Pseudomonas aeruginosa ExoT ADP-ribosylates CT10 Regulator of Kinase (Crk) Proteins
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m304290200
ISSN1083-351X
Autores Tópico(s)Bacterial biofilms and quorum sensing
ResumoPseudomonas aeruginosa ExoT is a type III cytotoxin that functions as an anti-internalization factor with an N-terminal RhoGAP domain and a C-terminal ADP-ribosyltransferase domain. Although ExoT RhoGAP stimulates actin reorganization through the inactivation of Rho, Rac, and Cdc42, the function of the ADP-ribosylation domain is unknown. The present study characterized the mammalian proteins that are ADP-ribosylated by ExoT, using two-dimensional SDS-PAGE and matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) analysis. ExoT ADP-ribosylated two cytosolic proteins in cell lysates upon type III delivery into cultured HeLa cells. MALDI-TOF mass spectrometry analysis identified the two proteins as Crk-I and Crk-II that are Src homology 2–3 domains containing adaptor proteins, which mediate signal pathways involving focal adhesion and phagocytosis. ExoT ADP-ribosylated recombinant Crk-I at a rate similar to the ADP-ribosylation of soybean trypsin inhibitor by ExoS. ExoS did not ADP-ribosylate Crk-I. ADP-ribosylation of Crk-I may be responsible for the anti-phagocytosis phenotype elicited by ExoT in mammalian cells. Pseudomonas aeruginosa ExoT is a type III cytotoxin that functions as an anti-internalization factor with an N-terminal RhoGAP domain and a C-terminal ADP-ribosyltransferase domain. Although ExoT RhoGAP stimulates actin reorganization through the inactivation of Rho, Rac, and Cdc42, the function of the ADP-ribosylation domain is unknown. The present study characterized the mammalian proteins that are ADP-ribosylated by ExoT, using two-dimensional SDS-PAGE and matrix-assisted laser desorption ionization/time of flight (MALDI-TOF) analysis. ExoT ADP-ribosylated two cytosolic proteins in cell lysates upon type III delivery into cultured HeLa cells. MALDI-TOF mass spectrometry analysis identified the two proteins as Crk-I and Crk-II that are Src homology 2–3 domains containing adaptor proteins, which mediate signal pathways involving focal adhesion and phagocytosis. ExoT ADP-ribosylated recombinant Crk-I at a rate similar to the ADP-ribosylation of soybean trypsin inhibitor by ExoS. ExoS did not ADP-ribosylate Crk-I. ADP-ribosylation of Crk-I may be responsible for the anti-phagocytosis phenotype elicited by ExoT in mammalian cells. Exoenzyme S (ExoS) 1The abbreviations used are: Exo, exoenzyme; Crk, CT10 regulator of kinase; MALDI-TOF, matrix-assisted laser desorption ionization/time of flight; HA, hemagglutinin; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; IEF, isoelectric focusing; CHO, Chinese hamster ovary; PNS, post-nuclear supernatant; GST, glutathione S-transferase; m.o.i., multiplicity of infection; SH, Src homology; SBTI, soybean trypsin inhibitor; MS, mass spectrometry; PGK-1, phosphoglycerate kinase 1. was originally isolated from spent culture medium of Pseudomonas aeruginosa as a protein aggregate that had ADP-ribosyltransferase activity distinct from exotoxin A (1Bjorn M.J. Pavlovskis O.R. Thompson M.R. Iglewski B.H. Infect. Immun. 1979; 24: 837-842Crossref PubMed Google Scholar). The ExoS aggregate consisted primarily of two proteins with molecular masses of 53 and 49 kDa (2Kulich S.M. Frank D.W. Barbieri J.T. Infect. Immun. 1993; 61: 307-313Crossref PubMed Google Scholar). The 53- and 49-kDa proteins were subsequently shown to be encoded by separate genes, termed exoT and exoS, respectively (3Kulich S.M. Yahr T.L. Mende-Mueller L.M. Barbieri J.T. Frank D.W. J. Biol. Chem. 1994; 269: 10431-10437Abstract Full Text PDF PubMed Google Scholar, 4Yahr T.L. Barbieri J.T. Frank D.W. J. Bacteriol. 1996; 178: 1412-1419Crossref PubMed Scopus (105) Google Scholar). ExoT and ExoS share 76% amino acid identity and are bi-functional proteins with two independent enzymatic activities, which include a RhoGAP domain and an ADP-ribosyltransferase domain. The RhoGAP domain is encoded within the N terminus of ExoT and ExoS and causes actin reorganization as a GTPase-activating protein for the Rho GTPases (5Goehring U.M. Schmidt G. Pederson K.J. Aktories K. Barbieri J.T. J. Biol. Chem. 1999; 274: 36369-36372Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 6Krall R. Schmidt G. Aktories K. Barbieri J.T. Infect. Immun. 2000; 68: 6066-6068Crossref PubMed Scopus (139) Google Scholar, 7Kazmierczak B.I. Engel J.N. Infect. Immun. 2002; 70: 2198-2205Crossref PubMed Scopus (69) Google Scholar). Both ExoT and ExoS utilize an active site Arg (Arg-149 for ExoT and Arg-146 for ExoS) for expression of RhoGAP activity (5Goehring U.M. Schmidt G. Pederson K.J. Aktories K. Barbieri J.T. J. Biol. Chem. 1999; 274: 36369-36372Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar, 8Garrity-Ryan L. Kazmierczak B. Kowal R. Comolli J. Hauser A. Engel J.N. Infect. Immun. 2000; 68: 7100-7113Crossref PubMed Scopus (149) Google Scholar, 9Geiser T.K. Kazmierczak B.I. Garrity-Ryan L.K. Matthay M.A. Engel J.N. Cell. Microbiol. 2001; 3: 223-236Crossref PubMed Scopus (58) Google Scholar). Expression of RhoGAP activity has been implicated in the inhibition of phagocytosis in polarized epithelial cells and macrophage-like cells (8Garrity-Ryan L. Kazmierczak B. Kowal R. Comolli J. Hauser A. Engel J.N. Infect. Immun. 2000; 68: 7100-7113Crossref PubMed Scopus (149) Google Scholar). The ADP-ribosyltransferase domain is encoded within the C terminus of ExoT and ExoS. ExoS ADP-ribosylates numerous proteins, including members of the Ras protein family (10Coburn J. Dillon S.T. Iglewski B.H. Gill D.M. Infect. Immun. 1989; 57: 996-998Crossref PubMed Google Scholar, 11Coburn J. Gill D.M. Infect. Immun. 1991; 59: 4259-4262Crossref PubMed Google Scholar). ADP-ribosylation of Ras at Arg-41 interferes with the ability to bind the guanine nucleotide exchange factor, uncoupling Ras signal transduction (12Ganesan A.K. Mende-Mueller L. Selzer J. Barbieri J.T. J. Biol. Chem. 1999; 274: 9503-9508Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Expression of ADP-ribosyltransferase activity is dependent upon the binding of a eukaryotic protein termed FAS (Factor Activating ExoS), later shown to be a 14-3-3 protein (13Coburn J. Kane A.V. Feig L. Gill D.M. J. Biol. Chem. 1991; 266: 6438-6446Abstract Full Text PDF PubMed Google Scholar, 14Fu H. Coburn J. Collier R.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 2320-2324Crossref PubMed Scopus (217) Google Scholar). Scanning mutagenesis showed that Glu-381 contributed to expression of ADP-ribosyltransferase activity of ExoS (15Liu S. Kulich S.M. Barbieri J.T. Biochemistry. 1996; 35: 2754-2758Crossref PubMed Scopus (46) Google Scholar, 16Radke J. Pederson K.J. Barbieri J.T. Infect. Immun. 1999; 67: 1508-1510Crossref PubMed Google Scholar). Subsequent studies showed that ExoS was a bi-glutamic acid transferase, where Glu-381 functioned in a catalytic capacity and Glu-379 was required for efficient ADP-ribosyltransferase activity (16Radke J. Pederson K.J. Barbieri J.T. Infect. Immun. 1999; 67: 1508-1510Crossref PubMed Google Scholar). In contrast, ExoT does not efficiently ADP-ribosylate proteins that are ADP-ribosylated by ExoS (17Liu S. Yahr T.L. Frank D.W. Barbieri J.T. J. Bacteriol. 1997; 179: 1609-1613Crossref PubMed Google Scholar). Thus, ExoT was considered to have limited capacity to express ADP-ribosyltransferase activity. Engel and co-workers (8Garrity-Ryan L. Kazmierczak B. Kowal R. Comolli J. Hauser A. Engel J.N. Infect. Immun. 2000; 68: 7100-7113Crossref PubMed Scopus (149) Google Scholar) recently observed that the mutation of catalytic Arg (149) of ExoT partially diminished the anti-internalization activity, and P. aeruginosa expressing ExoT(R149K) retained some capacity to stimulate cell rounding and disruption of the actin cytoskeleton. Sundin et al. (18Sundin C. Henriksson M.L. Hallberg B. Forsberg A. Frithz-Lindsten E. Cell. Microbiol. 2001; 3: 237-246Crossref PubMed Scopus (36) Google Scholar) also reported that ExoT reorganized the actin cytoskeleton without interfering with Ras signal transduction and that ExoT(R149A) stimulated a morphological change of cultured cells. These data suggested that expression of ADP-ribosyltransferase activity by ExoT contributed to modulation of the actin cytoskeleton. This promoted an investigation on the ADP-ribosyltransferase activity of ExoT, which resulted in the identification of two mammalian adaptor proteins that ExoT efficiently ADP-ribosylates. ExoT Mutagenesis—DNA encoding ExoT-HA was engineered into pEGFPN1 vector (6Krall R. Schmidt G. Aktories K. Barbieri J.T. Infect. Immun. 2000; 68: 6066-6068Crossref PubMed Scopus (139) Google Scholar). HA was used as a reporter epitope tag. Mutations were engineered into ExoT by Quick-change Mutagenesis (Stratagene), using pEGFP-ExoT-HA as template. The following primers were utilized: ExoT(R149K)-HA, 5′-gcg acg gcg ccc tga aat cgc tgg cca ccg c-3′ and 5′-gcg gtg gcc agc gat ttc agg gcg ccg tcg c-3′; and ExoT(E383D/E385D)-HA, 5′-ctt gtc gta gag gat atc ctg atc atc gcc ctc gat cga-3′ and 5′-tcg atc gag ggc gat gat cag gat atc ctc tac gac aag-3′. ExoT(R149K/E383D/E385D)-HA was engineered using the primers for ExoT(R149K) and using DNA encoding ExoT(E383D/E385D)-HA as template. Mutated DNA was sequenced to confirm the presence of the mutation and that additional mutations were not introduced into the template. Subsequently, DNA encoding ExoT-HA and mutated ExoT-HA in the pEGFPN1 vector were subcloned into pUCP vector at NsiI and BamHI sites, which allowed for expression from the exoS promoter (19Knight D.A. Finck-Barbancon V. Kulich S.M. Barbieri J.T. Infect. Immun. 1995; 63: 3182-3186Crossref PubMed Google Scholar). Crk Expression Vector—pCMV-Sport6, containing Crk cDNA, was purchased from American Type Culture Collection (GenBank™ code BC008506). cDNA encoding Crk-I was amplified by PCR using the primers 5′-gtt ccg cgt gga tcc cat atg gcg ggc aac ttc ga-3′ and 5′-atc tgc agg cgg ccg cgt cga cga ctc aaa gct tcc gac ctc caa tca ga-3′ to introduce a 5′ BamHI site in-frame with the GST fusion component of pGEX4T and a SalI site 3′ to the tga stop codon. After restriction endonuclease digestion, the PCR product was cloned into the BamHI and SalI sites of pGEX4T. pGEX-Crk-I was transferred into Escherichia coli BL21(DE3) for protein expression. P. aeruginosa PA103 (ΔexoU,exoT::Tc) transformed with pUCP encoding ExoS-HA, ExoT-HA, or the mutated forms of ExoT-HA were cultured at 37 °C in low calcium media (20Vallis A.J. Yahr T.L. Barbieri J.T. Frank D.W. Infect. Immun. 1999; 67: 914-920Crossref PubMed Google Scholar) to A 540 = 4–5. Cultures were centrifuged at 10,000 rpm for 10 min (SS-34) to pellet bacteria, and the proteins in spent culture medium were precipitated (56% final concentration of saturated ammonium sulfate, v/v). The precipitate was collected and suspended in HG-1 buffer (20 mm PIPES, 2 mm Na-ATP, 1mm MgSO4, 150 mm potassium glutamate, and 2 mm EGTA). Secreted ExoS-HA, ExoT-HA, or the mutated forms of ExoT-HA were detected by ECL-Western blot analysis (SuperSignal, Pierce), using mouse α-HA IgG (Covance) as the primary antibody and goat α-mouse-IgG-horseradish peroxidase (Pierce) as the secondary antibody. The density of HA reactive bands were measured in an Alpha Imager. Concentrations of the secreted ExoS-HA, ExoT-HA, and the mutated forms of ExoT-HA were calculated, using known concentration of recombinant ExoS(Δ51–72)-HA as standard in an Alpha Imager. Concentrated culture supernatants containing 3 ng of the secreted toxins were incubated with 20–40 μg of CHO cells or HeLa cell lysates with 1 μm NAD+ ([32P]adenylate phosphate-NAD+). The reaction was stopped by either adding SDS sample buffer or 90% acetone (final concentration, v/v). Samples treated with SDS sample buffer were subjected to one-dimensional SDS-PAGE followed by autoradiography of dried gels, whereas acetone-precipitated samples were subjected to analysis by two-dimensional SDS-PAGE. Analytical Analysis of ADP-ribosylated Proteins—Cell lysates were precipitated with 90% acetone (final concentration, v/v) at –20 °C for at least 2 h. Precipitated proteins were suspended in isoelectric focusing (IEF) rehydration buffer (40 mm Tris base, 9 m urea, 4% CHAPS, 2% IPG buffer (Amersham Biosciences), 1% protease inhibitor mixture (Sigma)). Cell lysates in rehydration buffer were loaded onto Immobiline DryStrips (Amersham Biosciences, 5–20 μg of proteins for 7-cm pH 3–10 strips, and 10–40 μg of proteins for 7-cm pH 4–7 strips). After 12 h of passive rehydration, proteins were focused in PROTEAN IEF Cell (Bio-Rad) with the following program: step 1, 500 V for 1 V-h; step 2, 3500 V for 2800 V-h (slow voltage ramping); step 3, 3500 V for 8000 V-h; and step 4, holding at 500 V. Preparative Analysis for Protein Identification—0.6 mg of a cytosolic fraction of HeLa cells was incubated with ExoT with 1 μm NAD+ ([32P]adenylate phosphate-NAD+) at 37 °C for 1 h. Proteins in the reaction mixture were precipitated with 90% acetone. Precipitated proteins were suspended in 300 μl of IEF rehydration buffer and loaded onto an Immobiline DryStrip (18-cm pH 4.5–5.5 strips). After 12 h of passive rehydration, proteins were focused in PROTEAN IEF Cell (Bio-Rad) with the following program: step 1, 300 V for 1 h; step 2, 400 V for 1 h; step 3, 500 V for 1 h; step 4, 3500 V for 3 h; step 5, 5000 V for 72 h; and step 6, holding at 500 V. Analysis of Two-dimensional SDS-PAGE by Two-dimensional Software—Silver-stained two-dimensional SDS-PAGE gel containing two-dimensional SDS-PAGE standards (Bio-Rad) or corresponding autoradiographic images with marked two-dimensional SDS-PAGE standards were scanned into PD-quest two-dimensional software (Bio-Rad) or Phoretix two-dimensional software (Nonlinear Dynamics) to determine molecular weight and pI of proteins of interest and to align proteins that were ADP-ribosylated by ExoS and ExoT. Tentanolysin Assay—CHO or HeLa cells were infected at 37 °C with the indicated strain of P. aeruginosa at m.o.i. = 8:1 (bacteria/mammalian cell) for 3.5–5 h. The cell monolayer was washed and then incubated with tetanolysin (0.4 μg/ml) in 6 ml of HG-1 buffer at 4 °C for 7–10 min to permeabilize the plasma cell membrane (21Riese M.J. Goehring U.M. Ehrmantraut M.E. Moss J. Barbieri J.T. Aktories K. Schmidt G. J. Biol. Chem. 2002; 277: 12082-12088Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). After permeabilization, cells were incubated with HG-1 buffer containing 20 nm NAD+ ([32P]adenylate phosphate-NAD+) at 37 °C for 20–25 min. Cells were washed, harvested in HB2 buffer, and fractionated (see below) to obtain membrane and cytosolic fractions. The subcellular fractions were subjected to one-dimensional SDS-PAGE or two-dimensional SDS-PAGE followed by Western blot or autoradiography. Subtraction Assay—CHO or HeLa cells were infected at 37 °C with various strains of P. aeruginosa at m.o.i. = 8:1 (bacteria/mammalian cell) for 3.5–5 h. Infected cells were harvested, lysed, and fractionated. The cell fractions from infected cells were incubated with secreted ExoT with 1 μm NAD+ ([32P]adenylate phosphate-NAD+) for 30 min. Samples were subjected to two-dimensional SDS-PAGE followed by autoradiography. CHO or HeLa cells (85-mm dishes) were harvested in 0.6 ml of HB2 buffer (250 mm sucrose, 3 mm imidazole (pH 7.4), 0.5 mm EDTA, 1% protease inhibitor mixture) and lysed by passage through a 1-ml syringe, using a 25-gauge needle 14–20 times. A post-nuclear supernatant (PNS) was obtained by centrifugation of cell lysate at 250 × g for 5 min. Membrane and cytosolic fractions were obtained by centrifugation of the PNS at 100,000 × g for 30 min. One day prior to infection, cultured cells were transfected with 50 ng of pEGFPN1 (LipofectAMINE-Plus, Invitrogen), and enhanced green fluorescent protein was used as a cytosolic marker for cellular fractionation. The GST-Crk-I fusion protein was affinity-purified from E. coli using glutathione-Sepharose 4B as described by the manufacturer (Amersham Biosciences). GST-Crk-I was cleaved by thrombin, and the cleaved GST and uncleaved GST-Crk-I were removed by incubating with glutathione-Sepharose 4B. Thrombin was removed in the same incubation using p-aminobenzamidine-agarose (Sigma). Resins were pelleted by centrifugation at 250 × g for 10 min. The soluble fraction, Crk-I, was stored in aliquots at –80 °C. Purified Crk-I and soybean trypsin inhibitor (SBTI) were quantified by measuring Coomassie-stained samples subjected to SDS-PAGE, using bovine serum albumin as a standard. Specific Activity—The ADP-ribosylation activity of ExoS for SBTI and ExoT for Crk-I was determined by a linear velocity assay. Secreted ExoS or ExoT (5 nm) was incubated with SBTI or Crk-I (3 μm), respectively, with 50 mm Tris-HCl (pH 7.4), 0.1 mm NAD+ ([32P]adenylate phosphate-NAD+), 500 nm FAS, and 0.2 μg/μl bovine serum albumin. The reactions were stopped at 2, 4, 8, and 16 min by adding SDS sample buffer. Samples were applied to SDS-PAGE, followed by Coomassie staining. The radioactive bands were excised and subjected to scintillation counting. Stoichiometry—ExoT or ExoS (45 nm) was incubated with Crk-I or SBTI (3 μm), respectively, with 500 nm FAS and 0.1 mm NAD+ ([32P]-adenylate phosphate-NAD+) for 1, 3, and 5 h. Stoichiometry of the NAD+/target protein was determined at the time where incorporation of NAD+ to target protein did not increase with extended reaction time. P. aeruginosa Type III Delivered ExoT-stimulated Morphological Changes in HeLa Cells—Several forms of ExoT were engineered for type III expression in P. aeruginosa from the pexoS promoter in the broad host range plasmid, pUCP. P. aeruginosa PA103 (ΔexoU, exoT::Tc) was used as the host for these experiments, because it does not express known type III cytotoxins. Initial experiments showed that P. aeruginosa expressed and secreted similar amounts of ExoT, ExoT(R149K) (RhoGAP-deficient), ExoT(E383D/E385D) (ADP-ribosyltransferase-deficient), and ExoT(R149K/E383D/E385D) (RhoGAP- and ADP-ribosyltransferase deficient) (data not shown). Whereas type III delivered ExoT and ExoT(E383D/E385D) stimulated HeLa cell rounding, type III delivered ExoT(R149K) stimulated an intermediate change in HeLa cell morphology relative to ExoT and ExoT(E383D/E385D) (Fig. 1, upper panel). The morphological change stimulated by ExoT(R149K) was dependent on ADP-ribosyltransferase activity, because type III delivered ExoT(R149K/E383D/E385D), which was defective in both RhoGAP and ADP-ribosyltransferase activities, did not stimulate morphological changes in HeLa cells. Expression and subcellular distribution of the type III delivered ExoT and the ExoT mutants in HeLa cells (Fig. 1, lower panel) were similar and found in both the membrane and cytosolic fractions. This indicated that aberrant expression or subcellular localization was not responsible for the morphological changes elicited by the expression of ADP-ribosyltransferase activity of ExoT and prompted an evaluation of host proteins that ExoT ADP-ribosylated. Type III delivered ExoT(R149K) also stimulated an intermediate morphological change in CHO cells relative to ExoT and ExoT(E383D/E385D) (data not shown). ExoT ADP-ribosylated a Subset of Proteins in HeLa Cells— Proteins that ExoT ADP-ribosylated were determined in HeLa cell lysates by one-dimensional SDS-PAGE (Fig. 2A) and two-dimensional SDS-PAGE (Fig. 2B). Incubation of the HeLa cell lysate with [32P]adenylate phosphate NAD+ identified one 43-kDa protein that was ADP-ribosylated by an endogenous ADP-ribosyltransferase in the lysate. In addition to the 43-kDa endogenously ADP-ribosylated protein, HeLa cell lysates incubated with supernatants from P. aeruginosa or supernatants from P. aeruginosa transformed with the pUCP vector control contained a 97-kDa ADP-ribosylated protein, which represented the ADP-ribosylated elongation factor-2 by exotoxin A. HeLa cell lysates incubated with supernatants containing either ExoT or ExoT(R149K) displayed identical profiles of ADP-ribosylated proteins in one-dimensional and two-dimensional SDS-PAGE with ExoT being auto-ADP-ribosylated and three proteins (labeled T1, T2, and T3) being ADP-ribosylated. In contrast, HeLa cell lysates incubated with the ADP-ribosyltransferase-deficient form of ExoT, ExoT(E383D/E385D), did not display auto-ADP-ribosylated ExoT or radiolabeled T1, T2, or T3. Subcellular fractionation of the HeLa cell lysates in in vitro ADP-ribosylation assay showed that T1, T2, and T3 were present in the cytosolic fraction (data not shown). A subtraction assay was utilized to determine whether T1, T2, and T3 were ADP-ribosylated by type III delivered ExoT in HeLa cells (Fig. 3). In the subtraction assay, HeLa cells were infected with P. aeruginosa PA103 (ΔexoU, exoT::Tc) expressing the indicated form of ExoT for 4 h. Cell lysates prepared from the infected cells were then incubated with ExoT and [32P]adenylate-phosphate NAD+ to [32P]ADP-ribosylate host proteins that were not ADP-ribosylated during the infection phase of the assay. Two-dimensional SDS-PAGE analysis showed that three radiolabeled proteins had electrophoretic mobility identical to T1, T2, and T3 in cell lysates from the mock infection or infection with P. aeruginosa expressing ExoT(E383D/E385D), suggesting that these three proteins had not been ADP-ribosylated during the infection with P. aeruginosa expressing ExoT(E383D/E385D). In contrast, T1 and T2 were not available for in vitro ADP-ribosylation in cell lysates from the cells infected with P. aeruginosa expressing ExoT or ExoT(R149K), indicating that both T1 and T2 had been ADP-ribosylated during the infection. Only a portion of T3 was available for in vitro ADP-ribosylation in these lysates, indicating that only a portion of T3 had been ADP-ribosylated during the infection. Additional experiments showed that extending the infection time to 6 h reduced the amount of T3 that was available for in vitro ADP-ribosylation, suggesting that T3 was a late target for ADP-ribosylation by type III delivered ExoT (data not shown). Therefore, T1 and T2 appear to be early targets for ADP-ribosylation by type III delivered ExoT. T1 and T2 Are Crk-I and Crk-II, Respectively—HeLa cell lysates were incubated with ExoT and [32P]adenylate phosphate NAD+ and subjected to preparative two-dimensional SDS-PAGE, using 18-cm IEF strips with a pH gradient between 4.5 and 5.5. Overlaying the autoradiogram (Fig. 4B) with the silver-stained gel (Fig. 4A) identified three protein spots that were radioactive, representing T1, T2, and T3. The silver-stained spots for T1 and T2 from 12 gels were collected, tryptic digested, and identified as Crk-I and Crk-II by MALDI-MS/PSD analysis, respectively (Table I). Crk-I and Crk-II are the translation products of alternative splicing of the human CRK gene (22Matsuda M. Tanaka S. Nagata S. Kojima A. Kurata T. Shibuya M. Mol. Cell. Biol. 1992; 12: 3482-3489Crossref PubMed Scopus (247) Google Scholar). Whereas the predicted molecular mass was 23 kDa for Crk-I and 34 kDa for Crk-II (Table I), the determined molecular mass has been reported to be 28 kDa for Crk-I and 42 kDa for Crk-II (22Matsuda M. Tanaka S. Nagata S. Kojima A. Kurata T. Shibuya M. Mol. Cell. Biol. 1992; 12: 3482-3489Crossref PubMed Scopus (247) Google Scholar), similar to the molecular mass determined in the present study (28 kDa for Crk-I and 40 kDa for Crk-II). The predicted pI values for Crk-I and Crk-II were 5.3 and 5.4, respectively, whereas the determined pI values for ADP-ribosylated Crk-I and Crk-II were 4.9 and 5.0, respectively. Because one ADP-ribosylation decreases a protein pI by ∼0.2 pH unit, the difference between predicted pI and determined pI suggested that there were 1–2 ADP-ribosylations per Crk protein. Crk-I and Crk-II have one SH2 and one SH3 domains, whereas Crk-II has an additional SH3 domain at its C terminus. Presumably, the ADP-ribosylation sites of ExoT are localized in the common regions between Crk-I and Crk-II.Table IT1 and T2 are splice variants of the Crk proteinsTwo-dimensional SDS-PAGE MALDI-MS/PSD analysisaMALDI-MS/post-source decay (PSD) was performed by John Leszyk (University of Massachusetts Medical School, Laboratory for Proteomic Mass Spectrometry).SpotCalculated (kDa; pI)Protein IDMass MatchesPSD peptidesPredicted (kDa; pI)SpeciesT127.6; 4.9Crk-I14/19 (73%)1. (R)SSWYWGR(L)23.0; 5.3Homo sapiensT239.6; 5.0Crk-II14/20 (70%)1. (R)ALFDFNGNDEEDLPFKKGDILR(I)33.8; 5.4Homo sapiensT328.6; 5.2PGK-11. (K)ALESPERPFLAILGGAK(V)44.7; 8.3Homo sapiens2. (K)ITLPVDFVTADKFDENAK(T)a MALDI-MS/post-source decay (PSD) was performed by John Leszyk (University of Massachusetts Medical School, Laboratory for Proteomic Mass Spectrometry). Open table in a new tab T3 was resolved on 18-cm IEF strips with pH gradient 5–6 followed by preparative two-dimensional SDS-PAGE. Silver-stained T3 was collected, tryptic digested, and subjected to MALDI-MS/PSD analysis. MALDI-TOF analysis did not generate sufficient peptides (two major peptides) to identify T3 from data bases, but the MS/MS data derived from post-source decay analysis of the two peptides identified T3 as phosphoglycerate kinase 1 (PGK-1 or primer recognition protein 2) (Table I). The determined molecular weights and pI values for ADP-ribosylated T3 in two-dimensional SDS-PAGE are 28.6 kDa and 5.21, respectively, whereas the predicted molecular weights and pI values for PGK-1 are 44.7 kDa and 8.3, respectively. This indicates that T3 may be a proteolytic product of PGK-1. The location of the two peptides within the primary amino acid sequence of PGK-1 suggests that T3 is not a known splice variant. In addition to catalyzing the conversion of 3-phospho-d-glycerate and ATP to 1,3-diphospho-d-glycerate and ADP, PGK-1 also plays a structural role through association with microtubules (23Walsh J.L. Keith T.J. Knull H.R. Biochim. Biophys. Acta. 1989; 999: 64-70Crossref PubMed Scopus (148) Google Scholar). Whereas a majority of PGK-1 is in the cytoplasm of HeLa cells, PGK-1 complexes with annexin II (primer recognition protein 1) on the nuclear matrix to form a primer recognition complex, which plays a role in lagging strand DNA synthesis (24Vishwanatha J.K. Jindal H.K. Davis R.G. J. Cell Sci. 1992; 101: 25-34Crossref PubMed Google Scholar). Recombinant Crk Was Specifically ADP-ribosylated by ExoT but Not ExoS—To address the specificity of ExoT for the ADP-ribosylation of Crk, recombinant Crk-I was expressed as a GST fusion protein (Fig. 5A). Following proteolytic digestion, affinity purification was used to obtain a purified form of Crk-I (Fig. 5A). SBTI was used as substrate for ExoS. Previous studies (17Liu S. Yahr T.L. Frank D.W. Barbieri J.T. J. Bacteriol. 1997; 179: 1609-1613Crossref PubMed Google Scholar) had shown that ExoT ADP-ribosylated SBTI at ∼0.2% the rate of ExoS. Consistent with previous results, ExoS ADP-ribosylated SBTI at a velocity that was much greater than ExoT (Fig. 5B), whereas ExoT, but not ExoS, ADP-ribosylated Crk-I in a FAS-dependent reaction (Fig. 5B). ADP-ribosylation of Crk-I required ExoT ADP-ribosyltransferase activity because the ADP-ribosyltransferase-deficient mutated protein, ExoT-(E383D/E385D), did not ADP-ribosylate Crk-I. Linear Velocity for ADP-ribosylation of Crk-I by ExoT Is Similar to ADP-ribosylation Rate of SBTI by ExoS—By using Crk-I as ExoT substrate and SBTI as ExoS substrate, the specific activities of ExoS and ExoT in a linear velocity assay (Table II) were determined to be similar. At saturation, ∼0.3 mol of NAD+ had been incorporated per mol of Crk-I, which did not increase with extended incubations. In contrast, under similar conditions, ∼1 mol of NAD+ was incorporated per mol of SBTI by ExoS and continued to increase over extended incubations. The accuracy for determining the number of ADP-ribose bound to each protein is limited by several assumptions that were made during the determination, including the use of Coomassie staining to establish protein concentrations. These data suggest that although there appeared to be a limited number of sites for ExoT to ADP-ribosylate Crk-I, ExoS was capable of ADP-ribosylating multiple sites on SBTI.Table IIRate and stoichiometry for the ADP-ribosylation of Crk-I by ExoT and SBTI by ExoSEnzyme/substrateSpecific activityaExoS and ExoT (5 nm) were incubated with SBTI or Crk-I (3 μm), respectively, in the presence of 0.1 mm [32P]-adenylate phosphate NAD+ with or without FAS (500 nm) for 2, 4, 8, and 16 min. The reactions were stopped with SDS sample buffer and subjected to SDS-PAGE followed by Coomassie Blue staining and autoradiography. The radioactive bands were excised and subjected to scintillation counting to measure the incorporation of radiolabel. The specific activity was determined as mol of NAD+ incorporation per mol of enzyme per min.StoichiometrybExoS and ExoT (45 nm) was incubated with SBTI or Crk-I (3 μm), respectively, with the presence of NAD+ (0.1 mm) and FAS (500 nm). The stoichiometry NAD+/target protein was determined at saturation points where incorporation of NAD+ did not increase with extended reaction time for ExoT/Crk-I. NAD+/substrate+FAS-FASExoT/Crk-I7.8 ± 1.3Not detectedcNot detected. ADP-ribosylation activity was not greater than a control reaction that did not contain indicated toxin.0.34 ± 0.04ExoS/SBTI10.5 ± 1.5Not detected1.15 ± 0.15a ExoS and ExoT (5 nm) were incubated with SBTI or Crk-I (3 μm), respectively, in the presence of 0.1 mm [32P]-adenylate phosphate NAD+ with or without FAS (500 nm) for 2, 4, 8, and 16 min. The reactions were stopped with SDS sample buffer and subjected to SDS-PAGE followed by Coomassie Blue staining and autoradiography. The radioactive bands were excised and subjected to scintillation counting to measure the incorporation of radiolabel. The specific activity was determi
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