UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series no. 851 ISSN: 0346-6612 ISBN: 91-7305-505-0 - From the Departments of Molecular Biology Umeå University, Umeå, Sweden

CELLULAR TARGETS OF PSEUDOMONAS AERUGINOSA TOXIN EXOENZYME S

Akademisk avhandling som med vederbörligt tillstånd från rektorsämbetet vid Umeå Universitet för avläggande av doktorsexamen i cellbiologi offentligen kommer att försvaras i hörsal Betula, byggnad 6M, Norrlands Universitetssjukhus fredagen den 31 oktober 2003, klockan 09.00

av

Maria Henriksson

Fakultetsopponent:

Professor Lars Rönnstrand, Institutionen för Experimentell Klinisk Kemi,

Lunds Universitet, MAS

ABSTRACT Cellular targets of Pseudomonas aeruginosa toxin Exoenzyme S

Maria Henriksson, Department of Molecular Biology, Umeå University, Sweden

Pseudomonas aeruginosa is an opportunistic pathogen that can cause life-threatening infections in immunocompromised patients. It uses a type III secretion dependent mechanism to translocate toxic effector proteins directly into the eukaryotic cell. The enzymatic activity of two of these toxins, Exoenzyme S (ExoS) and Exoenzyme T (ExoT), have been studied in this thesis. ExoS is a bi-functional toxin known to contain a C-terminal ADP- ribosyltransferase activity, which has been shown to modify members of the Ras family in vitro. The N-terminal of ExoS contains a GTPase Activating Protein (GAP) domain, which shows specificity towards Rho proteins in vitro. ExoT shows high homology (76%) towards ExoS and has also been reported to contain ADP-ribosyltransferase activity in vitro. To study the biological effect of the two toxins, we inserted ExoS or ExoT into eukaryotic cells using the heterologous type III secretion system of Yersinia pseudotuberculosis. We found that Ras was ADP-ribosylated in vivo and this modification altered the ratio of GTP/GDP bound directly to Ras. We also found that ExoS could ADP-ribosylate several members of the Ras superfamily in vivo, modulating the activity of those proteins. In contrast, ExoT showed no ADP-ribosylation activity towards any of the GTPases tested. This suggests that ExoS is the major ADP-ribosyltransferase modulating small GTPase function encoded by P. aeruginosa. Furthermore, we have demonstrated that the GAP activity of ExoS abolishes the activation of RhoA, Cdc42 and Rap1 in vivo, and that ExoT shows GAP activity towards RhoA in vitro.

The ADP-ribosyltransferase activity of ExoS is dependent on the eukaryotic protein 14-3-3. 14-3-3 proteins interact with ExoS in a phospho-independent manner. We identified the amino acids 424DALDL428 on ExoS to be necessary for the specific interaction between ExoS and 14-3-3. Deletion of these five amino acids abolishes the ADP-ribosylation of Ras and hence the cytotoxic effect of P. aeruginosa on cells. Thus the 14-3-3 binding motif on ExoS appears to be critical for both the ADP-ribosylation activity and the cytotoxic action of ExoS in vivo.

2

TABLETABLE OF OF CONTENTS CONTENTS

ABSTRACT ...... 2

PAPERS IN THIS THESIS...... 5

ABBREVIATIONS ...... 6

INTRODUCTION...... 7

Pseudomonas aeruginosa...... 7 Infection and virulence...... 7

The Ras superfamily ...... 10 Ras superfamily members ...... 11

Signalling through Ras...... 12 Guanine nucleotide Exchange Factors (GEFs) for Ras...... 13 GTPase Activating Proteins (GAPs) for Ras ...... 14 Downstream of Ras ...... 15 The Raf pathway ...... 15 The PI-3 kinase pathway...... 17 The RalGDS pathway...... 18

Signals from RhoA, Rac1 and Cdc42 ...... 18

The signalling molecules Rap1 and Rap2 ...... 21

The abundant protein 14-3-3...... 21

ExoS, ExoT and eukaryotic signal transduction ...... 22 ExoS, ExoT and their ADP-ribosylated target molecules...... 22 ExoS, ExoT and the GAP activity...... 23 ExoS and 14-3-3...... 23

The Yersinia pseudotuberculosis model system...... 24

AIM...... 26

RESULT AND DISCUSSION...... 27

Ras inhibition by ADP-ribosylation in vivo (Paper I) ...... 27

ExoT elicits cytotoxicity without interfering with Ras (Paper II) ...... 28

3

ExoS acts towards members of the Ras superfamily in vivo (paper III)...... 29 ExoS activity on Ras subfamily ...... 30 ExoS activity on Rho subfamily...... 31

14-3-3 binding is required for the inhibition of Ras by ExoS(Papers IV and V) ...... 32

CONCLUSIONS...... 33

ACKNOWLEDGEMENT...... 34

REFERENCES...... 36

4

PAPERSPAPERS IN IN THIS THIS THESIS THESIS

This thesis is based on the following publications referred to in the text by their roman numerals (I-V).

Paper I Henriksson, M.L., Rosqvist, R., Telepnev, M., Wolf-Watz, H. and Hallberg, B. (2000). Ras effector pathway activation by epidermal growth factor is inhibited in vivo by exoenzyme S ADP-ribosylation of Ras. Biochem J. 347, 217-222.

Paper II Sundin C, Henriksson, M.L., Hallberg, B., Forsberg, A., Frithz-Lindsten, E. (2001). Exoenzyme T of Pseudomonas aeruginosa elicits cytotoxicity without interfering with Ras signal transduction. Cell Microbiol. Apr;3(4):237-46.

Paper III Henriksson, M.L., Sundin, C., Jansson, A.L., Forsberg, Å. Palmer, R.H. and Hallberg, B. (2002). Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein (GAP) activities towards small GTPases in vivo. Biochem J. 367, 617-628.

Paper IV Henriksson, M.L., Trollér, U. and Hallberg, B. (2000). 14-3-3 proteins are required for the inhibition of Ras by exoenzyme S. Biochem J. 349, 697-701.

Paper V Henriksson, M.L., Francis, M., Peden, A., Aili, M., Stefansson, K., Palmer, R.H., Aitken, A. and Hallberg, B. (2002). A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo. Eur J Biochem 269(20), 4921-4929.

5

ABBREVIATIONSABBREVIATIONS

ADP adenosine diphosphate cAMP cyclic adenosine monophosphate CNF1 Cytotoxic Necrotizing Factor 1 Crk CT10-regulator of kinase C-TAK1 Cdc25C-associated kinase 1 EGF Epidermal Growth Factor Erk Extracellular signal-regulated kinase E-son Elofsson F-actin filamentous actin FAS Factor Activating exoenzyme S G-actin globular (monomeric) actin GAP GTPase Activating Proteins GDI Guanine nucleotide Dissociation factors GDP guanine diphosphate GEF Guanine nucleotide Exchange Factor Grb2 growth factor receptor-bound protein 2 GRF Guanine nucleotide Releasing Factor GRP Guanine nucleotide Releasing Protein GSK3 Glycogen Synthase Kinase 3 GTP guanine triphosphate KSR Kinase Suppressor of Ras LPA lysophosphatidic acid MAPK Mitogen-Activated Protein Kinase MEK Mitogen-activated protein kinase/Erk Kinase MP1 MEK Partner 1 NAD Nicotinamide Adenine Dinucleotide NF1 Neurofibromin 1 PAK p21-associated kinase PDGF Platelet-Derived Growth Factor PDK PI-3K Dependent Kinase PH pleckstrin homology PI-3K phosphoinositide 3-kinase PI-4-P5K phosphatidyl inositol 4-phosphate 5-kinase PKB Protein Kinase B PLD phospholipase D POB1 Partner for RalBP1 PI(4,5)P2 phosphatidyl inositol 4,5-bisphosphate PI(3,4,5)P3 phosphatidyl inositol 3,4,5-triphosphate RalBP1 Ral Binding Protein 1 RalGDS Ral GDP Dissociation Stimulator Shc Src homology 2 domain containing Sos Son-of-Sevenless WASP Wiskott-Aldrich Syndrom Protein

6 INTRODUCTION ______

INTRODUCTIONINTRODUCTION

Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that infects immunocompromised persons, including, for example, patients with severe burn injuries, leukaemia or AIDS [1-3]. Cystic fibrosis patients are susceptible to chronic respiratory infection and this is associated with high rates of morbidity and death [4]. P. aeruginosa is an extra-cellular, Gram-negative bacterium that survives in almost any environment and is notorious for its nutritional and ecological flexibility. Since P. aeruginosa is found in so many habitats, it has become one of the leading causes of hospital-acquired infections. Multiple drug efflux pumps or production of antibiotic modifying enzymes provides P. aeruginosa resistance to most antibiotics, which makes sustained infection even more difficult to treat [5, 6].

Infection and virulence

To initiate infection, P. aeruginosa usually requires a break in the first-line defence, such as burn wounds or alteration of the immunological defence mechanisms. The capacity of P. aeruginosa to produce widespread and often overwhelming infections is due to an arsenal of secreted virulence factors. Many of these secreted factors are suggested to be controlled by a regulatory circuit involving a cell-to-cell signalling system that allows factor production to occur in a coordinated, cell-density-dependent manner [7]. P. aeruginosa has been shown to possess a type III secretion system by which the bacterium transfers virulence factors into eukaryotic cells. Type III secretion systems are protein secretion pathways that are used by many Gram-negative pathogens and are activated by host cell contact [8, 9]. Proteins are secreted without cleavage of a signal peptide, although the information for secretion is located in the amino-terminal portion of the protein [9]. In addition to P. aeruginosa, Salmonella, Shigella, Yersinia, E. coli and many other Gram- negative pathogens use conserved type III secretion systems to secrete proteins thought to be virulence factors [10]. The translocated virulence factors from P. aeruginosa identified so far

7 INTRODUCTION ______

are Exoenzyme Y (ExoY), Exoenzyme U (ExoU), Exoenzyme S (ExoS) and Exoenzyme T (ExoT). Notably, it appears that clinical isolates contain either the exoS or the exoU gene, while almost all forms of P.aeruginosa harbour exoT and exoY [11, 12], which indicates that ExoT and ExoY are important for the bacterial induced pathology.

ExoY is an adenylate cyclase that is translocated into the eukaryotic cell by the type III secretion system. The essential residues for adenylate cyclase activity are conserved in ExoY and both CyaA (Bordetell pertussis) and EF (Bacillus anthracis). The activity of both CyaA and EF are dependent on calmodulin, but the ExoY activity has been shown to be dependent on an unknown eukaryotic protein distinct from calmodulin. Cells intoxicated with ExoY show a rounded morphology that correlates with increased cAMP levels [13].

ExoU (PepA) is also secreted by the type III secretion system. The ExoU activity and its target(s) have for a long time been unrecognized. ExoU expression is correlated with acute cytotoxicity and bacterial-mediated epithelial cell damage in a mouse model of acute pneumonia [12, 14]. Recently, it has been reported that ExoU possesses lipase activity and disrupts the membranes of the infected host cell [15].

ExoS was the first type III effector of P. aeruginosa to be isolated and characterized and upon infection of host cells a decrease in DNA synthesis and viability was reported [16- 19].

E381 1 453

Secr./Transl. GAP ADP-ribosyltransferase

Figure 1: Schematic drawing of ExoS

ExoS is a 453 amino acid long bi-functional enzyme that induces actin microfilament disruption [17] (Fig 1). Recently it has been shown that the amino-terminal (aa 96-232) is a

8 INTRODUCTION ______

GTPase Activating Protein (GAP) for members of the Rho family in vitro [20]. The carboxy- terminal has been shown to be cytotoxic to eukaryotic cells [17]. It contains an ADP- ribosyltransferase activity (aa 233-453), which covalently transfers ADP-ribose from NAD to eukaryotic target proteins [16]. It has previously been suggested that this enzymatic activity induces programmed cell death in the infected cell [21]. A point mutation at glutamic acid 381 decreases the enzymatic activity approximately 2000 times [22]. The first target found for ExoS in vitro was Ras [23], which later was shown to be ADP-ribosylated on Arg41 and Arg128 by ExoS [24, 25]. The ADP-ribosyltransferase activity of ExoS has been shown to be dependent on a eukaryotic co-factor named FAS, for Factor Activating exoenzyme S [16, 26]. FAS has been identified as a member of the eukaryotic 14-3-3 family [27], which is involved in many eukaryotic signal transduction pathways (see below). Amino acids 51-72 of ExoS harbour a membrane localization domain (MLD), which localises the toxin to membrane regions inside the eukaryotic cell [28].

ExoT shows 76 % homology at the amino acid level when compared to ExoS [29]. Like ExoS, ExoT has been reported to contain a carboxy-terminal ADP-ribosyltransferase activity, however ExoT only possesses 0.2-1% of 14-3-3 dependent ADP-ribosyltransferase activity in vitro as compared to ExoS. The candidate active site residue E385 is homologous to E381 in ExoS [29, 30]. The amino terminal part of ExoT also displays high homology to ExoS, which suggests that ExoT also harbours GAP activity.

P. aeruginosa also produce virulence factors, not translocated by the type III apparatus, which contribute to the overall virulence of the bacterium. These factors can cause extensive tissue damage, bloodstream invasion and dissemination, described as follows: Exotoxin A catalyses the ADP-ribosylation and inactivation of elongation factor 2, leading to inhibition of protein synthesis and cell death [31]. Exotoxin A is responsible for local tissue damage, bacterial invasion [32] and immunosuppression [33]. P. aeruginosa produces two hemolysins: phospholipase C and rhamnolipid. These proteins may act synergistically to break down lipids and contribute to tissue invasion [34]. Rhamnolipid also keeps fluid/nutrient channels open in the biofilms produced by the bacteria [35].

9 INTRODUCTION ______

Proteases are also assumed to contribute to P. aeruginosa infections. LasA and LasB elastase destroy elastin-containing human lung-tissue and cause pulmonary haemorrhages in invasive infections [36]. LasB also degrades fibrin and collagen and inactivates immunoglobulins G and A and complement components which not only destroy tissue components, but also interfere with host defence mechanisms [37]. Type IV pili are important for the ability of P. aeruginosa to adhere to eukaryotic cells, and therefore also for the colonization of the host [38]. In addition, type IV pili mediate motility on surfaces, referred to as “twitching motility” [39]. Pili also play a role in the initiation of biofilm formation [39, 40]. Alginate/biofilm formation allows P. aeruginosa to grow encapsulated in a slime layer consisting of bacteria and the polysaccharide alginate. The biofilm makes the bacteria more resistant to all kinds of environmental stress [41].

The Ras superfamily

The Ras superfamily of proteins is a group of 20-25 kDa proteins that are a central point for many signal transduction pathways in the eukaryotic cell. The ras (rat sarcoma) oncogenes were initially discovered as the transforming genes of the Harvey and Kirsten murine sarcoma viruses [42, 43], and ras was one of the first oncogenes to be implicated in human cancer [44]. Basically, all members of the Ras super family bind guanine nucleotides with high affinity, GDP when inactive and GTP when active, and they possess intrinsic GTPase activity [45] (Fig 2). Exchange of GDP for GTP results in an allosteric GDP change in two key regions of the protein, the switch I and Ras switch II, exposing them for binding of effector proteins GAP GEF [46]. Different proteins tightly regulate the cycle between Ras the inactive and the active form and thus allow the Ras GTP proteins to function as molecular switches in the cell. The proteins that promote exchange of GDP to GTPare called Effector proteins Guanine nucleotide Exchange Factors (GEFs) [47]. The Figure 2: Regulation of Ras protein proteins that increase the intrinsic GTPase activity are activity called GTPase Activating Proteins (GAPs) [45].

10 INTRODUCTION ______

Ras superfamily members

The superfamily consists of over 60 mammalian members and can be divided into several subfamilies (see Table 1): Ras, Rho, Rab, ARF, Ran and Kir/Rem/Rad. Many Ras-like proteins have also been identified in other eukaryotes like fungi, flies, frogs and nematodes. The Ras superfamily members regulate many different cellular functions in the eukaryotic cell.

Table 1 Summary of the Ras super family members

Subfamily Members Ras (HRas, KRas4A/B, NRas), Rap (Rap1A/B, Rap2A/B), RRas like (RRas, TC21, Ras RRas3), Ral (RalA/B), Rheb, M-Ras, Rin, Rit Rho (RhoA-C), RhoG, RhoH, Rac (Rac1-3), Cdc42 (Cdc42, TC10), Rnd (Rnd1-3), Rho RhoD, Chp, Rif, Wrch1

Rab Rab1-63

ARF ARF1-6, Arl 1-8

Ran Ran

Kir/Rem/Rad Rad, Gem, Kir, Rem1/2, Ges

The Ras family interacts with a wide spectrum of regulators and downstream effectors producing diverse cellular responses including proliferation, differentiation, apoptosis and cell cycle progression [48]. H-Ras, K-Ras4A/B and N-Ras are grouped together under the name p21 Ras or classical Ras proteins. The Rho family is involved in the control and reorganization of the actin cytoskeleton. The most widely recognized members of the Rho family are Cdc42, Rac1 and RhoA. In fibroblasts, Cdc42 regulates the formation of actin-containing microspikes or filopodia. Rac1 regulates the formation of lamellipodia and membrane ruffles. Activation of RhoA leads to formation of focal contacts and stress fibre formation. Rho proteins have also been reported to be involved in G1 cell cycle progression [49]. Rab proteins are the largest branch of the Ras superfamily. In mammals over 60 Rab proteins are known. They are localized to the cytoplasmic face of all organelles involved in intracellular transport and regulate or facilitate the assembly of SNARE complexes [49].

11 INTRODUCTION ______

ARF (ADP-ribosylation factor) proteins are required for maintaining the integrity of organelle structure and intracellular transport. The best characterized is ARF1, which is localized to the Golgi complex and is a regulator of nonclathrin and clathrin coat recruitment [50, 51]. ARF6 is the least conserved ARF member. It controls the integrity of peripheral membranes and appears to cycle between the plasma membrane and recycling endosomes [49, 52]. Ran is an essential factor in nuclear transport [49]. It both defines compartmental identity and specifies directionality of nuclear-cytoplasmatic transport [53]. Ran appear also to be involved in other cellular activities, such as nuclear assembly, mitotic cell-cycle regulation [54] and spindle assembly [55, 56]. Kir/Rem/Rad are the latest contribution to the Ras superfamily. They differ from the other Ras-like GTPases in several ways. They lack, for example, the carboxy-terminal modification motif, CAAX [57-60]. The functions of these proteins are not well described, but there are reports suggesting a connection to the cytoskeleton [59-64].

Signalling through Ras

The four members of the p21 Ras proteins (H-Ras, N-Ras, K-Ras4A and K-Ras4B) are ubiquitously expressed. The amino acid difference are located in the C-terminal part of the proteins, but the functional differences are still not well understood. It seems that the differences between the different Ras proteins are due to different functions, rather than random drift, since K-Ras is conserved between species as diverse as carp, opossum and human. It has also been reported that K-Ras alone is essential for development, while embryos lacking either N-Ras or H-Ras seem perfectly normal [65]. The Ras proteins become associated with the inner side of the plasma membrane after posttranslational modifications, and this localisation is important for Ras activity [66]. All four proteins have a carboxy-terminal CAAX motif, where a cysteine is followed by two aliphatic residues and one random amino acid. This sequence is required to initiate the posttranslational modifications. The cysteine is first prenylated, followed by proteolysis of the C-terminal tripeptide, AAX, with the now C-terminal cysteine carboxy-methylated [67]. Palmitylation of one or two cysteines in the hypervariable region of H-Ras, N-Ras and K-Ras 4A serve as an additional signal required for translocation [68]. K-Ras 4B lacks sites for

12 INTRODUCTION ______

palmitylation, but has multiple basic residues that are thought to stabilize membrane localization by interacting with negatively charged groups in the membrane [67]. In short, these modifications lead to a Ras protein that is more hydrophobic and hence has a higher affinity for membranes.

Guanine nucleotide Exchange Factors (GEFs) for Ras

Ras is part of a multi-component signalling network which gets activated transiently by different receptors and activates many downstream targets. The proteins responsible for nucleotide exchange, and hence the activation of Ras, are the GEFs. The GEFs identified upstream of Ras are GRF (Guanine nucleotide Releasing Factor) 1-2, GRP (Guanine nucleotide Releasing Protein) 1-4 and Son-of-Sevenless 1/2 (Sos) (see Table 2)

Table 2 Summary of p21 Ras proteins and respective GEF

H-Ras N-Ras K-Ras 4A K-Ras 4B

Sos1 + + + +

Sos2 +

GRF1 + - -

GRF2 +

GRP1 + + +

GRP2 - + +

GRP3 +

GRP4 +

The GEFs contain many different domains, making it possible for them to respond to many different signals, including for example, phosphorylation of lipids, calcium fluxes and generation of diacylglycerol (DAG). GRF1 and 2 are both expressed in the brain and seem to be H-Ras specific [69]. They are calmodulin-binding through an IQ domain, and are activated by calcium [70]. GRF1 has also been implicated in activation of Ras through heterotrimeric G-protein coupled signals [71].

13 INTRODUCTION ______

The GRPs have both calcium and DAG binding domains. GRP1 is localized in T- and B- cells, while GRP3 may only be found in B-cells. GRP4 seems to be mast cell specific [72].

Sos (∼150 kDa) is the best characterized GEF for Ras, and is ubiquitously expressed [73]. Sos mediates Ras activation from several stimuli including receptor tyrosine kinases, tyrosine kinase coupled receptors and heterotrimeric G-protein coupled receptors. Upon extracellular stimuli, Sos is recruited to the plasma membrane by the growth factor receptor-bound protein 2 (Grb2), which recognizes tyrosine phosphate docking sites located on receptors themselves or in receptor substrate proteins, like Shc [74-81]. Localized to the membrane, Sos is in close contact to Ras, resulting in Ras activation [82, 83] (Fig 3).

EGF

Ras Shc GDP Sos Ras Grb2 GTP

Figure 3: Activation of Ras upon EGF stimulation

GTPase Activating Proteins (GAPs) for Ras

To control the output of the signals upon extracellular stimulation the Ras proteins finally have to be inactivated by hydrolysis of the GTP into a GDP. The intrinsic GTPase activity of each small G protein is relatively slow and the activity is thereby stimulated by GAPs. There are five GAPs known to inactivate Ras: p120GAP, neurofibromin1 (NF1), GAP1m, GAPIII and GapI(IP4BP) (also called R-RasGAP) [84-89]. p120GAP was the first identified Ras GAP. It is a cytoplasmic protein increasing the hydrolysis of the GTP bound to active Ras [90-93]. p120GAP also interacts with p190GAP, which primarily functions as a GAP for Cdc42, Rac1 and RhoA [94]. This provides evidence that p120GAP may serve dual roles as both a negative regulator of Ras and as an effector that facilitates Ras regulation of Rho protein function.

14 INTRODUCTION ______

NF-1 possesses GTPase-activating activity and can complement the loss of IRA function in S. cerevisiae, indicating that it might be the mammalian homologue of the yeast protein IRA1 and IRA2 [95]. NF-1 is the human protein defective in von Recklinghausen neurofibromatosis [96, 97]. GAP1m and GAP1(IP4BP) both interact with inositol 1,3,4,5-tetrakisphosphate (IP4) [98, 99]. GAP1m gets recruited to the plasma membrane from the cytosol upon EGF stimulation [100]. GAP1(IP4BP) on the other hand resides entirely at the plasma membrane regulating Ras activity [98].

Downstream of Ras

Together, GEFs and GAPs tightly regulate the downstream signalling output of the Ras proteins. Activation results in a GTP-bound Ras that can interact with a variety of downstream effector proteins. Of these, three have been studied in this thesis: Raf, PI3-kinase and RalGDS.

The Raf pathway Activated Ras binds to Raf serine/threonine kinases and causes their translocation to the cell membrane where Raf activation takes place [101]. The kinase Raf-1 (c-Raf) and its close relatives B-raf and A-raf are the best characterized downstream effectors of Ras. All three Raf isoforms share Ras as a common upstream activator and MEK as the only known downstream substrate [102, 103]. Raf-1 interacts with 14-3-3 in unstimulated cells [104-107], which keeps Raf-1 in an inactive state [108]. This interaction is needed for recruitment of Raf-1 to the plasma membrane where it interacts with activated Ras [109]. Once at the plasma membrane, phosphatidylserine (PS) acts to promote the interaction with Ras by displacing 14-3-3 to permit full Raf-1 activation [110, 111]. Finally, 14-3-3 may recycle Raf-1 back to the cytosol [112]. Still, the activation of Raf is not fully understood. Active Raf-1 activates MEK by phosphorylation of two serine residues in the activation loop of the MEK kinase [103]. MEK is a dual-specific kinase that can phosphorylate both threonine and tyrosine residues [113]. MEK activates MAP kinases, Erk1 and Erk2, via phosphorylation of a Thr-Glu-Tyr motif in the activation loop [114-116]. Activated Erks then phosphorylate and activate different

15 INTRODUCTION ______

molecules such as transcription factors, cytoskeletal proteins, kinases or phosphatases (Fig 4) [117]. A number of proteins have recently been identified and suggested to be involved in the regulation and stabilization of the MAP kinase pathway. One of these proteins is the scaffold protein Kinase Suppressor of Ras (KSR), which appears to organize the MAP kinase modules and ensure the efficiency and fidelity of the signal transduction [118]. Upon extracellular stimulation, KSR gets dephosphorylated by PP2A, allowing the KSR complex to localize with activated Ras at the plasma membrane where it facilitates the signalling through Raf, MEK and Erk [119, 120]. KSR is sequestered by 14-3-3 in the cytoplasm in the resting cell through a serine phosphorylation mediated by C-TAK1 [120]. Another protein which seems to be important for MAP kinase signalling is MEK Partner 1(MP1). MP1 links MEK with Erk upon Ras activation [121]. This might ensure that the kinase cascade is proceeded. MP1 interacts with a small protein called p14, and this interaction results in MP1 localization to late endosomes [122]. This process seems to be required for efficient signalling in the Erk cascade after EGF stimulation [122]. Negative regulators of the MAP kinase pathway have recently been identified, such as Raf Kinase Inhibitory Protein (RKIP), increasing our understanding of the complexity of the signalling cascade [123]. RKIP acts like a competitor and thereby disrupts the physical interaction between Raf-1 and MEK [124].

Ras GTP 14-3-3 Raf

MP1 14-3-3 MEK p14 Ksr Erk

C-TAK1

transcription

Figure 4: Signalling downstream of Ras through the Raf pathway

16 INTRODUCTION ______

The PI-3 kinase pathway

PI-3-kinase catalyses the phosphorylation of PI(4,5)P2 to yield PI(3,4,5)P3 in response to many growth factors and cytokines. PI-3-kinase and its lipid products act on pathways that control cell proliferation and cell survival [125]. Most PI-3-kinases consist of two subunits, the regulatory p85 [126, 127], which interacts with the activated receptor and the catalytic p110, which has been shown to interact with RasGTP in some cellular systems [128, 129]. Upon extracellular stimulation and PI-3-kinase activation, the substrate of PI-3-kinase,

PtdIns(3,4,5)P3, directly interacts with the pleckstrin homology (PH) domain of PKB/Akt, causing its translocation to the plasma membrane and enabling other kinases to phosphorylate and activate PKB/Akt [130-133]. Three isoforms of PKB/Akt has been identified (Akt1, Akt2 and Akt3) and they show high homology on the amino acid level [134, 135]. Although they have different tissue distribution, they appear to be equal in activation and activity [134, 135]. PKB/Akt gets phosphorylated at two sites, Thr308 and Ser 473 [136]. The protein responsible for the Thr308 phosphorylation is PI-3-kinase Dependent Kinase 1 (PDK1) [137, 138]. PDK1 contains a PH domain and the interaction with PtdIns(3,4,5)P3 localizes PDK1 in close proximity to with PKB/Akt, thereby allowing PDK1 to phosphorylate and activate PKB/Akt [139]. The identity of the kinase that phosphorylates Ser473 is still under investigation. Recently, it has been suggested that there is a kinase, called PDK2, which uses PKCζ as an adaptor molecule when phosphorylating Ser473 [140]. Once phosphorylated and activated, PKB/Akt phosphorylates several downstream targets, for example Forkhead, leading to cell survival [141] (Fig 5) and glycogen synthase kinase-3 (GSK3) which influences cellular metabolism [142].

PIP2 PIP3 Ras P PIP3 GTP PI3-K K P D B K 1

-3 -3 14 Forkhead Forkhead

Survival Apoptosis

Figure 5: Signalling through the PI-3K pathway

17 INTRODUCTION ______

The RalGDS pathway Three nucleotide exchange factors for Ral, named RalGDS, Rgl and Rlf, are recruited to the plasma membrane by activated Ras. These have been shown to stimulate Ral activation in a Ras-dependent manner [143, 144]. RalGDS also interacts with the amino-terminal of PDK1 which enhances the specific activity of the RalGEF in response to EGF [145]. The function of the ubiquitously expressed small GTPases RalA and RalB has just begun to be understood. Active Ral interacts with its downstream target Ral binding protein1 (RalBP1), which has been suggested to be a GAP protein for Cdc42 and Rac1, but not for RhoA [146, 147]. Another signal transduction pathway where Ral might be involved is in the activation of phospholipase D (PLD) [148], which results in the hydrolysis of phosphatidylcholine, generating phosphatidate and choline [149]. RalBP1 also connects Ral to the Partner for RalBP1 (POB1) [150]. Ral, RalBP1 and POB1 appear to be involved in the regulation of internalisation of the EGF and insulin receptors through the interaction between Eps15, epsin and POB1 at the coated pit [151-153]. These results suggest that Ral, RalBP1 and POB1 transmit the signal from the EGF receptor, via Ras and RalGDS, resulting in the regulation of ligand-dependent receptor mediated endocytosis [153] (Fig 6).

Ral Ras GDP Ral GTP GTP RalGDS

RalBP GDP Cdc42 POB1 GDP Rac

Receptor endocytosis Actin cytoskeleton

Figure 6: Signalling through the RalGDS pathway

Signals from RhoA, Rac1 and Cdc42

Originally, Rho proteins have been classified as regulators of the actin cytoskeleton architecture, but it is becoming more and more obvious that they interact with a variety of

18 INTRODUCTION ______

targets and influence a diverse range of cellular responses. They drive many forms of cellular motility, particularly extension of the leading edge of migrating cells [154] and transporting pathogenic microorganisms through host cell cytoplasm [155]. This thesis will focus on the actin portion of Rho signalling, and will leave the other signalling cascades unexamined.

As the Rho proteins belong to the Ras superfamily, they of course are regulated by GEFs and GAPs. Additionally they interact with guanine nucleotide dissociation inhibitors (GDIs) [156, 157]. GDIs preferentially bind GDP-bound Rho proteins and prevent spontaneous release of the nucleotide, thereby maintaining the GTPases in an inactive state. Upon extracellular stimulation, the GDP-bound form of Rho proteins is released from GDI by an unknown mechanism, and is converted into a GTP-bound form by a GEF. The GTPases are then recruited from the cytoplasm to the plasma membrane where they interact with downstream effectors.

Cdc42 Rac Rho

N-WASP PAK PI-4-P5K ROCK Dia

Arp2/3 LIMK MLC Profilin

ADF/Cofilin

Filopodia Stress fibres Focal adhesion Lamellipodia

Figure 7: Signalling pathways in actin rearrangments involving Rho GTPases

Rho A has at least two effectors that appear to be required for formation of stress fibres and focal adhesions: ROCK and Dia [158-160]. First, ROCK phosphorylates the myosin light chain (MLC) [161, 162] and then prevents its dephosphorylation by inhibiting MLC phosphatase through phosphorylation of the myosin-binding subunit (MBS) of MLC phosphatase [163, 164]. This results in an increase in MLC phosphorylation and promotes the assembly of actomyosin filaments [162, 163, 165]. ROCK can also phosphorylate LIM kinase

19 INTRODUCTION ______

(LIMK) which inhibits the actin depolymerizing protein ADF/cofilin, leading to stabilization of actin structures [166, 167]. Dia interacts with profilin, a G-actin-binding protein [168]. Through this interaction, Dia contributes to actin polymerization and F-actin organization into stress fibres [169]. Activation of both Dia and ROCK are required to induce stress fibres [169, 170] (Fig 7 and 8).

Rac1 and Cdc42 appear to have common downstream target proteins [171, 172]. They bind and activate N-WASP. Active N-WASP increases actin polymerization through interaction with Arp2/3, profilin and G-actin [173, 174] (Fig 7). Rac and Cdc42 also mediate activation of LIM kinase through activation of PAK [175]. LIM kinase phosphorylates ADF/cofilin leading to an inactive ADF/cofilin that no longer can depolymerise actin filaments [166, 167] (Fig 7 and 8). Rac also interacts with PI-4-P5K, leading to actin polymerization [176] (Fig 7). The resulting output from all these signals is generally summarized as follows: Cdc42 induces filopodia [177], Rac lamellipodia [178] and finally Rho assembles focal adhesions and stress fibres [179].

Membrane pushed forward Extra cellular stimuli by growing filaments Extra cellular stimuli

n o ti A a g Capping limits T n Cdc42 P o h l elongation y E dr ol Rac/Rho ys Cdc42 is a nd Dia P i di ADF/cofilin actin ss oc inhibition ia ti LIMK on PAK ADF/cofilin severs ADF/cofilin and depolymerize Arp2/3 complex ADP-ATP exchange WASP

Figure 8: Model depicting actin dynamics (Adapted from Pollard et. al 2000[180] )

20 INTRODUCTION ______

The signalling molecules Rap1 and Rap2

Rap1 is activated by stimulation of various transmembrane receptors, including receptor tyrosine kinases, cytokine receptors cell-adhesion molecules and others [181]. Several second messenger pathways, including calcium, diacylglycerol (DAG) and cAMP, are also able to activate Rap1 [181-184]. It has an effector domain almost identical to that of Ras, suggesting that both proteins interact with the same/similar effectors. Indeed, Rap1 appears to suppress Ras function by interacting with the Ras effector Raf [185, 186], but evidence that Rap1 functions independently of Ras is emerging [187].

Rap2 shows 60% identity to Rap1, but still very little is known about this protein [188].

The abundant protein 14-3-3

14-3-3 proteins are some of the most abundant proteins in the eukaryotic cell. They are involved in many different signal transduction pathways and play an important role in the transmission of these signals. The 14-3-3 proteins form a highly conserved family of acidic dimeric proteins. They have been found in all eukaryotic organisms studied, ranging from mammals to yeast [189-191]. Many organisms contain multiple isoforms; in mammals seven isoforms have been found (β,γ, ε, η, σ, τ, and ζ). In many, but not all cases, 14-3-3 interacts with phosphorylated proteins and in this way, the 14-3-3 proteins play a role in various cellular processes including signal transduction, cell cycle regulation, apoptosis, stress response and cytoskeleton organisation. The function of the 14-3-3 proteins is still not completely understood, but the general areas of activity may be summarized as follows: (a) 14-3-3 proteins regulate the activity of enzymes [192], (b) 14-3-3 proteins may act as localization anchors, controlling the subcellular localization of proteins [193], (c) 14-3-3 proteins can function as adaptor molecules or scaffolds, thus stimulating protein-protein interactions [194]. The structures of the ζ and τ isoforms have been described [195, 196]. Each monomer in the U-shaped dimer is composed of nine antiparallel α-helices. The helices form walls around a mainly negatively charged groove, which is large enough to accommodate two helices of binding partners. The residues that line the interior of the target protein-binding cleft are highly conserved between the different isoforms. More variable residues are distributed over

21 INTRODUCTION ______

the outer surface of the protein. The N-terminal domains form the dimerization interface and the floor of the cleft. Different motifs have been identified in proteins that bind to 14-3-3. Many of these contain a phosphoserine (pS) residue flanked by an arginine and proline: RSXpSXP [197, 198], RX1-

2SX2-3pS [199], RX(Y/F)XpSXP or RXpSX(S/T)XP [198]. It appears that this phosphoserine motif provides a general mechanism for 14-3-3/ligand interactions. Interestingly, they also have been reported to bind to the unrelated GHSL [200], the R18 peptide PHCVPRDLSWLDLEANMCLP [201] and to the phosphothreonine (pT)-containing YpTV motif [202], suggesting an even broader function for this group of proteins.

ExoS, ExoT and eukaryotic signal transduction

Many bacterial toxins have been observed to alter eukaryotic signal transduction by specifically targeting members of the Ras superfamily. For example, C3 from Clostridium botulinum ADP-ribosylates RhoA-C at asparagine (Asn) 41 and thereby inhibits Rho signalling [203]. Clostridium difficile toxins A and B also inhibit Rho proteins - RhoA-C, Rac and Cdc42 – through glycosylation of threonine 37 [204]. On the other hand, the E. coli toxin CNF1 deamidation of glutamine 63 Rho results in a constitutively active Rho protein [205]. Recently it has been suggested that ExoS also could be added to this group of bacterial toxins targeting Ras superfamily members.

ExoS, ExoT and their ADP-ribosylated target molecules

ExoS has been suggested to have several target molecules for its ADP-ribosyltransferase activity in vitro, including the intermediate filament protein vimentin [206] and H-Ras, Rap1A, Rap2, Ral, Rab3 and Rab4 [207, 208]. Recently it was suggested that Ras was the in vivo target for ExoS in eukaryotic cells [209] and that ExoS ADP-ribosylates Ras preferentially at two sites, arginine 41 and arginine 128 [24, 25]. When this study was initiated, the effect and the consequences of the ADP-ribosylation of Ras were unknown, but better understanding of this process may help to elucidate the mechanism of cytotoxicity of P. aeruginosa infections.

22 INTRODUCTION ______

Although ExoT and ExoS show high homology on the amino acid level, the ADP- ribosylating activity of ExoT has been reported to be only 0.2-1% of ExoS in vitro [29, 30]. The cellular target for ExoT has not yet been found. Clinical isolates investigated by Fleitzig 1997 showed that all strains harbour the exoT gene, but not always the exoS gene, which implicates a unique role for ExoT, distinct from ExoS [11]. Previous studies show that ExoT, like ExoS, alters the cell morphology of infected cells [210]. This suggests that the toxin has a specific activity inside the eukaryotic cell.

ExoS, ExoT and the GAP activity

Early studies showed that eukaryotic cells infected with ExoS show rounded cell morphology due to disrupted actin microfilaments and thereby possible resistance to phagocystosis [17]. It was later found that the amino-terminal portion of ExoS harbours an activity with this effect on the eukaryotic cell, and this could be reversed by treatment with CNF-1, which constitutively activates RhoA [211]. Rescuing the cells by activating RhoA indicate that the amino-terminal of ExoS might act as a GAP for Rho proteins inside the eukaryotic cell. [211]. In vitro GAP assay studies with recombinant proteins suggested that the N-terminal part of ExoS harboured GAP activity towards RhoA, Rac1 and Cdc42 [20]. These studies also identified arginine 146 as an essential amino acid for this enzymatic activity [20].

Previous studies showed that ExoT, like ExoS, alters the cell morphology of infected cells [210]. Since ExoT has high homology to ExoS, with arginine 149 as homolog to arginine 146 on ExoS, ExoT may also exhibit GAP activity.

ExoS and 14-3-3

ExoS requires a soluble eukaryotic protein, which has been named FAS (Factor Activating exoenzyme S), in order to ADP-ribosylate its substrates in vitro [26]. The requirement for a eukaryotic protein for enzymatic activity may be a device to identify the eukaryotic environment and to ensure that the bacterial enzymes cannot function internally and harm the toxin producing bacteria. It was later found that FAS is a member of the 14-3-3 protein family [27]. As mentioned earlier, 14-3-3 preferentially interacts with serine phosphorylated proteins. Interestingly, ExoS is not known to be phosphorylated [212]. The positive amino

23 INTRODUCTION ______

acids Lys49, Arg56, Lys120 and Arg127 in the basic groove of 14-3-3 have been suggested as crucial for the activation of ExoS [213]. This suggests that negatively charged residues on ExoS may be needed for the interaction to occur. Lys49 has also been reported to be necessary for 14-3-3 interaction with Raf-1 [214], suggesting that diverse ligands may share a common site on 14-3-3 proteins.

The Yersinia pseudotuberculosis model system

The type III secretion system, as mentioned above, functions to deliver proteins directly from the bacterium into the cytosol of the eukaryotic cell. The proteins secreted via this system are not processed during secretion, and the sequence targeting proteins for secretion is located in the N-terminal of the protein [215]. The secreted proteins need small cytoplasmic proteins, chaperones, which specifically interact with the type III system to ensure stabilization and efficient secretion [216-221]. Immediately downstream from the secretion signal resides the recognition sequence for translocation into the host cell [222]. The delivery of proteins is dependent on physical contact between the bacterium and the eukaryotic cell and the translocation process appears to be polarized [8]. The type III apparatus of Yersinia pseudotuberculosis is one of the most intensively studied among the Gram-negative pathogens. At least 20 components co-operate to form a channel through the bacterial double envelope that permits the secretion of virulence factors in one step from the cytoplasm to the extracellular environment [9]. At least three translocator proteins, LcrV, YopB and YopD are needed for the translocation of Yop proteins into the host cell [223-225]. Through several independent methods, it has been proposed that a complex of LcrV, YopB and YopD forms a pore in the eukaryotic cell membrane through which the other virulence factors are delivered into the eukaryotic cytosol [223, 226-229]. The translocation apparatus of P. aeruginosa show high similarity to the one of Y. pseudotuberculosis. The similarities are seen both at the level of the individual protein components as well as for genetic organisation [19, 216, 230]. Individual selected components of the secretion system have been shown to be interchangeable between P. aeruginosa and Y. pseudotuberculosis [231]. In paper I-III, we have used the type III secretion system of a genetically defined secretion and translocation mutant of Y. pseudotuberculosis to express and translocate ExoS

24 INTRODUCTION ______

into the eukaryotic cell. It has been shown that the type III secretion system of Y. pseudotuberculosis can secret and translocate ExoS into the target cell with the same mode of delivery as for its normal cytotoxins [17]. The advantage to use this approach is that it enabled us to study the effect(s) of ExoS in the absence of other toxins and proteases secreted by P. aeruginosa. In paper V, we used a different variant of bacteria to translocate ExoS into the eukaryotic cell. The Y. pseudotuberculosis used in paper I-III expresses and delivers ExoS proteins with high efficiency, at levels higher then P. aeruginosa. To reduce the expression and translocation of ExoS from the bacteria into the target cell we constructed a Y. pseudotuberculosis strain which expresses and tranlocates ExoS under the control of an arabinose inducible promoter [232]. By growing the bacteria in the presence of 0.1% arabinose in the culture media we could induce a reduced expression and translocation of ExoS into eukaryotic cells, similar to P. aeruginosa wild-type levels.

25 AIMS ______

AIMSAIM

The investigations in this thesis were designed to study the enzymatic activity of ExoS on eukaryotic cell signal transduction through examination of the ADP-ribosyltransferase activity and the GAP-activity in cell culture systems.

The specific aims were as follows: to identify the target molecule(s) for both ExoS activities in the eukaryotic cell, to analyse the effect(s) of signal transduction downstream these targets after infection, to study the effect of ExoT on eukaryotic cells.

A second general aim was to study the binding motif with which ExoS interacts with 14-3-3 inside the eukaryotic cell.

Specific aims here were as follows: to use mutational analysis of ExoS to find the specific amino acids necessary for 14-3- 3 interaction, to analyse toxicity in vivo of ExoS mutants which are unable to interact with 14-3-3.

26 RESULTS AND DISCUSSION ______

RESULTSRESULT AND DISCUSSION

Ras inhibition by ADP-ribosylation in vivo (Paper I)

With Ras proposed as a target for P. aeruginosa toxin ExoS [23], this study was designed to analyse the functional consequences of ADP-ribosyltransferase modification of Ras in vivo. In this study we have used the Yersinia pseudotuberculosis model system to translocate wild type ExoS and different ExoS variants into the eukaryotic cell. The mutants that were used are shown in Figure 9. In short, we have used a point mutant, ExoS(E381A), which has roughly 2000 times lower enzymatic activity and two deletion variants, lacking either the C-terminal GAP domain, ExoS(∆98-232), or both lacking the GAP domain and containing the point mutation, ExoS(E381A, ∆98-232). In this way, the important domains of ExoS could be studied separately to confirm their participation in ADP-ribosylation process.

E381 1 453 ExoS wt

Secr./Transl. GAP ADP-ribosyltransferase

98 232 E381A 1 453 ExoS(E381A,∆98-232)

E381A 1 453 ExoS(E381A)

98 232 1 453 ExoS(∆98-232)

Figure 9: Schematic drawing of wild-type ExoS and the ExoS variants used in this study

27 RESULTS AND DISCUSSION ______

In time course experiments a mobility shift of Ras by SDS-PAGE was seen already after 15 minutes upon infection with bacteria expressing wild-type ExoS (Fig 2, Paper I), indicating that Ras is indeed modified in vivo. This is in agreement with McGuffie et. al [209] who showed Ras modification after infection with the P. aeruginosa strain 388. ADP-ribosylation of Ras has been suggested to correlate with inhibition of DNA synthesis [209], which indicates that Ras modification might effect proliferative signals from Ras. Indeed, after 32 metabolic labelling of cells with [ P]Pi, followed by infection with bacteria expressing ExoS and EGF stimulation, we observed a dramatic decrease in GTP-bound Ras (Fig 3, Paper I). Using a pulldown experiment with the Ras-binding domain of Raf the inactivation of Ras was further established (Fig 4, Paper I). Raf is the first molecule in the kinase cascade that leads to activation of the MAP kinases Erk1 and Erk2. Another signalling pathway induced by EGF stimulation is the PI-3 kinase pathway, which results in the activation of PKB/Akt (see Introduction). Infection with bacteria expressing wild-type ExoS or ExoS(∆98-232) abolished the phosphorylation of Erk1/2 and PKB/Akt, as compared to non-infected cells (Fig 6, Paper I). From these experiments we conclude that ExoS is able to block activation of Ras upon stimulation with EGF, and that it is the carboxy-terminal part of ExoS that ADP-ribosylates and inhibits Ras in vivo.

ExoT elicits cytotoxicity without interfering with Ras (Paper II)

ExoT shows a high degree of homology with ExoS, but no target protein(s) have been found. Clinical isolates all harbour the exoT gene, but not always the exoS gene, which suggests a unique function for ExoT in pathogenesis [11]. The conservation of the exoT gene across strains, and the fact that it co-exists with ExoS, suggests that ExoT has an important role in infection. HeLa cells infected with bacteria expressing ExoT followed by phalloidin staining showed that ExoT disrupts the actin microfilaments (Fig 3, Paper II), which agrees with previous transfection studies [233]. Since it has been suggested that ExoT inhibits internalization and that arginine 149 is essential for this activity [234, 235], ExoT might be a GAP for the small GTPases of the Rho subfamily. Indeed, in a filter-binding assay we observed that wild-type ExoT increased the intrinsic hydrolysis of RhoA (fig 6, paper II) [233]. Substitution of arginine 149 abolishes, as suggested, the GAP activity (Fig 6, Paper II). Interestingly though, a GAP deficient ExoT still causes a morphological change of eukaryotic

28 RESULTS AND DISCUSSION ______

cells, indicating an existing ADP-ribosyltransferase target for ExoT (Fig 7, Paper II). ExoS has been shown to ADP-ribosylate and inhibit Ras signal transduction (paper I) [209], but ExoT did not target Ras in vivo in this experiment (Fig 5, Paper II). Recently, ExoT was shown to ADP-ribosylate CrkI and CrkII [236]. The Crk proteins are thought to have a central role in mediating actin skeleton related events, like phagocytosis, and their modification might connect ExoT mediated anti-internalization to its ADP-ribosyltransferase activity.

ExoS acts towards members of the Ras superfamily in vivo (paper III)

To understand the effects of ExoS and ExoT on cellular function, we have focused on the identification of signalling molecules that are putative in vivo targets for ExoS and ExoT. These two toxins show high homology at the amino acid level and are often co-ordinately expressed [12, 29, 230]. However, it has been observed that clinical isolates do not always contain both the exoS and the exoT gene, suggesting that these two proteins have different targets and functions in the eukaryotic cell [11]. It has been established that Ras is an in vivo target for ExoS ADP-ribosyltransferase activity [209] (Paper I). Since there is high homology among the small GTP-binding proteins we investigated if other members of the Ras superfamily could be significantly ADP-ribosylated by ExoS. We used both Y. pseudotuberculosis and P. aeruginosa to express and translocate ExoS and ExoT into HeLa cells. The results showed that ExoS indeed modified H-Ras, N- Ras, K-Ras, R-Ras, Rap1, Rap2, RalA, Rac1, Rab5, Rab8, Rab11 and Cdc42 in vivo (Fig 2 and 3, Paper III). This was later confirmed by Fraylick et. al [237, 238] who used 2- dimentional electrophoresis to identify ADP-ribosylated target molecules. The infection with bacteria expressing ExoT resulted in no detectable in vivo modification of any of the small GTPases tested (Fig 2 and 3, Paper III). From these result we conclude that ExoS is the major ADP-ribosylating toxin encoded by P. aeruginosa directed against the Ras superfamily members. Since we detected ADP-ribosylation of several members of the Ras superfamily members, we wished to extend the test and see whether or not ExoS modulates the activity of these small GTPases in vivo. Pulldown assays were employed using specific downstream target proteins for the various GTPases investigated. Proteins chosen for further studies were RalA, Rap1, Rap2, Rac1, RhoA and Cdc42. The ExoS variants used to detect the two different activities of

29 RESULTS AND DISCUSSION ______

ExoS were ExoS wt, ExoS(E381A) (containing only GAP activity) and ExoS(∆98-232) (only ADP-ribosylation) (Fig 9).

ExoS activity on Ras subfamily

The ADP-ribosylation of Ras, RalA, Rap1 and Rap2 clearly inhibited the co-precipitation of the small GTPases (Fig 5, 6, 7, Paper III) and thus the activation of these proteins upon extra- cellular stimulation. It has recently been suggested that the ADP-ribsylation inhibits the interaction between Ras, Rap1 and their respective GEF in vitro [239, 240], which could explain why these proteins are held in their GDP-bound state even after stimulation. We extended our analysis to cells transiently transfected with HA-tagged Rap2 in the presence or absence of HA-tagged PDZ-GEF, an exchange factor for Rap1 and Rap2. As observed earlier, infection with ExoS modified HA-Rap2, and a decrease in binding to GST-RalGDS was observed, even in the presence of the GEF (Fig 10).

Figure 10: HeLa cells transiently transfected with HA-Rap2 and HA-PDZ-GEF followed by infection with ExoS variants as indicated. (A) Western blot analysis of whole cell lysate using an anti-HA antibody. (B) GST-RalGDS pulldown followed by western blot using an anti-HA antibody.

Infection with bacteria expressing and translocating ExoS(E381A) reflected the GAP activity of ExoS. In Figure 7, Paper III, it was obvious that Rap1 showed reduced activation upon

30 RESULTS AND DISCUSSION ______

infection with ExoS(E381A) and thus ExoS exhibits GAP activity toward this small GTPase. Rap2, on the other hand, seems totally unaffected by ExoS GAP activity. It is notable that ExoS shows differential GAP activity towards the Rap proteins, since Rap1 and Rap2 exhibit 60% identity at the amino acid level.

ExoS activity on Rho subfamily

The members of the Rho subfamily have been associated with the regulation of the actin cytoskeleton. They are suggested as important factors in several cellular processes, including migration and phagocytosis. Several bacterial toxins appear to block mechanisms regulating phagocytosis [241-244] protecting the bacteria against the immune system. The amino- terminal of ExoS disrupts the actin microfilaments and induces phagocytosis resistance [17, 245] and recently, the amino-terminal of ExoS was proposed to be a GAP for RhoA, Rac1 and Cdc42 in vitro [20]. The amino-terminal of ExoS also shows high homology to other suggested GAP toxins, like YopE. In Figures 9 and 10, Paper III, the pulldown assays show a reduction in RhoA and Cdc42 activation after infection with bacteria expressing either wild-type ExoS or ExoS(E381A), indicating that ExoS functions as a GAP protein for RhoA and Cdc42 in vivo. When studying the activation of Rac1, we observed a decrease in GTP-bound Rac1 after infection with ExoS(E381A), which would suggest that ExoS also functions as a Rac-GAP (Fig 8, Paper III). Unexpectedly, infection with ExoS wild-type produced no such reduction, and a rather slight increase in activity of Rac1 was obeserved. Thus, full-length ExoS appears to activate Rac1 in vivo. In cells infected with ExoS(∆98-232) which only contain the ADP- ribosyltransferase domain, a dramatic increase in active Rac1 was detected (Fig 8, Paper III). It is clear that ExoS wild-type can ADP-ribosylate Rac1, but this modification does not appear to alter Rac1 activity in vivo. The GAP domain of ExoS appears to function as a Rac- GAP in vivo, but this does not seem to have any biological relevance since this activity is not seen with full-length ExoS. It should be noted that this experiment highlights a drawback with using deletion variants of ExoS. The above results from two ExoS proteins, which are variants of the bi-functional enzyme ExoS, should be compared with the wild-type ExoS protein, which does not significantly modulate Rac1 activity in vivo, though it is clearly able to ADP- ribosylate Rac1.

31 RESULTS AND DISCUSSION ______

14-3-3 binding is required for the inhibition of Ras by ExoS(Papers IV and V)

14-3-3 is the eukaryotic protein suggested to be required for ADP-ribosylation of ExoS targets in vitro [26, 27]. In the cell, 14-3-3 interacts with various proteins and this binding is mostly phosphorylation dependent [197, 246]. However, phosphoserine recognition does not account for all 14-3-3-ligand interactions, so a different motif must exist for unphosphorylated proteins like ExoS. By employing a GST-fusion protein pulldown approach, we localized the 27 most C-terminal amino acids of ExoS to be important for 14-3- 3 binding (Fig 1, Paper IV). Transfection of cells verified that this interaction is important also in vivo, as ExoS lacking the interaction motif is significantly less cytotoxic then ExoS wt (Table 1, Paper IV). The best studied example of a nonphosphorylated interaction with 14-3-3 is with an artificial peptide, R18, which was isolated from a phage display library as having high affinity for 14-3-3 proteins. The motif WLDLE in R18 interacts with lysine 49 on 14-3-3 [201]. Lys49 seems important for ligand binding, since Raf and ExoS interaction with 14-3-3 also depends on this specific residue [214]. During our investigation for a consensus binding site on ExoS for 14-3-3 interaction, we noted that ExoS contains a DALDL sequence, at amino acid 424-428, which is similar to WLDLE in R18 (Table 2, Paper V). Amino acid substitution variants of ExoS in a pull down experiment showed that DALDL indeed was required for interaction between ExoS and 14-3-3 (Fig1, Paper V). Substitutions can always result in conformational changes in the original protein structure, so we performed a competition analysis with a peptide, denoted E-son, spanning the area of interest from amino acid 415-432 (Table 2, Paper V). In in vitro assays we observed that E-son not only was able to block the interaction between ExoS and 14-3-3, but was also capable of inhibiting ExoS ADP-ribosyltransferase activity (Fig 2 and 3, Paper V). It has previously been demonstrated that translocation of ExoS into eukaryotic cells causes rounding up of the cells followed by detachment from the surface [17]. When we infected HeLa cells with the Y. pseudotuberculosis arabinose strain expressing wild-type ExoS we noticed the expected morphological change and the following modification of Ras (Fig 5, Paper V). Infection with ExoS variants lacking the DALDL motif showed no cytotoxic response and no ADP- ribosylation of Ras (Fig 5, Paper V). Thus, the 14-3-3 binding motif of ExoS – DALDL – appears to be necessary for both the ADP-ribosylation activity and the cytotoxic action of ExoS in vivo.

32 CONCLUSIONS ______

CONCLUSIONSCONCLUSIONS

The C-terminal domain of ExoS ADP-ribosylates many members of the Ras superfamily in vivo, including H-Ras, N-Ras, K-Ras, R-Ras, Rap1, Rap2, RalA, Rac1, Rab5, Rab8, Rab11 and Cdc42

ADP-ribosylation by ExoS inactivates Ras, Rap1, Rap2, RalA in vivo

The N-terminal domain of ExoS functions as a GAP for RhoA, Cdc42 and Rap1 in vivo

ExoT ADP-ribosyltransferase activity does not seem to target members of the Ras superfamily in vivo

ExoT functions as a GAP for RhoA in vitro

The amino acids 424DALDL428 are necessary for the interaction between ExoS and 14- 3-3

ExoS interaction with 14-3-3 is required to elicit cytotoxicity in vivo.

33 ACKNOWLEDGEMENTS ______

ACKNOWLEDGEMENTSACKNOWLEDGEMENT

Many people have passed my way from day one at cmb until now at the pathology department and many of them have in some way contribute to this thesis – or just been great people to chat with during incubations and fika! I specifically want to thank:

My supervisor Bengt who somehow has believed in me all this time, even though I really am lazy….I guess I must have chosen the best supervisor at cmb!! Thanks for teaching me lots about cell signalling, western blots, wine, golf, gossip and many other important things in life! …and, we are unbeatable at bowling, aren’t we!?!

Ruth, me co-supervisor and idol. All your brilliant help over the years made my work so much better. It is not fair that you are only one year older then me but so much smarter!! I truly enjoyed our whale floating – wanna go again?? ☺

At the last couple of years I finally got “group BH PhD student fellows” (yippie!!) who made everything more enjoyable at work: Charlotte and Anna (I know you’ve left, I just still count you in!) for laughs and fika and for being there listening to my problems and non-problems and sharing ordinary PhD student-days. You’re great!!! Emma, Pawel and Lubna for being nice persons listening to my endless talks about nothing. I hope you will stay!

Christina, Caroline, Gaurav, Anna, Margareta (thank your mother too for the flowers!) and Sofie for all fantastic cakes at journal club!! You make Friday mornings sooooo much more fun!

Through the years several persons have passed “group BH” (and left ) and I think they all need a BIG thank you ☺. Henke (the best badminton/party partner you could ever have!) Ullis (too bad you don’t know how to appreciate snow and darkness) Amir (we had some really good times not working) Dan (the absolutely most enthusiastic, hard working student ever – doping??) Tooba and Kristina (I can’t say how happy I am that you made all those constructs!!)

Through the years I have shared room with a bunch of people that made my days brighter: Sara (may first and dearest room mate, I have missed you since you left!) Jeanette and Frida (we never repainted the room!), Jenny (I do miss our gossip hunts on aftonbladet.se), Fredrik (the perfect example of how to keep sane with no sleep)

Our collaborators in Edinburgh, Scotland, Professor Alastair Aitken and Dr Alex Peden for excellent contribution to our 14-3-3 project.

Matt for great bug making skills and our long talks about all and nothing.

Hans W, Roland R and Max being invaluable Yersinia experts through the whole project and thanks for having time with my endless questions.

Our partners at FOI, Åke, Elisabet and Lotta, for help with bugs and bug-knowledge.

My friend Elisabet for excellent help with the statistics.

34 ACKNOWLEDGEMENTS ______

Johnny, Ethel and Eva-Christine for making impossible things possible. You guys have saved my days many many times!

All the ladies at the media department who make our life so much easier – you are brilliant!!!

All past and present persons at the old cmb for making it a great and memorable place and forcing me to improve my English.

All my new colleges at the pathology department and the whole floor 2, building 6M for making me feel welcomed.

Ylva for your help with all thesis/dissertation strategies.

My wonderful ËFRIENDSË outside the lab for connecting me to the real world, keeping me somewhat normal ☺. Now it’s time to party!!

Saga, Sten, Marie, Jerker, (Henrik), Richard and Lina for being great and big-hearted persons!

My mother, father and sister Cissi (+ huge family), for supporting in me through all this, even though you never really understood what I have been doing.

David, the sweetest boy on earth, for making your mother happy. It is a pleasure to rediscover the world with you!

Per-Erik for being there, sharing it all ♥

Financial support for this work was provided by J.C. Kempe Memorial Grant Foundation, Swedish Society för Medical Research (SSMF), Foundation Lars Hiertas Memory and the Royal Swedish Academy of Sciences.

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