The Drosophila Toll Signaling Pathway

Susanna Valanne, Jing-Huan Wang and Mika Rmet

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J Immunol 2011; 186:649-656; ;

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

The Drosophila Toll Signaling Pathway

Susanna Valanne,* Jing-Huan Wang,* and Mika Ramet*,

he Toll pathway was initially identified in a series of

genetic screens for genes involved in early Drosophila
embryonic development. These screens were based on
the revolutionary saturation mutagenesis screen developed by
C. Nusslein-Volhard and E.F. Wieschaus, who identified 15
genes that control embryonic segmentation (1). This approach
earned them, together with E.B. Lewis, the Nobel Prize in
Medicine in 1995 (http://nobelprize.org/nobel_prizes/medicine/
laureates/1995/). Subsequent genetic screens led to the discovery
of genes important in the dorsal-ventral (DV) patterning of the
embryo (i.e., the dorsal group of genes, including Toll, tube,
pelle, cactus, the NF-kB homolog dorsal, and seven genes upstream of Toll) (2).
Because NF-kB was implied to be involved in mammalian
immunity, and because the moth Hyalophora cecropia expresses
an NF-kBlike immunoresponsive factor (3), it gradually
became evident that parallels between the signaling pathways
in Drosophila embryonic development and activation of the

*Laboratory of Experimental Immunology, Institute of Medical Technology, University

of Tampere, 33014 Tampere, Finland; and Department of Pediatrics, Tampere University 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 for
Pediatric Research, the Sigrid Juselius Foundation, and the Emil Aaltonen Foundation
(to M.R.), the Foundation of the Finnish Anti-Tuberculosis Association (to S.V.), the
Tampere Tuberculosis Foundation, Competitive Research Funding of the Pirkanmaa
Hospital District, and Biocenter Finland (to M.R. and S.V.).
Address correspondence and reprint requests to Prof. Mika Ramet, Laboratory of Experimental Immunology, Institute of Medical Technology, University of Tampere,
33014 Tampere, Finland. E-mail address: mika.ramet@uta.fi
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002302

immune system exist (4). Hultmark and colleagues (5) first

identified Toll (Toll-1) as an activator of the immune response
in a Drosophila cell line in 1995. Around the same time, a human homolog of Toll was identified and mapped to chromosome 4p14 (6). Soon after this, a compelling in vivo study in
Drosophila demonstrated that the DV regulatory gene cassette
signaling from the Toll ligand spatzle to cactus is involved in the
antifungal response in Drosophila (7). The first mammalian
TLR was described 1 y later in 1997 (8). This was shortly
followed by the characterization of five human TLRs (9) establishing the role of the Drosophila Toll pathway as an evolutionarily conserved signaling cascade. However, mammalian TLRs
are believed to have no role in development (10), whereas the
Drosophila Toll pathway is involved both in immunity (7) and
developmental processes (2, 11, 12).
The Toll pathway in the immune response

The Drosophila immune system is composed of humoral

and cellular components. A Gram-positive or fungal infection
triggers the activation of the Toll pathway, which leads to the
systemic production of antimicrobial peptides (AMPs) (13,
14). The antifungal peptide Drosomycin appears to be the
principal target of the Toll humoral response. The Toll pathway also plays a role in the cellular immune response, which
includes the phagocytosis of microbes, and the encapsulation
and killing of parasites (15). Infecting Drosophila with the
parasitic wasp Leptopilina boulardi activates a cellular immune
response (16), which is manifested by increased production of
circulating plasmatocytes, and the differentiation of a group
of plasmatocytes into another specialized class of hemocyte,
the lamellocyte. Lamellocytes participate in the encapsulation
and killing of the parasite. Mutations in the gene cactus,
a gain-of-function mutation in the Toll receptor gene, or
the constitutive expression of dorsal can induce lamellocyte
differentiation and cause the formation of melanotic tumor
phenotypes (12, 17). Moreover, the Toll signaling pathway
together with other pathways has been found to control hemocyte proliferation and hemocyte density (16, 18). In Drosophila larvae, Toll signaling is required for melanization (19).
In gain-of-function Toll mutant flies, or cactus mutant flies
Abbreviations used in this article: AMP, antimicrobial peptide; DAP, diaminopimelic
acid; 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 receptor 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-processing
enzyme; Spz, Spa(e)tzle; TAB, TGF-bactivated kinase 1 binding protein; TAK1, TGFbactivated kinase 1; TIR, Toll/IL-1R; TRAF, TNFR-associated factor.
Copyright 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

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The identification of the Drosophila melanogaster Toll

pathway cascade and the subsequent characterization of
TLRs have reshaped our understanding of the immune
system. Ever since, Drosophila NF-kB signaling has
been actively studied. In flies, the Toll receptors are
essential for embryonic development and immunity. In
total, nine Toll receptors are encoded in the Drosophila
genome, including the Toll pathway receptor Toll. The
induction of the Toll pathway by Gram-positive bacteria
or fungi leads to the activation of cellular immunity as
well as the systemic production of certain antimicrobial peptides. The Toll receptor is activated when the
proteolytically cleaved ligand Spatzle binds to the receptor, eventually leading to the activation of the NF-kB
factors Dorsal-related immunity factor or Dorsal. In
this study, we review the current literature on the Toll
pathway and compare the Drosophila and mammalian
NF-kB pathways. The Journal of Immunology, 2011,
186: 649656.

650

that exhibit melanotic tumor phenotypes, the Toll-responsive

NF-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-related
immunity factor (Dif) in this context (17).
The cellular response can also affect the activation of the Toll
pathway. Under normal conditions, Spn77Ba, a protease inhibitor of the serpin family, inhibits a phenol oxidase protease
cascade. It was reported that tracheal melanization resulting
from Spn77Ba disruption induces the systemic expression of
the antifungal Drosomycin via the Toll pathway (20). Such
signaling between local and systemic immune responses may
be an alarm mechanism that prepares the host in case a pathogen breaches the epithelial barrier (20).
Drosophila Toll receptors

Spatzle activation

To activate the Drosophila Toll pathway either in development or in immunity, extracellular recognition factors initiate
protease cascades leading to the activation of the Toll receptor
ligand Spatzle ( or Spaetzle, Spatzle [Spz]) (27, 28). In nonsignaling conditions, the prodomain of Spz masks a predominantly hydrophobic C-terminal Spz region. Activation
induces proteolysis, which causes a conformational change
exposing determinants that are critical for binding of the Toll
receptor (29). Interestingly, the prodomain remains associated
with the C terminus and is only released when the Toll extracellular domain binds to the complex (30). Two models for
the binding of Spz to Toll have been suggested, the first of
which implies that one Spz dimer binds to two Toll receptors
(31). In a newer model, two Spz dimers, each binding to the
N terminus of one of the two Toll receptors, trigger a conformational change in the Tolls to activate downstream signaling
(32) (Fig. 1).

Spz is synthesized and secreted as an inactive precursor

consisting of a prodomain and a C-terminal region (C-106)
(33). In DV patterning, Spz is processed into its active
C-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 protease
cascade to activate Easter (36). In microbe recognition, Spzprocessing enzyme (SPE) is responsible for Spz cleavage (37).
The current model for activation of SPE contains three upstream cascades depending on the activating microorganism
(Fig. 1). Two protease cascades leading to the activation of
Gram-positivespecific serine protease (Grass) are initiated
by cell wall components of both fungi (b-glucan) and Grampositive bacteria (Lysine-type peptidoglycan) (38). Grass
was originally identified to be specifically involved in the
recognition of Gram-positive bacteria (39), but was later
shown to be important also for the recognition of fungal
components (38). In addition, four other serine proteases,
namely spirit, spheroide, and sphinx1/2, were identified in
response to both fungi and Gram-positive bacteria (39).
Upstream of Grass, a modular serine protease (ModSP),
conserved in insect immune reactions, plays an essential role
in integrating signals from the recognition molecules Gramnegative binding protein (GNBP) 3 and PGN recognition
protein (PGRP)-SA to the Grass-SPE-Spatzle cascade (40).
A third protease cascade leading to the activation of SPE is
mediated by the protease Persephone, which is proteolytically matured by the secreted fungal virulence factor PR1
(41) and Gram-positive bacterial virulence factors (38).
Similar detection mechanisms have been suggested to occur
in mammals, in which TLRs or Nod-like receptors directly
detect virulence factors or endogenous proteins released by
damaged cells (42, 43).
The recognition of the Gram-positive bacterial lysine-type
peptidoglycan and/or the b-glucan from fungal cell walls is
mediated by extracellular recognition factors. GNBP3 is responsible for yeast recognition (41). The other identified factors, namely GNBP1, PGRP-SA, and PGRP-SD, appear to
mainly recognize Gram-positive bacteria. Upon Gram-positive
bacterial recognition, PGRP-SA and GNBP1 physically interact and form a complex (4446). Thereafter, activated GNBP1
hydrolyzes the Lys-type PGN and produces new glycan reducing ends, which are presented to PGRP-SA (47). In contrast,
Buchon et al. (40) showed that full-length GNBP1 had no
enzymatic activity. They suggested a role for GNBP1 as a linker
between PGRP-SA and ModSP. PGRP-SD functions as a receptor for Gram-positive bacteria with partial redundancy to
the PGRP-SAGNBP1 complex (48). It appears that PGRPSD can also recognize diaminopimelic acid (DAP)-type PGNs
from Gram-negative bacteria, thereby activating the Toll
pathway (49).
The core Toll signaling pathway

After binding the processed Spz, the activated Toll receptor

binds to the adaptor protein MyD88 via intracellular TIR
domains (5052). Upon this interaction, MyD88, an adaptor
protein, Tube, and the kinase Pelle are recruited to form
a MyD88-Tube-Pelle heterotrimeric complex through death
domain (DD)-mediated interactions (5254). MyD88 and
Pelle do not come into contact with each other; instead,
two distinct DD surfaces in the adaptor protein Tube

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To date, nine genes encoding Toll-related receptors have been

identified in the Drosophila genome. Toll, or Toll-1, was the
first Toll identified and is responsible for AMP induction via
the Toll pathway. All Drosophila Toll receptors share a similar
molecular structure, with an ectodomain mainly composed of
leucine-rich repeat and cystein-rich flanking motifs. Phylogenetically, Toll-5 is the closest relative to Toll (21). In contrast
to other Tolls, Toll-9 has only one cystein-rich motif between
the transmembrane domain and leucine-rich repeats, a structure very similar to mammalian TLRs (22). Drosophila Tolls
and the IL-1Rs in mammals share a cytosolic homology domain called Toll/IL-1R (TIR) domain, which interacts with
adaptor molecules, thereby activating downstream events
(22).
As all mammalian TLRs are involved in the immune response, it is tempting to speculate the involvement of other
Drosophila Tolls in immunity. Some Tolls could well play
roles in immunological events; for example, Toll-5 may induce Drosomycin and Metchnikowin expression (21, 23, 24).
In addition, Toll-5 has been shown to interact with the intracytoplasmic domains of Toll and Pelle, leading to the activation of Dorsal-dependent transcription in a synergistic
manner with Toll (24). Also, Toll-9 has been reported to
activate the constitutive expression of Drosomycin (25), and
for this, Toll-9 may take advantage of the Toll signaling pathway components (26).

BRIEF REVIEWS: THE DROSOPHILA TOLL PATHWAY

The Journal of Immunology

651

separately bind MyD88 and Pelle (52). Recently, a highly

conserved Pelle/IL-1Rassociated kinase (IRAK) interacting
protein Pellino was shown to act as a positive regulator of
Toll signaling (55). Drosophila Pellino mutants have impaired
Drosomycin expression and reduced survival against Grampositive bacteria (55). As all Pellinos contain a RING domain,
it is tempting to speculate that Drosophila Pellino may ubiquitinate Pelle in a similar fashion as mammalian Pellinos
polyubiquitinate IRAK1 (56).
From the oligomeric MyD88-Tube-Pelle complex, the signal proceeds to the phosphorylation and degradation of the
Drosophila IkB factor Cactus. In nonsignaling conditions,
Cactus is bound to the NF-kB transcription factor(s) Dorsal
and/or Dif in a context-dependent manner, inhibiting their
activity and nuclear localization. So, the nuclear translocation
of both Dorsal and Dif requires Cactus degradation (57). To
be degraded, Cactus needs to be phosphorylated, and although it has not been directly shown, it is possible that this
is achieved by Pelle, because its kinase activity is required for

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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 fulllength Spz. In the immune response, three protease
cascades lead to the activation of SPE to cleave fulllength Spz; the Persephone (PSH) cascade senses
virulence factors and is activated by live Grampositive bacteria and fungi. The other two cascades
are activated by pattern recognition receptors binding cell wall components from Gram-positive bacteria 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 critical for binding of Toll. Spz binding to the Toll
receptor initiates intracellular signaling.

Cactus phosphorylation (58). Also, in a recent screening (59)

in which 476 dsRNA were targeted against all the known and
predicted Drosophila kinases, Pelle was found to be the only
kinase implicated in Cactus phosphorylation. Cactus needs
to be phosphorylated in two distinct N-terminal motifs (60)
that resemble IkB kinase (IKK) targets, yet the Drosophila
IKK-b (Ird5) or IKK-g (Kenny) are not involved in the
Toll/Cactus pathway (61, 62). After phosphorylation, nuclear translocation of Dorsal/Dif leads to activation of the
transcription of several sets of target genes. The Drosophila
core 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 and
adult Drosophila, Dorsal is expressed in the fatbody, and both
its expression level (63) and nuclear localization (17) are enhanced 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-related
sequence of AMP genes (63). Dorsal can activate the dip-

652

BRIEF REVIEWS: THE DROSOPHILA TOLL PATHWAY

tericin promoter in vitro (67), and, moreover, bacterial culture supernatants can stimulate nuclear translocation of Dorsal in vivo in dissected fatbodies in a hemolymph-dependent
manner (19). Also, Dorsal activity is required to restrict the
infectivity of Pseudomonas aeruginosa in adult Drosophila, providing evidence for Dorsal function in resistance against microorganisms (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 antifungal peptide gene Drosomycin in Drosophila adults, whereas
Dorsal and Dif seem to be redundant in larvae (71, 72). Furthermore, Dif and Dorsal can form heterodimers in vitro (67),
and in a Drosophila macrophage-like S2 cell line, Dorsal seems
to play a more important role in Drosomycin promoter activation than does Dif (73).
RNA interference screening for new components of the Toll pathway

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

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FIGURE 2. Comparison of Drosophila Imd, Toll, and mammalian TLR signaling pathways. Homologies between signaling components are depicted by similar
shape. 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 MyD88dependent 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.

The Journal of Immunology

Synergistic activation of the Drosophila immune-responsive pathways

It is clear that the Drosophila Toll pathway plays a key role in

Gram-positive bacterial and fungal infections (80). In turn,
Imd signaling is initiated by the PGRP-LCmediated
recognition of mainly a DAP-type PGN from Gram-negative bacteria (74, 81). Imd pathway activation ultimately leads
to the activation of the NF-kB factor Relish (8284), its
translocation to the nucleus, and the transcriptional activation
of a group of target genes including AMPs (13, 14) (Fig. 2).
Although the Drosophila immunity pathways get selectively
activated to a certain degree (85), synergistic interactions
between the Toll and the Imd pathways have gradually
become evident (73, 8688). For example, although the
bacterial branch of the Toll pathway is mainly activated
by a Lys-type PGN, the crystal structure of the Toll pathway
mediator PGRP-SD suggests binding to a DAP-type PGN
rather than a Lys-type one (49). Moreover, in a Drosophila
cell line, Relish RNAi reduces the expression of the Toll10b
-induced Drosomycin reporter gene (86), and the Drosomycin
reporter can be synergistically activated by Toll10b and Gramnegative bacteria (73). Furthermore, the expression of Drosomycin and Defensin are best induced by the Relish/Dif and the
Relish/Dorsal heterodimers, respectively (89). In vivo, postinfection with Escherichia coli, the double mutants for Dif and
the Imd pathway component kenny die earlier than kenny
mutants (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 transcriptional regulation of many Drosophila AMP genes depends
on 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 Ushaped were recently identified as regulators of the Toll pathway in S2 cells (73). Serpent is the major GATA transcription
factor in the larval fat body, and synergy between Relish and
Serpent in the activation of the full immune response in larvae
has been shown (92). Moreover, evidence is presented for
dGATAe-mediated immune responses in the gut (93). It
appears that, in most cases, Rel proteins and GATA factors
act in concert to activate immune responses. Also, at least full
Metchnikowin expression requires DEAF-1 (78).
Comparison of the Drosophila Toll and Imd pathways to mammalian
TLR signaling

To date, 10 TLRs have been identified in humans and 12 in

mice. The significance of TLRs was unknown until the mouse
Tlr4 gene was identified as essential for LPS signaling (94).
TLRs have since been shown to act as pattern recognition
receptors for bacterial-, viral-, and self-derived products
(reviewed in Ref. 95). When the signal is transduced, Tolls
and TLRs associate with MyD88 via their intracytoplasmic
TIR domains, activating the homologous protein kinases Pelle
(in Drosophila) and IRAK (in mammals) (22). A recent study
provides evidence for orthology between Tube and IRAK4 as
well as Pelle and IRAK1 (96). In contrast, it has also been
suggested that Drosophila Tube is at least functionally equivalent, and maybe distantly related in sequence, to the human
TLR pathway adaptor protein MyD88 adaptor-like (97). In
mammals, six MyD88, four IRAK4, and four IRAK2 DDs
form a helical oligomer complex called Myddosome for
downstream signaling (98). A similar three-component system, albeit with a different stoichiometry, is used in Drosophila: dimers of MyD88, Tube, and Pelle are needed for
complex formation (54). In mammals, signal transmission
downstream of MyD88 triggers the cooperation of several
IRAKs, after which the IRAK complex interacts with
TNFR-associated factor (TRAF) 6, which mediates the signal
forward, via ubiquitination events, to the TGF-bactivated
kinase 1 (TAK1) and TAK1 binding protein (TAB) complexes.
TRAF homologs have been identified in the Drosophila
genome, but they do not appear to participate in immune
signaling (52, 86).
It appears that downstream from TAK1/TAB, the mammalian TLR pathway and the Drosophila Imd pathway, rather
than the Toll pathway, share homologous components (95,
99). In mammals, the signal bifurcates at the level of a complex containing TAK1 and TABs, where one signal leads to
the phosphorylation of the IKK complex and another via
MAPKs to the activation of the JNK pathway and the eventual nuclear translocation of AP-1. The IKK complex phosphorylates 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 the
Drosophila Imd pathway, two signals from a complex
containing Tak1, Tab2, and inhibitor of apoptosis 2 (86)
are transmitted, one to the JNK pathway and one to the
IKK complex, which phosphorylates the Rel protein Relish.
After this, the caspase death-related Ced-3/Nedd2-like protein (DREDD) cleaves the C-terminal inhibitory domain of
Relish (100). As was recently reported, DREDD is also

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to several kilobases are readily internalized and processed by

Drosophila S2 cells, which makes RNAi in S2 cells a very
feasible tool for identifying genes involved in various processes
(76, 77). In general, robust degradation of target RNA is
obtained without a need for any transfection reagents. RNAi
screening strategies have revealed several new important findings related to Drosophila Toll signaling. Recently, deformed
epidermal autoregulatory factor-1 (DEAF-1), which was first
identified as a transcription factor that binds to Metchnikowin
and Drosomycin promoters (78), was confirmed to be required
for full Drosomycin expression as well as for defending against
fungal infections (79). Moreover, endocytic machinery components, including Myopic, were indicated to play a role in the
endocytosis of the Toll receptor upon pathway activation (59).
In a recent genome-wide RNAi screen in S2 cells, G Proteincoupled receptor kinase 2 (Gprk2) was identified as a regulator
of the Toll pathway (73). Gprk2 was found to be important
against a Gram-positive bacterial infection as well as in Toll
pathway-mediated hemocyte activation in Drosophila in vivo.
Gprk2 interacts with Cactus in S2 cells, but is not involved in
Cactus degradation, adding a new level of complexity to Drosophila Toll/Cactus regulation (73). Other genes identified in
the screen include a Friend of GATA factor U-shaped and
Toll activation mediating protein (TAMP; CG15737), with
a previously unknown function. RNAi knockdown of ush or
TAMP was shown to reduce the activity of the Drosomycin
reporter in S2 cells in vitro as well as Drosomycin expression in
vivo in infected flies. However, the molecular mechanisms of
the effect of these components on the Toll pathway remain to
be investigated (73).

653

654

Conclusions
Since the initial discovery of the Toll pathway in fruit fly
development 25 y ago, research in the field has firmly established the role of Toll signaling in immunity as well. In recent
years, studies on microbe recognition and events upstream of
Spz activation have revealed new components of the pathway.
In addition, large-scale RNAi screens on the core intracellular
pathway have revealed new essential components, putative conserved mechanisms, and cooperation of the fly immune pathways.
Mammalian TLR signaling mechanisms share similarities
with the Drosophila Toll pathway, but also important differences exist; for example, the Toll receptor is a cytokine receptor, whereas TLRs are pattern recognition receptors. Also,
among the nine Drosophila Tolls, a clear immunological role
has only been assigned to Toll, whereas the others have putative roles in development. In contrast, all mammalian TLRs
appear to have roles in immunity. Future work on the Drosophila Toll and other immune response pathways will undoubtedly continue to increase our understanding of these
conserved NF-kB mechanisms in mammals.

Acknowledgments
We thank Dr. Helen Cooper for revising the language of the manuscript.

Disclosures
The authors have no financial conflicts of interest.

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involved in the cleavage of the Imd protein (101). The Drosophila Toll and Imd pathways are compared with related
mammalian TLR pathways in Fig. 2.
Events downstream of MyD88 in the Drosophila Toll
pathway appear somewhat different from the mammalian
MyD88-dependent TLR pathways. The IKK complex is not
involved in the phosphorylation and degradation of the IkB
protein Cactus. However, conserved mechanisms in downstream parts of the Toll pathway and mammalian NF-kB
signaling are evident. The Drosophila Gprk2 protein, which
was shown to be involved in Toll pathway regulation and to
interact with Cactus (73), is homologous to the murine
GRK5, which was recently implicated in TNF-ainduced NFkB 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-dependent
way to recognize nucleic acids from, for example, viruses (95,
104). Upon activation, the signal is propagated via several
cytoplasmic IRAK proteins leading to the phosphorylation
and nuclear translocation of IFN regulatory factor 7 (95).
Interestingly, it was recently reported (59) that the Drosophila
Mop (myopic) and Hrs (hepatocyte growth factor-regulated
tyrosine kinase substrate), which are critical components of
the endocytosis complex, colocalize with the Toll receptor in
endosomes. Also, the Bro1 domain in Mop, which points to
endosomal localization, is required for Toll signaling. So, it is
plausible that endocytosis has an evolutionarily conserved role
in Drosophila Toll and mammalian TLR signaling (59).

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