relish RNAi reduces the expression of the Toll10b

-induced Drosomycin reporter gene (86), and the Drosomycinreporter can be synergistically activated by Toll10b and Gram-negative bacteria (73). Furthermore, the expression of Droso-mycin and Defensin are best induced by the Relish/Dif and theRelish/Dorsal heterodimers, respectively (89). In vivo, post-infection with Escherichia coli, the double mutants for Dif andthe Imd pathway component kenny die earlier than kennymutants (90). The same holds for the Relish,spz, and Relish,Toll double mutants compared with Relish mutants (88).In addition to kB binding sites for Rel proteins, the tran-

scriptional regulation of many Drosophila AMP genes dependson GATA binding sites in their promoter proximal regions

(91). Drosophila has five GATA factors, namely Pannier(dGATAa), Serpent (dGATAb), Grain (dGATAc), dGATAd,and dGATAe. Pannier and a Friend of GATA factor U-shaped were recently identified as regulators of the Toll path-way in S2 cells (73). Serpent is the major GATA transcriptionfactor in the larval fat body, and synergy between Relish andSerpent in the activation of the full immune response in larvaehas been shown (92). Moreover, evidence is presented fordGATAe-mediated immune responses in the gut (93). Itappears that, in most cases, Rel proteins and GATA factorsact in concert to activate immune responses. Also, at least fullMetchnikowin expression requires DEAF-1 (78).

Comparison of the Drosophila Toll and Imd pathways to mammalianTLR signaling

To date, 10 TLRs have been identified in humans and 12 inmice. The significance of TLRs was unknown until the mouseTlr4 gene was identified as essential for LPS signaling (94).TLRs have since been shown to act as pattern recognitionreceptors for bacterial-, viral-, and self-derived products(reviewed in Ref. 95). When the signal is transduced, Tollsand TLRs associate with MyD88 via their intracytoplasmicTIR domains, activating the homologous protein kinases Pelle(in Drosophila) and IRAK (in mammals) (22). A recent studyprovides evidence for orthology between Tube and IRAK4 aswell as Pelle and IRAK1 (96). In contrast, it has also beensuggested that Drosophila Tube is at least functionally equiv-alent, and maybe distantly related in sequence, to the humanTLR pathway adaptor protein MyD88 adaptor-like (97). Inmammals, six MyD88, four IRAK4, and four IRAK2 DDsform a helical oligomer complex called Myddosome fordownstream signaling (98). A similar three-component sys-tem, albeit with a different stoichiometry, is used in Droso-phila: dimers of MyD88, Tube, and Pelle are needed forcomplex formation (54). In mammals, signal transmissiondownstream of MyD88 triggers the cooperation of severalIRAKs, after which the IRAK complex interacts withTNFR-associated factor (TRAF) 6, which mediates the signalforward, via ubiquitination events, to the TGF-bactivatedkinase 1 (TAK1) and TAK1 binding protein (TAB) complexes.TRAF homologs have been identified in the Drosophilagenome, but they do not appear to participate in immunesignaling (52, 86).It appears that downstream from TAK1/TAB, the mam-

malian TLR pathway and the Drosophila Imd pathway, ratherthan the Toll pathway, share homologous components (95,99). In mammals, the signal bifurcates at the level of a com-plex containing TAK1 and TABs, where one signal leads tothe phosphorylation of the IKK complex and another viaMAPKs to the activation of the JNK pathway and the even-tual nuclear translocation of AP-1. The IKK complex phos-phorylates IkB, leading to its ubiquitination and degradation.This results in the nuclear translocation of NF-kB factor(s)and the activation of transcription (95). Similarly, in theDrosophila Imd pathway, two signals from a complexcontaining Tak1, Tab2, and inhibitor of apoptosis 2 (86)are transmitted, one to the JNK pathway and one to theIKK complex, which phosphorylates the Rel protein Relish.After this, the caspase death-related Ced-3/Nedd2-like pro-tein (DREDD) cleaves the C-terminal inhibitory domain ofRelish (100). As was recently reported, DREDD is also

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involved in the cleavage of the Imd protein (101). The Dro-sophila Toll and Imd pathways are compared with relatedmammalian TLR pathways in Fig. 2.Events downstream of MyD88 in the Drosophila Toll

pathway appear somewhat different from the mammalianMyD88-dependent TLR pathways. The IKK complex is notinvolved in the phosphorylation and degradation of the IkBprotein Cactus. However, conserved mechanisms in down-stream parts of the Toll pathway and mammalian NF-kBsignaling are evident. The Drosophila Gprk2 protein, whichwas shown to be involved in Toll pathway regulation and tointeract with Cactus (73), is homologous to the murineGRK5, which was recently implicated in TNF-ainduced NF-kB signaling via direct interaction with IkB (102). Furthermore,the GRK5 knockout mice have attenuated LPS response,suggesting an evolutionarily conserved role for Gprk2/GRK5 (103).In mammals, TLR7/TLR7, TLR9/TLR9, and TLR7/TLR8

act on the endosomal membrane also in a MyD88-dependentway to recognize nucleic acids from, for example, viruses (95,104). Upon activation, the signal is propagated via severalcytoplasmic IRAK proteins leading to the phosphorylationand nuclear translocation of IFN regulatory factor 7 (95).Interestingly, it was recently reported (59) that the DrosophilaMop (myopic) and Hrs (hepatocyte growth factor-regulatedtyrosine kinase substrate), which are critical components ofthe endocytosis complex, colocalize with the Toll receptor inendosomes. Also, the Bro1 domain in Mop, which points toendosomal localization, is required for Toll signaling. So, it isplausible that endocytosis has an evolutionarily conserved rolein Drosophila Toll and mammalian TLR signaling (59).

ConclusionsSince the initial discovery of the Toll pathway in fruit flydevelopment 25 y ago, research in the field has firmly estab-lished the role of Toll signaling in immunity as well. In recentyears, studies on microbe recognition and events upstream ofSpz activation have revealed new components of the pathway.In addition, large-scale RNAi screens on the core intracellularpathway have revealed new essential components, putative con-served mechanisms, and cooperation of the fly immune path-ways.Mammalian TLR signaling mechanisms share similarities

with the Drosophila Toll pathway, but also important differ-ences exist; for example, the Toll receptor is a cytokine re-ceptor, whereas TLRs are pattern recognition receptors. Also,among the nine Drosophila Tolls, a clear immunological rolehas only been assigned to Toll, whereas the others have pu-tative roles in development. In contrast, all mammalian TLRsappear to have roles in immunity. Future work on the Dro-sophila Toll and other immune response pathways will un-doubtedly continue to increase our understanding of theseconserved NF-kB mechanisms in mammals.

AcknowledgmentsWe thank Dr. Helen Cooper for revising the language of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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