Serine/threonine acetylation of TGFβ-activated kinase
(TAK1) by Yersinia pestis YopJ inhibits innate
Nicholas Paquettea,b,1, Joseph Conlona,1,2, Charles Sweeta,3, Florentina Rusa, Lindsay Wilsona, Andrea Pereiraa,
Charles V. Rosadinia, Nadege Goutagnya, Alexander N. R. Weberc, William S. Laned, Scott A. Shaffere,
Stephanie Maniatise, Katherine A. Fitzgeralda, Lynda Stuartb, and Neal Silvermana,4
aDivision of Infectious Disease, Department of Medicine, andeProteomics and Mass Spectrometry Facility, University of Massachusetts Medical School,
Worcester, MA 01605;bProgram of Developmental Immunology, Department of Pediatrics, Massachusetts General Hospital/Harvard Medical School, Boston,
MA 02114;cToll-Like Receptors and Cancer, German Cancer Research Center, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany; anddMass Spectrometry
and Proteomics Resource Laboratory, Center for Systems Biology, Harvard University, Cambridge, MA 02138
Edited by Frederick M. Ausubel, Harvard Medical School and Massachusetts General Hospital, Boston, MA, and approved May 24, 2012 (received for review
June 9, 2010)
The Gram-negative bacteria Yersinia pestis, causative agent of
plague, is extremely virulent. One mechanism contributing to
Y. pestis virulence is the presence of a type-three secretion system,
which injects effector proteins, Yops, directly into immune cells of
the infected host. One of these Yop proteins, YopJ, is proapoptotic
and inhibits mammalian NF-κB and MAP-kinase signal transduc-
tion pathways. Although the molecular mechanism remained elu-
sive for some time, recent work has shown that YopJ acts as
a serine/threonine acetyl-transferase targeting MAP2 kinases.
Using Drosophila as a model system, we find that YopJ inhibits
one innate immune NF-κB signaling pathway (IMD) but not the
other (Toll). In fact, we show YopJ mediated serine/threonine acet-
ylation and inhibition of dTAK1, the critical MAP3 kinase in the
IMD pathway. Acetylation of critical serine/threonine residues in
the activation loop of Drosophila TAK1 blocks phosphorylation of
the protein and subsequent kinase activation. In addition, studies
in mammalian cells show similar modification and inhibition of
hTAK1. These data present evidence that TAK1 is a target for
Drosophila immunity|innate immunity
has been shown to be both proapoptotic and to inhibit proin-
flammatory signal transduction (1–5). However, the precise tar-
gets and mechanism used by YopJ to interfere with these
signaling pathways have been controversial. Initially YopJ was
proposed to act as an ubiquitin-like protein protease, cleaving
the ubiquitin-like protein SUMO from unidentified conjugated
targets (6). Subsequently, it was argued that YopJ acted as
a deubiquitinase, removing critical polyubiquitin chains from the
essential NF-κB/innate immune signaling pathway protein
TRAF6 (7, 8). Recent biochemical evidence from two groups
strongly argues that YopJ has a completely novel and unpre-
dicted function, that of a serine/threonine acetyltransferase. In
this role, YopJ acetylates serines and threonines on various mi-
togen-activated protein kinase kinases (MAP2Ks). This acetyla-
tion blocks the phosphorylation and activation of these kinases,
thus neutralizing these signaling pathways (9, 10). Interestingly,
the Yersinia enterocolitica homolog, YopP, has been shown to
inactivate the mammalian MAP3 kinase, hTAK1; however, the
mechanism of suppression is not yet established (11, 12).
Unlike mammals, Drosophila lack a fully developed adaptive
immune system and instead rely largely on two innate immune
signaling pathways, the Toll and IMD pathways, to control an-
timicrobial peptide gene expression and other defense responses.
Infection by many Gram-positive bacteria (with Lysine-type
peptidoglycan) or by fungi leads to the proteolytic cleavage of
opJ is one of six effector proteins (Yops) injected into the
host cell cytoplasm during a Yersinia pestis infection. YopJ
pro-Spätzle. Once cleaved, active Spätzle then binds the receptor
Toll, which initiates a signaling cascade through the adaptor
proteins MyD88 and Tube to the kinase Pelle (13, 14). As
a consequence, the Drosophila IκB homolog Cactus is phos-
phorylated, ubiquitinated, and degraded by the proteosome,
which allows the NF-κB proteins DIF and Dorsal to translocate
into the nucleus and activate antimicrobial peptide gene syn-
thesis. Conversely, the IMD immune signaling pathway is acti-
vated by DAP-type peptidoglycan (PGN), common to Gram-
negative bacteria and certain Gram positives (15). DAP-type
PGN is recognized by two receptors, PGRP-LC and PGRP-LE,
which signal the caspase-8–like DREDD to cleave imd protein.
Cleaved-IMD is then K63-polyubiquitinated through its associ-
ation with the E3 ligase DIAP2 (16). These K63-polyubiquitin
chains are proposed to then recruit and activate the downstream
MAP3 kinase dTAK1, as reported for mammalian NF-κB sig-
naling pathways (17, 18). Once activated, dTAK1 initiates two
downstream arms of the IMD pathway. In the Relish/NF-κB
arm, dTAK1 phosphorylates and activates the IKK complex
leading to the subsequent phosphorylation and activation of the
NF-κB protein Relish, which drives the expression of a battery of
antimicrobial peptide genes (19, 20). In the second arm of the
IMD pathway, dTAK1 also activates the JNK kinase Hemip-
terous, which, in turn, phosphorylates the JNK homolog Basket,
leading to the activation of AP1 transcription factors and in-
duction of various immune genes (19, 21).
Both the Drosophila IMD and Toll pathways show homology
to mammalian innate immune signaling pathways, and either
could be targets of YopJ-mediated inhibition. However, we find
that only the IMD pathway is sensitive to YopJ and that the
presence of YopJ results in the serine/threonine acetylation of
dTAK1 and subsequent inhibition of this kinase. This inhibition
is sufficient to block downstream signaling to both the JNK and
Relish/NF-κB branches of the IMD pathway. Furthermore, we
demonstrate similar YopJ-mediated inhibition of hTAK1 in
Author contributions: N.P., J.C., C.S., L.W., A.P., C.V.R., and N.S. designed research; N.P.,
J.C., C.S., F.R., L.W., A.P., C.V.R., S.A.S., and S.M. performed research; N.P., N.G., A.N.R.W.,
W.S.L., S.A.S., and S.M. contributed new reagents/analytic tools; N.P., J.C., C.S., L.W.,
W.S.L., S.A.S., K.A.F., L.S., and N.S. analyzed data; and N.P. and N.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1N.P. and J.C. contributed equally to this work.
2Present address: The Takeda Oncology Company, Cambridge, MA 02139.
3Present address: Chemistry Department, United States Naval Academy, Annapolis,
4To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| July 31, 2012
| vol. 109
| no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1008203109
mammalian cells. These data demonstrate that the MAP3 kinase
TAK1 is a target of YopJ-mediated inhibition and suggests that
the broad innate immune inhibitory activity of YopJ results from
its ability to inactivate this MAP3K.
YopJ Blocks the IMD Innate Immune Signaling Pathway. To develop
Drosophila as a model system for the study of YopJ-mediated
innate immune inhibition, S2* stable cell lines inducibly ex-
pressing either wild-type YopJ (YopJWT) or a catalytically in-
active mutant (C172A, YopJCA) under the control of the
metallothionein promoter were generated. In these cells, the
addition of copper sulfate induced YopJ expression, whereas in
the absence of copper, no protein was detected (Fig. 1A, Lower).
These cells were stimulated with Spätzle-C106 (22) or DAP-type
PGN to activate the Toll or IMD pathways, respectively. Toll
signaling was monitored by Northern blotting for Drosomycin
message, whereas IMD signaling was monitored by probing for
Diptericin (and, to a lesser degree, Drosomycin) (Fig. 1A). YopJ
had no effect on the Spätzle-induced expression of Drosomycin,
whereas PGN-induced Diptericin and Drosomycin expression
was dramatically inhibited. The failure to induce Diptericin was
clearly linked to the presence of YopJWT, because YopJCAwas
not inhibitory and without copper pretreatment Diptericin in-
duction was robust. Together, these results demonstrate that
YopJ is a potent inhibitor of IMD, but not Toll, signaling in
For studies in whole animals, we generated YopJ transgenic
flies. Using the dual GMR/UAS promoter system (pGUS; ref.
23) (Fig. S1A), we generated flies constitutively expressing wild-
type or mutant YopJ in the eye, and at any other location/time
with appropriate Gal4 “drivers.” To monitor the effect of YopJ
on the humoral systemic immune response, the fat body-specific
yolk-Gal4 driver was used. After infection with live Escherichia
coli, control animals (female yolk-gal4 driver flies or YopJWTflies
containing no Gal4 driver) showed robust IMD pathway activa-
tion, as assayed by Northern blotting for Diptericin. However, the
yolk-gal4;UAS-YopJWTfemales exhibited markedly reduced Dip-
tericin expression (Fig. S1B), consistent with our cell culture
data. Together these observations show that YopJ blocks IMD
signaling in both tissue culture and whole animals.
Interestingly, moderate expression of YopJWTin the de-
veloping eye imaginal disk (from the GMR element in the pGUS
transgene) led to a rough and reduced eye phenotype, which was
not observed in YopJCAflies (Fig. S1C). Multiple transgenic
insertion lines were generated for both wild-type and mutant
YopJ. Although the penetrance of the rough eye phenotype
varied among the YopJWTlines (as shown in Fig. 1C, columns 1
and 2), no rough/reduced eye phenotype was present in any lines
expressing YopJCA. The rough and reduced eyes found in
YopJWTflies were further enhanced by driving higher levels of
YopJWTvia an additional GMR-GAL4 driver (Fig. S1C, Lower).
Unlike the Toll pathway, the IMD pathway is not involved in
development and most IMD pathway mutants are viable to
adulthood, with normal eyes. Therefore, it seems likely that the
developmental defects seen in YopJWTflies are a consequence
of inhibition of other MAP kinase pathways important for
eye development. Further work will be required to identify
Having established that YopJWTinhibits the IMD signaling
pathway, we next sought to determine which component(s) of
this signaling pathway are targeted. We have shown that the
cleavage and subsequent ubiquitination of the imd protein are
crucial events during signaling (16). Because YopJ was proposed
to act as a ubiquitin-protein protease (7, 8), we examined
whether either of these IMD modifications were altered in stable
cell lines expressing YopJ. After stimulation with PGN, com-
parable levels of IMD cleavage and ubiquitination were detected
in both the YopJWTand YopJCAexpressing cells, in the presence
or absence of copper, indicating that YopJ does not function as
an IMD-specific ubiquitin-protein protease (Fig. 1B).
However, downstream signaling events in both the JNK and
NF-κB arms of the IMD pathway were clearly inhibited by YopJ.
For example, the Drosophila JNK pathway, as assayed by
IB: -JNK (FL)
IP: -FLAG (YopJ)
IB: -FLAG (YopJ)
CA WT CA WT CA WT CA WT CA WT CA
functions between IMD and JNK. (A) S2* cells stably expressing YopJWTor
YopJCAunder control of the metallothionein promoter were pretreated with
copper (to activate expression of YopJ) or not, before stimulation with
Spätzle (Left) or DAP-type PGN (Right). Activation of immune signaling was
monitored by Northern blotting for Diptericin and Drosomycin RNA. (B)
Expression of YopJWTor YopJCAwas induced with the addition of copper
sulfate in S2* cells before stimulation with PGN. Ubiquitination of IMD was
monitored by IMD immunoprecipitation followed by immunoblotting for
ubiquitin (Upper). Anti-IMD blotting was used as a loading control and also
to examine PGN-induced IMD cleavage. Anti-FLAG probing was used to
verify the presence/absence of FLAG-YopJ.
highlights phosphorylated IMD, and
products. (C) Phospho-JNK was monitored in whole-cell lysates. Full-length
JNK blot serves as a loading control. (D) IKK activity was monitored in S2*
cells expressing YopJWTor YopJCAafter stimulation with PGN. IKKγ blot serves
as an immunoprecipitation control.
YopJ inhibits Drosophila IMD but not Toll immune signaling and
marks unmodified full-length
marks the cleaved-IMD
Paquette et al. PNAS
| July 31, 2012
| vol. 109
| no. 31
immunoblotting for PGN-induced phospho-JNK, was blocked by
YopJWT(Fig. 1C). In the NF-κB/Relish branch, activation of the
Drosophila IKK complex was also inhibited by YopJWT, but not
by YopJCA(Fig. 1D). Together with the inhibition of AMP gene
induction, these data indicate that YopJ acts by inhibiting the
IMD pathway downstream of IMD cleavage and ubiquitination
but upstream or at the level of JNK phosphorylation and IKK
activation. Although Orth and colleagues have argued that both
NF-κB and JNK signaling are inhibited by YopJ-mediated
acetylation of multiple MAP2 kinases (10), an alternate possi-
bility is that YopJ also inhibits a common upstream protein that
is required for both NF-κB and JNK activation, such as dTAK1.
YopJ Inhibits dTAK1 Activation. To examine whether YopJ may be
directly inhibiting the kinase dTAK1, we next coexpressed both
dTAK1 and YopJ (WT or CA) in double-stable S2* cells. In
these cells, dTAK1 and IKK kinase activities were monitored
with IP-kinase assays by using recombinant MKK6K82A(a
mammalian TAK1 target) or recombinant Relish as substrates,
respectively. In S2* cell lines, overexpression of dTAK1 was
sufficient to activate both dTAK1 and the downstream IKK
complex (Fig. 2A, rows 1 and 4). When both dTAK1 and YopJWT
were coexpressed, the activity of both kinases was inhibited, whereas
coexpression of YopJCAresulted in no discernible reduction in
signal. Interestingly, dTAK1, expressed alone or in combination
with YopJCA, migrated as a tight doublet at a molecular mass
just below 105 kDa, whereas in concert with YopJWT, dTAK1
migrated noticeably faster, closer to 85 kDa (Fig. 2A, row 2).
When optimized for better resolution, immunoblot analysis
showed that overexpressed dTAK1 alone, or in the presence
of YopJCA, migrates as a doublet at ∼95 and 105 kDa. In the
presence of YopJWT, dTAK1 runs faster at a molecular mass of
∼85 kDa (Fig. 2A, row 3). The predicted molecular mass for
Drosophila TAK1 is 76 kDa. Together, these data strongly in-
dicate that YopJ directly interferes with the activity of dTAK1
and that this interference is likely the result of posttranslational
To determine whether the observed Drosophila TAK1 bands
represent phosphorylated forms, we treated lysates with λ-pro-
tein phosphatase. Phosphatase treatment resolved the doublets
observed in the dTAK1 alone and dTAK1 with YopJCAsamples
from ∼95/105 kDa to ∼85/93 kDa. In the presence of YopJWT,
no change in the migration of dTAK1 was detected with phos-
phatase treatment (Fig. 2B). These data show that active
dTAK1, (when expressed alone or with the inactive YopJCA) is
phosphorylated; however in the presence of YopJWT, dTAK1 is
inactive and unphosphorylated.
To analyze the effect of YopJ on endogenous dTAK1, IP-ki-
nase assays were undertaken with an anti-dTAK1 antibody (16).
Cells expressing YopJWTshowed little PGN-induced TAK1 ki-
nase activity, whereas cells similarly expressing YopJCAshowed
activity comparable to parental S2* cells (Fig. 2C). Although our
anti-dTAK1 antibody is effective for immunopreciptiation, as
monitored by endogenous dTAK1 IP-kinase assays, it is not very
useful for immunoblotting (16). Therefore, we used an RNAi
approach to validate this assay. RNAi treatment targeting
dTAK1 was able to inhibit endogenous dTAK1 kinase activity in
this IP-kinase assay and accumulation of phospho-JNK after
immune induction (Fig. S2). Together, the results with both
endogenous or overexpressed dTAK1 strongly suggest that YopJ
interferes with this MAP3 kinase.
YopJ Acetylates dTAK1. An alignment of Drosophila and mam-
malian TAK1 shows that three phospho-acceptor sites found in
the activation loop of mammalian TAK1 (T184, T187, and S192)
and most of the surrounding residues are highly conserved
(Fig. 3A). In mammals, alanine substitution of any of these serine
or threonine residues is sufficient to ablate kinase activity and
block downstream signaling (24–28). To identify the phosphor-
ylated residues of Drosophila TAK1 and map the molecular
changes induced by YopJ, FLAG-TAK1 was isolated from
lysates prepared from cells expressing dTAK1 alone or in com-
bination with YopJWTor YopJCA. These samples were then
subjected to microcapillary reverse-phase HPLC nano-electrospray
tandem mass spectrometry (MS/MS).
Within the activation loop of Drosophila TAK1, a single phos-
phorylation, at S176 (which aligns to S192 of the mammalian pro-
tein), was identified by MS/MS (Fig. 3B). A number of other
phosphorylation sites were found outside the activation loop (see
Table S1 for the full list of phosphoresidues). Interestingly, no
phosphorylation was detected in the activation loop of dTAK1
when coexpressed with YopJWT, and overall phosphorylation was
greatly reduced. When coexpressed with YopJCA, dTAK1 was
phosphorylated on a number of residues, including all of the sites
identified when TAK1 was expressed alone and some additional
expressed in S2* cells alone or in the presence of YopJWTor YopJCA, and
activity was monitored by IP-kinase assays with catalytically inactive MKK6
serving as a substrate (row 1). As a control, immunoprecipitated FLAG-TAK1
were immunoblotted with anti-FLAG to verify kinase capture and banding
pattern (row 2). To more clearly observe YopJ-mediated alteration in the
dTAK1 banding pattern, FLAG-TAK1 was immunobloted directly from
lysates under optimized conditions (row 3). IKK activity was similarly moni-
tored by immunoprecipitation using an endogenous IKKγ antisera and the
substrate Relish (row 4). Immuoprecipitated IKKγ samples were immuno-
blotted with IKKγ antisera to verify capture (row 5). The presense of FLAG-
YopJ was also monitored by FLAG IP/immunoblot (row 6). (B) Anti-FLAG
(TAK1) immunoblot of lysates from S2* cells expressing FLAG-TAK1 alone or
in the presence of YopJWTor YopJCAwere treated (or not) with λ-phos-
phatase. (C) Activation of endogenous dTAK1 was monitored by IP-kinase
assays, with rMKK6 as substrate, from S2* cells expressing either YopJWTor
YopJCA. Anti-FLAG immunoblot serves as a control for the presence of YopJ.
YopJ inhibits Drosophila TAK1 kinase activity. (A) dTAK1 was over-
| www.pnas.org/cgi/doi/10.1073/pnas.1008203109Paquette et al.
sitesoutsidetheactivation loop (Table S1).The relevance ofthese
to note that when expressed with YopJCA, dTAK1 remains fully
functional. Given that YopJ is thought to act as a serine/threonine
acetyl-transferase (9, 10), mass spectrometry was also used to de-
tect acetylation. In the presence of YopJWT, dTAK1 showed clear
and S176, all within the activation loop and at residues highly
conserved with mammalian TAK1 (Fig. 3B). However, dTAK1
alone or in the presence YopJCAdisplayed no detectible acetyla-
tion (see Table S2 for a full list of acetyl residues). Together, these
data suggest that acetylation of dTAK1 threonines T168 and T171
leaves it in an unphosphorylated state.
Previous studies with mammalian TAK1 indicate that the
residues equivalent to Drosophila TAK1 T168, T171, and S176
(in mammalian TAK1: T184, T187, and S192) play important
roles in the kinase activity of hTAK1 (24–28). To elucidate the
role that these residues play in Drosophila TAK1 activity, we
generated various substitution mutations at these sites. As shown
previously, overexpression of wild-type dTAK1 is sufficient to
activate dTAK1 kinase activity, as monitored by cold-IP-kinase
assay (e.g., immunoblotting with anti-pMKK6; Fig. 3C). In-
terestingly, substitution of T168 to alanine did not affect kinase
activity, and this mutant is still subject to YopJ-mediated in-
hibition, likely because T171 is still a target for acetylation and,
as such, is sufficient for inhibition of the kinase. Conversely,
substituting either T171 or S176 to alanine renders dTAK1 in-
active (Fig. 3C), indicating that these residues are necessary for
proper dTAK1 function. These data are consistent with studies
of mammalian TAK1 that indicate the corresponding residues,
T187 and S192, are essential for activation of the kinase, whereas
T184 plays a minor role (24–28). In an attempt to bypass YopJ-
mediated acetylation of these residues, we also generated a sec-
ond set of Drosophila TAK1 mutants in which these serine and
threonines were changed to a phosphomemetic acidic residue.
Unfortunately, substitution of T168, T171, or S176 to glutamic
acid rendered TAK1 inactive (Fig. 3D). All together, this analysis
of dTAK1 substitution mutants indicates that residues T171 and
S176 are essential for kinase activity, and that substitution or
acetylation of these residues impairs the function of dTAK1.
YopJ Inhibits and Acetylates Mammalian TAK1. With the discovery
that YopJ inactivates Drosophila TAK1, we sought to determine
whether YopJ had a similar effect on mammalian TAK1. To that
end, transient expression of wild-type mammalian TAK1 in hu-
man 293T cells resulted in kinase activation and autophosphor-
ylation, as monitored by cold-kinase assay using MKK6 substrate
or immunoblotting for phospho-hTAK1, respectively (Fig. 4A).
Interestingly, when wild-type YopJ was coexpressed with
hTAK1, the kinase failed to activate, similar to the effects ob-
served with YopJ and Drosophila TAK1. As controls, an inactive
WT CA- WT CA--- WT CA
Drosophila TAK1 activation loops. The established phosphorylation sites on
mammalian TAK1 are indicated (P). (B) Summary of tandem MS identifica-
tion of Drosophila TAK1 activation loop. Phosphorylated (P) and acetylated
(Ac) residues are indicated. All phospho-peptide and acetyl-peptide residues
can be found in Tables S1–S3. In both A and B, conserved residues are shaded
in gray. (C) Activity of dTAK1 alanine substitutions in the presence or ab-
sence of YopJ assayed by IP/cold kinase assay (row 1). Immunoblots for FLAG-
TAK1 and FLAG-YopJ verify the presence of the respective proteins (rows 2
and 3). (D) Activity of dTAK1 glutamic acid substitutions by IP/cold kinase
assay [IP/KA-followed by immunoblot for phospho-MKK6 (S207)] (row 1).
FLAG immunoblot verifies the expression of dTAK1 protein (row 2).
YopJ acetylates Drosophila TAK1. (A) Alignment of human and
IB: α-GFP (YopJ)IB: α-GFP (YopJ)
IB: α-pTAK(T187) IB: α-pTBK1(S172)
YFP-YopJ YFP-YopJCA WT--
WT WTWT WTK63W
K63W hTAK1 was transiently expressed in human HEK 293T cells in the
presence or absence of YFP-tagged YopJ. hTAK1 activity was monitored by
cold IP-kinase assay (row 1). Phospho- and total hTAK1 were also monitored
by IP/immunoblot (rows 2 and 3). (B) Alignment of hTAK1 and dTAK1 acti-
vation loops. Conserved residues are boxed (gray). Acetylated residues, as
detected by MS/MS, are marked (Ac). Small Ac indicates residues that
showed ambiguous acetylation. (C) hTAK1 (Left) or TBK1 (Right) were
expressed in human HEK 293T cells in the presence or absence of wild-type
(WT) or inactive (CA) YopJ. Kinase activities of hTAK1 and TBK1 were
assayed by IP kinase assay and with phosphospecific hTAK1 or TBK1 immu-
noblotting. Total substrate amounts were detected by Coomassie blue
staining; note the shift in IRF3 protein upon phosphorylation. As controls,
total immunoprecipitated protein was detected by FLAG immunoblot,
whereas YopJ levels were confirmed by GFP immunoblot.
YopJ inhibits and acetylates mammalian kinases. (A) Wild-type or
Paquette et al.PNAS
| July 31, 2012
| vol. 109
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hTAK1 mutant (TAKK63W) also failed to autophosphorylate or
activate, whereas the coexpression of YopJCAhad no effect.
To confirm that YopJ acetylates mammalian TAK1, hTAK1
was expressed in the presence or absence of YopJ and then
analyzed by tandem mass spectrometry. Similar to dTAK1,
hTAK1 showed no acetylation when expressed alone; however,
in the presence of YopJ, T184 and T187 within the activation
loop of hTAK1 were acetylated (Fig. 4B). Because phosphory-
lation of T187 is critical for activation of hTAK1 (24–28), acet-
ylation of this residue likely blocks phosphorylation and
activation of the kinase. A number of other residues in the ac-
tivation loop and beyond were also acetylated in the presence of
YopJ, although definitive localization of some of the sites could
not be unambiguously identified (Table S3).
Furthermore, we tested the YopJ-dependent acetylation of
a number of mammalian MAP2 kinases downstream of TAK1
in mammalian systems. Previously, YopJ has been shown to
acetylate MKK6 on ser207 and thr211, the activating phos-
phoacceptor sites in the activation loop (10). Consistent with
published findings, YopJ blocked MKK6 phosphorylation on
ser207 (Fig. S3A, Top). In addition to acetylating ser207 and
thr211, YopJ also acetylates a neighboring lysine (lys210) (10).
Although this lysine modification is dispensable for the inhibitory
effect of YopJ, it provides a unique opportunity to detect YopJ-
mediated acetylation by immunoblotting with an acetyl-lysine
antibody (Fig. S3A, Middle). MKK4 and MKK7, the MAP2Ks
that specifically activate JNK, also contain at least one lysine
in their activation motif. Exploiting these nearby lysines revealed
that YopJ acetylates both MKK4 and MKK7 (Fig. S3B). Con-
sistent with this acetylation, YopJWT, blocked JNK phosphory-
lation by MKK7 overexpression (Fig. S3C). Although YopJ has
been shown to interact with MKK4 or MKK7 (29, 30), these data
provide direct evidence for YopJ-mediated acetylation and in-
hibition of these JNK-specific MAP2Ks. Our results argue that
that YopJ is capable of modifying both a MAP3 kinase (i.e.,
TAK1) and MAP2 kinases (i.e., MKK4 and MKK7) in a single
pathway (i.e., JNK) (Fig. 5). We therefore propose that this two-
pronged attack underlies the potent inhibitory effects YopJ
exerts on multiple signaling pathways.
To date, previous work has failed to identify a kinase not af-
fected by the acetyltransferase activity of YopJ. Our prior study
on YopJ demonstrated that RIG-I–mediated IRF3 activation is
impervious to YopJ-mediated inhibition (7). In this pathway,
TBK1 is an essential IRF3 kinase (31–33). Thus, the ability of
YopJ to interfere with hTAK1 and TBK1 kinase activities was
compared directly. Consistent with the inability of YopJ to dis-
rupt RIG-I signaling, it failed to inhibit TBK1 kinase activity
while clearly blocking hTAK1 (Fig. 4C, row 1). Furthermore,
phosphorylation of the TBK1 activation loop was not blocked by
YopJ, whereas hTAK1 phosphorylation was inhibited (Fig. 4C,
row 4). These results suggest that, despite the broad inhibitory
profile of YopJ, this effector targets a subset of host kinases.
Previous studies in mammalian systems have identified a number
of targets of YopJ inhibitory activity. In one study, the YopJ
homolog, YopP, was shown to inhibit mammalian TAK1; how-
ever, no mechanism was identified (11, 12). A number of in vitro
studies identified the MAP2 kinase family as targets of YopJ-
mediated serine/threonine acetylation (9, 10). Through these
acetylations, it was argued that YopJ is capable of inhibiting both
MAPK (ERK pathway in particular) and NF-κB signaling. The
data presented here demonstrate that the MAP3 kinase TAK1 is
also potently targeted by YopJ-mediated acetylation. This acet-
ylation inhibits the critical autophosphorylation of TAK1, si-
multaneously blocking innate immune-induced NF-κB and JNK
signaling of both Drosophila and mammals (Fig. 5).
We show that YopJ is able to inhibit Drosophila IMD but not
Toll innate immune signaling by blocking the activity of dTAK1,
after PGN stimulation or overexpression of dTAK1. However,
upstream signaling events, such as IMD cleavage and ubiquiti-
nation, remain intact in the presence of YopJ. Instead, YopJ
acetylates multiple serine and threonine residues in the dTAK1
activation loop, inhibiting autophosphorylation of this kinase.
Furthermore, YopJ also inhibits the activation of mammalian
TAK1. Because TAK1 belongs to the more divergent MAP3
kinases, these findings raise the possibility that YopJ may target
other MAP3 kinases as well. However, YopJ did not inhibit the
kinase TBK1 and, therefore, is not an inhibitor of all kinases. We
also demonstrate that YopJ is also capable of acetylating and
inhibiting the MAP2 kinases MKK4, MKK6, and MKK7. When
put in context with previous work showing YopJ acetylation of
mammalian IKK and MKKs (9, 10), our data provide evidence
that YopJ is able to redundantly inhibit the JNK, p38, and NF-
κB innate immune signaling pathways at critical MAP2 and
MAP3 kinases (Fig. 5).
The precise mechanism of YopJ-mediated immune inhibi-
tion remains controversial. Previous work, from our group and
others, suggests that YopJ acts as an ubiquitin protein pro-
tease, cleaving ubiquitin from conjugated substrates (7, 8),
whereas experiments herein demonstrate that YopJ functions
as a serine/threonine acetyl-transferase, acetylating critical
residues on TAK1. Our earlier studies primarily concluded
that YopJ-inhibited innate immune signaling upstream of IKK
activation, consistent with our results here. We, and others,
further argued that YopJ may have ubiquitin protease activity
based solely on cotransfection studies. Here, we show in the
context of ligand-induced innate immune signaling that Dro-
sophila TAK1 is inhibited by YopJ, and that TAK1 is a direct
target of YopJ-mediated acetylation, strongly arguing that this
acetyltransferase activity is the critical activity responsible for
the immunosuppressive function of YopJ. However, Zhou et al.
present biochemical data that recombinant YopJ has ubiquitin
protease activity (8). Therefore, it remains possible that YopJ
has two enzymatic activities. Although we demonstrate that
IMD ubiquitination is not perturbed in the presence of YopJ,
IMD may not be a suitable substrate for YopJ-mediated
Our approach, with the Drosophila model system, has many
advantages over that previously described. First, this work was
undertaken exclusively in live cells or animals stably expressing
YopJ, an approach that has proven difficult within the context of
(IKK /IKK )
inhibition, from our work and others, in both the Drosophila IMD (Left) and
mammalian TNF/TLR (Right) signaling pathways. Proteins known to be
acetylated by YopJ are marked with ‘Ac’ (red).
YopJ inhibits MAP2 and MAP3 kinases. A model of YopJ-mediated
| www.pnas.org/cgi/doi/10.1073/pnas.1008203109Paquette et al.
mammalian cells because of the proapoptotic nature of YopJ
(2). Using the inducibility of the Drosophila metallothionein
promoter system, we developed stable cell lines, which provide
reliable protein expression and overall consistency. Second, the
proteins analyzed by MS/MS were produced and isolated from
animal cells. The identification of TAK1 as the target for YopJ-
mediated inhibition is also more in line with the observations
regarding the ability of YopJ to inhibit both proinflammatory
NF-κB signaling and MAPK pathways (1–4, 9, 10). In fact, while
this work was under revision, Meinzer et al. reported that Yersina
pseduotuberculosis YopJ acetylates TAK1, similar to our findings
(34). Lastly, YopJ-mediated inhibition of hTAK1 in a mamma-
lian context validates our model system approach. Targeted in-
activation of TAK1 allows for a much broader and more efficient
method of innate immune pathway inhibition. Perhaps in concert
with inhibition of MAP2 kinases, YopJ is able to effectively in-
hibit immune pathways in both mammals and insects.
Materials and Methods
Cell Culture. S2* cells were cultured in Schneiders media (Gibco) suppli-
mented with 10% (vol/vol) FBS (Valley Biomedical), 1% (vol/vol) glutamine
(Gibco), and 0.2% Pen/Strep (Gibco). Cells were treated with 1 μM 20-
hydroxyecdysone (Sigma) for 24–30 h before stimulation with 100 ng/mL
peptidoglycan (Invivogen) or 5 mM Spätzle (22).
RNA Analysis. Total RNA was isolated with the TRIzol reagent (Invitrogen) as
described (35) and expression of Diptericin and Rp49 was analyzed by
Northern blot analysis followed by autoradiography.
Protein and Immunoprecipitation Assays. Proteins were precipitated and an-
alyzed as described (7, 16). See SI Materials and Methods for more details.
Kinase Assays. Kinases were assayed as described (16, 35, 36), with mod-
GST-IRF3 aa380-427 was used as a substrate for TBK1 as described (32).
Peptide Identification by Tandem Mass Spectrometry. See SI Materials and
Methods for more details.
ACKNOWLEDGMENTS. N.P. was supported by New England Regional Center
of Excellence Grant U54AI057159. N.S. and L.S. were supported by National
Institutes of Health Grants AI060025 and AI053809, and 1R01AI079198, re-
spectively. N.S. was also supported by the Ellison Medical Foundation.
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| vol. 109
| no. 31