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: [email protected]
(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

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

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

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

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

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

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-

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)

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

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

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

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

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

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

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

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
+++
+++
++

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

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

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

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
+

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

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

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

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

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

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-

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

(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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