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

Post-translational modification of the deubiquitinating enzyme otubain 1 modulates active RhoA levels and susceptibility to Yersinia invasion Mariola J. Edelmann, Holger B. Kramer, Mikael Altun and Benedikt M. Kessler Department of Clinical Medicine, University of Oxford, UK Introduction The genus Yersinia consists of three pathogenic species that are agents of a variety of diseases, one of which was historically the cause of major pandemics. These include the bubonic plague caused by Yersinia pestis, mesenteric adenitis and septicaemia caused by Yersinia pseudotuberculosis and gastroenteritis caused Keywords deubiquitinating enzymes; otubain 1; phosphorylation; RhoA; YpkA Correspondence B. M. Kessler, Henry Wellcome Building for Molecular Physiology, Nuffield Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Fax: +44 1865 287 787 Tel: +44 1865 287 799 E-mail: 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)byanti bait coimmunoprecipitation (MI:0006) l MINT-7717229: rhoA (uniprotkb:P61586) physically interacts (MI:0915) with OTUB1 (uni- protkb: Q96FW1)byaffinity chromatography technology (MI:0004) l MINT-7717075, MINT-7717207, MINT-7717193, MINT-771 7170: ypkA (uniprotkb:Q56921) physically interacts ( MI:0915) with OTUB1 (uniprotkb:Q96FW1)byanti tag coimmunopre- cipitation ( MI:0007) l MINT-7717390: ypkA (uniprotkb:Q56921) physically interacts (MI:0914) with OTUB1 (uni- protkb: Q96FW1) and RhoA (uniprotkb:P61586)byanti tag coimmunoprecipitation (MI:0007) Abbreviations HA-Ub-Br2, hemagglutinin-tagged ubiquitin-bromide; MOI, multiplicity of infection; OTUB1, otubain 1; Rac1, ras-related C3 botulinum toxin substrate 1; RhoA, ras homolog gene family member A; USP, ubiquitin-specific protease; Yop, Yersinia outer protein; YpkA ⁄ YopO, Yersinia serine ⁄ threonine kinase. FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2515 by Yersinia enterocolitica [1]. Even though the plague is not a major health concern today, cases are reported annually. Moreover, Y. pestis was weaponized in the former Soviet Union [2] and there are reports of emerging multidrug resistant strains [3]. Pathogenic Yersiniae are typically taken up through ingestion and first reach the intestine. The Yersinia surface protein invasin binds to b1 integrins on the apical surface of M cells, which facilitates translocation across the epithelium [4,5]. The pathogenicity and virulence of Yersiniae is mainly based on the plasmid-encoded type III secretion system that encodes for six effector proteins, which are injected into the host cell (primarily monocytes) to modulate the physiology of the infected cell and to prevent uptake and killing (reviewed in [6]). An additional chromosomally encoded Ysa type - III secretion system has been described in Y. enterocol- itica [7,8]. The injection of effector proteins promotes Yersinia growth and survival in lymphoid follicles (Peyer’s patches) underlying the intestinal epithelium and controls antibacterial activities of immune cells located at these sites. Four of these Yersinia outer pro- teins (Yops) are engaged in modifying the cytoskele- ton: YopE, YopH, YopT and YpkA [9–11]. YpkA, an essential virulence factor, is a serine ⁄ threonine kinase that phosphorylates actin [12], binds the deubiquitinat- ing enzyme otubain 1 (OTUB1) [13,14], the small G protein subunit Gaq [15] and interacts with mem- bers of the Rho family of small GTPases, ras homo- logue gene family member A (RhoA) and ras-related C3 botulinum toxin substrate 1 (Rac1) [16]. Although the interaction with actin, in particular G-actin, has been shown to be crucial for YpkA serine ⁄ threonine kinase activity, the functional relevance of the interac- tion with OTUB1 remains to be determined [12,13,17]. YpkA-mediated phosphorylation of Gaq impairs guanine nucleotide binding and subsequently inhibits Gaq-mediated signalling pathways including RhoA activation and cytoskeletal rearrangements in the host cell [15]. In addition, a crystallography-based study revealed that YpkA mimics host guanine nucleotide dissociation inhibitors (GDIs), thereby blocking nucle- otide exchange in RhoA and Rac1, a process that is crucial for virulence in Yersinia [18]. YpkA therefore uses several ways to interfere with the function of small GTPases, which appears to be essential for Yersinia pathogenesis [19]. The Rho family of small G proteins represents a large group of the Ras superfamily of GTPases. More than 20 proteins of this class have been described to date, among which RhoA, Rac1 and Cdc42 are well characterized, particularly their role in cytoskeletal regulation. Specifically, RhoA is involved in the formation of stress fibres and focal adhesion com- plexes [20–23]. Yersinia is not the only pathogen that affects the function of small GTPases such as RhoA [24], indicating that interference with the function of small GTPases is of prime importance in bacterial pathogenesis because microbes have evolved a number of virulence factors that modulate the function of these proteins. In this study, we show for the first time that suscep- tibility to bacterial invasion by Yersinia can be altered by changing expression of otubain 1 (OTUB1), a host cell-encoded deubiquitinating enzyme that belongs to the ovarian tumour domain-containing protein family. This effect is dependent on the catalytic activity of OTUB1 and its ability to stabilize the active form of RhoA prior to invasion. YpkA and OTUB1 modulate the stability of RhoA in opposing ways, therefore leading to cytoskeletal rearrangements that may be involved in bacterial uptake. During this process, OTUB1 was found to be phosphorylated, a post-trans- lational modification that modulates its ability to stabi- lize RhoA. These findings provide a novel entry point for the manipulation of host cell interactions with Yersinia and perhaps other enterobacteria by deubiqui- tination. Results OTUB1 controls cell susceptibility to Yersinia invasion Yersinia virulence factors are injected into target host cell molecules to manipulate signalling pathways dur- ing invasion in order to prevent uptake and killing. In addition to actin, other host cell proteins have been shown to bind to the virulence factor YpkA, including OTUB1 [13]. In order to investigate the role of OTUB1 in Yersinia invasion of HEK293T cells, we established a cell culture invasion assay, in which the effects of overexpression and knockdown of OTUB1 could be monitored. Bacterial uptake into HEK293T cells was measured using a gentamicin-based invasion assay (Fig. 1). Cells transfected with wild-type OTUB1 were infected with Y. enterocolitica and the number of intracellular bacteria compared with the quantity observed in cells overexpressing either a catalytically inactive mutant C91S or an empty vector. We observed that susceptibility to Y. enterocolitica inva- sion was significantly increased upon overexpression of wild-type OTUB1 in HEK293T cells, an effect that was not seen when the catalytically inactive OTUB1 mutant (C91S) was expressed (Fig. 1A). A marked increase in susceptibility was also observed upon OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al. 2516 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS overexpression of OTUB1 and invasion with Y. pseudo- tuberculosis. Conversely, OTUB1 knockdown signifi- cantly attenuates Yersinia invasion (Fig. 1B). We repeated the OTUB1 knockdown experiment in pri- mary human monocytes, which are among the first cells targeted for Yersinia invasion in vivo, and this also resulted in decreased invasion efficiency (Fig. 1C). These differences could not be accounted for by changes in cell viability or cell growth given the con- trols and time frame of the experiment. To confirm the initial observation by an alternate method, we used a double fluorescence staining technique that enables visualization of extracellular and intracellular bacteria in the same cell [25]. The results concurred with the data from the gentamicin-based invasion assay. The ratio of intracellular to extracellular bacteria was much higher in the case of cells overexpressing OTUB1 com- pared with control cells or cells overexpressing a cata- lytically inactive mutant of OTUB1 (CS91S, Fig. 2A). Increased susceptibility to Yersinia in the presence of overexpressed OTUB1 was observed as early as 15 min after invasion, and decreased over time, proba- bly because of intracellular elimination. Taken together, our results indicated that it was the efficiency of bacterial uptake, not the proliferation of bacteria within the host cell that is modulated by OTUB1 (Fig. 2B). Post-translationally modified OTUB1 interacts with the virulence factor YpkA Previous evidence suggested that the Yersinia-encoded virulence factor YpkA interacts with OTUB1 in vitro [13], providing a potential molecular entry point to explain this effect. We therefore aimed to validate this result and examine whether this interaction also occurs during bacterial invasion in living cells. To test whether YpkA interacts with OTUB1, wild-type YpkA and an inactive kinase mutant D267A were overexpressed in HEK293T cells, followed by YpkA OTUB1-HA Ctrl (EV) 84.8 5.1 OTUB1 wt 175.5 10.3 OTUB1 C91S 77.5 3.7 Infection (relative to control) PDI α-HA α-PDI OTUB1 PDI OTUB1 PDI siRNA OTUB1 150 9.8 1.2 0.8 0.4 0 Ctrl (EV) 150.8 7.8 Ctrl2 (sc) 142.3 9.2 siRNA OTUB1 62.5 5.0 1.2 0.8 0.4 0 2.4 1.8 1.2 0.6 0 α-OTUB1 α-PDI α-OTUB1 α-PDI Ctrl (sc) 243.5 1.3 P < 0.001 ABC P < 0.001 P < 0.001 P < 0.001 P < 0.001 n = 4n = 6n = 10 Mean SD (+/–) # Colonies Mean SD (+/–) Mean SD (+/–) Infection (relative to control) Infection (relative to control) Fig. 1. OTUB1 controls susceptibility to invasion by Yersinia enterocolitica. (A) HEK293T cells were transfected with empty vector (EV), wild-type OTUB1- HA or the C91S mutant, followed by invasion with Yersinia enterocolitica (MOI 60 : 1). Gentamicin was added after 1 h to kill extracellular bacteria. After 2 h, cells were lysed and dilutions plated and cultured for 2 days at 27 °C. Susceptibility to invasion was mea- sured as the ratio between the numbers of colonies for OTUB1 (black bar), C91S mutant (grey bar) relative to the number obtained in the control (white bar, set to as 1.0). Ten independent experiments were performed and the P-values are displayed as calculated using the Student’s t-test. The mean and standard deviations of the absolute numbers of observed colonies are indicated. (B) Number of colonies obtained relative to control when HEK293T cells were either transfected with empty vector (EV, white bar), transfected with negative scram- bled control (sc, grey bar) or OTUB1 shRNA (black bar) for 24 h prior to Yersinia invasion. Six independent experiments were performed and the P-values are displayed, as calculated using the Student’s t-test. The mean and standard deviations of absolute numbers of the observed colonies are indicated. (C) Number of colonies obtained relative to control from primary monocytes that were previously isolated from human peripheral blood mononuclear cells and were either transfected with negative scrambled control or transfected with OTUB1 shRNA for 24 h (black bar) prior to invasion with Yersinia. Four independent experiments were performed and the P-values are displayed, as calculated using the Student’s t-test. The mean and standard deviations of the absolute numbers of observed colonies are indicated. M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2517 immunoprecipitation and separation by SDS ⁄ PAGE. This was compared with a control immunoprecipitate from cells transfected with empty vector, and the pres- ence of OTUB1 was assessed by immunoblotting. We observed that endogenous OTUB1 and YpkA are part of the same protein complex (Fig. 3A). Inactivation of the YpkA kinase activity by a D267A mutation did not abolish this interaction. Moreover, this interaction was also observed with endogenous YpkA present in host cells during bacterial invasion (Fig. 3B). We noted that multiple forms of OTUB1 can be detected, as described previously [26,27], and that the form of OTUB1 that co-immunoprecipitated with YpkA has an apparent molecular mass of 37 kDa, corroborating the findings of a previous study [13]. However, the majority of endogenous OTUB1 protein is detected at its expected molecular mass, 31 kDa (Fig. 3A, left). We also observed increased levels of this higher molec- ular mass form of OTUB1 in infected HEK293T cells compared with control (Fig. 3C). Nevertheless, the appearance of this form did not depend on YpkA kinase activity (Fig. 3D). We therefore examined whether this corresponds to the previously identified alternative spliced form of OTUB1 referred to as ARF-1, which has an apparent molecular mass of 35 kDa [26]. Overexpression of HSV-tagged ARF-1 was detected by anti-HSV, but not by OTUB1 immu- noblotting, indicating that our antibody does not rec- ognize ARF-1 (Fig. S1). We therefore hypothesized that this form of OTUB1 may be post-translationally modified, leading to a change in apparent molecular mass and enhancing interaction with YpkA. Consis- tent with this, treatment with protein phosphatase sug- gested that the 37 kDa form of OTUB1 may contain multiple phosphorylation sites, based on the observed differential migration pattern (Fig. 3E). To further shed light on the role of these OTUB1 modifications in the invasion process, we embarked on identification Ctrl (EV) AB OTUB1 15 min 30 min 60 min TRITC – intracellular bacteria FITC – extracellular bacteria Ctrl (EV) OTUB1 OTUB1 C91S 1.8 1.5 1.2 0.9 0.6 0.3 0 P < 0.001 P = 0.017 P = 0.023 Intracellular/extracellular bacteria Fig. 2. OTUB1 expression levels affect bacterial uptake but not intracellular proliferation. (A) HEK293T cells were transfected either with empty vector (EV), wild-type OTUB1- HA or the C91S mutant and after 24 h infected with Yersinia pseudotuberculosis (MOI 60 : 1) for 15, 30 and 60 min, followed by fixing and staining for extracellular bacteria using fluorescein isothiocyanate (FITC)-labelled Yersinia antibodies (green). Cells were then permeabilized and stained with tetramethyl rhodamine iso-thiocyanate (TRITC)-labelled Yersinia antibodies to label intracellular bacteria (red), followed by analysis using confocal microscopy. Pictures of the 30-min time point are shown. Control cells (upper, EV) and cells overexpressing OTUB1- HA (lower, OTUB1) have different ratios of intracellular (tetramethyl rhodamine iso-thiocyanate-stained, lower left compartment) versus extracellular bacteria (fluorescein isothiocyanate-stained, upper right compartment). The nuclei were visual- ized using 4¢,6-diamidino-2-phenylindole staining (blue). (B) OTUB1- HA -overexpressing cells are characterized by a higher ratio of intracellu- lar ⁄ extracellular bacteria in comparison with OTUB1- HA C91S mutant or control cells. This difference occurred as early as 15 min after invasion with Yersinia. Three independent experiments were performed for the statistical analysis, and relative ratios between intracellular (red) versus total ⁄ extracellular (green) bacteria are shown as well as the P-values calculated using the Student’s t-test. OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al. 2518 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS using a tandem mass spectrometry approach (LC- MS ⁄ MS). Endogenous OTUB1 was isolated from HEK293T cells, separated by SDS ⁄ PAGE and the stained material subjected to in-gel trypsin digestion and analysis by LC-MS ⁄ MS (Fig. 4A). An OTUB1- derived N-terminal peptide containing three phos- phorylation sites, Ser16, Ser18 And Tyr26 was identi- fied. In addition, OTUB1 which was overexpressed in HEK293T cells was isolated and analysed in a similar manner, revealing a different N-terminal peptide that contained the same phosphorylated residues (Fig. 4B). Based on these results, OTUB1 mutants were gener- ated in which Ser16, Ser18 and Tyr26 were replaced with glutamic acid in order to mimic the negative charge caused by phosphorylation (S16E, S18E and Y26E). This approach was successfully used to imitate phospho-serine and -threonine residues, but is to some extent less ideal for phospho-tyrosines [28]. Interestingly, we observed that the OTUB1 Y26E and S18E mutants exerted increased affinity to YpkA in co-immunoprecipitation experiments, thereby resem- bling the increased binding of the 37 kDa form of OTUB1 to YpkA (Fig. 5A). This is consistent with the notion that phosphorylation of OTUB1 affects the interaction with YpkA, although the regulation might be more complex, because the OTUB1 S16E ⁄ S18E ⁄ Y26E triple mutant did not show any increased bind- ing to YpkA. Mimicry of OTUB1 phosphorylation modulates susceptibility to Yersinia invasion If the interaction between OTUB1 and YpkA were relevant for increased susceptibility to invasion, one would expect that modification of OTUB1 may have an effect on this process. To examine this, we repeated Ctrl 10:1 MOI 37 kDa OTUB1 - Infected 37 kDa 25 kDa hc lc 50 kDa - * YpkAFLAG YpkAFLAG 37 kDa 20 kDa 50 kDa 100 kDa hchc lc lc OTUB1 37 kDa EVEV WT WTD267A D267A YpkA YpkA Input OTUB1 α-OTUB1 37 kDa 25 kDa Y. pseudotuberculosis MOI 10:1 α-OTUB1 WB: α-FLAG IP: α-FLAG (YpkA-FLAG) A CD E B IP: α-endogenous YpkA in infected cells WB: α-OTUB1WB: α-OTUB1 wt D270A - 31 kDa OTUB1 37 kDa OTUB1 CIP Phosphatase +– α-OTUB1 37 kDa 25 kDa Exp. 1 Exp. 2 Fig. 3. Interaction between OTUB1 and YpkA in living cells and during Yersinia invasion. (A) Empty vector (EV), wild-type YpkA- FLAG , or the YpkA- FLAG inactive kinase mutant D267A were transfected into HEK293T cells. After 24 h, cell extracts were prepared and YpkA material immunoprecipitated using anti-FLAG Ig. Association with endogenous OTUB1 was demonstrated by immunoblotting using OTUB1 antibo- dies in the presence of YpkA wild-type and D267A inactive kinase mutant. (B) HEK293T cells were infected with Y. pseudotuberculosis for 2 h. Cell extracts were prepared and YpkA immunoprecipitated using YpkA antibodies. In infected cells, association with endogenous OTUB1 was demonstrated by anti-OTUB1 immunoblotting (hc, heavy chain; lc, light chain; *, a smaller form of OTUB1 was also detected). (C) Modification of OTUB1 during Yersinia invasion. HEK293T were infected with Y. enterocolitica at an MOI of 10 : 1 for 2 h, followed by cell lysis, separation by SDS ⁄ PAGE and anti-OTUB1 immunoblotting. (D) Modification of OTUB1 does not depend on YpkA kinase activity. HEK293T cells were left untreated or infected with Y. pseudotuberculosis wild-type (wt) and YpkA kinase inactive mutant at an MOI of 10 : 1 for 2 h. Cell extracts were prepared and two forms of OTUB1 (31 kDa unmodified form and 37 kDa modified form) were visualized using OTUB1 antibodies. (E) OTUB1 37 kDa form is phosphorylated. HEK293T cells were lysed and incubated with calf intestinal phospha- tase (CIP) for 1 h, resulting in the appearance of multiple forms between 27 and 37 kDa, which indicates the presence of several phosphory- lation sites. OTUB1 was visualized using anti-OTUB1 immunoblotting. Two independent experiments are shown. M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2519 [M+3H] 3+ 934.1 kDa Ion counts [%] m/z 50 kDa 37 kDa 25 kDa - OTUB1 IP A B ESI-Ion trap MS/MS analysis of endogenous OTUB1 290.1 y2 418.1 y3 546.3 y4 617.3 y5 764.4 y6 877.5 y7 953.5 b16 ++ 1018.51 b17 ++ 1092.1 b18 ++ 1255.5 b21 ++ 1313.1 y21 ++ - 97 1606.8 b13 - 64 1722.7 b14 1851.0 b15 0 0.5 1.0 1.5 4 x10 400 600 800 1000 1200 1400 1600 1800 961.9 b16 ++ 1191.1 y10 1230.6 y20 ++ pY pS 200 400 600 800 1000 1200 1400 1600 1800 0 100 [M+3H] 3+ 966.56 kDa 764.39 y6 617.34 y5 338.16 b4 290.17 y2 175.13 y1 211.16 b2 418.23 y3 451.25 b5 546.31 y4 678.35 y11++ 877.49 y7 1192.62 y10 1077.60 y9 985.52 y17++ 1355.69 y11 1426.69 y12 1539.86 y13 1814.83 y15 1699.89 y14 948.55 y8 268.18 b3 1049.9 y18++ PLGSDSEGVNCLAYDEAIMAQQDR y6 y2y3y4y7 y5 b3 y1y8y9y10y11y12y14 y 15 y13 y17y18 b2 b5 b4 3613 Intensity [%] 50 kDa 37 kDa 25 kDa - OTUB1 -HA QTOF MS/MS analysis of overexpressed OTUB1-HA m/z pY pS HA IP 14 L G S D S E G V N C L A Y D E A I M A Q Q D R 36 y6 y2y3y4y7 y5y10 y20y21 b21b18b15 b17b16b13 b14 P P P PP P Fig. 4. Detection of OTUB1 phosphorylation using MS. (A) Detection of endogenous phosphorylated OTUB1. HEK293T cells were lysed, followed by immunoprecipitation of OTUB1. As a control, lysate was incubated with agarose without the antibody. Immunoprecipitated material was analysed by SDS ⁄ PAGE and silver staining, and the large band corresponding to the expected molecular mass of OTUB1 as well as the area above (rectangle) was excised and digested with trypsin. Digested material was analysed by a nano-LC Ion Trap mass spec- trometer. For the peptide 14–36 ([M + 2H] 2+ , 934.1 Da) containing the phosphorylated tyrosine and two serines, the b- and y-fragment ion series are shown. (B) Detection of phosphorylated OTUB1 in an overexpression model. Control or HEK293T cells overexpressing OTUB1- HA wild-type were lysed, followed by immunoprecipitation of OTUB1. Eluted material was analysed by SDS ⁄ PAGE gel and Coomassie Blue staining, and the band corresponding to a modified OTUB1 (rectangle) was excised and digested with trypsin. The peptide mixture was analysed by a nano-UPLC-QTOF tandem mass spectrometer. For the peptide 13-36 ([M + 2H] 2+ , 966.6 Da) containing the phosphorylated tyrosine and two serines, the b- and y-fragment ion series detected are shown. OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al. 2520 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 P < 0.001 Ctrl (EV) wt S16E S18E Y26E S16E S18E S16E S18E Y26E - wt S16E S18EY26E S16E S18E Y26E S16E S18E OTUB1 α-HA α-PDI PDI OTUB1 OTUB1 0 0.5 1.0 1.5 2.0 2.5 OTUB1 + probe OTUB1 WT S16E S18EC91S Y26E S16E S18E S16E S18E Y26E Ctrl (EV) +++++ +++ 0 0.5 1 1.5 WT Y26ECtrl (EV) Y26F Exp 1 Exp 2 ++++ OTUB1 Labelling ratio (labelled/unlabelled) α-HA Probe (HA-Ub-Br2) OTUB1 + probe OTUB1 2.0 2.5 OTUB1 OTUB1 Infection (relative to control) wtS16E S18E C91SY26E S16E S18E S16E S18E Y26E YpkACtrl ++++++++– α-HA FLAG IP OTUB1 α-FLAG α-HA α-PDI OTUB1 PDI YpkA OTUB1 Input YpkAA B C α-HA α-HA Fig. 5. OTUB1 modification controls its function and its effect on Yersinia invasion. (A) Binding of YpkA to OTUB1 depends on OTUB1 modifi- cation. Empty vector (EV control), OTUB1- HA wild-type, catalytically inactive mutant (C91S) or mutants mimicking phosphorylated OTUB1- HA (S16E, S18E, Y26E) were co-expressed with YpkA- FLAG in HEK293T cells. Cells were lysed and YpkA- FLAG immunoprecipitated with anti-FLAG Ig. Binding of OTUB1 mutants to YpkA was measured by immunoblotting using HA antibodies. OTUB1 expression levels as well as the loading control (PDI) were shown in the input, whereas YpkA- FLAG was visualized in immmunoprecipitated material. One representative out of three experiments is shown. (B) OTUB1 modification affects bacterial invasion. HEK293T cells were transfected either with empty vector (EV control), wild-type OTUB1- HA or mutants mimicking phosphorylated OTUB1 listed in Fig. 5A followed by invasion with Y. enterocolitica (MOI 60 : 1). Gentamicin was added after 1 h to kill extracellular bacteria. After 2 h, cells were lysed and dilutions plated and cultured for 2 days at 27 °C. The number of colonies for OTUB1 and the OTUB1 mutants were counted and presented relative to the number obtained for the control (EV). The P-values were calculated using a Student’s t-test. Expression of OTUB1 in infected cells is shown using anti-OTUB1 western blotting and the loading control using anti-PDI western blotting. (C) Mimicry of phosphorylation on Tyr 26 interferes with OTUB1 func- tion. HEK293T cells were transfected either with empty vector (EV), HA-tagged wild-type OTUB1, catalytically inactive OTUB1 C91S, mutants mimicking phosphorylated OTUB1 (see above) or the Y26F mutant. Cells were lysed and extracts incubated with an HA-tagged ubiquitin Br2 probe to measure OTUB1 activity as described previously [30]. As a control, cells were treated the same way but without addition of the probe. OTUB1 and OTUB1–probe adduct were visualized by immunoblotting using HA antibodies and quantified for two experiments (black and grey bars). The intensities of the corresponding bands were measured and the ratio between them is shown (labelling ratio), reflecting reactivity towards the probe. Two independent experiments are shown. M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2521 the gentamicin-based invasion assay with cells overex- pressing the OTUB1 mutants that mimic phosphoryla- tion. Overexpression of the OTUB1 mutants S16E, S18E, Y26E, S16E ⁄ S18E and S16E ⁄ S18E ⁄ Y26E abol- ished the observed increase in susceptibility to invasion seen with wild-type OTUB1 (Fig. 5B) or the S16A and Y26F control mutants (data not shown), thereby con- firming that modification of OTUB1 has an impact on the magnitude of Yersinia invasion. Because no effect on invasion was seen with the catalytically inactive mutant C91S OTUB1 (Fig. 1), we set out to test whether the constructed proteins mimicking phosphor- ylated OTUB1 were functional by monitoring their reaction with the deubiquitinating enzyme-specific probe, hemagglutinin-tagged ubiquitin-bromide (HA- Ub-Br2), which was previously shown to covalently bind active OTUB1 [29,30]. Interestingly, the OTUB1 Y26E mutant did not react with the HA-Ub-Br2 active-site probe, whereas all other mutants were able to do so (Fig. 5C). We conclude that phosphorylation of OTUB1, in particular at Tyr26, modulates OTUB1 function by interfering with its enzymatic activity, ubiquitin binding or substrate recognition. Next, we examined whether OTUB1 phosphorylation may be attributed to the Ser ⁄ Thr kinase activity of YpkA directly. Recombinant OTUB1 and immunopre- cipitated YpkA expressed in HEK293T cells were incubated in a radioactive in vitro kinase assay. Recombinant OTUB1 was weakly phosphorylated by YpkA, consistent with previous findings, but to a much lesser degree than the control protein myelic basic protein (Fig. S2A). By contrast, OTUB1 isolated from cell lysates was not phosphorylated by YpkA at a detectable level, although wild-type YpkA was read- ily autophosphorylated and therefore active (Fig. S2B). These results indicate that modification of OTUB1 by phosphorylation has an effect on OTUB1-mediated Yersinia bacterial uptake, but did not resolve the relevance of YpkA’s Ser ⁄ Thr kinase activity in this process. OTUB1-mediated susceptibility to invasion is modulated by the YpkA GTPase-binding domain YpkA consists of several domains including a serine ⁄ threonine kinase and a GTPase-binding domain, both of which contribute to virulence [6] (Fig. 6A). In order to dissect which of these functionalities contribute to OTUB1-mediated susceptibility to invasion, we used Yersinia strains that either had mutations in the kinase (ypkA D270A ) or GTPase-binding domain (Yersinia con- tact A mutant strain) [18]. OTUB1-mediated suscepti- bility to invasion with the Yersinia ypkA D270A strain was unaltered, but was compromised with the con- tact A mutant strain (Fig. 6B). These results show that the YpkA GTPase-binding domain, but not the Ser ⁄ Thr kinase activity, interferes with susceptibility to Yersinia invasion provoked by overexpression of OTUB1 in host cells. Previous experiments have demonstrated an interac- tion between YpkA and the small GTPases RhoA or Rac1 [16,18]. Our data suggest that the ability of YpkA to bind GTPases may be critical for the OTUB1-mediated increased Yersinia uptake. We there- fore tested whether YpkA and RhoA interact in vitro and whether this protein complex includes OTUB1. YpkA was immunoprecipitated and the presence of OTUB1 and RhoA examined by immunoblotting (Fig. 6C). YpkA, OTUB1 and RhoA were found to be part of the same complex. Moreover, OTUB1 is asso- ciated with RhoA in the absence of YpkA, as demon- strated by co-immunoprecipitation of OTUB1 and RhoA (Fig. 6C, lane 3). OTUB1 stabilizes active RhoA The existence of all three components in the same complex and the association between OTUB1 and RhoA suggested that OTUB1 might play a role in modulating the ubiquitination status and stability of RhoA. In order to investigate this, we expressed both proteins in HEK293T cells and examined the polyubiq- uitination status and the stability of RhoA by immu- noprecipitation ⁄ western blotting experiments (Fig. 7A– C). When OTUB1 was overexpressed, the total amount of RhoA increased marginally. The same observation was made for endogenous RhoA levels which were elevated upon overexpression of OTUB1 (Fig. 7A). However, levels of endogenous active (GTP-bound) RhoA isolated from noninfected cells using a rhotekin- based pulldown were stabilized considerably by OTUB1, but not by a catalytically inactive OTUB1 C91S mutant (Fig. 7B). This was not accounted for by an increase in RhoA activation through its guanine nucleotide exchange factor LARG, for which a mar- ginal increase was noted in the presence of wild-type and catalytically inactive OTUB1 (Fig. 7B, lower). A more striking effect was observed when immunoprecip- itated RhoA was incubated with recombinant OTUB1 in vitro (Fig. 7C). The experiment was performed by expressing a constitutively active RhoA (Q63L mutant) to enrich for polyubiquitinated material. The quantity of ubiquitinated RhoA was significantly decreased in presence of wild-type OTUB1, whereas levels of unmodified RhoA increased with time. The catalyti- cally inactive mutant OTUB1 C91S was unable to OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al. 2522 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS deubiquitinate RhoA (Fig. 7C right). These results clearly indicate that OTUB1 is responsible for stabil- ization of active RhoA and that it is dependent on the deubiquitinating activity of the enzyme (Fig. 7B). Correlation between RhoA stabilization and enhanced susceptibility to Yersinia invasion Because RhoA has been shown previously to be impli- cated in modulating host–pathogen interactions by regulating cell morphology and uptake [31,32], our results raised the question of whether OTUB1-medi- ated enhanced susceptibility to invasion may involve RhoA. To examine this in further detail, we first tested whether levels of the GDP- or GTP-bound form of RhoA are affected during invasion. A rhotekin-based pulldown assay was used to isolate the active form of RhoA from infected and noninfected cells. The amount of active RhoA is substantially increased when OTUB1 was overexpressed, but not during invasion (Fig. 7D). Therefore, overexpression of OTUB1 does stabilize active RhoA prior to, but not after, invasion. Co-transfection experiments revealed that YpkA alone counteracts OTUB1-mediated stabilization of RhoA (Fig. 7D), therefore identifying two factors that have an opposing effect on RhoA function and stability. Finally, to underscore the relevance of OTUB1-medi- ated stabilization of RhoA in enhanced susceptibility to invasion, we tested whether OTUB1 mutants mimicking phosphorylation were able to stabilize 2.5 2.0 1.5 1.0 0 0.5 2.5 2.0 0 1.5 1.0 0.5 Yersinia wtYersinia D270A Yersinia wt Yersinia Contact A mutant n = 6n = 3 OTUB1-HA YpkA-FLAG RhoA-Myc +++ +++ ++ IP: HA IP: FLAG + + +++ + α -HA α -FLAG α -Myc - - - - - - OTUB1 YpkA RhoA N term. domain Kinase domain GTPase binding domain Actin activation domain *** 559591072 * 599 4345111 A C B 815 732 P < 0.001 P < 0.001 P = 0.003 P = 0.015 EV (ctrl) C91S EV (ctrl) OTUB1 OTUB1 C91S P < 0.001 P < 0.001 EV (ctrl) OTUB1 OTUB1 OTUB1 OTUB1 C91S EV (ctrl) OTUB1 OTUB1 C91S Infection (relative to control) Infection (relative to control) Fig. 6. OTUB1-mediated susceptibility to invasion requires YpkA and its GTPase-binding domain, but not its serine ⁄ threonine kinase activity. (A) Scheme of the domains present in YpkA. Mutated amino acid positions in the mutant strains used in this study are indicated. (B) Increased susceptibility to Yersinia invasion is not dependent on YpkA-mediated phosphorylation. Control HEK293T cells, HEK293T cells overexpressing OTUB1- HA or OTUB1- HA C91S were infected with either wild-type Y. pseudotuberculosis or Y. pseudotuberculosis mutants containing an inactive kinase domain (D270A) or the YpkA contact A mutant (unable to bind GTPases). Cells were collected after 3 h, lysed and cell extracts plated on agar plates. Colonies were counted after 2 days of incubation at 27 °C and the colony numbers were displayed as ratios relative to the control. Experiments were performed at least three times and the P-values were calculated using a Student’s t-test. For each strain, susceptibility to invasion was measured as the ratio between the numbers of colonies for OTUB1 (black bar), C91S mutant (grey bar) relative to the number obtained in untransfected cells (EV, white bar, set to as 1.0). (C) YpkA is in a complex with RhoA and OTUB1. Cells were co-transfected with OTUB1- HA , RhoA- myc and YpkA- FLAG , followed by immunoprecipitation with HA or FLAG antibodies. OTUB1- HA , RhoA- myc and YpkA- FLAG were visualized by immunoblotting using OTUB1, RhoA or FLAG antibodies, respectively. M. J. Edelmann et al. OTUB1 affects susceptibility to Yersinia invasion FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS 2523 active RhoA (Fig. 7E). Overexpression of the OTUB1 mutants S16E, S18E, Y26E, S16E ⁄ S18E and S16E ⁄ S18E ⁄ Y26E did not rescue active RhoA levels to the same extent as observed with wild-type OTUB1, thereby corroborating their effect on enhanced suscep- tibility to invasion (Fig. 5B). 01530 030 RhoA +++ min Poly Ub RhoA Q63L RhoA Q63L +++ –– α-RhoA α-Ub EV Ctrl OTUB1 C91S Poly Ub RhoA Q63L Active RhoA Inactive RhoA (Ft) EV OTUB1 InfectionControl Active RhoA Inactive RhoA (ft) YpkA-FLAG Ctrl HA plasmid Ctrl FLAG plasmid YpkA-FLAG OTUB1-HA + ++ + ++ α-RhoA α-RhoA α-HA EV OTUB1 α-FLAG + OTUB1-HA RhoA-myc - + + α-myc α-HA α-PDI α-RhoA OTUB1-HA RhoA-endog + OTUB1 endogenous OTUB1-HA α-OTUB1 PDI RhoA OTUB1-HA α-RhoA α-RhoA α-PDI α-LARG α-HA LARG OTUB1-HA OTUB1-HA Ctrl (EV) A C DE B ++ Ctrl (EV) ++ Input PDI Input OTUB1 wt S16E, S18E, Y26E Y26E S16E, S18E S16E S18E Active RhoA Inactive RhoA (ft) PDI OTUB1-HA Input α-RhoA α-RhoA α-HA α-PDI - 01530 RhoA min01530 α-RhoA Poly Ub RhoA Q63L α-OTUB1 OTUB1 OTUB1 OTUB1 C91S OTUB1 Fig. 7. OTUB1 stabilizes active RhoA. (A) Protein lysates from HEK293T cells co-transfected with RhoA wild-type and OTUB1- HA wild-type or control plasmid (EV) were subjected to RhoA detection by immunoblotting. Levels of RhoA (transfected RhoA- myc , left; endogenous RhoA, right) were increased if cells were co-transfected with OTUB1- HA but not in the presence of empty vector (EV). Loading control is shown using anti-PDI western blotting. (B) OTUB1 stabilizes active RhoA. Endogenous active RhoA was isolated using Rhotekin-coupled beads from HEK293T cells overexpressing either empty vector (EV), OTUB1- HA wild-type or C91S mutant. LARG, PDI (loading control) and OTUB1- HA wild- type were visualized using western blotting of the input material. (C) OTUB1 deubiquitinates RhoA in vitro. Purified ubiquitylated RhoA isolated from HEK293T cells previously transfected with the constitutively active mutant RhoA- myc QL63 was incubated with recombinant wild-type OTUB1 (both panels) the catalytically inactive mutant C91S (right) for 0, 15 and 30 min at 37 °C. RhoA deubiquitination was visualized by anti- ubiquitin and anti-RhoA immunoblotting. (D) OTUB1-mediated stabilization of active RhoA is impaired during invasion and if co-expressed with YpkA. Active RhoA was enriched using recombinant Rhotekin from HEK293T cells overexpressing empty vector (EV), OTUB1- HA wild-type, infected or not with Y. pseudotuberculosis for 3 h (left) or from HEK293T cells overexpressing YpkA- FLAG alone or together with OTUB1- HA or the control plasmids (HA and FLAG plasmids, right). The loading control (PDI), OTUB1- HA and YpkA- FLAG were visualized by western blotting of the input material. (E) Mimicry of OTUB1 phosphorylation impairs its ability to stabilize active RhoA. Active RhoA was enriched using recombi- nant Rhotekin from HEK293T cells overexpressing empty vector (EV), OTUB1- HA wild-type or the mutants S16E, S18E, Y26E, S16E ⁄ S18E and S16E ⁄ S18E ⁄ Y26E mimicking OTUB1 phosphorylation. The loading control (PDI) and OTUB1- HA were visualized by western blotting of the input material. Moreover, RhoA in the flow through material (ft) was also visualized. One out of two experiments is shown. OTUB1 affects susceptibility to Yersinia invasion M. J. Edelmann et al. 2524 FEBS Journal 277 (2010) 2515–2530 ª 2010 The Authors Journal compilation ª 2010 FEBS [...]... absence of bacterial invasion OTUB1 prolongs the lifetime of the active (GTP-bound) form of RhoA, because this form is rapidly ubiquitinated and turned over [42,43] Increased susceptibility to invasion provoked by OTUB1 overexpression 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... initial OTUB1 mutants S16E, S16A, S18E, Y26E and Y26F were generated using the OTUB1 pcDNA 3 .1 construct containing a C-terminal HA-TEVSBP 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... 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 FEBS Journal 277 (2 010 ) 2 515 –2530 ª 2 010 The Authors Journal compilation ª 2 010 FEBS M J Edelmann et al site-directed mutagenesis kit by Stratagene (La Jolla, CA, USA) The initial OTUB1 mutants... 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... FEBS Journal 277 (2 010 ) 2 515 –2530 ª 2 010 The Authors Journal compilation ª 2 010 FEBS 2529 OTUB1 affects susceptibility to Yersinia invasion 31 32 33 34 35 36 37 38 39 40 M J Edelmann et al members of the deubiquitinating enzyme family Chem Biol 9, 11 49 11 59 Kazmierczak BI, Jou TS, Mostov K & Engel JN (20 01) Rho GTPase activity modulates Pseudomonas aeruginosa internalization by epithelial cells Cell... 7E) Modification of Y26 in OTUB1 interfered 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 sustain susceptibility to invasion (compare Figs 5B and 7E), indicating that controlling active RhoA levels. .. 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 susceptibility to bacterial invasion Alternatively, these modifications may also change OTUB1 deubiquitination activity, affinity to or recognition of substrates Consistent with this, we observed that only wild-type OTUB1 was able to stabilize active RhoA, ... Cornelis GR (2002) Role of Yops and adhesins in resistance of Yersinia entrocolitica to phagocytosis Infect Immun 70, 416 5– 417 6 11 Cornelis GR (2002) Yersinia type III secretion: send in the effectors J Cell Biol 15 8, 4 01 408 12 Juris SJ, Rudolph AE, Huddler D, Orth K & Dixon JE (2000) A distinctive role for the Yersinia protein kinase: actin binding, kinase activation, and cytoskeleton disruption Proc... 2 010 The Authors Journal compilation ª 2 010 FEBS 2525 OTUB1 affects susceptibility to Yersinia invasion M J Edelmann et al 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... Microbiol 15 , 437–440 20 Tzima E (2006) Role of small GTPases in endothelial cytoskeletal dynamics and the shear stress response Circ Res 98, 17 6 18 5 21 Ridley AJ & Hall A (19 92) The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors Cell 70, 389–399 22 Ridley AJ (19 97) The GTP-binding protein Rho Int J Biochem Cell Biol 29, 12 25 12 29 . b18 ++ 12 55.5 b 21 ++ 13 13 .1 y 21 ++ - 97 16 06.8 b13 - 64 17 22.7 b14 18 51. 0 b15 0 0.5 1. 0 1. 5 4 x10 400 600 800 10 00 12 00 14 00 16 00 18 00 9 61. 9 b16 ++ 11 91. 1 y10 12 30.6 y20 ++ pY pS 200. kDa 764.39 y6 617 .34 y5 338 .16 b4 290 .17 y2 17 5 .13 y1 211 .16 b2 418 .23 y3 4 51. 25 b5 546. 31 y4 678.35 y 11+ + 877.49 y7 11 92.62 y10 10 77.60 y9 985.52 y17++ 13 55.69 y 11 1426.69 y12 15 39.86 y13 18 14.83 y15 16 99.89 y14 948.55 y8 268 .18 b3 10 49.9 y18++ PLGSDSEGVNCLAYDEAIMAQQDR y6