Tài liệu Báo cáo khoa học: Post-translational modification of the deubiquitinating enzyme otubain 1 modulates active RhoA levels and susceptibility to Yersinia invasion pptx

Post-translational modification of the deubiquitinating
enzyme otubain 1 modulates active RhoA levels and
susceptibility to Yersinia invasion
Mariola J. Edelmann, Holger B. Kramer, Mikael Altun and Benedikt M. Kessler
Department of Clinical Medicine, University of Oxford, UK
Introduction
The genus Yersinia consists of three pathogenic species
that are agents of a variety of diseases, one of which
was historically the cause of major pandemics. These
include the bubonic plague caused by Yersinia pestis,
mesenteric adenitis and septicaemia caused by
Yersinia pseudotuberculosis and gastroenteritis caused
Keywords
deubiquitinating enzymes; otubain 1;
phosphorylation; RhoA; YpkA
Correspondence
B. M. Kessler, Henry Wellcome Building for
Molecular Physiology, Nuffield Department
of Clinical Medicine, University of Oxford,
Roosevelt Drive, Oxford OX3 7BN, UK
Fax: +44 1865 287 787
Tel: +44 1865 287 799
E-mail:
(Received 24 November 2009, revised 17
March 2010, accepted 29 March 2010)
doi:10.1111/j.1742-4658.2010.07665.x
Microbial pathogens exploit the ubiquitin system to facilitate infection and
manipulate the immune responses of the host. In this study, susceptibility
to Yersinia enterocolitica and Yersinia pseudotuberculosis invasion was
found to be increased upon overexpression of the deubiquitinating enzyme
otubain 1 (OTUB1), a member of the ovarian tumour domain-containing

protein family. Conversely, OTUB1 knockdown interfered with Yersinia
invasion in HEK293T cells as well as in primary monocytes. This effect
was attributed to a modulation of bacterial uptake. We demonstrate that
the Yersinia-encoded virulence factor YpkA (YopO) kinase interacts with a
post-translationally modified form of OTUB1 that contains multiple phos-
phorylation sites. OTUB1, YpkA and the small GTPase ras homologue
gene family member A (RhoA) were found to be part of the same protein
complex, suggesting that RhoA levels are modulated by OTUB1. Our
results show that OTUB1 is able to stabilize active RhoA prior to invasion,
which is concomitant with an increase in bacterial uptake. This effect is
modulated by post-translational modifications of OTUB1, suggesting a
new entry point for manipulating Yersinia interactions with the host.
Structured digital abstract
l
MINT-7717124: ypkA (uniprotkb:Q05608) physically interacts (MI:0915) with OTUB1 (uni-
protkb:
Q96FW1)byanti bait coimmunoprecipitation (MI:0006)
l
MINT-7717229: rhoA (uniprotkb:P61586) physically interacts (MI:0915) with OTUB1 (uni-
protkb:
Q96FW1)byaffinity chromatography technology (MI:0004)
l
MINT-7717075, MINT-7717207, MINT-7717193, MINT-771 7170: ypkA (uniprotkb:Q56921)
physically interacts (
MI:0915) with OTUB1 (uniprotkb:Q96FW1)byanti tag coimmunopre-
cipitation (
MI:0007)
l
MINT-7717390: ypkA (uniprotkb:Q56921) physically interacts (MI:0914) with OTUB1 (uni-
protkb:

Q96FW1) and RhoA (uniprotkb:P61586)byanti tag coimmunoprecipitation (MI:0007)
Abbreviations
HA-Ub-Br2, hemagglutinin-tagged ubiquitin-bromide; MOI, multiplicity of infection; OTUB1, otubain 1; Rac1, ras-related C3 botulinum toxin
substrate 1; RhoA, ras homolog gene family member A; USP, ubiquitin-specific protease; Yop, Yersinia outer protein; YpkA ⁄ YopO, Yersinia
serine ⁄ threonine kinase.
FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2515
by Yersinia enterocolitica [1]. Even though the plague
is not a major health concern today, cases are reported
annually. Moreover, Y. pestis was weaponized in the
former Soviet Union [2] and there are reports of
emerging multidrug resistant strains [3]. Pathogenic
Yersiniae are typically taken up through ingestion and
first reach the intestine. The Yersinia surface protein
invasin binds to b1 integrins on the apical surface of
M cells, which facilitates translocation across the
epithelium [4,5]. The pathogenicity and virulence of
Yersiniae is mainly based on the plasmid-encoded
type III secretion system that encodes for six effector
proteins, which are injected into the host cell (primarily
monocytes) to modulate the physiology of the infected
cell and to prevent uptake and killing (reviewed in
[6]). An additional chromosomally encoded Ysa type -
III secretion system has been described in Y. enterocol-
itica [7,8]. The injection of effector proteins promotes
Yersinia growth and survival in lymphoid follicles
(Peyer’s patches) underlying the intestinal epithelium
and controls antibacterial activities of immune cells
located at these sites. Four of these Yersinia outer pro-
teins (Yops) are engaged in modifying the cytoskele-
ton: YopE, YopH, YopT and YpkA [9–11]. YpkA, an

essential virulence factor, is a serine ⁄ threonine kinase
that phosphorylates actin [12], binds the deubiquitinat-
ing enzyme otubain 1 (OTUB1) [13,14], the small
G protein subunit Gaq [15] and interacts with mem-
bers of the Rho family of small GTPases, ras homo-
logue gene family member A (RhoA) and ras-related
C3 botulinum toxin substrate 1 (Rac1) [16]. Although
the interaction with actin, in particular G-actin, has
been shown to be crucial for YpkA serine ⁄ threonine
kinase activity, the functional relevance of the interac-
tion with OTUB1 remains to be determined [12,13,17].
YpkA-mediated phosphorylation of Gaq impairs
guanine nucleotide binding and subsequently inhibits
Gaq-mediated signalling pathways including RhoA
activation and cytoskeletal rearrangements in the host
cell [15]. In addition, a crystallography-based study
revealed that YpkA mimics host guanine nucleotide
dissociation inhibitors (GDIs), thereby blocking nucle-
otide exchange in RhoA and Rac1, a process that is
crucial for virulence in Yersinia [18]. YpkA therefore
uses several ways to interfere with the function
of small GTPases, which appears to be essential for
Yersinia pathogenesis [19].
The Rho family of small G proteins represents a
large group of the Ras superfamily of GTPases. More
than 20 proteins of this class have been described to
date, among which RhoA, Rac1 and Cdc42 are well
characterized, particularly their role in cytoskeletal
regulation. Specifically, RhoA is involved in the
formation of stress fibres and focal adhesion com-

plexes [20–23]. Yersinia is not the only pathogen that
affects the function of small GTPases such as RhoA
[24], indicating that interference with the function of
small GTPases is of prime importance in bacterial
pathogenesis because microbes have evolved a number
of virulence factors that modulate the function of
these proteins.
In this study, we show for the first time that suscep-
tibility to bacterial invasion by Yersinia can be altered
by changing expression of otubain 1 (OTUB1), a host
cell-encoded deubiquitinating enzyme that belongs to
the ovarian tumour domain-containing protein family.
This effect is dependent on the catalytic activity of
OTUB1 and its ability to stabilize the active form of
RhoA prior to invasion. YpkA and OTUB1 modulate
the stability of RhoA in opposing ways, therefore
leading to cytoskeletal rearrangements that may be
involved in bacterial uptake. During this process,
OTUB1 was found to be phosphorylated, a post-trans-
lational modification that modulates its ability to stabi-
lize RhoA. These findings provide a novel entry point
for the manipulation of host cell interactions with
Yersinia and perhaps other enterobacteria by deubiqui-
tination.
Results
OTUB1 controls cell susceptibility to Yersinia
invasion
Yersinia virulence factors are injected into target host
cell molecules to manipulate signalling pathways dur-
ing invasion in order to prevent uptake and killing. In

addition to actin, other host cell proteins have been
shown to bind to the virulence factor YpkA, including
OTUB1 [13]. In order to investigate the role of
OTUB1 in Yersinia invasion of HEK293T cells, we
established a cell culture invasion assay, in which the
effects of overexpression and knockdown of OTUB1
could be monitored. Bacterial uptake into HEK293T
cells was measured using a gentamicin-based invasion
assay (Fig. 1). Cells transfected with wild-type OTUB1
were infected with Y. enterocolitica and the number of
intracellular bacteria compared with the quantity
observed in cells overexpressing either a catalytically
inactive mutant C91S or an empty vector. We
observed that susceptibility to Y. enterocolitica inva-
sion was significantly increased upon overexpression of
wild-type OTUB1 in HEK293T cells, an effect that
was not seen when the catalytically inactive OTUB1
mutant (C91S) was expressed (Fig. 1A). A marked
increase in susceptibility was also observed upon
OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al.
2516 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS
overexpression of OTUB1 and invasion with Y. pseudo-
tuberculosis. Conversely, OTUB1 knockdown signifi-
cantly attenuates Yersinia invasion (Fig. 1B). We
repeated the OTUB1 knockdown experiment in pri-
mary human monocytes, which are among the first
cells targeted for Yersinia invasion in vivo, and this
also resulted in decreased invasion efficiency (Fig. 1C).
These differences could not be accounted for by
changes in cell viability or cell growth given the con-

trols and time frame of the experiment. To confirm the
initial observation by an alternate method, we used a
double fluorescence staining technique that enables
visualization of extracellular and intracellular bacteria
in the same cell [25]. The results concurred with the
data from the gentamicin-based invasion assay. The
ratio of intracellular to extracellular bacteria was much
higher in the case of cells overexpressing OTUB1 com-
pared with control cells or cells overexpressing a cata-
lytically inactive mutant of OTUB1 (CS91S, Fig. 2A).
Increased susceptibility to Yersinia in the presence of
overexpressed OTUB1 was observed as early as
15 min after invasion, and decreased over time, proba-
bly because of intracellular elimination. Taken
together, our results indicated that it was the efficiency
of bacterial uptake, not the proliferation of bacteria
within the host cell that is modulated by OTUB1
(Fig. 2B).
Post-translationally modified OTUB1 interacts
with the virulence factor YpkA
Previous evidence suggested that the Yersinia-encoded
virulence factor YpkA interacts with OTUB1 in vitro
[13], providing a potential molecular entry point to
explain this effect. We therefore aimed to validate this
result and examine whether this interaction also
occurs during bacterial invasion in living cells. To test
whether YpkA interacts with OTUB1, wild-type
YpkA and an inactive kinase mutant D267A were
overexpressed in HEK293T cells, followed by YpkA
OTUB1-HA

Ctrl
(EV)
84.8
5.1
OTUB1
wt
175.5
10.3
OTUB1
C91S
77.5
3.7
Infection (relative to control)
PDI
α-HA
α-PDI
OTUB1
PDI
OTUB1
PDI
siRNA
OTUB1
150
9.8
1.2
0.8
0.4
0
Ctrl
(EV)

150.8
7.8
Ctrl2
(sc)
142.3
9.2
siRNA
OTUB1
62.5
5.0
1.2
0.8
0.4
0
2.4
1.8
1.2
0.6
0
α-OTUB1
α-PDI
α-OTUB1
α-PDI
Ctrl
(sc)
243.5
1.3
P < 0.001
ABC
P < 0.001

P < 0.001
P < 0.001
P < 0.001
n = 4n = 6n = 10
Mean
SD (+/–)
# Colonies
Mean
SD (+/–)
Mean
SD (+/–)
Infection (relative to control)
Infection (relative to control)
Fig. 1. OTUB1 controls susceptibility to invasion by Yersinia enterocolitica. (A) HEK293T cells were transfected with empty vector (EV),
wild-type OTUB1-
HA
or the C91S mutant, followed by invasion with Yersinia enterocolitica (MOI 60 : 1). Gentamicin was added after 1 h to
kill extracellular bacteria. After 2 h, cells were lysed and dilutions plated and cultured for 2 days at 27 °C. Susceptibility to invasion was mea-
sured as the ratio between the numbers of colonies for OTUB1 (black bar), C91S mutant (grey bar) relative to the number obtained in the
control (white bar, set to as 1.0). Ten independent experiments were performed and the P-values are displayed as calculated using the
Student’s t-test. The mean and standard deviations of the absolute numbers of observed colonies are indicated. (B) Number of colonies
obtained relative to control when HEK293T cells were either transfected with empty vector (EV, white bar), transfected with negative scram-
bled control (sc, grey bar) or OTUB1 shRNA (black bar) for 24 h prior to Yersinia invasion. Six independent experiments were performed and
the P-values are displayed, as calculated using the Student’s t-test. The mean and standard deviations of absolute numbers of the observed
colonies are indicated. (C) Number of colonies obtained relative to control from primary monocytes that were previously isolated from human
peripheral blood mononuclear cells and were either transfected with negative scrambled control or transfected with OTUB1 shRNA for 24 h
(black bar) prior to invasion with Yersinia. Four independent experiments were performed and the P-values are displayed, as calculated using
the Student’s t-test. The mean and standard deviations of the absolute numbers of observed colonies are indicated.
M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion
FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2517

immunoprecipitation and separation by SDS ⁄ PAGE.
This was compared with a control immunoprecipitate
from cells transfected with empty vector, and the pres-
ence of OTUB1 was assessed by immunoblotting. We
observed that endogenous OTUB1 and YpkA are part
of the same protein complex (Fig. 3A). Inactivation of
the YpkA kinase activity by a D267A mutation did
not abolish this interaction. Moreover, this interaction
was also observed with endogenous YpkA present in
host cells during bacterial invasion (Fig. 3B). We
noted that multiple forms of OTUB1 can be detected,
as described previously [26,27], and that the form of
OTUB1 that co-immunoprecipitated with YpkA has
an apparent molecular mass of 37 kDa, corroborating
the findings of a previous study [13]. However, the
majority of endogenous OTUB1 protein is detected at
its expected molecular mass, 31 kDa (Fig. 3A, left).
We also observed increased levels of this higher molec-
ular mass form of OTUB1 in infected HEK293T cells
compared with control (Fig. 3C). Nevertheless, the
appearance of this form did not depend on YpkA
kinase activity (Fig. 3D). We therefore examined
whether this corresponds to the previously identified
alternative spliced form of OTUB1 referred to as
ARF-1, which has an apparent molecular mass of
35 kDa [26]. Overexpression of HSV-tagged ARF-1
was detected by anti-HSV, but not by OTUB1 immu-
noblotting, indicating that our antibody does not rec-
ognize ARF-1 (Fig. S1). We therefore hypothesized
that this form of OTUB1 may be post-translationally

modified, leading to a change in apparent molecular
mass and enhancing interaction with YpkA. Consis-
tent with this, treatment with protein phosphatase sug-
gested that the 37 kDa form of OTUB1 may contain
multiple phosphorylation sites, based on the observed
differential migration pattern (Fig. 3E). To further
shed light on the role of these OTUB1 modifications
in the invasion process, we embarked on identification
Ctrl (EV)
AB
OTUB1
15 min 30 min
60 min
TRITC – intracellular bacteria
FITC – extracellular bacteria
Ctrl (EV)
OTUB1
OTUB1 C91S
1.8
1.5
1.2
0.9
0.6
0.3
0
P < 0.001 P = 0.017 P = 0.023
Intracellular/extracellular bacteria
Fig. 2. OTUB1 expression levels affect bacterial uptake but not intracellular proliferation. (A) HEK293T cells were transfected either with
empty vector (EV), wild-type OTUB1-
HA

or the C91S mutant and after 24 h infected with Yersinia pseudotuberculosis (MOI 60 : 1) for 15, 30
and 60 min, followed by fixing and staining for extracellular bacteria using fluorescein isothiocyanate (FITC)-labelled Yersinia antibodies
(green). Cells were then permeabilized and stained with tetramethyl rhodamine iso-thiocyanate (TRITC)-labelled Yersinia antibodies to label
intracellular bacteria (red), followed by analysis using confocal microscopy. Pictures of the 30-min time point are shown. Control cells (upper,
EV) and cells overexpressing OTUB1-
HA
(lower, OTUB1) have different ratios of intracellular (tetramethyl rhodamine iso-thiocyanate-stained,
lower left compartment) versus extracellular bacteria (fluorescein isothiocyanate-stained, upper right compartment). The nuclei were visual-
ized using 4¢,6-diamidino-2-phenylindole staining (blue). (B) OTUB1-
HA
-overexpressing cells are characterized by a higher ratio of intracellu-
lar ⁄ extracellular bacteria in comparison with OTUB1-
HA
C91S mutant or control cells. This difference occurred as early as 15 min after
invasion with Yersinia. Three independent experiments were performed for the statistical analysis, and relative ratios between intracellular
(red) versus total ⁄ extracellular (green) bacteria are shown as well as the P-values calculated using the Student’s t-test.
OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al.
2518 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS
using a tandem mass spectrometry approach (LC-
MS ⁄ MS). Endogenous OTUB1 was isolated from
HEK293T cells, separated by SDS ⁄ PAGE and the
stained material subjected to in-gel trypsin digestion
and analysis by LC-MS ⁄ MS (Fig. 4A). An OTUB1-
derived N-terminal peptide containing three phos-
phorylation sites, Ser16, Ser18 And Tyr26 was identi-
fied. In addition, OTUB1 which was overexpressed in
HEK293T cells was isolated and analysed in a similar
manner, revealing a different N-terminal peptide that
contained the same phosphorylated residues (Fig. 4B).
Based on these results, OTUB1 mutants were gener-

ated in which Ser16, Ser18 and Tyr26 were replaced
with glutamic acid in order to mimic the negative
charge caused by phosphorylation (S16E, S18E and
Y26E). This approach was successfully used to imitate
phospho-serine and -threonine residues, but is to
some extent less ideal for phospho-tyrosines [28].
Interestingly, we observed that the OTUB1 Y26E and
S18E mutants exerted increased affinity to YpkA in
co-immunoprecipitation experiments, thereby resem-
bling the increased binding of the 37 kDa form of
OTUB1 to YpkA (Fig. 5A). This is consistent with the
notion that phosphorylation of OTUB1 affects the
interaction with YpkA, although the regulation might
be more complex, because the OTUB1 S16E ⁄ S18E ⁄
Y26E triple mutant did not show any increased bind-
ing to YpkA.
Mimicry of OTUB1 phosphorylation modulates
susceptibility to Yersinia invasion
If the interaction between OTUB1 and YpkA were
relevant for increased susceptibility to invasion, one
would expect that modification of OTUB1 may have
an effect on this process. To examine this, we repeated
Ctrl
10:1
MOI
37 kDa
OTUB1
-
Infected
37 kDa

25 kDa
hc
lc
50 kDa
-
*
YpkAFLAG
YpkAFLAG
37 kDa
20 kDa
50 kDa
100 kDa
hchc
lc
lc
OTUB1
37 kDa
EVEV WT WTD267A D267A
YpkA
YpkA
Input
OTUB1
α-OTUB1
37 kDa
25 kDa
Y. pseudotuberculosis
MOI 10:1
α-OTUB1
WB: α-FLAG
IP: α-FLAG (YpkA-FLAG)

A
CD E
B
IP: α-endogenous YpkA in
infected cells
WB: α-OTUB1WB: α-OTUB1
wt
D270A
-
31 kDa OTUB1
37 kDa OTUB1
CIP Phosphatase
+–
α-OTUB1
37 kDa
25 kDa
Exp. 1
Exp. 2
Fig. 3. Interaction between OTUB1 and YpkA in living cells and during Yersinia invasion. (A) Empty vector (EV), wild-type YpkA-
FLAG
, or the
YpkA-
FLAG
inactive kinase mutant D267A were transfected into HEK293T cells. After 24 h, cell extracts were prepared and YpkA material
immunoprecipitated using anti-FLAG Ig. Association with endogenous OTUB1 was demonstrated by immunoblotting using OTUB1 antibo-
dies in the presence of YpkA wild-type and D267A inactive kinase mutant. (B) HEK293T cells were infected with Y. pseudotuberculosis for
2 h. Cell extracts were prepared and YpkA immunoprecipitated using YpkA antibodies. In infected cells, association with endogenous
OTUB1 was demonstrated by anti-OTUB1 immunoblotting (hc, heavy chain; lc, light chain; *, a smaller form of OTUB1 was also detected).
(C) Modification of OTUB1 during Yersinia invasion. HEK293T were infected with Y. enterocolitica at an MOI of 10 : 1 for 2 h, followed by
cell lysis, separation by SDS ⁄ PAGE and anti-OTUB1 immunoblotting. (D) Modification of OTUB1 does not depend on YpkA kinase activity.

HEK293T cells were left untreated or infected with Y. pseudotuberculosis wild-type (wt) and YpkA kinase inactive mutant at an MOI of
10 : 1 for 2 h. Cell extracts were prepared and two forms of OTUB1 (31 kDa unmodified form and 37 kDa modified form) were visualized
using OTUB1 antibodies. (E) OTUB1 37 kDa form is phosphorylated. HEK293T cells were lysed and incubated with calf intestinal phospha-
tase (CIP) for 1 h, resulting in the appearance of multiple forms between 27 and 37 kDa, which indicates the presence of several phosphory-
lation sites. OTUB1 was visualized using anti-OTUB1 immunoblotting. Two independent experiments are shown.
M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion
FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2519
[M+3H]
3+
934.1 kDa
Ion counts [%]
m/z
50 kDa
37 kDa
25 kDa
-
OTUB1 IP
A
B
ESI-Ion trap MS/MS analysis of endogenous OTUB1
290.1
y2
418.1
y3
546.3
y4
617.3
y5
764.4
y6

877.5
y7
953.5
b16
++
1018.51
b17
++
1092.1
b18
++
1255.5
b21
++
1313.1
y21
++
-
97
1606.8
b13 - 64
1722.7
b14
1851.0
b15
0
0.5
1.0
1.5
4

x10
400 600 800 1000 1200 1400 1600 1800
961.9
b16
++
1191.1
y10
1230.6
y20
++
pY
pS
200 400 600 800 1000 1200 1400 1600 1800
0
100
[M+3H]
3+
966.56 kDa
764.39
y6
617.34
y5
338.16
b4
290.17
y2
175.13
y1
211.16
b2

418.23
y3
451.25
b5
546.31
y4
678.35
y11++
877.49
y7
1192.62
y10
1077.60
y9
985.52
y17++
1355.69
y11
1426.69
y12
1539.86
y13
1814.83
y15
1699.89
y14
948.55
y8
268.18
b3

1049.9
y18++
PLGSDSEGVNCLAYDEAIMAQQDR
y6 y2y3y4y7 y5
b3
y1y8y9y10y11y12y14
y
15
y13
y17y18
b2 b5
b4
3613
Intensity [%]
50 kDa
37 kDa
25 kDa
-
OTUB1
-HA
QTOF MS/MS analysis of overexpressed OTUB1-HA
m/z
pY
pS
HA IP
14
L G S D S E G V N C L A Y D E
A
I M A Q Q D R
36

y6 y2y3y4y7 y5y10
y20y21
b21b18b15 b17b16b13 b14
P
P
P
PP P
Fig. 4. Detection of OTUB1 phosphorylation using MS. (A) Detection of endogenous phosphorylated OTUB1. HEK293T cells were lysed,
followed by immunoprecipitation of OTUB1. As a control, lysate was incubated with agarose without the antibody. Immunoprecipitated
material was analysed by SDS ⁄ PAGE and silver staining, and the large band corresponding to the expected molecular mass of OTUB1 as
well as the area above (rectangle) was excised and digested with trypsin. Digested material was analysed by a nano-LC Ion Trap mass spec-
trometer. For the peptide 14–36 ([M + 2H]
2+
, 934.1 Da) containing the phosphorylated tyrosine and two serines, the b- and y-fragment ion
series are shown. (B) Detection of phosphorylated OTUB1 in an overexpression model. Control or HEK293T cells overexpressing OTUB1-
HA
wild-type were lysed, followed by immunoprecipitation of OTUB1. Eluted material was analysed by SDS ⁄ PAGE gel and Coomassie Blue
staining, and the band corresponding to a modified OTUB1 (rectangle) was excised and digested with trypsin. The peptide mixture was
analysed by a nano-UPLC-QTOF tandem mass spectrometer. For the peptide 13-36 ([M + 2H]
2+
, 966.6 Da) containing the phosphorylated
tyrosine and two serines, the b- and y-fragment ion series detected are shown.
OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al.
2520 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS
0.6
0.8
1.0
1.2
1.4
1.6

1.8
2.0
2.2
2.4
P < 0.001
Ctrl
(EV)
wt S16E S18E Y26E S16E
S18E
S16E
S18E
Y26E
-
wt S16E S18EY26E S16E
S18E
Y26E
S16E
S18E
OTUB1
α-HA
α-PDI
PDI
OTUB1
OTUB1
0
0.5
1.0
1.5
2.0
2.5

OTUB1 + probe
OTUB1
WT S16E S18EC91S Y26E S16E
S18E
S16E
S18E
Y26E
Ctrl (EV)
+++++ +++
0
0.5
1
1.5
WT Y26ECtrl (EV) Y26F
Exp 1
Exp 2
++++
OTUB1
Labelling ratio
(labelled/unlabelled)
α-HA
Probe (HA-Ub-Br2)
OTUB1 + probe
OTUB1
2.0
2.5
OTUB1 OTUB1
Infection (relative to control)
wtS16E S18E C91SY26E S16E
S18E

S16E
S18E
Y26E
YpkACtrl
++++++++–
α-HA
FLAG IP
OTUB1
α-FLAG
α-HA
α-PDI
OTUB1
PDI
YpkA
OTUB1
Input
YpkAA
B
C
α-HA
α-HA
Fig. 5. OTUB1 modification controls its function and its effect on Yersinia invasion. (A) Binding of YpkA to OTUB1 depends on OTUB1 modifi-
cation. Empty vector (EV control), OTUB1-
HA
wild-type, catalytically inactive mutant (C91S) or mutants mimicking phosphorylated OTUB1-
HA
(S16E, S18E, Y26E) were co-expressed with YpkA-
FLAG
in HEK293T cells. Cells were lysed and YpkA-
FLAG

immunoprecipitated with anti-FLAG
Ig. Binding of OTUB1 mutants to YpkA was measured by immunoblotting using HA antibodies. OTUB1 expression levels as well as the
loading control (PDI) were shown in the input, whereas YpkA-
FLAG
was visualized in immmunoprecipitated material. One representative out of
three experiments is shown. (B) OTUB1 modification affects bacterial invasion. HEK293T cells were transfected either with empty vector (EV
control), wild-type OTUB1-
HA
or mutants mimicking phosphorylated OTUB1 listed in Fig. 5A followed by invasion with Y. enterocolitica (MOI
60 : 1). Gentamicin was added after 1 h to kill extracellular bacteria. After 2 h, cells were lysed and dilutions plated and cultured for 2 days at
27 °C. The number of colonies for OTUB1 and the OTUB1 mutants were counted and presented relative to the number obtained for the
control (EV). The P-values were calculated using a Student’s t-test. Expression of OTUB1 in infected cells is shown using anti-OTUB1
western blotting and the loading control using anti-PDI western blotting. (C) Mimicry of phosphorylation on Tyr 26 interferes with OTUB1 func-
tion. HEK293T cells were transfected either with empty vector (EV), HA-tagged wild-type OTUB1, catalytically inactive OTUB1 C91S, mutants
mimicking phosphorylated OTUB1 (see above) or the Y26F mutant. Cells were lysed and extracts incubated with an HA-tagged ubiquitin Br2
probe to measure OTUB1 activity as described previously [30]. As a control, cells were treated the same way but without addition of the
probe. OTUB1 and OTUB1–probe adduct were visualized by immunoblotting using HA antibodies and quantified for two experiments (black
and grey bars). The intensities of the corresponding bands were measured and the ratio between them is shown (labelling ratio), reflecting
reactivity towards the probe. Two independent experiments are shown.
M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion
FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2521
the gentamicin-based invasion assay with cells overex-
pressing the OTUB1 mutants that mimic phosphoryla-
tion. Overexpression of the OTUB1 mutants S16E,
S18E, Y26E, S16E ⁄ S18E and S16E ⁄ S18E ⁄ Y26E abol-
ished the observed increase in susceptibility to invasion
seen with wild-type OTUB1 (Fig. 5B) or the S16A and
Y26F control mutants (data not shown), thereby con-
firming that modification of OTUB1 has an impact on
the magnitude of Yersinia invasion. Because no effect

on invasion was seen with the catalytically inactive
mutant C91S OTUB1 (Fig. 1), we set out to test
whether the constructed proteins mimicking phosphor-
ylated OTUB1 were functional by monitoring their
reaction with the deubiquitinating enzyme-specific
probe, hemagglutinin-tagged ubiquitin-bromide (HA-
Ub-Br2), which was previously shown to covalently
bind active OTUB1 [29,30]. Interestingly, the OTUB1
Y26E mutant did not react with the HA-Ub-Br2
active-site probe, whereas all other mutants were able
to do so (Fig. 5C). We conclude that phosphorylation
of OTUB1, in particular at Tyr26, modulates OTUB1
function by interfering with its enzymatic activity,
ubiquitin binding or substrate recognition. Next, we
examined whether OTUB1 phosphorylation may be
attributed to the Ser ⁄ Thr kinase activity of YpkA
directly. Recombinant OTUB1 and immunopre-
cipitated YpkA expressed in HEK293T cells were
incubated in a radioactive in vitro kinase assay.
Recombinant OTUB1 was weakly phosphorylated by
YpkA, consistent with previous findings, but to a
much lesser degree than the control protein myelic
basic protein (Fig. S2A). By contrast, OTUB1 isolated
from cell lysates was not phosphorylated by YpkA at
a detectable level, although wild-type YpkA was read-
ily autophosphorylated and therefore active (Fig. S2B).
These results indicate that modification of OTUB1 by
phosphorylation has an effect on OTUB1-mediated
Yersinia bacterial uptake, but did not resolve the
relevance of YpkA’s Ser ⁄ Thr kinase activity in this

process.
OTUB1-mediated susceptibility to invasion is
modulated by the YpkA GTPase-binding domain
YpkA consists of several domains including a serine ⁄
threonine kinase and a GTPase-binding domain, both
of which contribute to virulence [6] (Fig. 6A). In order
to dissect which of these functionalities contribute to
OTUB1-mediated susceptibility to invasion, we used
Yersinia strains that either had mutations in the kinase
(ypkA
D270A
) or GTPase-binding domain (Yersinia con-
tact A mutant strain) [18]. OTUB1-mediated suscepti-
bility to invasion with the Yersinia ypkA
D270A
strain
was unaltered, but was compromised with the con-
tact A mutant strain (Fig. 6B). These results show that
the YpkA GTPase-binding domain, but not the Ser ⁄
Thr kinase activity, interferes with susceptibility to
Yersinia invasion provoked by overexpression of
OTUB1 in host cells.
Previous experiments have demonstrated an interac-
tion between YpkA and the small GTPases RhoA or
Rac1 [16,18]. Our data suggest that the ability of
YpkA to bind GTPases may be critical for the
OTUB1-mediated increased Yersinia uptake. We there-
fore tested whether YpkA and RhoA interact in vitro
and whether this protein complex includes OTUB1.
YpkA was immunoprecipitated and the presence of

OTUB1 and RhoA examined by immunoblotting
(Fig. 6C). YpkA, OTUB1 and RhoA were found to be
part of the same complex. Moreover, OTUB1 is asso-
ciated with RhoA in the absence of YpkA, as demon-
strated by co-immunoprecipitation of OTUB1 and
RhoA (Fig. 6C, lane 3).
OTUB1 stabilizes active RhoA
The existence of all three components in the same
complex and the association between OTUB1 and
RhoA suggested that OTUB1 might play a role in
modulating the ubiquitination status and stability of
RhoA. In order to investigate this, we expressed both
proteins in HEK293T cells and examined the polyubiq-
uitination status and the stability of RhoA by immu-
noprecipitation ⁄ western blotting experiments (Fig. 7A–
C). When OTUB1 was overexpressed, the total amount
of RhoA increased marginally. The same observation
was made for endogenous RhoA levels which were
elevated upon overexpression of OTUB1 (Fig. 7A).
However, levels of endogenous active (GTP-bound)
RhoA isolated from noninfected cells using a rhotekin-
based pulldown were stabilized considerably by
OTUB1, but not by a catalytically inactive OTUB1
C91S mutant (Fig. 7B). This was not accounted for by
an increase in RhoA activation through its guanine
nucleotide exchange factor LARG, for which a mar-
ginal increase was noted in the presence of wild-type
and catalytically inactive OTUB1 (Fig. 7B, lower). A
more striking effect was observed when immunoprecip-
itated RhoA was incubated with recombinant OTUB1

in vitro (Fig. 7C). The experiment was performed by
expressing a constitutively active RhoA (Q63L mutant)
to enrich for polyubiquitinated material. The quantity
of ubiquitinated RhoA was significantly decreased in
presence of wild-type OTUB1, whereas levels of
unmodified RhoA increased with time. The catalyti-
cally inactive mutant OTUB1 C91S was unable to
OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al.
2522 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS
deubiquitinate RhoA (Fig. 7C right). These results
clearly indicate that OTUB1 is responsible for stabil-
ization of active RhoA and that it is dependent on the
deubiquitinating activity of the enzyme (Fig. 7B).
Correlation between RhoA stabilization and
enhanced susceptibility to Yersinia invasion
Because RhoA has been shown previously to be impli-
cated in modulating host–pathogen interactions by
regulating cell morphology and uptake [31,32], our
results raised the question of whether OTUB1-medi-
ated enhanced susceptibility to invasion may involve
RhoA. To examine this in further detail, we first tested
whether levels of the GDP- or GTP-bound form of
RhoA are affected during invasion. A rhotekin-based
pulldown assay was used to isolate the active form
of RhoA from infected and noninfected cells. The
amount of active RhoA is substantially increased when
OTUB1 was overexpressed, but not during invasion
(Fig. 7D). Therefore, overexpression of OTUB1 does
stabilize active RhoA prior to, but not after, invasion.
Co-transfection experiments revealed that YpkA alone

counteracts OTUB1-mediated stabilization of RhoA
(Fig. 7D), therefore identifying two factors that have
an opposing effect on RhoA function and stability.
Finally, to underscore the relevance of OTUB1-medi-
ated stabilization of RhoA in enhanced susceptibility
to invasion, we tested whether OTUB1 mutants
mimicking phosphorylation were able to stabilize
2.5
2.0
1.5
1.0
0
0.5
2.5
2.0
0
1.5
1.0
0.5
Yersinia wtYersinia D270A
Yersinia wt
Yersinia
Contact A mutant
n = 6n = 3
OTUB1-HA
YpkA-FLAG
RhoA-Myc
+++
+++
++

IP: HA
IP: FLAG
+
+
+++
+
α
-HA
α
-FLAG
α
-Myc
-
-
-
-
-
-
OTUB1
YpkA
RhoA
N term. domain Kinase domain GTPase binding domain Actin activation domain
***
559591072
*
599
4345111
A C
B
815 732

P < 0.001 P < 0.001
P = 0.003
P = 0.015
EV
(ctrl) C91S
EV
(ctrl)
OTUB1 OTUB1
C91S
P < 0.001
P < 0.001
EV
(ctrl)
OTUB1 OTUB1
OTUB1 OTUB1
C91S
EV
(ctrl)
OTUB1 OTUB1
C91S
Infection (relative to control)
Infection (relative to control)
Fig. 6. OTUB1-mediated susceptibility to invasion requires YpkA and its GTPase-binding domain, but not its serine ⁄ threonine kinase activity.
(A) Scheme of the domains present in YpkA. Mutated amino acid positions in the mutant strains used in this study are indicated.
(B) Increased susceptibility to Yersinia invasion is not dependent on YpkA-mediated phosphorylation. Control HEK293T cells, HEK293T cells
overexpressing OTUB1-
HA
or OTUB1-
HA
C91S were infected with either wild-type Y. pseudotuberculosis or Y. pseudotuberculosis mutants

containing an inactive kinase domain (D270A) or the YpkA contact A mutant (unable to bind GTPases). Cells were collected after 3 h, lysed
and cell extracts plated on agar plates. Colonies were counted after 2 days of incubation at 27 °C and the colony numbers were displayed
as ratios relative to the control. Experiments were performed at least three times and the P-values were calculated using a Student’s t-test.
For each strain, susceptibility to invasion was measured as the ratio between the numbers of colonies for OTUB1 (black bar), C91S mutant
(grey bar) relative to the number obtained in untransfected cells (EV, white bar, set to as 1.0). (C) YpkA is in a complex with RhoA and
OTUB1. Cells were co-transfected with OTUB1-
HA
, RhoA-
myc
and YpkA-
FLAG
, followed by immunoprecipitation with HA or FLAG antibodies.
OTUB1-
HA
, RhoA-
myc
and YpkA-
FLAG
were visualized by immunoblotting using OTUB1, RhoA or FLAG antibodies, respectively.
M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion
FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2523
active RhoA (Fig. 7E). Overexpression of the OTUB1
mutants S16E, S18E, Y26E, S16E ⁄ S18E and
S16E ⁄ S18E ⁄ Y26E did not rescue active RhoA levels to
the same extent as observed with wild-type OTUB1,
thereby corroborating their effect on enhanced suscep-
tibility to invasion (Fig. 5B).
01530 030
RhoA
+++

min
Poly Ub
RhoA Q63L
RhoA Q63L
+++ ––
α-RhoA
α-Ub
EV
Ctrl
OTUB1 C91S
Poly Ub
RhoA Q63L
Active RhoA
Inactive RhoA (Ft)
EV OTUB1
InfectionControl
Active RhoA
Inactive RhoA (ft)
YpkA-FLAG
Ctrl HA plasmid
Ctrl FLAG plasmid
YpkA-FLAG
OTUB1-HA
+
++
+
++
α-RhoA
α-RhoA
α-HA

EV OTUB1
α-FLAG
+
OTUB1-HA
RhoA-myc
-
+
+
α-myc
α-HA
α-PDI
α-RhoA
OTUB1-HA
RhoA-endog
+
OTUB1 endogenous
OTUB1-HA
α-OTUB1
PDI
RhoA
OTUB1-HA
α-RhoA
α-RhoA
α-PDI
α-LARG
α-HA
LARG
OTUB1-HA
OTUB1-HA
Ctrl (EV)

A
C
DE
B
++
Ctrl (EV)
++
Input
PDI
Input
OTUB1
wt
S16E, S18E, Y26E
Y26E
S16E, S18E
S16E
S18E
Active RhoA
Inactive RhoA (ft)
PDI
OTUB1-HA
Input
α-RhoA
α-RhoA
α-HA
α-PDI
-
01530
RhoA
min01530

α-RhoA
Poly Ub
RhoA Q63L
α-OTUB1
OTUB1
OTUB1
OTUB1 C91S
OTUB1
Fig. 7. OTUB1 stabilizes active RhoA. (A) Protein lysates from HEK293T cells co-transfected with RhoA wild-type and OTUB1-
HA
wild-type or
control plasmid (EV) were subjected to RhoA detection by immunoblotting. Levels of RhoA (transfected RhoA-
myc
, left; endogenous RhoA,
right) were increased if cells were co-transfected with OTUB1-
HA
but not in the presence of empty vector (EV). Loading control is shown using
anti-PDI western blotting. (B) OTUB1 stabilizes active RhoA. Endogenous active RhoA was isolated using Rhotekin-coupled beads from
HEK293T cells overexpressing either empty vector (EV), OTUB1-
HA
wild-type or C91S mutant. LARG, PDI (loading control) and OTUB1-
HA
wild-
type were visualized using western blotting of the input material. (C) OTUB1 deubiquitinates RhoA in vitro. Purified ubiquitylated RhoA isolated
from HEK293T cells previously transfected with the constitutively active mutant RhoA-
myc
QL63 was incubated with recombinant wild-type
OTUB1 (both panels) the catalytically inactive mutant C91S (right) for 0, 15 and 30 min at 37 °C. RhoA deubiquitination was visualized by anti-
ubiquitin and anti-RhoA immunoblotting. (D) OTUB1-mediated stabilization of active RhoA is impaired during invasion and if co-expressed with
YpkA. Active RhoA was enriched using recombinant Rhotekin from HEK293T cells overexpressing empty vector (EV), OTUB1-

HA
wild-type,
infected or not with Y. pseudotuberculosis for 3 h (left) or from HEK293T cells overexpressing YpkA-
FLAG
alone or together with OTUB1-
HA
or
the control plasmids (HA and FLAG plasmids, right). The loading control (PDI), OTUB1-
HA
and YpkA-
FLAG
were visualized by western blotting of
the input material. (E) Mimicry of OTUB1 phosphorylation impairs its ability to stabilize active RhoA. Active RhoA was enriched using recombi-
nant Rhotekin from HEK293T cells overexpressing empty vector (EV), OTUB1-
HA
wild-type or the mutants S16E, S18E, Y26E, S16E ⁄ S18E and
S16E ⁄ S18E ⁄ Y26E mimicking OTUB1 phosphorylation. The loading control (PDI) and OTUB1-
HA
were visualized by western blotting of the input
material. Moreover, RhoA in the flow through material (ft) was also visualized. One out of two experiments is shown.
OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al.
2524 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS
Discussion
This study describes a role for the host cell-encoded
deubiquitinating enzyme, OTUB1, in modulating cell
susceptibility to bacterial invasion. OTUB1 has been
shown to disassemble lys48-linked polyubiquitin chains
[14,29,33], and is involved in anergy induction in
CD4
+

lymphocytes through its interaction with the E3
ligase gene related to anergy induction in lymphocytes
(GRAIL) and ubiquitin-specific protease (USP)8 [26].
However, OTUB1 is expressed ubiquitously in most
tissues, which suggests its involvement in other cell
biological processes not restricted to lymphoid tissues.
Indeed, OTUB1 was suggested to stabilize estrogen
receptor alpha levels in breast and endometrial cancer
cells [27]. In addition, previous evidence suggested that
OTUB1 may be linked to Yersinia invasion based on
its reported interaction with the Yersinia-encoded viru-
lence factor YpkA, and it was also proposed that
OTUB1 may be a substrate for its serine ⁄ threonine
kinase activity, at least in vitro [13]. Consistent with
this, we found YpkA to be present in the same protein
complex as OTUB1 in living cells and during bacterial
invasion, as assessed by co-immunoprecipitation.
Moreover, we confirmed that YpkA can phosphorylate
OTUB1 in vitro (Fig. S1A). As observed previously, we
also noted that the form of OTUB1 interacting with
YpkA has an approximate molecular mass of 37 kDa,
which is different from its expected molecular mass of
31 kDa (Fig. 3). Our data demonstrate that endoge-
nous OTUB1 is modified by phosphorylation in living
cells (Fig. 4). However, our results question the in vivo
relevance of OTUB1 phosphorylation by YpkA which
has been observed in vitro. First, a 37 kDa form of
OTUB1 can be detected in addition to its normally
expected size at 31 kDa in HEK293T cells indepen-
dently of bacterial invasion (Fig. 3A, input material) or

YpkA expression (Fig. 3C,E). Second, OTUB1, as a
37 kDa polypeptide, was also found in a complex with
the inactive kinase mutant YpkA D267A (Fig. 3A).
Third, invasion with the Yersinia mutant strain express-
ing an inactive YpkA kinase (D270A) did not affect
OTUB1-mediated susceptibility to invasion (Fig. 6B).
Fourth, we did not observe YpkA-mediated phosphor-
ylation of OTUB1 that was isolated from HEK293T
cells, but noted a slight increase in YpkA autophospho-
rylation in the presence of OTUB1 (Fig. 1B, lower).
Our results indicate that OTUB1 phosphorylation is an
YpkA-independent event that is, however, crucial for
their interaction. Further investigation using MS con-
firmed the presence of three phosphorylated residues in
endogenous and overexpressed OTUB1 isolated from
HEK293T cells (Fig. 4) consistent with the fact that
multiple endogenous forms of OTUB1 were observed
(Fig. 3E). Sequence analysis did not reveal any typical
kinase consensus sites, so it is currently unknown what
physiological process and which kinases are involved in
OTUB1 phosphorylation. These modifications alone do
not fully account for the apparent molecular mass shift
observed with the 37 kDa form of endogenous OTUB1
(see Fig. 4, left), indicating that this form may harbour
additional post-translational modifications that escaped
our detection. However, OTUB1 mutants mimicking
phosphorylation appear to have similar biochemical
properties as the naturally occurring 37 kDa form of
OTUB1, in particular the S18E and Y26E variants,
both of which exert increased affinity to YpkA. The

effect of these modifications on OTUB1 binding to
YpkA did not fully account for loss of increased sus-
ceptibility to bacterial invasion. Alternatively, these
modifications may also change OTUB1 deubiquitina-
tion activity, affinity to or recognition of substrates.
Consistent with this, we observed that only wild-type
OTUB1 was able to stabilize active RhoA, whereas
mutations mimicking phosphorylation abrogated this
effect (Fig. 7E). Modification of Y26 in OTUB1 inter-
fered with active site labelling by the Ub-Br2 probe,
suggesting impaired deubiquitination function, whereas
mutations at positions S16 and S18 may alter substrate
binding. The lack of stabilizing active RhoA by
OTUB1 mutants correlated with their inability to sus-
tain susceptibility to invasion (compare Figs 5B and
7E), indicating that controlling active RhoA levels is
important for the magnitude of invasion, in line with
previous observations [32]. Phosphorylation of deubiq-
uitinating enzymes may be common and has been
observed previously for USP7 [34], USP8 [35] and
CYLD, the latter of which is also functionally altered
through this modification [36].
YpkA is a multifunctional protein that interferes
with host cell functions at several levels during Yersinia
invasion [19]. In addition to its serine ⁄ threonine kinase
activity [13,15,37] and binding to actin [12], YpkA has
been shown to interact with small GTPases and inhibit
nucleotide exchange in Rac1 and RhoA, mimicking the
guanidine nucleotide dissociation inhibitors of the host
[16,18]. Full virulence of Yersinia depends on all of

these properties mediated by YpkA, because mutations
or deletions in either the kinase or GTP-binding
domains reduce the pathogenicity of these strains
[18,37,38]. By contrast, null mutations in ypkA in
Y. pseudotuberculosis appear to be similar to wild-type
in their virulence, a trait that is thought to result from
a possible compensatory mechanism evolving in this
strain [18,39,40]. Our findings are consistent with the
former observation in that the wild-type strain had the
M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion
FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2525
highest invasion efficiency (data not shown), and our
results suggest a link between the GTPase-binding
capacity of YpkA and OTUB1-mediated increase in
susceptibility to infection (Fig. 6). The binding of small
GTPases has been reported to be independent of the
kinase activity of YpkA [16]. In line with this, we
detected RhoA in immunopurified YpkA and OTUB1
complexes. We also noted an interaction between
OTUB1 and RhoA in the absence of YpkA (Fig. 6C)
and therefore hypothesized that RhoA may be a sub-
strate for deubiquitination by OTUB1, a process that
may be modulated by YpkA during invasion. Indeed,
we showed that RhoA is stabilized by OTUB1
(Fig. 7A–D). This can be achieved either by induction
of RhoA mRNA expression, deubiquitination of RhoA
itself or promoting activation of RhoA, possibly
through the manipulation of RhoA-specific guanine
nucleotide exchange factors. Real-time PCR analysis
showed that OTUB1 overexpression did not alter

RhoA mRNA levels (Fig. S3). Distinguishing between
the two latter possibilities proved to be more challeng-
ing. Our results indicate that RhoA can be deubiquiti-
nated by OTUB1 directly, but not by the catalytically
inactive mutant (Fig. 7C). This is supported by the fact
that the catalytically inactive OTUB1 mutant also fails
to stabilize RhoA (Fig. 7B). However, we cannot
exclude any other mechanisms, such as deubiquitina-
tion and stabilization of RhoA-specific guanine nucle-
otide exchange factors, although LARG does not
appear to be significantly affected by OTUB1 (Fig. 7B)
[41]. YpkA interferes with this process by reducing
active RhoA levels (Fig. 7D). In general, YpkA does
not appear to have any effect on OTUB1 deubiquiti-
nating activity (data not shown), but it may bind and
sequester post-translationally modified OTUB1 and
GDP bound RhoA to interfere with active RhoA for-
mation, and perhaps provoke premature degradation
of RhoA during bacterial invasion.
Our results indicate that in absence of bacterial
invasion OTUB1 prolongs the lifetime of the active
(GTP-bound) form of RhoA, because this form is rap-
idly ubiquitinated and turned over [42,43]. Increased
susceptibility to invasion provoked by OTUB1 over-
expression seems to be dependent on stabilization of
active RhoA by OTUB1 prior to bacterial invasion.
The accumulated pool of active RhoA contributes to
an enhanced uptake in the early phase of invasion, con-
sistent with the involvement of the microtubule system
and GTPases in this process [32]. Once YpkA is present

in the infected host cell, further active RhoA formation
is blocked (Fig. 7D) [6], which may prevent further
bacterial uptake. Interestingly, we observed high levels
of OTUB1-mediated bacterial uptake that decreased
after prolonged invasion times (Fig. 2B). This may
reflect a decrease in the efficiency of bacterial uptake
once intracellular bacteria are present possibly com-
bined with intracellular elimination. RhoA as well as
Rac1 and Cdc42 are involved in modulating cytoskele-
tal rearrangements and endocytosis [44], further con-
firming that OTUB1-mediated stabilization of RhoA
could affect bacterial entry into the host. In line with
this, OTUB1 overexpression appears to also affect the
stability of Rac1 and Cdc42 (unpublished data). YpkA
and other Yersinia-encoded virulence factors target
small GTPases to limit bacterial uptake in order to pre-
vent internalization and killing. In addition, a different
GTPase targeted by YpkA-mediated phosphorylation,
Gaq, may also be implicated in limiting bacterial
uptake [15,45]. YpkA may therefore use both its kinase
and guanine nucleotide dissociation inhibitor domains
to interfere with RhoA activation more effectively [19].
In summary, our findings reveal a new aspect of the
complex interplay in host–pathogen interactions and
demonstrate a physiological role of the deubiquitinat-
ing enzyme OTUB1 in Yersinia invasion. OTUB1 as a
potential key player in regulating RhoA stability may
represent a novel pharmacological target for yersinio-
sis, but may also be linked to the biology of RhoA-
mediated regulation of cell morphology, adhesion and

migration in general.
Materials and methods
Cell lines and reagents
The HEK293T cell line was maintained in Dulbecco’s
modified Eagle’s medium (DMEM) containing 10% fetal
bovine serum, 1% glutamine and 100 lgÆmL
)1
streptomycin
and penicillin in a humidified atmosphere of 5% CO
2
at
37 °C. Primary peripheral blood mononuclear cells were
prepared from buffy coats (National Blood Centre,
London, UK) using a standard lymphoprep-based method
(Axis-Shield PoC AS, Dundee, UK) and subsequent isola-
tion of monocytes using CD14 microbeads (Miltenyi Biotec,
Bergisch Gladbach, Germany) was carried out according to
the manufacturer’s instructions. Chemicals were purchased
from Sigma-Aldrich (St Louis, MO, USA), unless indicated
otherwise. The antibodies used in this study are described
in the Supporting information.
DNA constructs
The cDNA for human OTUB1 and OTUB1-
HA
C91S was
obtained as described previously [29]. The OTUB1-
HA
S16E, S16A S18E, Y26E, Y26F, S16E ⁄ S18E and S16E ⁄
S18E ⁄ Y26E mutants were created using the QuikChange II
OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al.

2526 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS
site-directed mutagenesis kit by Stratagene (La Jolla, CA,
USA). The initial OTUB1 mutants S16E, S16A, S18E,
Y26E and Y26F were generated using the OTUB1
pcDNA 3.1 construct containing a C-terminal HA-TEV-
SBP tag and the primers described in Table S1. The
S16E ⁄ S18E double mutant was generated using the OTUB1
S16E mutant construct as a template. The
S16E ⁄ S18E ⁄ Y26E triple mutant was generated using the
OTUB1 S16E ⁄ S18E construct as a template. All OTUB1
mutant constructs were verified by sequencing.
The siRNAs specific for OTUB1 and a negative control
(scrambled, SI 03650318, All Stars negative control) were
purchased from Qiagen (Crawley, UK) and tested for their
ability to knockdown endogenous OTUB1 (data not
shown). The best siRNA (OTUB1_3 SI00676053) that has
no reported off-target effects (information by the manufac-
turer) was used for this study. The RhoA-specific constructs
RhoA-
myc
L63 (Q63L) and wild-type RhoA (both in pEXV
Amp-R) were generously provided by M. Olson (Glasgow,
UK). The YpkA-
FLAG
wild-type and D267A constructs
were a kind gift from L. Navarro and J.E. Dixon (UCLA,
Los Angeles, CA, USA). The OTUB1 ARF-1 construct
was a gift from C.G. Fathman (Stanford University, Palo
Alto, CA, USA).
Yersinia strains

The Yersinia strains used in this study were Yersinia pseu-
dotuberculosis YPIII (pIB102) wt (Km-R), YPIII (pIB44),
YPIII (pIB47) YpkA D270A (Tc-R), contact A mutant
strain of Yersinia pseudotuberculosis (IP2777) containing
Y591A, N595A and E599A mutations and Yersinia entero-
colitica (Ye 8081). Strains were cultured in Lysogeny broth
(Sigma-Aldrich, St Louis, MO, USA) at 27 °C overnight at
200 rpm.
Transfection assays
HEK293T cells were grown to confluence in DMEM con-
taining 10% fetal bovine serum, 1% glutamine and 1%
penicillin ⁄ streptomycin. Cells were then transferred to 150,
100, 50 mm or six-well tissue culture dishes at a concentra-
tion of 0.4 · 10
6
mL
)1
and grown overnight at 37 °C. The
cells were then washed with NaCl ⁄ P
i
and the transfection
was performed using SuperFect reagent (Qiagen), according
to the manufacturer’s protocol, followed by an overnight
incubation at 37 °C. For experiments in which overexpres-
sed proteins were subsequently deubiquitylated in vitro, cells
were treated with 10 lm proteasome inhibitor MG132
(Sigma-Aldrich) for 6 h in order to interfere with proteaso-
mal degradation and accumulate polyubiquitylated
proteins. For siRNA studies, cells were prepared in a
similar way as for gene overexpression, but transfections

were performed with HiperFect (Qiagen), and the cells were
grown for 24 h at 37 °C.
Primary monocytes isolated from a buffy coat (National
Blood Centre) were transfected using the Amaxa Nucleofec-
tor system (Amaxa ⁄ Lonza, Cologne, Germany) with the
Human Monocyte Nucleofector Kit (Amaxa) according to
the instructions provided by the manufacturer, and grown
for 10 h post-transfection in Human Monocyte Nucleo-
fector Medium (Amaxa).
Bacterial invasion assay
HEK293T cells (5 · 10
6
per sample) were incubated for
12–16 h after transfection, washed with NaCl ⁄ P
i
and incu-
bated in DMEM without fetal bovine serum and antibiotics
during invasion. A Y. pseudotuberculosis or Y. enterocolitica
overnight culture at 27 ° C was diluted 1 : 20 and incubated
further for 2 h at 27 °C, and then for 1 h at 37 °C. Bacteria
were washed in NaCl ⁄ P
i
and used to infect cells at an mul-
tiplicity of infection (MOI) of 60 : 1 for 1 h at 37 °C. In
order to obtain comparable MOIs for the Y. pseudotubercu-
losis wild-type and different mutant strains, dilutions series
of bacterial cultures were used to infect cells. Thereafter,
cells were washed three times with NaCl ⁄ P
i
and further

incubated for 2 h at 37 °C in DMEM supplemented with
10% fetal bovine serum and 100 lgÆmL
)1
gentamicin in
order to kill extracellularly located bacteria. Invasion of
primary monocytes and U937 cells were conducted in the
same way using RPMI-1640 medium. In order to measure
cell susceptibility to invasion, cells were lysed in 0.5%
NP-40, 150 mm NaCl, 5 mm CaCl
2
,50mm Tris pH 7.4,
and the dilutions were plated Yersinia selective agar base
(Sigma-Aldrich) containing Yersinia selective supplement
(Sigma-Aldrich) and cultured for 2 days at 27 °C. Colony
numbers were counted and a statistical analysis (Student’s
t-test) was performed using sigmaplot software (Systat
Software Inc, Salisbury, UK).
Immunoblotting and immunoprecipitation
Cells (5 · 10
6
per sample) were lysed in 0.1% NP-40,
150 mm NaCl, 20 mm CaCl
2
,50mm Tris pH 7.4 contain-
ing a protease inhibitor cocktail (Roche Applied Science,
Basel, Switzerland). Samples were separated by SDS ⁄ PAGE
and subjected to immunoblotting as described in the Sup-
porting information. For immunoprecipitation protein
lysates (5 mg per sample) were first diluted in NET buffer
(50 mm Tris, 5 mm EDTA, 150 mm NaCl, 0.5% NP-40,

pH 7.4) to a protein concentration of 1 mgÆmL
)1
, and pre-
cleared with agarose-coupled Protein A beads (Sigma-
Aldrich) for 1 h at 4 °C. Immunoprecipitation was then
carried out either for 2 h or overnight at 4 °C.
Analysis of OTUB1 by tandem MS
For analysis of the endogenous OTUB1, HEK293T cells
were grown to confluence in DMEM in 175 cm
2
tissue
M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion
FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2527
culture flasks. The cells were then washed in NaCl ⁄ P
i
,
lysed in 0.1% NP-40, 150 mm NaCl, 20 mm CaCl
2
,
50 mm Tris pH 7.4 containing protease inhibitor cocktail
(Roche Applied Science) and 100 lm sodium orthovana-
date (Sigma-Aldrich). For immunoprecipitation, protein
lysates (100 mg per sample) were first diluted in NET
buffer to a protein concentration of 1 mgÆmL
)1
, and pre-
cleared with Protein A agarose (Sigma-Aldrich) beads for
1 h at 4 °C. Mouse OTUB1 mAb was added in dilution
1 : 1000 and the immunoprecipitation was then carried
out overnight at 4 °C, followed by incubation with Pro-

tein A agarose for 2 h at 4 °C to couple the beads. Mate-
rial was eluted using 100 mm glycine pH 2.5, precipitated
using chloroform and methanol, separated by SDS ⁄ PAGE
and visualized using silver staining, as described in
Edelmann et al. [29]. Gel bands that were unique to lanes
containing OTUB1 as well as the corresponding areas in
the control lane were excised and subjected to in-gel
digestion with trypsin and analysis by nano-LC-MS ⁄ MS
as described in [46]. The analysis of overexpressed
OTUB1 by LC-MS ⁄ MS is described in the supplementary
material.
RhoA activation assay
Cells (5 · 10
6
per sample) overexpressing either wild-type
OTUB1, the C91S mutant or an empty control vector
(pEF-IRES P) were lysed in 100 mm plates with cold lysis
buffer (25 mm Tris ⁄ HCl pH 7.4, 5 mm MgCL
2
, 1% NP-40,
1mm dithiothreitol, 5% glycerol) containing a protease
inhibitor cocktail (Pierce, Rockford, IL, USA). The protein
concentration was determined by a Lowry protein concen-
tration assay (BCA; Bio-Rad, Hemel Hempstead, UK) and
equal amounts of protein lysate were used for each reac-
tion. This was followed by immunoprecipitation of acti-
vated RhoA using Rhotekin and immobilized glutathione
discs according to the manufacturer’s instructions of the
EZ-Detect Rho Activation Kit (Pierce). Samples enriched
with active RhoA, as well as the flow-through, were sepa-

rated by SDS ⁄ PAGE and RhoA was detected via immuno-
blotting.
Fluorescence microscopy
Double staining of intra- and extracellular Yersinia was per-
formed essentially as described previously [25]. HEK293T
cells were seeded in a 12-well plate (2 · 10
5
well
)1
) on cover-
slips and grown for 12 h in DMEM supplemented with
10% fetal bovine serum, 1% glutamine and 1% penicil-
lin ⁄ streptomycin. Cells were then transfected using Super-
Fect (Qiagen) and infected on the following day. Prior to
invasion cells were washed with NaCl ⁄ P
i
and incubated in
DMEM without fetal bovine serum and antibiotics for
30 min. A Y. pseudotuberculosis overnight culture was
diluted 1 : 20 and incubated for another 2 h at 27 °C, and
then for 1 h at 37 °C. Bacteria were washed in NaCl ⁄ P
i
and
cells were infected with Yersinia at an MOI of 60 : 1 for the
indicated time course at 37 °C. Thereafter cells were washed
three times with NaCl ⁄ P
i
and fixed at room temperature
with 3% paraformaldehyde for 10 min. Fixed cells were
washed with cold NaCl ⁄ P

i
, blocked for 30 min with 1%
BSA, washed three times with NaCl ⁄ P
i
and incubated with
primary Y. pseudotuberculosis antibodies for 45 min. Cells
were washed and incubated with fluorescein isothiocyanate
conjugated rabbit secondary antibodies for 45 min in the
dark, followed by washing and permeabilization in 2%
Triton X-100 for 4 min. A second antibody-based staining
was performed using tetramethyl rhodamine iso-thiocyanate
rabbit secondary antibodies. Cells were washed and briefly
dried and mounted on coverslips in Vectashield HardSet
Mounting Medium with 4¢,6-diamidino-2-phenylindole
(Vector Laboratories, Burlingame, CA, USA), and sealed
with nail polish. The images were taken and analysed using
a confocal microscope (Zeiss LSM510 Meta Confocal Imag-
ing System, Jena, Germany). For the statistical analysis to
determine the ratio between intracellular and extracellular
bacteria the experiment was repeated three times and the
numbers of bacteria associated with at least 300 cells were
counted.
Acknowledgements
We would like to thank Dr B.W. Wren (UCL,
London) for providing us with the Yersinia enterocoli-
tica and Yersinia pseudotuberculosis strains,
Dr K. Tru
¨
lzsch (Max von Pettenkofer Institute, Ger-
many) for the anti-YpkA serum, Dr Roland Nordfelth

(Umea
˚
University, Sweden) for providing us with the
Yersinia YpkA D270A mutant strain, Prof. James R.
Bliska (University of California-Berkeley, USA) for
the Yersinia pseudotuberculosis contact A mutant
strain, Dr C. Garrison Fathman (Stanford University,
USA) for the OTUB1 ARF-1 construct, Dr M. Olson
(Glasgow, UK) for the generous gift of RhoA DNA
constructs and Dr L. Navarro and Dr J.E. Dixon
(UCLA, USA) for sending us YpkA wild-type and
D267A expression plasmids. We also thank
Dr C. Wright (University of Oxford, UK) for assis-
tance with the isolation of primary monocytes and
Dr A. Simmons and J. Baker (University of Oxford,
UK) for providing buffy coats from the National
Blood Centre (UK). BMK was supported by a MRC
New Investigation Award and is now supported by
the Biomedical Research Centre (NIHR), Oxford,
UK. MA is supported by the Swedish Research
Council, Lars Hiertas Minne, the Loo and Hans
Ostermans Foundation for Geriatric Research and the
Foundation for Geriatric Diseases at the Karolinska
Institutet, Stockholm, Sweden.
OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al.
2528 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS
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Supporting information
The following supplementary material is available:
Doc. S1. Additional methods.
Fig. S1. The 37 kDa form of OTUB1 expressed in
HEK293T cells does not correspond to OTUB1 ARF-1.
Fig. S2. YpkA does not phosphorylate OTUB1.
Fig. S3. OTUB1 does not affect RhoA mRNA expres-
sion.
Table S1. DNA sequences for primers used in this
study.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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copy-edited or typeset. Technical support issues arising
from supporting information (other than missing files)
should be addressed to the authors.
OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al.
2530 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS