Post-translational modification of the deubiquitinating enzyme otubain 1 modulates active RhoA...

Post-translational modification of the deubiquitinatingenzyme otubain 1 modulates active RhoA levels andsusceptibility to Yersinia invasionMariola 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

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 abstractl MINT-7717124: ypkA (uniprotkb:Q05608) physically interacts (MI:0915) with OTUB1 (uni-

protkb:Q96FW1) by anti bait coimmunoprecipitation (MI:0006)l MINT-7717229: rhoA (uniprotkb:P61586) physically interacts (MI:0915) with OTUB1 (uni-

protkb:Q96FW1) by affinity chromatography technology (MI:0004)l MINT-7717075, MINT-7717207, MINT-7717193, MINT-7717170: ypkA (uniprotkb:Q56921)

physically interacts (MI:0915) with OTUB1 (uniprotkb:Q96FW1) by anti tag coimmunopre-

cipitation (MI:0007)l MINT-7717390: ypkA (uniprotkb:Q56921) physically interacts (MI:0914) with OTUB1 (uni-

protkb:Q96FW1) and RhoA (uniprotkb:P61586) by anti 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 ⁄ threoninekinase 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)84.85.1

OTUB1wt

175.510.3

OTUB1C91S77.53.7

Infe

ctio

n (r

elat

ive

to c

ontr

ol)

PDI

α-HA

α-PDI

OTUB1

PDI

OTUB1

PDI

siRNAOTUB1

1509.8

1.2

0.8

0.4

0Ctrl(EV)150.8

7.8

Ctrl2(sc)

142.39.2

siRNAOTUB1

62.55.0

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)

243.51.3

P < 0.001A B CP < 0.001

P < 0.001

P < 0.001

P < 0.001n = 4n = 6n = 10

Mean SD (+/–) # Colonies

Mean SD (+/–)

Mean SD (+/–)

Infe

ctio

n (r

elat

ive

to c

ontr

ol)

Infe

ctio

n (r

elat

ive

to c

ontr

ol)

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


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)

A B

OTUB1

15 min 30 min 60 min

TRITC – intracellular bacteria

FITC – extracellular bacteria

Ctrl (EV)

OTUB1OTUB1 C91S

1.8

1.5

1.2

0.9

0.6

0.3

0

P < 0.001 P = 0.017 P = 0.023

Intr

acel

lula

r/ex

trac

ellu

lar

bact

eria

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.



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

Ctrl10:1MOI

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 D267AYpkA 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

C D E

B IP: α-endogenous YpkA ininfected cells

WB: α-OTUB1WB: α-OTUB1

wt

D27

0A

-

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+3H]3+ 934.1 kDa

Ion

co

un

ts [

%]

m/z

50 kDa

37 kDa

25 kDa

-OTUB1 IP

A

B

ESI-Ion trap MS/MS analysis of endogenous OTUB1

290.1y2

418.1 y3

546.3y4

617.3 y5

764.4y6

877.5y7

953.5 b16 ++

1018.51 b17++

1092.1 b18 ++

1255.5b21 ++

1313.1 y21 ++-97

1606.8b13 - 64

1722.7b14

1851.0b15

0

0.5

1.0

1.5

4x10

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 18000

100

[M+3H]3+ 966.56 kDa

764.39y6

617.34y5

338.16b4

290.17y2

175.13y1

211.16b2

418.23y3

451.25b5

546.31y4

678.35y11++

877.49y7

1192.62y10

1077.60y9

985.52y17++

1355.69y11 1426.69

y12

1539.86y13 1814.83

y15

1699.89y14

948.55y8

268.18b3

1049.9y18++

P L G S D S E G V N C L A Y D E A I M A Q Q D Ry6 y2y3y4y7 y5

b3

y1y8y9y10y11y12y14y15 y13y17y18

b2 b5 b4

3613

Inte

nsi

ty [

%]

50 kDa

37 kDa

25 kDa

-OTUB1-HA

QTOF MS/MS analysis of overexpressed OTUB1-HA

m/z

pY

pSHA IP

14L 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 y5y10y20y21

b21b18b15 b17b16b13 b14P PP

P P 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.



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 S16ES18E

S16ES18EY26E

- wt S16E S18E Y26E S16ES18EY26E

S16ES18E

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 S16ES18E

S16ES18EY26E

Ctrl (EV)

+ + ++ + ++ +0

0.5

1

1.5

WT Y26ECtrl (EV) Y26F

Exp 1

Exp 2

+ +++

OTUB1

Labe

lling

rat

io(la

belle

d/un

labe

lled)

α-HA

Probe (HA-Ub-Br2)

OTUB1 + probe

OTUB1

2.0

2.5

OTUB1 OTUB1

Infe

ctio

n (r

elat

ive

to c

ontr

ol)

wtS16E S18E C91SY26E S16ES18E

S16ES18EY26E

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.



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

(ypkAD270A) or GTPase-binding domain (Yersinia con-

tact A mutant strain) [18]. OTUB1-mediated suscepti-

bility to invasion with the Yersinia ypkAD270A 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



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 YersiniaContact A mutant

n = 6n = 3

OTUB1-HA

YpkA-FLAG

RhoA-Myc + + ++++

+ +

IP: HAIP: FLAG +

++ + +

+

α-HA

α-FLAG

α-Myc

--

-

---

OTUB1

YpkA

RhoA

N term. domain Kinase domain GTPase binding domain Actin activation domain

* ** 559591072 *599

4345111A C

B

815 732

P < 0.001 P < 0.001

P = 0.003 P = 0.015

EV(ctrl) C91S

EV(ctrl)

OTUB1 OTUB1C91S

P < 0.001

P < 0.001

EV(ctrl)

OTUB1 OTUB1 OTUB1 OTUB1C91S

EV(ctrl)

OTUB1 OTUB1C91S

Infe

ctio

n (r

elat

ive

to c

ontr

ol)

Infe

ctio

n (r

elat

ive

to c

ontr

ol)

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.



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).

0 15 30 0 30RhoA + + +

min

Poly UbRhoA Q63L

RhoA Q63L

+ + + – –

α-RhoA

α-Ub

EV

Ctrl

OTUB1 C91S

Poly UbRhoA 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-HARhoA-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

D E

B+ +

Ctrl (EV) + +

Input

PDI

Input

OTUB1 wt

S16

E, S

18E

, Y26

E

Y26

E

S16

E, S

18E

S16

E

S18

E

Active RhoA

Inactive RhoA (ft)

PDI

OTUB1-HAInput

α-RhoA

α-RhoA

α-HA

α-PDI

-

0 15 30

RhoA

min0 15 30

α-RhoA

Poly UbRhoA Q63L

α-OTUB1 OTUB1

OTUB1 OTUB1 C91SOTUB1

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.



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 ⁄ threoninekinase 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



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% CO2 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



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 · 106 mL)1 and grown overnight at 37 �C. Thecells were then washed with NaCl ⁄Pi 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 · 106 per sample) were incubated for

12–16 h after transfection, washed with NaCl ⁄Pi 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. Bacteriawere washed in NaCl ⁄Pi 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 ⁄Pi 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 CaCl2, 50 mm 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. Colonynumbers 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 · 106 per sample) were lysed in 0.1% NP-40,

150 mm NaCl, 20 mm CaCl2, 50 mm 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 cm2 tissue



culture flasks. The cells were then washed in NaCl ⁄Pi,

lysed in 0.1% NP-40, 150 mm NaCl, 20 mm CaCl2,

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 · 106 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 MgCL2, 1% NP-40,

1 mm 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 · 105 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 ⁄Pi 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 ⁄Pi 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 ⁄Pi and fixed at room temperature

with 3% paraformaldehyde for 10 min. Fixed cells were

washed with cold NaCl ⁄Pi, blocked for 30 min with 1%

BSA, washed three times with NaCl ⁄Pi 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. Trulzsch (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.



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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

by the authors. Such materials are peer-reviewed and

may be re-organized for online delivery, but are not

copy-edited or typeset. Technical support issues arising

from supporting information (other than missing files)

should be addressed to the authors.



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