Serine/threonine acetylation of TGF -activated kinase (TAK1) by Yersinia pestis YopJ inhibits innate immune signaling

Article (PDF Available)inProceedings of the National Academy of Sciences 109(31):12710-5 · July 2012with47 Reads
DOI: 10.1073/pnas.1008203109 · Source: PubMed
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 transduction pathways. Although the molecular mechanism remained elusive 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 acetylation 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 YopJ-mediated inhibition.

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Available from: Lindsay H Wilson, Jan 08, 2014
Serine/threonine acetylation of TGFβ-activated kinase
(TAK1) by Yersinia pestis YopJ inhibits innate
immune signaling
Nicholas Paquette
, Joseph Conlon
, Charles Sweet
, Florentina Rus
, Lindsay Wilson
, Andrea Pereira
Charles V. Rosadini
, Nadege Goutagny
, Alexander N. R. Weber
, William S. Lane
, Scott A. Shaffer
Stephanie Maniatis
, Katherine A. Fitzgerald
, Lynda Stuart
, and Neal Silverman
Division of Infectious Disease, Department of Medicine, and
Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School,
Worcester, MA 01605;
Program of Developmental Immunology, Department of Pediatrics, Massachusetts General Hospital/Harvard Medical School, Boston,
MA 02114;
Toll-Like Receptors and Cancer, German Cancer Research Center, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany; and
Mass 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 nd 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 modication and inhibition of
hTAK1. These data present evidence that TAK1 is a target for
YopJ-mediated inhibition.
Drosophila immunity
innate immunity
YopJ is one of six effector proteins (Yops) injected into the
host cell cytoplasm during a Yersinia pestis infection. YopJ
has been shown to be both proapoptotic and to inhibit proin-
ammatory signal transduction (15). 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 unidentied 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
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-8like 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 nd
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 sufcient 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 reage nts/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 conict of interest.
This article is a PNAS Direct Submission.
N.P. and J.C. contributed equally to this work.
Present address: The Takeda Oncology Company, Cambridge, MA 02139.
Present address: Chemistry Department, United States Naval Academy , Annapolis,
MD 21402.
To whom correspondence should be addressed. E-mail:
This article contains supporting information online at
July 31, 2012
vol. 109
no. 31
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 (YopJ
) or a catalytically in-
active mutant (C172A, YopJ
) 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 YopJ
, because YopJ
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
Drosophila cells.
For studies in whole animals, we generated YopJ transgenic
ies. Using the dual GMR/UAS promoter system (pGUS; ref.
23) (Fig. S1A), we generated ies 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-specic
yolk-Gal4 driver was used. After infection with live Escherichia
coli, control animals (female yolk-gal4 driver ies or YopJ
containing no Gal4 driver) showed robust IMD pathway activa-
tion, as assayed by Northern blotting for Diptericin. However, the
females 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 YopJ
in 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 YopJ
ies (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 YopJ
lines (as shown in Fig. 1C, columns 1
and 2), no rough/reduced eye phenotype was present in any lines
expressing YopJ
. The rough and reduced eyes found in
ies were further enhanced by driving higher levels of
via 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 YopJ
ies are a consequence
of inhibition of other MAP kinase pathways important for
eye development. Further work will be required to identify
these kinases.
Having established that YopJ
inhibits 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 modications 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 YopJ
and YopJ
expressing cells, in the presence
or absence of copper, indicating that YopJ does not function as
an IMD-specic 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
S2* YopJWT YopJ CA
+- -+--+
S2* YopJWT YopJ CA
IB: -Ub
+- -+--+
IP: -FLAG (YopJ)
IB: -FLAG (YopJ)
KA: p*-Relish
Cu PGNSpatzle
--++- -+ +-- ++
-- - -++++-- ++
Fig. 1. YopJ inhibits Drosophila IMD but not Toll immune signaling and
functions between IMD and JNK. (A) S2* cells stably expressing YopJ
under 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 YopJ
or YopJ
was 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. marks unmodied full-length
IMD, highlights phosphorylated IMD, and marks the cleaved-IMD
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 YopJ
or YopJ
after stimulation with PGN. IKKγblot serves
as an immunoprecipitation control.
Paquette et al. PNAS
July 31, 2012
vol. 109
no. 31
immunoblotting for PGN-induced phospho-JNK, was blocked by
(Fig. 1C). In the NF-κB/Relish branch, activation of the
Drosophila IKK complex was also inhibited by YopJ
, but not
by YopJ
(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 MKK6
mammalian TAK1 target) or recombinant Relish as substrates,
respectively. In S2* cell lines, overexpression of dTAK1 was
sufcient to activate both dTAK1 and the downstream IKK
complex (Fig. 2A, rows 1 and 4). When both dTAK1 and YopJ
were coexpressed, the activity of both kinases was inhibited, whereas
coexpression of YopJ
resulted in no discernible reduction in
signal. Interestingly, dTAK1, expressed alone or in combination
with YopJ
, migrated as a tight doublet at a molecular mass
just below 105 kDa, whereas in concert with YopJ
, 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 YopJ
, migrates as a doublet at 95 and 105 kDa. In the
presence of YopJ
, 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 YopJ
from 95/105 kDa to 85/93 kDa. In the presence of YopJ
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 YopJ
phosphorylated; however in the presence of YopJ
, 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 YopJ
showed little PGN-induced TAK1 ki-
nase activity, whereas cells similarly expressing YopJ
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 sufcient to ablate kinase activity and
block downstream signaling (2428). 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 YopJ
or YopJ
. 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 identied 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 YopJ
, and overall phosphorylation was
greatly reduced. When coexpressed with YopJ
, dTAK1 was
phosphorylated on a number of residues, including all of the sites
identied when TAK1 was expressed alone and some additional
KA: p*-MKK6
KA: p*-Relish
IB: α-FLAG(YopJ)
YopJC172A --
YopJWT -
+- -+
KA: p*-MKK6
S2* Yop J WT Yo p JCA
+-- +--
Fig. 2. YopJ inhibits Drosophila TAK1 kinase activity. (A) dTAK1 was over-
expressed in S2* cells alone or in the presence of YopJ
or YopJ
, 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 IKKγantisera and the
substrate Relish (row 4). Immuoprecipitated IKKγsamples were immuno-
blotted with IKKγantisera 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 YopJ
or YopJ
were 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 YopJ
. Anti-FLAG immunoblot serves as a control for the presence of YopJ.
| Paquette et al.
sites outside the activation loop (Table S1). The relevance of these
additional phosphorylation sites is unclear; however, it is important
to note that when expressed with YopJ
, 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 YopJ
, dTAK1 showed clear
acetylation of both T168 and T171 and possible acetylation of S167
and S176, all within the activation loop and at residues highly
conserved with mammalian TAK1 (Fig. 3B). However, dTAK1
alone or in the presence YopJ
displayed 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
(and possibly S167 and S176) by YopJ inactivates the kinase, which
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 (2428). 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 sufcient 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 sufcient 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 (2428). 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
KA/IB: α-p-MKK6
WT CA - WT CA - - -WT CA
WT T168A
KA/IB: α-p-MKK6
Fig. 3. YopJ acetylates Drosophila TAK1. (A) Alignment of human and
Drosophila TAK1 activation loops. The established phosphorylation sites on
mammalian TAK1 are indicated (P). (B) Summary of tandem MS identica-
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 S1S3. In both Aand 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 veries the expression of dTAK1 protein (row 2).
KA/IB: α-pMKK6
KA: p*-MKK6K82A
KA: p*-IRF3380-427
CB: p*-MKK6K82A CB: p*-IRF3380-427
IB: α-GFP (YopJ) IB: α-GFP (YopJ)
IB: α-pTAK(T187) IB: α-pTBK1(S172)
MKK6 -++++
YopJ CA WT ---
IB: α-pTAK1
Fig. 4. YopJ inhibits and acetylates mammalian kinases. (A) Wild-type or
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 phosphospecic 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 conrmed by GFP immunoblot.
Paquette et al. PNAS
July 31, 2012
vol. 109
no. 31
hTAK1 mutant (TAK
) also failed to autophosphorylate or
activate, whereas the coexpression of YopJ
had no effect.
To conrm 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 (2428), 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 denitive localization of some of the sites could
not be unambiguously identied (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 ndings, 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 modication 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 specically 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, YopJ
, 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-specic 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-Imediated IRF3 activation is
impervious to YopJ-mediated inhibition (7). In this pathway,
TBK1 is an essential IRF3 kinase (3133). 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
prole of YopJ, this effector targets a subset of host kinases.
Previous studies in mammalian systems have identied 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 identied (11, 12). A number of in vitro
studies identied 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 ndings 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 difcult within the context of
genes AMPs
Fig. 5. YopJ inhibits MAP2 and MAP3 kinases. A model of YopJ-mediated
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).
| Paquette 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 identication 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 proinammatory
NF-κB signaling and MAPK pathways (14, 9, 10). In fact, while
this work was under revision, Meinzer et al. reported that Yersina
pseduotuberculosis YopJ acetylates TAK1, similar to our ndings
(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 efcient
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 μM20-
hydroxyecdysone (Sigma) for 2430 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-
ications for cold kinase assays (see SI Materials and Methods for more details).
GST-IRF3 aa380-427 was used as a substrate for TBK1 as described (32).
Peptide Identication 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.
1. Monack DM, Mecsas J, Ghori N, Falkow S (1997) Yersinia signals macrophages to
undergo apoptosis and YopJ is necessary for this cell death. Proc Natl Acad Sci USA 94:
2. Monack DM, Mecsas J, Bouley D, Falkow S (1998) Yersinia-induced apoptosis in vivo
aids in the establishment of a systemic infection of mice. J Exp Med 188:21272137.
3. Palmer LE, Hobbie S, Galán JE, Bliska JB (1998) YopJ of Yersinia pseudotuberculosis is
required for the inhibition of macrophage TNF-alpha production and downregulation
of the MAP kinases p38 and JNK. Mol Microbiol 27:953965.
4. Palmer LE, Pancetti AR, Greenberg S, Bliska JB (1999) YopJ of Yersinia spp. is sufcient
to cause downregulation of multiple mitogen-activated protein kinases in eukaryotic
cells. Infect Immun 67:708716.
5. Schesser K, et al. (1998) The yopJ locus is required for Yersinia-mediated inhibition of
NF-kappaB activation and cytokine expression: YopJ contains a eukaryotic SH2-like
domain that is essential for its repressive activity. Mol Microbiol 28:10671079.
6. Orth K, et al. (2000) Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like
protein protease. Science 290:15941597.
7. Sweet CR, Conlon J, Golenbock DT, Goguen J, Silverman N (2007) YopJ targets TRAF
proteins to inhibit TLR-mediated NF-kappaB, MAPK and IRF3 signal transduction. Cell
Microbiol 9:27002715.
8. Zhou H, et al. (2005) Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit
NF-kappa B activation. J Exp Med 202:13271332.
9. Mittal R, Peak-Chew SY, McMahon HT (2006) Acetylation of MEK2 and I kappa B
kinase (IKK) activation loop residues by YopJ inhibits signaling. Proc Natl Acad Sci USA
10. Mukherjee S, et al. (2006) Yersinia YopJ acetylates and inhibits kinase activation by
blocking phosphorylation. Science 312:12111214.
11. Thiefes A, et al. (2006) The Yersinia enterocolitica effector YopP inhibits host cell
signalling by inactivating the protein kinase TAK1 in the IL-1 signalling pathway.
EMBO Rep 7:838844.
12. Haase R, Richter K, Pfafnger G, Courtois G, Ruckdeschel K (2005) Yersinia outer
protein P suppresses TGF-beta-activated kinase-1 activity to impair innate immune
signaling in Yersinia enterocolitica-infected cells. J Immunol 175:82098217.
13. Sun H, Bristow BN, Qu G, Wasserman SA (2002) A heterotrimeric death domain
complex in Toll signaling. Proc Natl Acad Sci USA 99:1287112876.
14. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA (1996) The dorsoventral
regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response
in Drosophila adults. Cell 86:973983.
15. Kaneko T, et al. (2004) Monomeric and polymeric gram-negative peptidoglycan but
not puried LPS stimulate the Drosophila IMD pathway. Immunity 20:637649.
16. Paquette N, et al. (2010) Caspase-mediated cleavage, IAP binding, and ubiquitination:
Linking three mechanisms crucial for Drosophila NF-kappaB signaling. Mol Cell 37:
17. Xia ZP, et al. (2009) Direct activation of protein kinases by unanchored polyubiquitin
chains. Nature 461:114119.
18. Xu M, Skaug B, Zeng W, Chen ZJ (2009) A ubiquitin replacement strategy in human
cells reveals distinct mechanisms of IKK activation by TNFalpha and IL-1beta. Mol Cell
19. Silverman N, et al. (2003) Immune activation of NF-kappaB and JNK requires Dro-
sophila TAK1. J Biol Chem 278:4892848934.
20. Ertürk-Hasdemir D, et al. (2009) Two roles for the Drosophila IKK complex in the
activation of Relish and the induction of antimicrobial peptide genes. Proc Natl Acad
Sci USA 106:97799784.
21. Boutros M, Agaisse H, Perrimon N (2002) Sequential activation of signaling pathways
during innate immune responses in Drosophila. Dev Cell 3:711722.
22. Weber AN, et al. (2003) Binding of the Drosophila cytokine Spätzle to Toll is direct
and establishes signaling. Nat Immunol 4:794800.
23. Brodsky MH, et al. (2000) Drosophila p53 binds a damage response element at the
reaper locus. Cell 101:103113.
24. Kishimoto K, Matsumoto K, Ninomiya-Tsuji J (2000) TAK1 mitogen-activated protein
kinase kinase kinase is activated by autophosphorylation within its activation loop. J
Biol Chem 275:73597364.
25. Yu Y, et al. (2008) Phosphorylation of Thr-178 and Thr-184 in the TAK1 T-loop is re-
quired for interleukin (IL)-1-mediated optimal NFkappaB and AP-1 activation as well
as IL-6 gene expression. J Biol Chem 283:2449724505.
26. Prickett TD, et al. (2008) TAB4 stimulates TAK1-TAB1 phosphorylation and binds
polyubiquitin to direct signaling to NF-kappaB. J Biol Chem 283:1924519254.
27. Qiao B, Padilla SR, Benya PD (2005) Transforming growth factor (TGF)-beta-activated
kinase 1 mimics and mediates TGF-beta-induced stimulation of type II collagen syn-
thesis in chondrocytes independent of Col2a1 transcription and Smad3 signaling. J
Biol Chem 280:1756217571.
28. Singhirunnusorn P, Suzuki S, Kawasaki N, Saiki I, Sakurai H (2005) Critical roles of
threonine 187 phosphorylation in cellular stress-induced rapid and transient activa-
tion of transforming growth factor-beta-activated kinase 1 (TAK1) in a signaling
complex containing TAK1-binding protein TAB1 and TAB2. J Biol Chem 280:
29. Orth K, et al. (1999) Inhibition of the mitogen-activated protein kinase kinase su-
perfamily by a Yersinia effector. Science 285:19201923.
30. Du F, Galán JE (2009) Selective inhibition of type III secretion activated signaling by
the Salmonella effector AvrA. PLoS Pathog 5:e1000595.
31. Fitzgerald KA, et al. (2003) IKKepsilon and TBK1 are essential components of the IRF3
signaling pathway. Nat Immunol 4:491496.
32. McWhirter SM, et al. (2004) IFN-regulatory factor 3-dependent gene expression is
defective in Tbk1-decient mouse embryonic broblasts. Proc Natl Acad Sci USA 101:
33. Zeng W, Xu M, Liu S, Sun L, Chen ZJ (2009) Key role of Ubc5 and lysine-63 poly-
ubiquitination in viral activation of IRF3. Mol Cell 36:315325.
34. Meinzer U, et al. (2012) Yersinia pseudotuberculosis effector YopJ subverts the Nod2/
RICK/TAK1 pathway and activates Caspase-1 to induce intestinal barrier dysfunction.
Cell Host Microbe 11:337351.
35. Silverman N, et al. (2000) A Drosophila IkappaB kinase complex required for Relish
cleavage and antibacterial immunity. Genes Dev 14:24612471.
36. Wang C, et al. (2001) TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature
Paquette et al. PNAS
July 31, 2012
vol. 109
no. 31
    • "His acetylations have not been widely reported in any system. A kinase substrate of YopJ may have a His modification, but its dependence on YopJ catalytic activity has yet to be validated (Paquette et al., 2012). Previous work using nuclear magnetic resonance (NMR) showed that a chemically induced acetylation of a Lys residue in ubiquitin occurred via an unstable acetylated His intermediate (Macdonald et al., 2000). "
    [Show abstract] [Hide abstract] ABSTRACT: Modifications of plant immune complexes by secreted pathogen effectors can trigger strong immune responses mediated by the action of nucleotide binding-leucine-rich repeat immune receptors. Although some strains of the pathogen Pseudomonas syringae harbor effectors that individually can trigger immunity, the plant's response may be suppressed by other virulence factors. This work reveals a robust strategy for immune suppression mediated by HopZ3, an effector in the YopJ family of acetyltransferases. The suppressing HopZ3 effector binds to and can acetylate multiple members of the RPM1 immune complex, as well as two P. syringae effectors that together activate the RPM1 complex. These acetylations modify serine, threonine, lysine, and/or histidine residues in the targets. Through HopZ3-mediated acetylation, it is possible that the whole effector-immune complex is inactivated, leading to increased growth of the pathogen.
    Full-text · Article · Nov 2015
    • "The types of the exported toxins differ among bacterial species as they appear tailored for specific hosts or niches within a host [4, 5]. While the molecular targets may vary, the secreted virulence factors generally fall into three functional categories: factors that act to subvert the host immune system [5][6][7][8][9][10][11][12], those that induce apoptosis [5, 11,[13][14][15][16][17][18][19][20], or, in case of intracellular bacteria, those that mediate engulfment by the host cell [4,[21][22][23][24][25]. Expression of T3SS-associated genes is usually timed to coincide with host infection. "
    [Show abstract] [Hide abstract] ABSTRACT: Pseudomonas aeruginosa employs a type three secretion system to facilitate infections in mammalian hosts. The operons encoding genes of structural components of the secretion machinery and associated virulence factors are all under the control of the AraC-type transcriptional activator protein, ExsA. ExsA belongs to a unique subfamily of AraC-proteins that is regulated through protein-protein contacts rather than small molecule ligands. Prior to infection, ExsA is inhibited through a direct interaction with the anti-activator ExsD. To activate ExsA upon host cell contact this interaction is disrupted by the anti-antiactivator protein ExsC. Here we report the crystal structure of the regulatory domain of ExsA, which is known to mediate ExsA dimerization as well as ExsD binding. The crystal structure suggests two models for the ExsA dimer. Both models confirmed the previously shown involvement of helix α-3 in ExsA dimerization but one also suggest a role for helix α-2. These structural data are supported by the observation that a mutation in α-2 greatly diminished the ability of ExsA to activate transcription in vitro. Additional in vitro transcription studies revealed that a conserved pocket, used by AraC and the related ToxT protein for the binding of small molecule regulators, although present in ExsA is not involved in binding of ExsD.
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    • "TAK1 also prevents RIP1 to interact with caspase-8 to trigger apoptosis (Bertrand et al., 2008). Two studies have shown that TAK1 can be directly acetylated by Y. pestis YopJ (Paquette et al., 2012) or Y. pseudotuberculosis YopJ (Meinzer et al., 2012). The acetylation of TAK1 may result in its inhibition, leading to the interaction between RIP1 and caspase-8 and the initiation of apoptosis. "
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