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• W2060690139 abstract "The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion. Two of the injected toxins, SopE and the 69% identical protein SopE2, are highly efficient guanine nucleotide exchange factors for the RhoGTPase Cdc42 of the host cell. However, it has been a puzzle why S. typhimurium might employ two toxins with redundant function. We hypothesized that SopE and SopE2 might have different specificities for certain host cellular RhoGTPases. In vitro guanine nucleotide exchange assays and surface plasmon resonance measurements revealed that SopE is an efficient guanine nucleotide exchange factor for Cdc42 and Rac1, whereas SopE2 was interacting efficiently only with Cdc42, but notwith Rac1. Affinity precipitation of Cdc42·GTP and Rac1·GTP from lysates and characteristic cytoskeletal rearrangements of infected tissue culture cells confirmed that SopE is highly efficient at activating Cdc42 and Rac1 in vivo, whereas SopE2 was efficiently activating Cdc42, but not Rac1. We conclude that the translocated effector proteins SopE and SopE2 allowS. typhimurium to specifically activate different sets of RhoGTPase signaling cascades. The bacterial enteropathogen Salmonella typhimurium employs a specialized type III secretion system to inject toxins into host cells, which trigger signaling cascades leading to cell death in macrophages, secretion of pro-inflammatory cytokines, or rearrangements of the host cell cytoskeleton and thus to bacterial invasion. Two of the injected toxins, SopE and the 69% identical protein SopE2, are highly efficient guanine nucleotide exchange factors for the RhoGTPase Cdc42 of the host cell. However, it has been a puzzle why S. typhimurium might employ two toxins with redundant function. We hypothesized that SopE and SopE2 might have different specificities for certain host cellular RhoGTPases. In vitro guanine nucleotide exchange assays and surface plasmon resonance measurements revealed that SopE is an efficient guanine nucleotide exchange factor for Cdc42 and Rac1, whereas SopE2 was interacting efficiently only with Cdc42, but notwith Rac1. Affinity precipitation of Cdc42·GTP and Rac1·GTP from lysates and characteristic cytoskeletal rearrangements of infected tissue culture cells confirmed that SopE is highly efficient at activating Cdc42 and Rac1 in vivo, whereas SopE2 was efficiently activating Cdc42, but not Rac1. We conclude that the translocated effector proteins SopE and SopE2 allowS. typhimurium to specifically activate different sets of RhoGTPase signaling cascades. guanine nucleotide exchange factor amino acid(s) O-(N-methylanthraniloyl)-GDP glutathioneS-transferase human umbilical vein endothelial cell Cdc42/Rac1-binding domain pleckstrin homology Dbl homology p21-activated kinase dithiothreitol phosphate-buffered saline bovine serum albumin Salmonella pathogenicity island I The RhoGTPase subfamily of the Ras superfamily of small GTP-binding proteins comprises more than 10 different proteins (1Ridley A.J. Hall A. GTPases. Oxford University Press, Oxford2000: 89-136Google Scholar). They act as molecular switches and cycle between GDP-bound (inactive) and GTP-bound (active) conformations (2Bourne H.R. Sanders D.A. McCormick F. Nature. 1991; 349: 117-127Crossref PubMed Scopus (2690) Google Scholar). Activation and inactivation of RhoGTPases is controlled by guanine nucleotide exchange factors (GEFs),1GTPase-activating enzymes, and guanine dissociation inhibitors (3Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1761) Google Scholar, 4Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1679) Google Scholar). The eukaryotic GEFs for Rho-GTPases share two common sequence motives, the DH (Dbl homology) and the PH (pleckstrin homology) domains, which are responsible for targeting, binding the RhoGTPases, and catalysis of RhoGTPase·GDP → RhoGTPase·GTP exchange (5Cerione R.A. Zheng Y. Curr. Opin. Cell Biol. 1996; 8: 216-222Crossref PubMed Scopus (466) Google Scholar). Only the active GTP-bound RhoGTPases can interact with downstream elements of signal transduction cascades mediating the cellular responses. GTPases of the Rho subfamily are central switches in the signaling cascades regulating motility, cellular adhesion, cell shape, cytokinesis, cell contraction, and gene expression (6Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5220) Google Scholar, 7Van Aelst L. D'Souza-Schorey C. Genes Dev. 1997; 11: 2295-2322Crossref PubMed Scopus (2097) Google Scholar). Each of the RhoGTPases can regulate a specific set of downstream signaling cascades, leading to activation of specific cellular functions (4Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1679) Google Scholar). For example, in Swiss 3T3 cells, activation of Cdc42 leads to the formation of filopodia, activation of Rac1 induces formation of lamellipodia, and activation of RhoA leads to the formation of stress fibers and focal adhesions (8Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3832) Google Scholar, 9Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3076) Google Scholar, 10Kozma R. Ahmed S. Best A. Lim L. Mol. Cell. Biol. 1995; 15: 1942-1952Crossref PubMed Scopus (883) Google Scholar, 11Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3735) Google Scholar). Thus, selective activation of specific RhoGTPases leads to specific cellular responses. Invasion of the Gram-negative bacterial enteropathogen Salmonella typhimurium (Salmonella enterica subspecies I serovar Typhimurium) into non-phagocytic mammalian cells is studied as a model system for the “trigger mechanism” of bacterial invasion (12Galán J.E. Curr. Opin. Microbiol. 1999; 2: 46-50Crossref PubMed Scopus (156) Google Scholar). For triggering invasion, S. typhimurium employs the specialized type III secretion system encoded in “Salmonellapathogenicity island I” (SPI1) to inject/translocate a set of at least nine different bacterial toxins (called “effector proteins”) into host cells (13Wood M.W. Rosqvist R. Mullan P.B. Edwards M.H. Galyov E.E. Mol. Microbiol. 1996; 22: 327-338Crossref PubMed Scopus (207) Google Scholar, 14Galyov E.E. Wood M.W. Rosqvist R. Mullan P.B. Watson P.R. Hedges S. Wallis T.S. Mol. Microbiol. 1997; 25: 903-912Crossref PubMed Scopus (232) Google Scholar, 15Collazo C.M. Galán J.E. Mol. Microbiol. 1997; 24: 747-756Crossref PubMed Scopus (256) Google Scholar, 16Hardt W.-D. Galán J.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9887-9892Crossref PubMed Scopus (185) Google Scholar, 17Jones M.A. Wood M.W. Mullan P.B. Watson R. Wallis T.J. Galyov E.E. Infect. Immun. 1998; 66: 5799-5804Crossref PubMed Google Scholar, 18Fu Y. Galán J.E. Mol. Microbiol. 1998; 27: 359-368Crossref PubMed Scopus (200) Google Scholar, 19Zhou D. Mooseker M.S. Galán J.E. Science. 1999; 283: 2092-2095Crossref PubMed Scopus (329) Google Scholar, 20Miao E.A. Scherer C.A. Tsolis R.M. Kingsley R.A. Adams L.G. Bäumler A.J. Miller S.I. Mol. Microbiol. 1999; 34: 850-864Crossref PubMed Scopus (226) Google Scholar, 21Bakshi C.S. Singh V.P. Wood M.W. Jones P.W. Wallis T.S. Galyov E.E. J. Bacteriol. 2000; 182: 2341-2344Crossref PubMed Scopus (138) Google Scholar, 22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar). Inside the host cell, the effector proteins activate signaling cascades leading to a variety of responses including cytoskeletal rearrangements and bacterial internalization/invasion (12Galán J.E. Curr. Opin. Microbiol. 1999; 2: 46-50Crossref PubMed Scopus (156) Google Scholar). It has been shown that Cdc42 is a key element in theSalmonella-induced signaling cascades leading to transcriptional activation and bacterial invasion: disruption of Cdc42 signaling by transfection with dominant negative Cdc42N17alleles interferes with bacterial invasion and activation of c-Jun kinase and p21-activated kinase (PAK) signaling (23Chen L.M. Hobie S. Galán J.E. Science. 1996; 274: 2115-2118Crossref PubMed Scopus (258) Google Scholar, 24Hardt W.-D. Chen L.M. Schuebel K.E. Bustelo X.R. Galán J.E. Cell. 1998; 93: 815-826Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 25Chen L.M. Bagrodia S. Cerione R.A. Galán J.E. J. Exp. Med. 1999; 189: 1479-1488Crossref PubMed Scopus (45) Google Scholar). Rac1 plays a less prominent role, and disruption of Rac1 signaling merely leads to reduced S. typhimurium invasion rates, whereas inhibition of RhoA signaling does not affect invasion at all (23Chen L.M. Hobie S. Galán J.E. Science. 1996; 274: 2115-2118Crossref PubMed Scopus (258) Google Scholar). This suggests that the translocated effector proteins of S. typhimurium may preferentially address certain RhoGTPase signaling pathways. Recent work has demonstrated that S. typhimurium (strain SL1344) relies mainly on a set of three translocated effector proteins to trigger invasion. A triple mutant S. typhimurium strain lacking SopB, SopE, and SopE2 is non-invasive, even though the SPI1 type III secretion system is still fully functional (22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar, 24Hardt W.-D. Chen L.M. Schuebel K.E. Bustelo X.R. Galán J.E. Cell. 1998; 93: 815-826Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 26Mirold S. Ehrbar K. Weissmüller A. Prager R. Tschäpe H. Rüssmann H. Hardt W.-D. J. Bacteriol. 2001; 183: 2348-2358Crossref PubMed Scopus (106) Google Scholar, 27Hardt W.-D. Urlaub H. Galán J.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2574-2579Crossref PubMed Scopus (182) Google Scholar, 28Hong K.H. Miller V.L. J. Bacteriol. 1998; 180: 1793-1802Crossref PubMed Google Scholar, 29Galán J.E. Zhou D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8754-8761Crossref PubMed Scopus (215) Google Scholar, 30Zhou D. Chen L.M. Hernandez L. Shears S.B. Galán J.E. Mol. Microbiol. 2001; 39: 248-259Crossref PubMed Scopus (311) Google Scholar). SopB has phosphatidyl inositol phosphatase activity and asopB− mutant is less invasive than wild typeS. typhimurium (28Hong K.H. Miller V.L. J. Bacteriol. 1998; 180: 1793-1802Crossref PubMed Google Scholar, 31Norris F.A. Wilson M.P. Wallis T.S. Galyov E.E. Majerus P.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14057-14059Crossref PubMed Scopus (359) Google Scholar). Until now, however, the molecular mechanism explaining the role of SopB in triggering invasion has been unclear. SopE and SopE2 of S. typhimurium are 69% identical (21Bakshi C.S. Singh V.P. Wood M.W. Jones P.W. Wallis T.S. Galyov E.E. J. Bacteriol. 2000; 182: 2341-2344Crossref PubMed Scopus (138) Google Scholar, 22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar,27Hardt W.-D. Urlaub H. Galán J.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2574-2579Crossref PubMed Scopus (182) Google Scholar). Besides the lack of any recognizable sequence similarity to proteins with DH or PH domains, both proteins are highly efficient GEFs for Cdc42 in vitro (22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar, 24Hardt W.-D. Chen L.M. Schuebel K.E. Bustelo X.R. Galán J.E. Cell. 1998; 93: 815-826Abstract Full Text Full Text PDF PubMed Scopus (665) Google Scholar, 32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In fact, the catalytic parameters of SopE-mediated guanine nucleotide exchange of Cdc42 are similar to those reported for the active domains of eukaryotic GEFs for members of the Ras superfamily (32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). The specificity of SopE and SopE2 for other GTPases of the Rho subfamily has not been studied in detail. If both bacterial virulence factors had different preferences for different RhoGTPases, SopE and SopE2 might provide S. typhimurium with a means to differentially activate specific signaling pathways inside the host cell. In the present study we have analyzed the specificity of SopE and SopE2 for the RhoGTPases Cdc42 and Rac1. Biochemical analyses of purified recombinant proteins and analysis of RhoGTPase activity in infected tissue culture cells revealed that SopE is an efficient activator for both Cdc42 and Rac1 in vitro and in vivo. In contrast, SopE2 efficiently activates Cdc42, but not Rac1. This demonstrates for the first time that expression of two homologous translocated effectors with GEF activity for RhoGTPases allows S. typhimurium to differentially activate specific signaling pathways within host cells. All S. typhimurium strains used in this study have been described (26Mirold S. Ehrbar K. Weissmüller A. Prager R. Tschäpe H. Rüssmann H. Hardt W.-D. J. Bacteriol. 2001; 183: 2348-2358Crossref PubMed Scopus (106) Google Scholar) and are isogenic derivatives of the virulent wild-type strain SL1344 (33Hoiseth S.K. Stocker B.A. Nature. 1981; 291: 238-239Crossref PubMed Scopus (1589) Google Scholar). M516 (SL1344, ΔsopB, sopE::aphT, sopE2::tetr) lacks the three major effector proteins necessary for tissue culture cell invasion (26Mirold S. Ehrbar K. Weissmüller A. Prager R. Tschäpe H. Rüssmann H. Hardt W.-D. J. Bacteriol. 2001; 183: 2348-2358Crossref PubMed Scopus (106) Google Scholar). Transformation of M516 with pM136 (pBAD24, which expresses SopE1–240-M45 under the control of the nativesopE promotor; Ref. 22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar) or with pM226 (pBAD24, which expresses SopE21–240-M45 under the control of the nativesopE2 promotor; Ref. 22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar) complements the invasion defect. For tissue culture cell infection experiments, the bacteria were grown in high salt media as described (22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar). Preparation of recombinant proteins was performed essentially as described (32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 34Friebel A. Hardt W.D. Methods Enzymol. 2000; 325: 82-91Crossref PubMed Google Scholar). Briefly, all proteins used in this study were overexpressed as GST fusion proteins, recovered from bacterial extracts by binding to glutathione-Sepharose 4B (Amersham Pharmacia Biotech) and either eluted with 20 mm glutathione (GST, GST-SopE78–240, GST-SopE269–240, GST-Cdc42Hs1–192, and GST-Rac11–191) or cleaved off the column by digestion with thrombin protease (SopE78–240 and SopE269–240, Cdc42Hs1–192 and Ha-Ras) or with factor Xa (Rac11–191). Proteins were concentrated by ultrafiltration (Mr cut-off 8000), snap-frozen in liquid nitrogen, and stored at −80 °C. Due to design of the expression vectors, the proteins carry the following additional amino acids. GST-SopE78–240 carries PGISGGGGGILEFEM between the thrombin cleavage site and Leu-78 of SopE; GST-SopE269–240 carries PGISGGGGGIL between the thrombin cleavage site and Gly-69 of SopE2; SopE78–240 carries the additional N-terminal amino acids GSPGISGGGGGILEFEM; SopE269–240 carries the additional N-terminal amino acids GSPGISGGGGGIL; GST- Rac11–191 carries GIDPGAT between the factor Xa recognition site and Met-1 of Rac1; Rac11–191 carries the N-terminal amino acids GIDPGAT; Ha-Ras carries the additional N-terminal amino acids GS; GST-Cdc42Hs1–192 carries RRASVGSKIISA between the thrombin recognition site and Met-1 of Cdc42. Cdc42Hs1–192 carries the N-terminal amino acids GSRRASVGSKIISA. The expression vector for the GST fusion protein with the Rac1 and Cdc42 binding region (aa 56–272) of human PAK1B was generously provided by E. Sander and J. G. Collard (35Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der Kammen R.A. Michiels F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (587) Google Scholar), and purification of the protein bound to glutathione-Sepharose beads was performed as described (35Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der Kammen R.A. Michiels F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (587) Google Scholar). To remove the associated GDP, GST-Cdc42Hs1–192 bound to a glutathione-Sepharose 4B column was washed with 10 ml of buffer D (50 mm Tris-HCl, pH 7.6, 100 mm NaCl, 2 mm EDTA, 2 mm DTT). Afterward, beads were incubated as a batch in the presence of a 2.5-fold molar excess of fluorescent mGDP (Molecular Probes, Netherlands) for 10 min at 22 °C in buffer D. After addition of excess MgCl2, mGDP·Cdc42Hs1–192 was cleaved off the column material using thrombin (Amersham Pharmacia Biotech) in buffer A (4 °C; overnight) and unbound mGDP was removed by gel filtration chromatography. Fractions containing the mGDP·Cdc42Hs1–192 complex were identified by fluorescence spectroscopy, pooled, concentrated by ultrafiltration (Millipore Ultrafree-15, Mr cut-off 8000), snap-frozen, and stored at −80 °C. For preparation of mGDP·Rac11–191, we devised a new method based on the high stability of the SopE·Rac1 complex (32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). 2 mg of GST-SopE78–240 was bound to a glutathione-Sepharose 4B column. Rac11–191 (in buffer A: 50 mmTris-HCl, pH 7.6, 100 mm NaCl, 5 mmMgCl2, 2 mm DTT) was applied to the column, and unbound Rac11–191 was removed by washing with 10 ml of buffer A. The bound Rac11–191 was eluted as mGDP·Rac11–191 complex using buffer A (20 °C) supplemented with 200 μm mGDP and purified and concentrated as described above for mGDP·Cdc42Hs1–192. Filter binding assays were performed in buffer B (50 mm Tris-HCl, pH 7.6, 50 mmNaCl, 5 mm MgCl2, 5 mm DTT) as described (22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar, 32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Association and dissociation reactions involving GST-Cdc42Hs1–192, GST-Rac11–191, SopE78–240, and SopE269–240 were analyzed in buffer E (10 mm HEPES/NaOH, pH 7.3, 150 mm NaCl, 5 mm MgCl2, 0.005% Igepal CA-630 (Sigma)) using surface plasmon resonance (BIAcore 2000 system) as described recently (32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Fluorescence measurements were performed at 20 °C in buffer F (40 mm HEPES/NaOH, pH 7.3, 100 mm NaCl, 5 mm MgCl2) on an Aminco Bowman series 2 fluorescence spectrometer (excitation: 366 nm; emission: 440 nm). Increasing concentrations of either mGDP·Rac11–191 or mGDP·Cdc42Hs1–192 were premixed with SopE78–240 or SopE269–240(final concentrations: 25 nm). Reactions were started by addition of unlabeled GDP (1 mm final concentration), and dissociation of mGDP was recorded as decreased fluorescence at 440 nm. To measure the amounts of Cdc42·GTP and Rac1·GTP in infected tissue culture cells, we performed affinity purification assays as described (35Sander E.E. van Delft S. ten Klooster J.P. Reid T. van der Kammen R.A. Michiels F. Collard J.G. J. Cell Biol. 1998; 143: 1385-1398Crossref PubMed Scopus (587) Google Scholar). Briefly, confluent COS7 cells grown in Dulbecco's modified Eagle's medium (5% fetal bovine serum) were infected with S. typhimurium at a multiplicity of infection of 50 bacteria/cell. Cells were washed with cold PBS and lysed in 1 ml of GST-fish buffer (10% glycerol, 50 mmTris-HCl, pH 7.6, 100 mm NaCl, 1% Igepal CA-630, 2 mm MgCl2 supplemented with “complete” protease inhibitor mixture (Roche Molecular Biochemicals)), lysates were cleared by centrifugation (4000 × g; 4 °C), and the activated Rac1/Cdc42 was recovered by binding to immobilized GST-PAK-cdc42/Rac interactive binding domain fusion protein (see above; 30 min, 4 °C). The beads were washed, and the amount of activated Cdc42·GTP and Rac1·GTP was determined by Western blot analysis using mouse-α-Rac1 (1:2500 in PBS, 5% nonfat milk; Upstate Biotechnology, Inc.) or mouse-α-Cdc42 (1:500, Transduction Laboratories) antibodies. SopE1–240-M45 and SopE21–240-M45 proteins were detected using mouse-αM45 antibody (1:100 in PBS, 5% nonfat milk; Ref. 36Obert S. O'Connor R.J. Schmid S. Hearing P. Mol. Cell. Biol. 1994; 2: 1333-1346Crossref Scopus (117) Google Scholar), a secondary horseradish peroxidase-conjugated α-mouse antibody (1:12000; Dianova) and the ECL Plus detection kit, as recommended by the manufacturer (Amersham Pharmacia Biotech). HUVECs were grown to confluence in endothelial cell growth medium (Promo Cell, endothelial cell growth supplement/H, 10% FCS) on gelatin-coated plastic coverslips (Thermanox, Nalge Nunc International) as described previously (37Aepfelbacher M. Essler M. Huber E. Sugai M. Weber P.C. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 1623-1629Crossref PubMed Scopus (96) Google Scholar). The culture medium was replaced with serum-free endothelial cell growth medium, and cells were infected with S. typhimurium (multiplicity of infection = 50) for 40 min, washed, fixed with PBS and 4% paraformaldehyde, permeabilized with PBS and 0.1% Triton X-100, and stained with rhodamine-phalloidin (Molecular Probes, 1:20 in PBS, 3% BSA). Bacteria were stained with α-Salmonella O-1,4,5,12(8) antiserum (Difco, 1:400 in PBS, 3% BSA) and a secondary α-rabbit fluorescein isothiocyanate conjugate (Sigma, 1:250 in PBS, 3% BSA). Coverslips were mounted and analyzed by fluorescence microscopy. Cells with obvious rearrangments in the actin cytoskeleton (∼35% of all cells) were evaluated and classified based on their cytoskeletal structure. Previous work had shown that SopE and SopE2 are efficient guanine nucleotide exchange factors for Cdc42 (22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar, 32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Guanine nucleotide exchange factor activity for other RhoGTPases has not been studied in detail. Here, we have compared the GEF-activity of SopE78–240 and SopE269–240 on Rac11–191 and Cdc42Hs1–192. Cdc42Hs1–192, Rac11–191, or Ha-Ras was loaded with [3H]GDP, and we determined the rates of SopE78–240- or SopE269–240-mediated [3H]GDP release using filter binding assays (Fig.1; see “Materials and Methods”). In line with earlier results (22Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.-D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (234) Google Scholar), 1 μmSopE78–240 and SopE269–240 catalyzed fast [3H]GDP release from Cdc42Hs1–192 (Fig. 1,a (●) and d (×)). In contrast, [3H]GDP release from Rac11–191 was much faster in the presence of SopE78–240 (Fig. 1 b, ●) than in the presence of 1 μmSopE269–240 (Fig. 1 e, ×). Therefore, SopE78–240 is a highly efficient GEF for Cdc42Hs1–192 and Rac11–191, whereas SopE269–240 acts equally efficient on Cdc42Hs1–192, but is much less active on Rac11–191 in vitro. To analyze the binding specificity of SopE and SopE2, we have measured the kinetics of formation and dissociation of the complexes between Cdc42 (or Rac1) and SopE2 (or SopE) using surface plasmon resonance. This technique allows one to study binding/dissociation kinetics by measuring the change in mass on the surface of a sensor chip. GST-Cdc42Hs1–192 or GST-Rac11–191 fusion protein (or GST as a control) was bound to a sensor chip, and we measured the kinetics of binding of SopE78–240 (or SopE269–240; 100 nm; Fig.2 a). The observed rates of complex formation between GST-Cdc42Hs1–192, GST-Rac11–191, SopE78–240, and SopE269–240 were dependent on the concentration of the RhoGTPase applied (Fig. 2 b; data not shown). From the binding curves, we calculated the kinetic constants for complex formation (ka) assuming simple one-step bimolecular association reactions:SopE78–240 binds with similar kinetics to GST-Cdc42Hs1–192 and to GST-Rac11–191 (Table I). In contrast, the association rate constant for formation of the GST-Cdc42Hs1–192·SopE269–240 complex is 7-fold higher than for the GST-Rac11–191·SopE269–240 complex.Table ISurface plasmon resonance measurement of the association/dissociation of SopE/SopE2 complexes with GST-Cdc42/GST-Rac1SopESopE2GST-Cdc42 ka (m−1s−1)1.6 ± 0.1 × 1054.0 ± 1.0 × 105 koff (s−1)4.5 ± 1.5 × 10−52.1 ± 0.5 × 10−5 KD (koff/ka) (m)2.8 × 10−105.2 × 10−11GST-Rac1 ka (m−1s−1)3.2 ± 0.5 × 1055.7 ± 1.4 × 104 koff(s−1)1.0 ± 0.2 × 10−41.3 ± 0.4 × 10−4 KD (koff/ka) (m)3.1 × 10−102.3 × 10−9 Open table in a new tab We have also analyzed the dissociation of the complexes (Table I). However, in the absence of GDP, dissociation was slow and the dissociation rate constants are prone to experimental error and should be regarded as rough estimates. The GST-Cdc42Hs1–192·SopE78–240 complex and the GSTRac11–191·SopE78–240complex are roughly equally stable. In contrast, dissociation of the GST-Rac11–191·SopE269–240 complex is 6-fold faster than dissociation of the GST-Cdc42Hs1–192·SopE269–240 complex (Table I). Overall, SopE78–240 binds with very similar equilibrium binding constants (KD =koff/ka) to GST-Cdc42Hs1–192 and to GST-Rac11–191(KD = 3.1 × 10−10m), whereas equilibrium binding of SopE269–240to GST-Cdc42Hs1–192 is 40-fold stronger than equilibrium binding to GST-Rac11–191 (Table I). In line with previous results for the GST-SopE78–240·Cdc42ΔC complex (32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), dissociation of all complexes between GST-RhoGTPases and SopE78–240 or SopE269–240 was accelerated more than 1000-fold in the presence of 20 μm GDP and the dissociation reactions were completed in less than 5 s (data not shown). Identical dissociation curves were obtained when we employed 20 μm GTP (data not shown). However, the dissociation kinetics in the presence of guanine nucleotides were too fast to allow an accurate analysis in order to detect differences between the dissociation rates of the complexes with GST-Cdc42Hs1–192and GST-Rac11–191. We have also analyzed the SopE78–240- and SopE269–240-mediated nucleotide exchange in multiple turnover kinetic experiments usingO-(N-methylanthraniloyl-GDP (mGDP), a fluorescent GDP derivative. The fluorescence of mGDP bound to Cdc42 is 4-fold higher than the fluorescence of unbound mGDP (32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 38Leonard D.A. Evans T. Hart M. Cerione R.A. Manor D. Biochemistry. 1994; 33: 12323-12328Crossref PubMed Scopus (69) Google Scholar, 39Rudolph M.G. Bayer P. Abo A. Kuhlmann J. Vetter I.R. Wittinghofer A. J. Biol. Chem. 1998; 273: 18067-18076Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). The kinetics and concentration dependence of mGDP dissociation from Cdc42Hs1–192·mGDP or Rac11–191·mGDP was followed by fluorescence spectrometry (Fig.3). In the SopE78–240-mediated nucleotide exchange reactions, Cdc42Hs1–192·mGDP nucleotide dissociation rate constants (v) reached a plateau at 20–40 μm and the Michaelis-Menten parameters (kcat = 5 ± 1 s−1 and Km = 6 ± 2 μm; Table II) were in the same order of magnitude as those reported for SopE78–240-mediated nucleotide exchange on Cdc42V12·mGDP (kcat = 0.95 ± 0.06 s−1 and Km = 4.5 ± 0.9 μm; Ref. 32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). It is unclear whether the slight differences might be attributable to effects of the G12V mutation of Cdc42 used in the earlier study (32Rudolph M.G. Weise C. Mirold S. Hillenbrand B. Bader B. Wittinghofer A. Hardt W.-D. J. Biol. Chem. 1999; 274: 30501-30509Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). SopE269–240 is an even more efficient GEF for Cdc42Hs1–192 than SopE78–240 (Table II).Table IIMultiple turnover measurements of SopE/SopE2-mediated nucleotide exchangeSopESopE2Cdc 42Rac1Cdc 42Rac1KM (μm)6 ± 2See Fig. 314 ± 3See Fig. 3kcat(s−1)5 ± 1See Fig. 319 ± 3See Fig. 3kobs/[GTPase] (m−1s−1)2-aDetermined from measurements at low RhoGTPase concentrations corresponding to linear ranges of the curves shown in Fig. 3.29 ± 5 × 10450 ± 15 × 10465 ± 8 × 1048 ± 1 × 1042-a Determined from measurements at low Rh" @default.
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• W2060690139 title "SopE and SopE2 from Salmonella typhimurium Activate Different Sets of RhoGTPases of the Host Cell" @default.
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