Acetylation of MEK2 and I?B kinase (IKK) activation
loop residues by YopJ inhibits signaling
Rohit Mittal†‡§, Sew-Yeu Peak-Chew†, and Harvey T. McMahon†
†Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom; and‡Department of Biological Sciences,
Tata Institute of Fundamental Research, Mumbai 400005, India
Communicated by Richard Henderson, Medical Research Council, Cambridge, United Kingdom, October 13, 2006 (received for review
September 15, 2006)
To overcome host defenses, bacterial pathogens of the genus
Yersinia inject specific effector proteins into colonized mammalian
cells. One such virulence factor, YopJ, inhibits the host inflamma-
tory response and induces apoptosis of immune cells by blocking
multiple signaling pathways, including the MAPK and NF-?B path-
by catalyzing the acetylation of two serine residues in the activa-
tion loop of the MAP kinase kinase, MEK2. This covalent modifi-
cation prevents the phosphorylation of these serine residues that
is required for activation of MEK2 and downstream signal propa-
gation. We also show that YopJ causes acetylation of a threonine
residue in the activation loop of both the ? and ? subunits of the
NF-?B pathway kinase, IKK. These results establish a hitherto
uncharacterized mode of action for bacterial toxins and suggest
the possibility that serine/threonine acetylation may occur even
under nonpathogenic conditions and may be a widespread protein
modification regulating protein function in eukaryotic cells.
inflammation ? MEK
especially with regard to signal transduction pathways that
impinge upon the activation of the innate immune system (1, 2).
Historically, plague has been one of the most devastating
diseases to humans, second only to smallpox. The bacillus
Yersinia pestis is the causative agent of plague, and two other
Yersinia species, Yersinia pseudotuberculosis and Yersinia entero-
colitica, cause septicaemic and gastrointestinal disorders (3).
These pathogens inject a bouquet of six effector proteins into the
mammalian cell cytosol using a type III secretion apparatus (4).
These Yersinia outer proteins (Yops) help the pathogen multiply
extracellularly in the host by preventing its phagocytosis and by
slowing down the onset of the inflammatory response (5). YopE,
YopT, and YopO target the Rho family of GTP-binding proteins
that control actin cytoskeleton dynamics whereas YopH dephos-
disassembly. Together, the action of these Yops contributes to
the resistance of Yersinia to undergo phagocytosis, a process
known to require remodeling of the actin cytoskeleton and of
focal adhesions. Suppression of phagocytosis enables Yersinia to
evade the macrophage defense network, thereby allowing them
to proliferate in Peyer’s patches as extracellular microcolonies.
The leucine-rich protein, YopM, has been shown to bind to
several host cell kinases, resulting in their activation (6). The
remaining outer protein, YopJ (also called YopP in Y. entero-
colitica) has emerged as an important agent that leads to the
reduced host inflammatory response characteristic of Yersinia
infections (5). Exposure of macrophages to lipopolysaccharide
leads to the activation of NF-?B and of several members of the
MAPK family that promote the production of proinflammatory
cytokines such as TNF-?. YopJ induces apoptosis in macro-
phages (7), and it was shown that inhibition of both MAPK and
NF-?B pathways was necessary for this effect (8), suggesting that
Yersinia might neutralize and eliminate macrophages without
nderstanding the mode of action of bacterial toxins has
provided insight into the working of mammalian cells
inducing an inflammatory response. Suppression of the inflam-
matory response is mediated in part by the inhibition of the
transcription factor NF-?B, the activation of which is central to
the onset of inflammation. YopJ has been shown to bind directly
to many members of the MAPK kinase superfamily and also to
IKK (I?B kinase), preventing signaling through MAP kinases as
well as through NF-?B (9).
Modification of MEK by YopJ. In an effort to understand the
biochemical basis of the effects of YopJ on cellular signal
transduction, we examined MAP kinase signaling in cultured
mammalian cells. The Erk MAP kinase cascade is a three-
component cascade in which the MAP kinases, Erk1/2, are
activated upon phosphorylation by the MAP kinase kinases,
MEK1/2, which are in turn, activated upon phosphorylation by
the MAP kinase kinase kinase, Raf (Fig. 1a). HeLa cells are a
well established system for the study of MAPK signaling and
show basal nonphosphorylated Erk1/2 and MEK1/2 levels after
serum starvation. Treatment of cells with EGF, phorbol ester
(TPA), or the ?2 adrenergic receptor agonist isoproterenol
(ISO) leads to transient activation of the MAPK cascade.
Treatment with 10 ?M ISO at 37°C causes an activation of Erk
and MEK within 5 min with a return to basal unphosphorylated
levels by 30 min (see Fig. 4, which is published as supporting
information on the PNAS web site). Treatment with EGF and
TPA also gave rise to robust activation of Erk1/2 and MEK1/2;
however, the return to baseline levels was not complete even 60
min after stimulation (see Fig. 4). It was thus decided to use ISO
activation for experiments involving a time-course of MAPK
YopJ has previously been shown to interact with MEK2 and
to inhibit its activation (9). We examined the effect of YopJ on
MAPK signaling by expressing either WT YopJ or the inactive
C172A mutant of YopJ in HeLa cells. MEK1/2 activation after
ISO stimulation was inhibited in cells expressing WT YopJ (Fig.
1b), whereas cells expressing the inactive C172A mutant of YopJ
showed the expected activation of MEK1/2 within 5 min of
agonist stimulation, with a return to basal unphosphorylated
levels by 30 min. Curiously, when the same cell lysates were
examined (Fig. 1b Lower) for total MEK1/2 protein levels by
using a MEK1/2 antibody (CST9122), no signal was detected in
cells expressing WT YopJ, whereas cells expressing the C172A
mutant of YopJ showed robust signals for total MEK1/2 protein.
This effect of YopJ on the immunodetection of total protein
levels was specific for MEK1/2 because total amounts of Erk1/2
Author contributions: R.M. and H.T.M. designed research; and R.M. and S.-Y.P.-C. per-
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Abbreviations: ISO, isoproterenol; amu, atomic mass unit; I?B, inhibitor of NF-?B; IKK, I?B
kinase; MEK1/2, mitogen-activated protein kinase kinase 1/2; Yop, Yersinia outer protein.
§To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
December 5, 2006 ?
vol. 103 ?
and of other control proteins like beta-actin and calnexin were
not observed to be different between YopJ WT and YopJ-
C172A cells (Fig. 1d). The effect of YopJ on the activation of
MEK1/2 was dose-dependent (Fig. 1c). Consistent with the
results of Fig. 1b, the dose dependence of MEK1/2 inhibition
correlated precisely with the loss of total MEK1/2 immunosignal
as detected by the CST9122 antibody. Taken at face value these
results would suggest that expression of YopJ in cells leads to
degradation of MEK1/2 proteins. This interpretation was clearly
inadmissible because previous studies had clearly shown that
whereas YopJ expression had an effect on the amount of
phosphoMEK it had no effect of on total level of MEK proteins
(9, 10). We began to suspect that expression of YopJ might be
resulting in the masking of the epitope on MEK1/2 recognized
by the CST9122 anti-MEK1/2 antibody. We thus probed lysates
of HeLa cells expressing WT or inactive YopJ using antibodies
directed against other epitopes on MEK1 and MEK2. Because
YopJ had been shown to interact with the MAPK kinases MKK3
and MKK4 (9), we also blotted with antibodies against MKK3
and MKK4 to examine whether any of them might show a
reduction in total protein levels. By using these antibodies, it was
determined that there was equal signal for total MEK protein
levels from cells expressing either WT or mutant YopJ proteins
(Fig. 1d). Under our experimental conditions, endogenous levels
of MKK3 protein could not be detected by using the anti-MKK3
The CST9122 MEK1/2 antibody used in Fig. 1 b and c was
directed against a 20-aa peptide (conserved in MEK1 and -2)
centered about amino acid 220 of MEK1 whereas the MEK1 and
MEK2 antibodies used in Fig. 1d were directed against peptides
taken from the amino and carboxyl termini of the respective
proteins (Cell Signaling Technology). It was thus clear that
expression of YopJ was not leading to degradation of MEK1/2
proteins but was causing a masking of the epitope of the
anti-MEK1/2 CST9122 antibody that was directed against a
peptide near the activation loops of the proteins. This observa-
tion was potentially very interesting because it suggested that
YopJ expression might prevent activation of MEK1/2 by causing
covalent modification of the activation loops of these kinases.
YopJ Leads to Acetylation of MEK2. To elucidate the nature of
modification, lysates from YopJ expressing cells were subjected
to treatment with phosphatase, reducing agents, mild acid and
mild alkali. However, none of these treatments restored the
MEK1/2 signal as detected by the CST9122 antibody. Prolonged
(5 h) incubation of the lysate in Laemmli Sample Buffer at 95°C
did restore the signal, confirming that YopJ was indeed adding
be noted here that given the nature of our readout wherein a loss
of signal on a Western blot was diagnostic of the modification,
YopJ must certainly be a very highly active enzyme in vivo and
that most of the MEK molecules in the cell must be modified by
YopJ. This effect of expressed YopJ on endogenous MEK1/2
was seen as early as 6 h after transfection of cells (data not
To identify the modification it was decided to isolate MEK
from YopJ expressing cells for analysis by mass spectrometry.
MEK2 bearing a myc epitope tag at its amino terminus and a
hexahistidine tag at the carboxyl terminus was overexpressed in
HeLa cells. It was verified from immunoblots by using the
MEK1/2 CST9122 antibody that cotransfection of YopJ was
fashion to endogenous MEK (data not shown). Myc-MEK2-
(His)6was prepared by Ni-NTA affinity from lysates of HeLa
cells coexpressing YopJ WT or a control plasmid. The prepared
samples were analyzed for the presence of modification by
Western blots by using anti-myc, anti-MEK2, and the discrimi-
nating anti-MEK1/2 CST9122 antibodies (Fig. 2a) and subjected
to analysis by mass spectrometry.
Analysis of tryptic digests of the two samples revealed a
peptide in MEK2 that was modified by YopJ. Triply charged
ions with a mass/charge (m/z) ratio of 792.0, corresponding
to the amino acid sequence of the activation loop
L210C*DFGVSGQLIDSMANSFVGTR231(where C* is carb-
amidomethylcysteine) was isolated for the control sample and
fragmented to confirm the sequence. The corresponding triply
an important signaling module that is shared by many signal transduction path-
ways; the MAPK pathway investigated in this study focuses on the MAP kinase
kinases MEK1/2 that, in turn, activate the MAP kinases Erk1/2; this and similar
pathways mediate the immediate host response to infection by Yersinia species.
(b) Immunoblots from HeLa cells transfected with either YopJ-wt or the C172A
mutant. Transfected cells were treated with 10 ?M ISO at 37°C for the indicated
pho-MEK, indicating that MEK activation is blocked by YopJ; in cells transfected
with the C172A mutant of YopJ MEK, activation is clearly seen with a return to
baseline levels by 30 min. (Lower) Loss of immunoreactivity with the CST9122
antibody against MEK1/2 in YopJ-wt expressing cells. (c) The block of MEK
of plasmid pSFV-YopJ were transfected into a 35 mm dish of HeLa cells. Twenty-
four hours after transfection, cells were serum-starved for 6 h and then treated
loss of MEK signal is specific for the MEK1/2 (CST9122) antibody. Lysates from
by using the indicated antibodies. Whereas the MEK1/2 (CST9122) antibody
discriminates between WT and C172A lanes, the other antibodies do not. This
that masks the epitope recognized by the MEK1/2 (CST9122) antibody.
MEK modification by YopJ. (a) The three-component MAPK cascade is
Mittal et al. PNAS ?
December 5, 2006 ?
vol. 103 ?
no. 49 ?
charged ion from the YopJ-expressing cells had an m/z ratio of
820.1, with an additional mass of 84 atomic mass units (amu)
(consistent with the addition of two acetyl groups). Peaks
within the fragmentation series of the modified peptide (m/z
820.1) were compared with the unmodified one (m/z 792.0).
The y6 and y10 ions of the modified peptide (Fig. 2b) have
masses that are, respectively, 42 amu and 84 amu greater than
the corresponding ions in the unmodified peptide (Fig. 2c).
This result indicates the addition of acetyl groups on the serine
residues at amino acid positions 222 and 226 of MEK2.
Subsequent ions in the series y7 to y9 showed a mass increase
of 42 amu, and in y11 to y12 an increase of 84 amu, supporting
the assignment of these acetylation sites. This finding provides
a mechanistic explanation for the ability of YopJ to inhibit
activation of MEK as Ser-222 and Ser-226 lie in the activation
loop of MEK2 and normally undergo phosphorylation by an
upstream kinase to be activated. Prior acetylation of the
hydroxyl groups of these serine residues leaves them incapable
of accepting phosphates, thereby not permitting MEK2
Additionally, it was observed that Thr-13 of MEK2 is also a
target for O-acetylation by YopJ (see Fig. 5, which is published
consequence of the acetylation of Thr-13 on MEK2 is currently
uncertain. We expect that this acetylation event does not have a
large impact on MEK activity because signaling by a constitu-
tively active mutant of the closely related MAP kinase kinase
MEK1 (made by replacing the activation loop serines with acidic
residues) was not inhibited by expression of YopJ (9).
YopJ has Acetyltransferase Activity. YopJ could either be an acetyl
transferase by itself or act as an adaptor between MEK2 and
some endogenous enzyme. We next examined whether acetyl
transferase activity was an inherent property of YopJ. Recom-
binant MEK2 expressed as a GST-fusion protein was purified
from Escherichia coli as were hexahistidine-tagged YopJ-wt and
YopJ-C172A. Acetyl transferase activity of WT YopJ is shown
in Fig. 2d where it can be seen that progressively higher amounts
of YopJ result in greater acetylation of GST-MEK2 in the
presence of14C-labeled acetyl CoA (where the radiolabel is on
the acetyl group). Control experiments were performed to verify
that GST was not a substrate for acetyl transfer under our assay
conditions. Fig. 2e shows a time course of the acetylation
reaction over 40 min of incubation of YopJ with MEK2 at 30°C.
It is to be noted that in addition to acetyl transfer to MEK2,
attachment of the radiolabel to WT YopJ is also seen. The
C172A mutant of YopJ does not serve as an acceptor of the
acetyl group and thus does not transfer it to the substrate MEK2.
These results demonstrate that acetyl transferase activity is an
inherent enzymatic property of YopJ.
YopJ Acetylates IKK? and IKK?. In addition to its effect on MAPK
signaling, YopJ is also known to exert an inhibitory effect on
NF-?B signaling (11). NF-?B is a collective designation for a
family of dimeric transcription factors. In resting cells NF-?B is
complexed with its inhibitor I?B and retained in the cytoplasm.
I?B can be phosphorylated by I?B kinase, IKK (Fig. 3a).
Phosphorylation of I?B leads to its ubiquitination and subse-
quent degradation. NF-?B is then freed to enter the nucleus and
affect transcription. IKK is a multisubunit complex comprising
two structurally similar catalytic subunits (IKK? and IKK?) and
a variable number of associated regulatory gamma subunits. The
canonical pathway of NF-?B activation relies on activation of
IKK? which has been shown to phosphorylate I?B proteins. The
kinase activity of IKK?, on the other hand, is required in the so
called noncanonical pathway of NF-?B activation that operates
proteins (52% sequence identity, ?70% sequence similarity)
210–231. The fragmentation of the modified peptide is shown in b and for the unmodified peptide in c. It can be seen that, whereas Cys-211 is carbamidom-
ethylated (CAM) in each case, Ser-222 and Ser-226 are O-acetylated (OAc) only in YopJ-coexpressing cells. (d) GST-MEK2 (0.5 ?g) is acetylated in vitro by YopJ-wt
in the presence of [14C]acetyl coenzymeA. Increasing amounts of YopJ lead to higher acetyl transfer. (e) Time course of acetylation of GST-MEK (0.5 ?g) by YopJ
(80 ng). Progressively greater acetyl transfer is seen over time. The inactive C172A mutant of YopJ does not show any activity in these assays.
www.pnas.org?cgi?doi?10.1073?pnas.0608995103 Mittal et al.
(14) and can be considered to be MAPKK-like molecules in that
they are activated by MAPKKK-like molecules (15) and by the
fact that the activation loops of MAPKK, IKK?, and IKK? all
contain two serine residues (or one serine and one threonine
residue) within the sequence SxxxS/T that are phosphorylated by
upstream kinases (14). Furthermore, a direct interaction of
MAPKKs and IKK? with YopJ has been reported (9).
We investigated the effect of YopJ expression on the NF-?B
pathway by examining the response of YopJ-transfected mam-
malian cells to the proinflammatory cytokine TNF-?. TNF-?
treatment induces a rapid activation of IKK which in turn
phosphorylates I?B leading to the ubiquitination and protea-
some-mediated degradation of I?B. Fig. 3b shows that expres-
cells are exposed to TNF-?. Phosphorylation and degradation of
I?B are markedly retarded in cells expressing WT YopJ when
compared with cells expressing the inactive C172A mutant of
YopJ, which, in turn, correlates well with delayed activation (by
phosphorylation) of IKK in these cells (Fig. 3b Bottom). These
results demonstrate that YopJ affects the activation of IKK in
Overexpression of either IKK? or IKK? is known to result
in autophosphorylation of their activation loops leading to
activation of their kinase activities (16, 17). We coexpressed
Flag-tagged IKK? or Flag-tagged IKK? along with YopJ in
mammalian cells. These cells were then examined for their
response to stimulation with TNF-? (see Fig. 6, which is
published as supporting information on the PNAS web site). It
was observed that total levels of I?B protein were lower in
IKK? plus YopJ-wt cells compared with IKK? plus YopJ-
C172A cells, whereas no such diminution in I?B levels was seen
in cells overexpressing IKK? (see Fig. 6 Upper). This obser-
vation is consistent with the fact that activation of IKK? and
not IKK? is required for I?B degradation (12). Furthermore,
it suggests that WT YopJ ‘‘protects’’ I?B from degradation
(presumably by affecting IKK activity), whereas inactive YopJ
does not. Fig. 3 c and d Upper shows that IKK? and IKK? are
fully autophosphorylated and activated in cells coexpressing
inactive YopJ. Activation of these cells with TNF-? does not
lead to any detectable increase in the amount of phosphory-
lated IKK? or IKK?. Interestingly it is observed (on these
same panels) that recognition of expressed IKK? and IKK? by
the phospho-specific antibody is abrogated in cells expressing
WT YopJ. As control, the total amounts of IKK? and IKK?
proteins were verified to be the same in cells expressing either
form of YopJ (Fig. 3 c and d Lower). This observation
immediately suggests that YopJ modifies IKK? and IKK?
within the activation loop, most probably by acetylating one or
more serine/threonine residues within it.
To identify the site(s) of modification we immunoprecipitated
Flag-tagged IKK? and Flag-tagged IKK? from cells coexpressing
YopJ. Mass spectrometric analysis (see Fig. 7, which is published as
supporting information on the PNAS web site) revealed that both
IKK? and IKK? were indeed acetylated on the activation loop. In
each case, the modification was on a threonine residue (indicated
in bold) located between the two (underlined) phospho-acceptor
serine residues (AKDVDQGSLC(OAc)T179SF in IKK? and
AKELDQGSLC(OAc)T180SF in IKK?). In addition to Thr-179,
IKK? was also found to be acetylated on Ser-119 in the kinase
domain and on Thr-722 located near the carboxyl terminus of
the significance of their modification is not presently clear.
The cysteine residue adjacent to the acetyl-acceptor threonine
residue in the activation loops of the catalytic IKK? and IKK?
subunits has previously been reported to be covalently modified
by cyclopentenone prostaglandins (18) and other thiol-reactive
agents (19, 20) resulting in the inhibition of IKK activation.
YopJ-mediated acetylation of the activation loop of the ?
and ? subunits of IKK also prevents activation of the kinase
activity of the IKK complex thus preventing phosphorylation
of I?B in response to exposure of cells to TNF-?. These results
are consistent with an earlier report (21) wherein it was
demonstrated that phosphorylation of the activation loop of
IKK? was inhibited in YopJ-expressing mammalian cells. Our
results thus provide a mechanistic basis for the observed
inhibition of the canonical NF-?B proinflammatory pathway
Taken together, our results establish that YopJ is an acetyl
transferase that catalyses O-acetylation of serine and threonine
residues in two important kinases involved in the innate immune
response of mammals, thereby inhibiting their activities.
Interestingly, the YopJ-like molecule from Salmonella typhi-
murium, AvrA, has been reported to inhibit the NF-?B pathway
(22) and also to inhibit the degradation of ?-catenin (23).
Phosphorylation of ?-catenin occurs within a motif that is highly
similar to the phosphorylation motif of I?B (14) and it has been
suggested that IKK? may phosphorylate and regulate the deg-
activate the NF-?B-signaling pathway by activating the I?B kinase (IKK) com-
plex. IKK activation results in the phosphorylation and subsequent ubiquitin-
in NF-?B translocation to the nucleus where it can regulate gene expression.
(b) HEK293 cells transfected with YopJ-wt or YopJ-C172A were stimulated
with TNF-?. I?B degradation is slowed down (Top) by the expression of
YopJ-wt (but not the C172A inactive mutant of YopJ). This effect is due to
reduced phosphorylation of I?B in these cells (Middle) caused by inhibition of
activation of IKK (Bottom) caused by WT YopJ. (c and d) Overexpression of
IKK? (c) or IKK? (d) results in their activation in cells expressing inactive
YopJ-C172A but this activation is blocked by WT YopJ (Upper). Total levels of
IKK? and IKK? are not affected by expression of YopJ (Lower).
YopJ modifies IKK? and IKK?. (a) Various proinflammatory stimuli
Mittal et al.PNAS ?
December 5, 2006 ?
vol. 103 ?
no. 49 ?
radation of ?-catenin (24). It is thus possible that YopJ-related
AvrA may also possess an acetyl transferase activity allowing it
to inactivate IKK? thereby exerting its effect on ?-catenin
Whereas acetylation of lysine residues in histones and tran-
scription factors has been widely described there have also been
reports of regulated acetylation of nonnuclear proteins (25).
Furthermore, it has been suggested that acetylation events may
temporally modulate IKK activity (26). Our elucidation of the
mechanism of action of the bacterial toxin YopJ proposes the
hypothesis that serine/threonine acetylation may be a wide-
spread mode of biochemical regulation of endogenous processes
in mammalian cells.
The analgesic and antiinflammatory effects of acetyl salicylic
acid (Aspirin) administration are attributed to the transfer of the
acetyl group to active site serine residues of cyclooxygenase
enzymes resulting in the inhibition of their enzymatic activities.
However, the cellular enzymes that (possibly) mediate the acetyl
transfer have not been identified. Interestingly, there are by now
numerous reports of cyclooxygenase-independent actions of
acetyl salicylic acid that impinge primarily upon MAP kinase-
we speculate that some of these effects might be due to the
transfer of acetyl groups to the active sites of MAPKKs/IKKs
catalyzed by yet uncharacterized serine/threonine acetyl
Materials and Methods
Reagents. Isoproterenol and EGF were from Sigma (Dorset,
U.K.), TPA was from Cell Signaling Technology (Danvers,
MA), TNF-? was from R & D Systems (Minneapolis, MN).
Primary antibodies against Erk, phosphoErk, MEK1/2, phos-
phosphoI?B, IKK?, IKK?, and phosphoIKK?/? were from
Cell Signaling Technology; primary antibodies against caln-
exin were from Affinity BioReagents (Golden, CO); and
primary antibodies against ?-actin were from Abcam (Cam-
Plasmid Constructs. Plasmids encoding WT YopJ (pSFV-YopJ)
and the inactive C172A mutant (pSFV-YopJ-CA) were the
kind gifts of K. Orth (University of Texas Southwestern
Medical Center, Dallas, TX); pCMV-MEK2 was provided by
K.-L. Guan (University of Michigan, Ann Arbor, MI); and
pCDNA3-Flag-IKK? and pCMV-Flag-IKK? were kindly pro-
vided by D. Ballard (Vanderbilt University Medical Center,
pCMV-myc-MEK2-(His)6coding for MEK2 with a hexahis-
tidine tag at its carboxyl terminus was constructed by ampli-
fying the MEK2 sequence with appropriate primers and
ligating into pCMV-myc (a modified pCMV5 plasmid). GST-
MEK2 was made by amplifying the coding region of MEK2 and
ligating in-frame with GST in the bacterial expression plasmid
pGEX6P. (His)6-YopJ-wt and (His)6-YopJ-C172A were made
by ligating the coding region of YopJ in-frame with the
amino-terminal hexahistidine tag of the bacterial expression
vector pET28. All constructs used were verified by plasmid
Cell Culture and Transfections. HeLa and HEK293 cells were
cultured in DMEM (Invitrogen) with 10% FBS. Transfections
were typically done with GeneJuice (Novagen) in 35-mm
dishes by using 1 ?g of plasmid DNA per dish. Twenty-four
hours after transfection, cells were serum-starved for 12–16 h
and then stimulated with either ISO (10 ?M) or TNF-? (20
ng/ml) for different times. Cells were harvested in Laemmli
Sample Buffer, and lysates were resolved by NuPAGE 4–12%
Bis-Tris Gels (Invitrogen). Western Blots were probed with
primary antibodies at a typical dilution of 1:10,000 (except
1:500 for the phosphoIKK?/? antibody) at 4°C for 16 h and
then incubated with horseradish peroxidise-linked secondary
antibodies at room temperature for an hour followed by ECL
(Amersham) detection on Kodak BioMax XAR film.
Identification of modification on MEK2. Ten micrograms of the
or along with 5 ?g of pSFV-YopJ-wt into each of three 10-cm
dishes by using GeneJuice (Novagen). Forty-eight hours after
transfection, cells were harvested, and hexahistidine-tagged
MEK2 was purified by using NiNTA affinity chromatography.
by SDS/PAGE and the gel stained by Coomassie blue. Gel slices
containing bands corresponding to MEK2 were excised and
washed, alkylated, and in-gel digested with trypsin (29). A
portion of the extracted tryptic peptides mixture was desalted
and concentrated by using a GELoader tip filled with Poros R2
sorbent (Perseptive Biosystems, Framingham, MA). The bound
peptides were eluted with 1 ?l of 60% acetonitrile/3% formic
acid directly into a nanospray capillary and then introduced into
an API QSTAR pulsar i hybrid quadrupole-time-of-flight mass
spectrometer (MDS Sciex, Ontario, Canada). Product ion scans
were carried out in positive ion-mode and MS survey scan for
peptides from m/z 600 to 1,500 were measured. Selected ions
were fragmented by collision-induced dissociation (CID) with
nitrogen in the collision cell and spectra of fragment ions
produced were recorded in the time-of-flight mass analyzer.
Identification of modification on IKK? and IKK?. Five micrograms of
plasmid encoding Flag-tagged IKK? were cotransfected along
with 5 ?g of either pSFV-YopJ-wt or pSFV-YopJ-C172A into
each of three 10-cm tissue culture dishes. Forty-eight hours after
was immunoprecipitated by using EZview Red Anti-Flag M2
affinity gel (Sigma). Immunoprecipitates were resolved on Nu-
PAGE 4–12% Bis-Tris Gels (Invitrogen). Protein bands corre-
sponding to modified and unmodified IKK? were excised from
a Coomassie blue-stained gel, washed, alkylated, and in-gel
digested with chymotrypsin.
Peptides from the in-gel digest mixtures were separated by
nanoscale liquid chromatography (LC Packings, Amsterdam,
The Netherlands) on a reverse-phase C18 column (150 ? 0.075
mm i.d., flow rate 0.15 ?l/min). The eluate was introduced
directly into a Q-STAR hybrid tandem mass spectrometer
(LC-MS/MS). The spectra were searched against a NCBI
nonredundant database with MASCOT MS/MS Ions search
(www.matrixscience.com). The modified peptides and acety-
lation sites were identified by manual inspection of the frag-
mentation series. The same procedure was used with trans-
fected Flag-IKK? (in place of IKK?) to identify the site of
modification in IKK?.
Acetyl Transferase Assays. GST-MEK2 (0.5 ?g) (purified by using
standard protocols for GST-tagged protein purification) was
incubated with varying amounts (0–200 ng) of (His)6-YopJ-wt or
(His)6-YopJ-C172A and 80 ?M [1-14C]acetyl coenzymeA (56
mCi/mmol; Amersham) (1 Ci ? 37 GBq) at 30°C for 40 min.
Reaction products were resolved on 4–12% SDS/PAGE gels.
Gels were stained with Coomassie blue, destained, soaked in
Amplify (Amersham) for 30 min, dried, and subjected to auto-
radiography for 72 h. For time-course experiments, 0.5 ?g of
GST-MEK2 was incubated with 80 ng of (His)6-YopJ-wt or
(His)6-YopJ-C172A and 80 ?M [1-14C]acetyl coenzymeA at
30°C for varying lengths of time. Reactions were stopped by the
www.pnas.org?cgi?doi?10.1073?pnas.0608995103Mittal et al.
addition of SDS/PAGE sample buffer and analyzed as described Download full-text
Note. While this study was being completed, Mukherjee et al. (30)
reported that YopJ acetylates MKK6 in the activation loop in a manner
similar to that reported in this study. Our results confirm that YopJ has
acetyl transferase activity, and, taken together, our studies show that, in
mammalian cells, this activity inhibits the MAP kinase- and NF-?B-
signaling pathways by targeting the activation loops of MAP kinase
kinases (MKKs) and IKKs.
R.M. thanks Sarada Raghavan, JyotiRanjan Mishra, S. Raghavi, and
Veronica Rodrigues for help and cooperation. R.M. acknowledges
grants from the British Council, India, and from The Royal Society
(International Incoming Fellowship) and is currently supported by the
Medical Research Council, U.K. (Career Development Fellowship).
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