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 2012with46 Reads
DOI: 10.1073/pnas.1008203109 · Source: PubMed
Abstract
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.
Serine/threonine acetylation of TGFβ-activated kinase
(TAK1) by Yersinia pestis YopJ inhibits innate
immune signaling
Nicholas Paquette
a,b,1
, Joseph Conlon
a,1,2
, Charles Sweet
a,3
, Florentina Rus
a
, Lindsay Wilson
a
, Andrea Pereira
a
,
Charles V. Rosadini
a
, Nadege Goutagny
a
, Alexander N. R. Weber
c
, William S. Lane
d
, Scott A. Shaffer
e
,
Stephanie Maniatis
e
, Katherine A. Fitzgerald
a
, Lynda Stuart
b
, and Neal Silverman
a,4
a
Division of Infectious Disease, Department of Medicine, and
e
Proteomics and Mass Spectrometry Facility, University of Massachusetts Medical School,
Worcester, MA 01605;
b
Program of Developmental Immunology, Department of Pediatrics, Massachusetts General Hospital/Harvard Medical School, Boston,
MA 02114;
c
Toll-Like Receptors and Cancer, German Cancer Research Center, Im Neuenheimer Feld 242, 69120 Heidelberg, Germany; and
d
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
Y
opJ 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 unidenti ed 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 ne w 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 conict of interest.
This article is a PNAS Direct Submission.
1
N.P. and J.C. contributed equally to this work.
2
Present address: The Takeda Oncology Company, Cambridge, MA 02139.
3
Present address : Che mistry Department, United States Naval Academy, Annapolis,
MD 21402.
4
To whom correspondence should be addressed. E-mail: neal.silverman@umassmed.edu.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1008203109/-/DCSupplemental.
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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.
Results
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
WT
) or a catalytically in-
active mutant (C172A, YopJ
CA
) 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
WT
, because YopJ
CA
was
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
WT
ies
containing no Gal4 driver) showed robust IMD pathway activa-
tion, as assayed by Northern blotting for Diptericin. However, the
yolk-gal4;UAS-YopJ
WT
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
WT
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
CA
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
WT
lines (as shown in Fig. 1C, columns 1
and 2), no rough/reduced eye phenotype was present in any lines
expressing YopJ
CA
. The rough and reduced eyes found in
YopJ
WT
ies were further enhanced by driving higher levels of
YopJ
WT
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
WT
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
WT
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
WT
and YopJ
CA
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
A
B
C
D
38
38
52
PGN
Cu
S2* YopJ
WT
YopJ
CA
-
--
++
+- -+--+
-++-++
IB:
-pJNK
IB:
-JNK (FL)
38
225
150
102
76
52
31
31
PGN
Cu
S2* YopJ
WT
YopJ
CA
IP: -IMD
IB:
-Ub
-
--
++
+- -+-- +
-++-++
IP:
-IMD
IB:
-IMD
IP:
-FLAG (YopJ)
IB:
-FLAG (YopJ)
IP: -FLAG
IB:
-FLAG(YopJ)
31
150
---+++
---+++
S2*
Cu
PGN
S2*
YopJ
WT
Y
opJ
CA
YopJ
WT
YopJ
CA
IP: -IKK
KA: p*-Relish
IP:
-IKK
IB: -IKK
52
38
31
Cells
Cu
YopJ
Cu
PGNSpatzle
Dipt
Dros
Rp49
IB:
-FLAG-YopJ
WT
--++--++-- ++
CA WT CA WT CA WT CAWT CA WT CA
-- --++++-- ++
Fig. 1. YopJ inhibits Drosophila IMD but not Toll immune signaling and
functions between IMD and JNK. (A) S2* cells stably expressing YopJ
WT
or
YopJ
CA
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
WT
or YopJ
CA
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
WT
or YopJ
CA
after stimulation with PGN. IKKγ blot serves
as an immunoprecipitation control.
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IMMUNOLOGY
immunoblotting for PGN-induced phospho-JNK, was blocked by
YopJ
WT
(Fig. 1C). In the NF-κB/Relish branch, activation of the
Drosophila IKK complex was also inhibited by YopJ
WT
, but not
by YopJ
CA
(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
K82A
(a
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
WT
were coexpressed, the activity of both kinases was inhibited, whereas
coexpression of YopJ
CA
resulted in no discernible reduction in
signal. Interestingly, dTAK1, expressed alone or in combination
with YopJ
CA
, migrated as a tight doublet at a molecular mass
just below 105 kDa, whereas in concert with YopJ
WT
, dTAK1
migrated noticeably faster, closer to 85 kDa (Fig. 2A, row 2).
When optimized for better resolution, immunoblot analysis
showed that overexpressed dTA K1 alone, or in the presence
of YopJ
CA
, migrates as a doublet at 95 and 105 kDa. In the
presence of YopJ
WT
, 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
modication(s).
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
CA
samples
from 95/105 kDa to 85/93 kDa. In the presence of YopJ
WT
,
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
CA
)is
phosphorylated; however in the presence of YopJ
WT
, 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
WT
showed little PGN-induced TAK1 ki-
nase activity, whereas cells similarly expressing YopJ
CA
showed
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 Drosophi la TAK1 and map the molecular
changes i nduced by YopJ, FLAG-TAK1 was isolated from
lysates prepared from cells expressing dTAK1 alone or in com-
bination with YopJ
WT
or YopJ
CA
. 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
WT
, and overall phosphorylation was
greatly reduced. When coexpressed with YopJ
CA
, dTAK1 was
phosphorylated on a number of residues, including all of the sites
identied when TAK1 was expressed alone and some additional
FLAG-TAK1
+
--
-
-
+
+
+
+
105
150
85
YopJ
C172A
YopJ
WT
IP: α-FLAG-TAK1
KA: p*-MKK6
IP: α-IKK
KA: p*-Relish
IP: α-FLAG
IB: α-FLAG(TAK1)
IP: α-FLAG
IB: α-FLAG(YopJ)
IP: α-IKK
IB: α-IKK
A
52
38
38
31
Lysate
IB: α-FLAG
Lysate
IB: α-FLAG
B
+λ-phosphatase
105
85
105
85
YopJ
C172A
--
+
--
+
YopJ
WT
-
-
+- -+
FLAG-TAK1
++
+
+++
++
+
-
--
IP: α-TAK
KA: p*-MKK6
IP: α-FLAG-YopJ
IB: α-FLAG-YopJ
PGN
Cu
S2*
YopJ
WT
YopJ
CA
++-++-
+-- +--
C
38
31
Fig. 2. YopJ inhibits Drosophila TAK1 kinase activity. (A) dTAK1 was over-
expressed in S2* cells alone or in the presence of YopJ
WT
or YopJ
CA
, 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 YopJ
WT
or YopJ
CA
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
WT
or
YopJ
CA
. Anti-FLAG immunoblot serves as a control for the presence of YopJ.
12712
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www.pnas.org/cgi/doi/10.1073/pnas.1008203109 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
CA
, 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
WT
, 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
CA
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
A
B
C
D
TAK1
YopJ
IP: FLAG
KA/IB: α-p-MKK6
IB: α-FLAG-TAK1
IB: α-FLAG-YopJ
-
-
WT CA - WT CA - - -WT CA
WT T168A
T171A
S176A
TAK1
IP: FLAG
KA/IB: α-p-MKK6
IB: α-FLAG-TAK1
-WT
T168E
T171E
S176E
38
38
105
85
105
85
31
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 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 veries the expression of dTAK1 protein (row 2).
IP: α-FLAG-hTAK1
KA/IB: α-pMKK6
IP: α-FLAG
KA: p*-MKK6
K82A
IP: α-FLAG
KA: p*-IRF3
380-427
CB: p*-MKK6
K82A
CB: p*-IRF3
380-427
IP/IB: α-FLAG
IB: α-GFP (YopJ) IB: α-GFP (YopJ)
IB: α-pTAK(T187) IB: α-pTBK1(S172)
hTAK1
MKK6
-++++
YopJ CA WT
---
FLAG-TAK1 FLAG-TBK1+++
YFP-YopJ YFP-YopJCAWT--
-+++
CAWT--
-
WT WT WT WT K63W
IP: α-FLAG-hTAK1
IB: α-pTAK1
IB: α-FLAG-hTAK1
IP: α-FLAG-hTAK1
38
76
76
102
A
B
C
38
31
102
102
76
76
5252
Ac
Ac
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
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vol. 109
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IMMUNOLOGY
hTAK1 mutant (TAK
K63W
) also failed to autophosphorylate or
activate, whereas the coexpression of YopJ
CA
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
WT
, 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.
Discussion
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 im mune inhibi-
tion remains controversial. Previous work, from our group and
others, suggests that YopJ acts as an ubiquitin protein pro-
tease, cleaving ubiqui tin from conjuga ted 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 conclud ed
that YopJ-inhibited innate immune signaling upstre am of IKK
activation, consistent with our results her e. 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 i s a direct
target of YopJ-mediated acetylation, strongly arguing that this
acetyltransferase activity is the critical activity responsib le for
the immunosupp ressive function of YopJ. However, Zhou et al.
present biochemical data that recombinant YopJ has ubiquitin
protease acti vity (8). T herefore, it remains possible t hat YopJ
has two enzymatic activities. Althou gh we demonstrate that
IMD ubiquitinati on is not perturbed in the presence of YopJ,
IMD may not be a suitable substrate for YopJ-mediated
deubiquitination.
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 dif
cult within the context of
PGRP-LC TNFR/TLR
TAK1
Ac
YopJ
Hep
(JNKK)
ird5/key
(IKK /IKK )
Bsk
(JNK)
AP1
Immune
genes
Relish
(NF-κB)
Relish
TAK1
Ac
MKK4
Ac
/7
Ac
Immune
genes
AP1
Immune
genes
AMPs
(Diptericin)
JNK
MKK6
Ac
p38
IKKα
Ac
/IKKβ
Ac
NF-κB
NF-κB
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).
12714
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www.pnas.org/cgi/doi/10.1073/pnas.1008203109 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.
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Paquette et al. PNAS
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    • "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.
    Full-text · Article · Aug 2015
    • "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. "
    [Show abstract] [Hide abstract] ABSTRACT: Yersinia pestis (Y. pestis), as the causative agent of plague, has caused deaths estimated to more than 200 million people in three historical plague pandemics, including the infamous Black Death in medieval Europe. Although infection with Yersinia pestis can mostly be limited by antibiotics and only 2000-5000 cases are observed worldwide each year, this bacterium is still a concern for bioterrorism and recognized as a category A select agent by the Centers for Disease Control and Prevention (CDC). The investigation into the host-pathogen interactions during Y. pestis infection is important to advance and broaden our knowledge about plague pathogenesis for the development of better vaccines and treatments. Y. pestis is an expert at evading innate immune surveillance through multiple strategies, several mediated by its type three secretion system (T3SS). It is known that the bacterium induces rapid and robust cell death in host macrophages and dendritic cells. Although the T3SS effector YopJ has been determined to be the factor inducing cytotoxicity, the specific host cellular pathways which are targeted by YopJ and responsible for cell death remain poorly defined. This thesis research has established the critical roles of caspase-8 and RIP kinases in Y. pestis-induced macrophage cell death. Y. pestis-induced cytotoxicity is completely inhibited in RIP1-/- or RIP3-/-caspase-8-/- macrophages or by specific chemical inhibitors. Strikingly, this work also indicates that macrophages deficient in either RIP1, or caspase-8 and RIP3, have significantly reduced infection-induced production of IL-1β, IL-18, TNFα and IL-6 cytokines; impaired activation of NF-κB signaling pathway and greatly compromised caspase-1 processing; all of which are critical for innate immune responses and contribute to fight against pathogen infection. Y. pestis infection causes severe and often rapid fatal disease before the development of adaptive immunity to the V bacterium, thus the innate immune responses are critical to control Y. pestis infection. Our group has previously established the important roles of key molecules of the innate immune system: TLR4, MyD88, NLRP12, NLRP3, IL-18 and IL-1β, in host responses against Y. pestis and attenuated strains. Yersinia has proven to be a good model for evaluating the innate immune responses during bacterial infection. Using this model, the role of caspase-8 and RIP3 in counteracting bacterial infection has been determined in this thesis work. Mice deficient in caspase-8 and RIP3 are very susceptible to Y. pestis infection and display reduced levels of pro-inflammatory cytokines in spleen and serum, and decreased myeloid cell death. Thus, both in vitro and in vivo results indicate that caspase-8 and RIP kinases are key regulators of macrophage cell death, NF-κB and caspase-1 activation in Yersinia infection. This thesis work defines novel roles for caspase-8 and RIP kinases as the central components in innate immune responses against Y. pestis infection, and provides further insights to the host-pathogen interaction during bacterial challenge.
    Article · Jul 2014 · PLoS ONE
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