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

enterocolitica inva-sion was significantly increased upon overexpresinva-sion of wild-type OTUB1 in HEK293T cells, an effect that was not seen when the catalytically inactive OTUB1 mutant

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: bmk@ccmp.ox.ac.uk

(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 ) 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.

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

Yersiniapathogenesis [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 killdur-ing 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 overexpresinva-sion 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

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

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

P < 0.001

P < 0.001

P < 0.001

n = 4

n = 6

n = 10

Mean

SD (+/–) # Colonies Mean

SD (+/–) Mean

SD (+/–)

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.

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)

OTUB1

TRITC – intracellular bacteria FITC – extracellular bacteria

Ctrl (EV) OTUB1 OTUB1 C91S

1.8

1.5

1.2

0.9

0.6

0.3

0

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-HAor 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

Ctrl 10:1 MOI

37 kDa

OTUB1

- Infected

37 kDa

25 kDa

hc

lc

50 kDa

-*

YpkA FLAG

YpkA FLAG

37 kDa

20 kDa

50 kDa

100 kDa

hc hc

OTUB1

37 kDa

EV

Input

OTUB1

α-OTUB1

37 kDa

25 kDa

Y pseudotuberculosis

MOI 10:1

α-OTUB1

WB: α-FLAG

IP: α-FLAG (YpkA-FLAG)

A

B IP: α-endogenous YpkA in infected cells

WB: α-OTUB1 WB: α-OTUB1

-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-FLAGinactive 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

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

961.9 b16 ++

1191.1 y10

1230.6 y20 ++

pY pS

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

y15

1699.89 y14

948.55 y8

268.18 b3

1049.9 y18++

P L G S D S E G V N C L A Y D E A I M A Q Q D R

y6 y4 y3 y2 y7 y5

b3

y1 y8

y9 y10 y11 y12 y14 y15 y13 y17 y18

b2 b5 b4

36 13

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 y4 y3 y2 y7 y5 y10

y20 y21

b21 b18 b15 b16 b17 b13 b14

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 S16E

S18E S16E S18E

Y26E S16E

OTUB1

α-HA

OTUB1

OTUB1

0 0.5 1.0 1.5 2.0 2.5

OTUB1 + probe OTUB1

WT C91S S16E S18E Y26E S16E

S18E S16E S18E Y26E Ctrl (EV)

0 0.5 1 1.5

Exp 1 Exp 2

+ +

OTUB1

α-HA

Probe (HA-Ub-Br2)

OTUB1 + probe

OTUB1

2.0 2.5

wt S16E S18E Y26E S16E C91S

S18E S16E S18E Y26E YpkA

Ctrl

–

α-HA

FLAG IP

OTUB1 α-FLAG

α-HA α-PDI

OTUB1 PDI

YpkA

OTUB1

Input

YpkA

A

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-HAwild-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-FLAGwas 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

Yersiniastrains 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 wt

Contact A mutant

n = 6

n = 3

OTUB1-HA YpkA-FLAG

+ + +

IP: HA IP: FLAG +

+

+

α-HA α-FLAG α-Myc

-OTUB1 YpkA RhoA

* *

*0 591559599*

434 5

1

B

EV

EV (ctrl) OTUB1 OTUB1 C91S

P < 0.001

P < 0.001

EV (ctrl)

C91S EV (ctrl) OTUB1 OTUB1 C91S

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)

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 Infection Control

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

RhoA

OTUB1-HA

α-RhoA α-RhoA

α-PDI α-LARG

α-HA

LARG OTUB1-HA

OTUB1-HA

Ctrl (EV)

A

C

B

Ctrl (EV) + +

Input

PDI

Input

OTUB1 wt S16E, S18E,

Y26E S16E, S18E S16E S18E

Active RhoA Inactive RhoA (ft)

PDI

OTUB1-HA

Input

α-RhoA α-RhoA α-HA α-PDI

RhoA

min

α- RhoA

Poly Ub RhoA Q63L

OTUB1

Fig 7 OTUB1 stabilizes active RhoA (A) Protein lysates from HEK293T cells co-transfected with RhoA wild-type and OTUB1-HAwild-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-HAwild-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-mycQL63 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-FLAGalone or together with OTUB1-HAor 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-HAwild-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.