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Originally published In Press as doi:10.1074/jbc.M504901200 on August 25, 2005

J. Biol. Chem., Vol. 280, Issue 43, 35953-35960, October 28, 2005
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Uncoupling Crk Signal Transduction by Pseudomonas Exoenzyme T*

Qing Deng, Jianjun Sun1, and Joseph T. Barbieri2

From the Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Received for publication, May 4, 2005 , and in revised form, August 9, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exoenzyme T (ExoT) is a bifunctional type III cytotoxin of Pseudomonas aeruginosa that possesses both Rho GTPase-activating protein and ADP-ribosyltransferase activities. The ADP-ribosyltransferase activity of ExoT stimulated depolymerization of the actin cytoskeleton independent of Rho GTPase-activating protein function, and ExoT was subsequently shown to ADP-ribosylate Crk (CT10 regulator of kinase)-I and Crk-II. Crk proteins are eukaryotic adaptor proteins comprising SH2 and SH3 domains that are components of the integrin signaling pathway leading to Rac1 and Rap1 functions. Mass spectroscopic analysis identified Arg20 as the site of ADP-ribosylation by ExoT. Arg20 is a conserved residue located within the SH2 domain that is required for interactions with upstream signaling molecules such as paxillin and p130cas. Glutathione S-transferase pull-down and far Western assays showed that ADP-ribosylated Crk-I or Crk-I(R20K) failed to bind p130cas or paxillin. This indicates that ADP-ribosylation inhibited the direct interaction of Crk with these focal adhesion proteins. Overexpression of wild-type Crk-I reduced cell rounding by ExoT, whereas expression of dominant-active Rac1 interfered with the ability of ExoT to round cells. Thus, the ADP-ribosylation of Crk uncouples integrin signaling by direct inhibition of the binding of Crk to focal adhesion proteins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that infects immunocompromised humans, including patients with cystic fibrosis (1). P. aeruginosa delivers four type III cytotoxins directly into host cells, exoenzyme (Exo)3 S, ExoT, ExoU, and ExoY (2). ExoU is a phospholipase (3), and ExoY is an adenylate cyclase (4). Both ExoS and ExoT are bifunctional cytotoxins, possessing an N-terminal Rho GTPase-activating protein (RhoGAP) domain and a C-terminal ADP-ribosyltransferase domain (5). The RhoGAP domains of ExoS and ExoT are essentially identical (6). RhoGAP activity stabilizes the transition state of the GTPase reaction and inactivates Rho, Rac, and Cdc42, leading to actin reorganization and cell rounding. Expression of the RhoGAP domain has been proposed to inhibit phagocytosis of P. aeruginosa by polarized epithelial cells and phagocytes (7). Expression of ADP-ribosyltransferase activities by ExoS and ExoT is dependent on direct binding to the mammalian 14-3-3 protein termed FAS (factor activating exoenzyme S) (8, 9). The ExoS ADP-ribosyltransferase domain is cytotoxic for mammalian cells and polysubstrate-specific. ExoS ADP-ribosylates numerous cellular proteins, including vimentin (10), Ras (8, 10), and ERM proteins (11), and undergoes auto-ADP-ribosylation. Expression of the ADP-ribosyltransferase domain of ExoT is not cytotoxic for mammalian cells. ExoT ADP-ribosylates a restricted subset of cellular proteins, including Crk (CT10 regulator of kinase) proteins (12).

Crk proteins and their downstream effectors are conserved throughout evolution, from Caenorhabditis elegans to humans. c-Crk is expressed as two distinct proteins of 28 and 40 kDa due to alternative splicing: Crk-I and Crk-II, respectively (13). Crk-I and Crk-II have identical SH2 and SH3 domains, with Crk-II possessing an additional SH3 domain separated by 60 amino acids. Both Crk-I and Crk-II are adaptor proteins involved in eukaryotic signal transduction pathways (13). The SH2 domain recognizes phosphorylated tyrosine, and the SH3 domain recognizes proline-rich sequences. Specifically, the Crk SH2 domain prefers the pYXXP binding motif (14), whereas the first SH3 domain recognizes the consensus sequence PXXPXK (15–17). The integrin receptor-activated p130cas (Crk-associated substrate)-Crk-DOCK180 pathway is involved in cell mobility and phagocytosis (18, 19). DOCK180 is a guanine nucleotide exchange factor for Rac1. Integrins compose a large family of transmembrane adhesion receptors. Once activated by ligands, integrin receptor {alpha}- and {beta}-subunits dimerize, recruiting focal adhesion complex proteins, including focal adhesion kinase and Src family kinases, as well as the scaffolding proteins paxillin and p130cas to the site of activation. Tyr31 and Tyr118 of paxillin are phosphorylated by either focal adhesion kinase or Src (20), whereas some of the 15 tyrosine residues within the substrate domain of p130cas are phosphorylated by Src kinase (21, 22), creating docking sites for Crk proteins (23). Crk is recruited to tyrosine-phosphorylated paxillin and p130cas via SH2 domain interaction, which brings SH3 domain-associated proteins such as DOCK180 to the site of activation. Activation of Rac1 by DOCK180 stimulates actin reorganization and eventually leads to cell migration and phagocytosis.

An early correlation between ExoT anti-internalization activity and Src-mediated phagocytosis showed that the deletion of Csk, a negative regulator of Src kinase in fibroblasts, attenuates ExoT-mediated anti-internalization activity (24). The ADP-ribosyltransferase domain of ExoT contributes to the depolymerization of the actin cytoskeleton (7). This, together with the identification of Crk-I and Crk-II as the targets of ExoT ADP-ribosylation (12), suggests that the ADP-ribosylation of Crk proteins contributes to the actin depolymerization activity of ExoT. In this study, the molecular basis for uncoupling the Crk signal transduction pathway by ExoT is investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and Reagents—Mouse monoclonal anti-paxillin antibody was purchased from BD Transduction Laboratories. Goat polyclonal anti-Crk-I/II antibody and rabbit polyclonal anti-p130cas antibody were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal anti-phosphotyrosine antibody (clone 4G10) was from Upstate Cell Signaling Solutions (Lake Placid, NY). Mouse monoclonal anti-glutathione S-transferase (GST) antibody was from Balco (Berkeley, CA). Sodium orthovanadate was purchase from Sigma.

Cell Culture and Bacterial Stains—HeLa cells were cultured in standard Dulbecco's modified Eagle's medium with 10% fetal bovine serum in a humidified incubator with 5% CO2 at 37 °C. P. aeruginosa strain PA103 ({Delta}exoU exoT::Tc) was transformed with pUCP encoding ExoT(R149K,E383D,E383D)-hemagglutinin (HA) or ExoT(R149K)-HA. The ability of P. aeruginosa to secrete these mutated forms of ExoT was verified as described previously (12).

In Vitro ADP-ribosylation of Crk-I—GST-Crk-I and ExoT proteins were purified as described previously (12). Recombinant GST-Crk-I (10 µg) or Crk-I (5 µg) was incubated with 100 µM [32P]NAD (3 µCi), 0.5 mM FAS, and 5 nM ExoT in 50 mM Tris-HCl (pH 7.4) in a final volume of 20 µl at room temperature for 2–4 h. The reaction was stopped by the addition of either SDS sample buffer or 90% (v/v) acetone (final concentration). 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 two-dimensional SDS-PAGE. Efficiency of ADP-ribosylation was calculated based on separation by two-dimensional isoelectric focusing (IEF)/SDS-PAGE.

Two-dimensional IEF/SDS-PAGE—Wild-type or ADP-ribosylated GST-Crk-I or Crk-I was mixed with 10 µg of two-dimensional marker (Bio-Rad) and 130 µl of IEF rehydration buffer (9 M urea, 4% CHAPS, 2% IPG buffer (Amersham Biosciences), 1% protease inhibitor (Sigma), and a trace amount of bromphenol blue) and loaded onto Immobiline DryStrip gels (7 cm, pH 4–7; Amersham Biosciences). After 12 h of passive rehydration, proteins were focused in a 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 16,000 V-h; and step 4, holding at 500 V.

Cleavage of GST-Crk-I—GST-Crk-I (1 mg) was incubated with thrombin (0.25 units/µl) in thrombin cleavage buffer (20 mM Tris-HCl (pH 8.5), 100 mM NaCl, 330 µM CaCl2, and 14 µg/µl dithiothreitol) in a final volume of 400 µl at 37 °C for 1.5 h. Cleaved GST, uncleaved GST-Crk-I, and thrombin were removed by incubating the reaction mixture with 250 µl of a 75% slurry of glutathione-Sepharose 4B beads (Amersham Biosciences) and 100 µl of a 50% slurry of p-aminobenzamidineagarose (Sigma) at room temperature for 30 min, followed by centrifugation at 500 x g for 5 min. The soluble fraction (Crk-I) was aliquoted and stored at –80 °C. Protein concentration was determined using the BCA protein assay kit (Pierce).

Identification of the ADP-ribosylation Site—Crk-I protein was ADP-ribosylated in vitro as described above and then precipitated with 90% (v/v) acetone (final concentration) at –20 °C. Precipitated proteins were suspended in IEF rehydration buffer and separated by two-dimensional IEF/SDS-PAGE. One protein spot containing ADP-ribosylated Crk-I (Crk-I-ADPr; 28 kDa, pI 5.3) was excised and digested with trypsin (1 unit/reaction) in 50 µl of 0.1 M NH4HCO3 (pH 8.0). Peptides were extracted for analysis by liquid chromatography (LC)-electrospray ionization tandem mass spectrometry (MS/MS).

Site-directed Mutagenesis—Reactions were performed with the QuikChange site-directed mutagenesis kit (Stratagene) using pGEX-Crk-I as a template. The following Crk-I(R20K) primers were utilized: 5'-gg tac tgg ggg agg ttg agt aag cag gag gcg gtg gcg ctg-3' and 5'-cag cgc cac cgc ctc ctg ctt act caa cct ccc cca gta cc-3'. Mutated DNA was verified by sequencing. DNAs encoding Crk-I and Crk-I(R20K) in the pGEX4T vector were subcloned into the pEGFPC1 vector at BamHI and SalI sites, which allowed expression in eukaryotic cells.

GST Pull-down Assay—Confluent lawns of HeLa cells (15-cm dishes) were pretreated with 1 mM Na3VO4 for 3 h to increase the phosphate content of proteins. Dishes were placed on ice and washed twice with 15 ml of cold phosphate-buffered saline containing 1 mM Na3VO4. Cells were lysed in 0.8 ml of cold Triton X-100 lysate buffer (10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, and 1% protease inhibitor) for 30 min on ice with occasional shaking. Cell lysate was centrifuged at 12,000 rpm for 10 min at 4 °C. The protein content of the cleared cell lysate was determined using the BCA protein assay kit. Equivalent amounts of cell lysate (4 mg) were precleared with 50 µl of a 75% slurry of glutathione-Sepharose 4B beads and then incubated with 100 µl of fresh glutathione-Sepharose 4B beads (75% slurry) and 10 µg of GST-Crk-I, GST-Crk-I-ADPr, or GST-Crk-I(R20K) for 2 h at 4°C. The sample was centrifuged at 850 x g for 5 min at 4 °C. The beads were washed three times with 1 ml of Triton X-100 lysate buffer, and the final pellet was suspended in 60 µl of SDS-PAGE sample buffer and boiled for 5 min to dissociate the complex. Samples containing equal amounts of protein were separated by 8% SDS-PAGE and transferred to polyvinylidene difluoride membrane (Millipore Corp.). Immunoblotting was performed using primary antibodies (anti-p130cas (1:1000), anti-paxillin (1:5000), anti-GST (0.2 µg/ml), and 4G10 (1:1000)) and an appropriate horseradish peroxidase-conjugated secondary antibody (Pierce) with enhanced chemiluminescence detection (SuperSignal, Pierce). From the cell lysate, ~43% of total GST-Crk-I was bound by the GST beads, whereas 1% of total paxillin and 1% of total p130cas were bound by the GST-Crk-I beads.

Far Western Assay—p130cas protein was immunoprecipitated from the HeLa cell lysate as described previously (25). The immunoprecipitates were washed three times with cold Src reaction buffer (50 mM HEPES (pH 7.4), 5 mM ATP, 5 mM MgCl2, 5 mM MnCl2, and 0.2 µM Na3VO4), suspended in 100 µl of Src reaction buffer with or without 0.15 units of active Src (Upstate Cell Signaling Solutions), and incubated at room temperature for 3 h. Reactions were then boiled in 60 µl of SDS sample buffer, subjected to 8% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane. Membranes were blocked in 2% bovine serum albumin-containing phosphate-buffered saline at room temperature for 30 min and then incubated for 3 h with 0.05 µg/ml GST-Crk-I, GST-Crk-I-ADPr, or GST-Crk-I(R20K); 2 µg/ml mouse anti-GST antibody; and 1 µg/ml horseradish peroxidase-conjugated anti-mouse antibody in 1% bovine serum albumin-containing phosphate-buffered saline at 4 °C in a final volume of 3 ml. Membranes were washed five times for 4 min each with 1% bovine serum albumin-containing phosphate-buffered saline plus 0.1% Triton X-100, followed by detection with enhanced chemiluminescence.

ADP-ribosylation of Host Proteins in HeLa Cells by Type III-delivered ExoT—HeLa cells (6-well plates) were transfected with 300 ng of the indicated green fluorescent plasmid (pGFP)-Crk-I constructs using Lipofectamine (Invitrogen) (26). Cells were infected with the indicated strain of P. aeruginosa at a multiplicity of infection (m.o.i.) of 8:1 bacteria/host cells for the indicated times and then permeabilized with tetanolysin (0.8 µg) in HG1 buffer (20 mM PIPES (pH 7.0), 2 mM sodium ATP, 1 mM magnesium acetate, 150 mM potassium glutamate, and 2 mM EDTA; 1.0-ml final volume) and incubated with 20 nM [32P]NAD (1.5 µCi) at 37 °C (12). Cells were washed and boiled in SDS sample buffer, and proteins were separated by 12% SDS-PAGE, followed by autoradiography or immunoblotting using the anti-GFP, anti-HA, or anti-actin primary antibody and the appropriate horseradish peroxidase-conjugated secondary antibody with enhanced chemiluminescence detection.



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  		FIGURE 1.
In vitro ADP-ribosylation of GST-Crk-I by ExoT. Recombinant GST-Crk-I protein was incubated alone (A) or with FAS, NAD, and ExoT{Delta}233 (ExoT ADP-ribosylation domain only) (B) at room temperature for 4 h. Reaction mixtures were mixed with two-dimensional marker proteins and separated by two-dimensional IEF/SDS-PAGE (first dimension, IEF, 7-cm Immobiline DryStrip gel with pH 4–7 linear gradient; second dimension, 12% SDS-PAGE), followed by staining with Coomassie Brilliant Blue. The two-dimensional marker proteins are circled and numbered: 1, conalbumin (76 kDa; pI 6.0, 6.3, and 6.6); 2, bovine serum albumin (66.2 kDa; pI 5.4 and 5.6); 3, actin (43 kDa; pI 5.0 and 5.1). Arrows indicate GST-Crk-I proteins and their calculated pI. The migration positions of molecular mass markers are shown on the left.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ExoT ADP-ribosylates Crk at Arg20—Although an early study identified Crk as a high affinity substrate for ADP-ribosylation by ExoT (12), neither the site(s) of ADP-ribosylation nor the effect of ADP-ribosylation of Crk on host signaling had been determined. As a first step in this characterization, the site that ExoT ADP-ribosylates in Crk was determined. Because it was difficult to resolve a shift in apparent molecular mass by one-dimensional gel electrophoresis, the electrophoretic properties of Crk and ADP-ribosylated Crk were measured by two-dimensional IEF/SDS-PAGE. Using the migration of marker proteins as reference, GST-Crk-I focused as a single spot at pH 5.9 (Fig. 1A), whereas GST-Crk-I-ADPr focused as a single spot at pH 5.7 (Fig. 1B). The acidic 0.2-unit pI shift of GST-Crk-I upon ADP-ribosylation suggests that Crk-I was ADP-ribosylated at a single site. Greater than 80% of total GST-Crk-I was ADP-ribosylated (Fig. 1B). Other experiments showed the incorporation of [32P]NAD into GST-Crk-I-ADPr, supporting that the acidic 0.2-unit pH shift represents the ADP-ribosylation of Crk (data not shown).

To resolve the effect of ADP-ribosylation on Crk function, the site of ADP-ribosylation in Crk-I protein was determined. To simplify the LC-MS/MS characterization, the GST tag was cleaved from GST-Crk-I with thrombin, and Crk-I was used as a substrate for ADP-ribosylation by ExoT. ADP-ribosylated Crk-I was determined to have a mass of 23,968 Da, which is 541 Da greater than the determined mass for Crk-I (TABLE ONE). This indicates that ExoT ADP-ribosylated Crk-I at a single amino acid. Crk-I-ADPr was subjected to two-dimensional IEF/SDS-PAGE, and the focused protein was digested with trypsin. LC-electrospray ionization MS/MS identified a single peptide (residues 18–31) with a 541-Da mass increase. Peptide sequencing showed that Arg20 contained a 541-Da mass addition, indicating this residue as the site of ADP-ribosylation (TABLES TWO and THREE).


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  		TABLE ONE
ExoT ADP-ribosylates Crk-I protein at a single site

Unmodified Crk-I protein and Crk-I that had been ADP-ribosylated (Crk-I-ADPr) with ExoT were subjected to LC-MS, and molecular masses were determined. Theoretic and determined masses are shown. The mass of Crk-I-ADPr is 541 Da more that that of unmodified Crk-I.

 


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  		TABLE TWO
Sequence of Crk-I protein

The peptide observed to possess a 541-Da increase from the predicted amino acid sequence is underlined. The SH2 domain is shown in boldface, and the SH3 domain is shown in italic. LC-MS/MS identified each of the tryptic peptides of Crk-I except the C-terminal tryptic peptide (amino acids 191-206). Arg20 is indicated by an asterisk.

 


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  		TABLE THREE
ExoT ADP-ribosylates Crk-I protein at Arg20

The ADP-ribosylated Crk-I tryptic peptide (amino acids 18-31) was analyzed by LC-MS/MS. Crk-I-ADPr was purified by two-dimensional SDS-PAGE, excised from a Coomassie Brilliant Blue-stained gel, and digested with trypsin. The determined sequence of the ADP-ribosylated Crk-I tryptic peptide (amino acids 18-31, positions 1-14) is shown as b and y ions. The position corresponding to Arg20 (indicated by an asterisk) showed a 541-Da mass increase. aa, amino acid. Italics indicate peptide mass plus 541 Da.

 
ExoT Does Not ADP-ribosylate Crk-I(R20K)—GST-Crk-I(R20K) was engineered and tested as a substrate for ADP-ribosylation by ExoT. Although ExoT efficiently ADP-ribosylated Crk-I, Crk-I(R20K) was not ADP-ribosylated (Fig. 2), indicating that Arg20 is the primary site for ADP-ribosylation and that there are no alternative sites for ADP-ribosylation within Crk-I. Next, the ability of type III-delivered (injected into host cells directly by the type III secretion system of P. aeruginosa) ExoT to ADP-ribosylate Crk-I(R20K) was tested. To analyze the effects of only the ADP-ribosyltransferase activity, experiments were performed with ExoT(R149K), which does not express RhoGAP activity. A time course showed that type III-delivered ExoT ADP-ribosylated transfected GFP-Crk-I and endogenous Crk-I at 3.5–4.5 h post-infection, with less GFP-Crk-I and endogenous Crk ADP-ribosylated 5 h post-infection (Fig. 3A). Thus, a 4-h post-infection time point was used in subsequent experiments to measure the ADP-ribosylation of Crk by ExoT. Under these conditions, type III-delivered ExoT(R149K) failed to ADP-ribosylate Crk-I(R20K) (Fig. 3B). Controls showed that ExoT(R149K,E383D,E383D) (defective in both RhoGAP and ADP-ribosyltransferase activities) did not ADP-ribosylate GFP-Crk-I or endogenous Crk-I or undergo auto-ADP-ribosylation. Actin was used as a loading control to ensure comparable loading of each sample. Measurement of GFP-Crk-I showed the presence of comparable amounts of GFP-Crk-I and GFP-Crk-I(R20K), whereas HA expression showed a similar delivery of ExoT(R149K) and ExoT(R149K,E383D,E383D). Both ExoT proteins had C-terminal HA epitopes for the detection of protein expression.



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  		FIGURE 2.
The ExoT ADP-ribosylation domain does not modify Crk-I(R20K) in vitro. Recombinant wild-type (WT) GST-Crk-I or GST-Crk-I(R20K) was incubated at room temperature alone (–) or with (+) ExoT{Delta}233 (ExoT ADP-ribosylation domain), FAS, and [32P]NAD for 2 h. Reactions were boiled in SDS-PAGE sample buffer and separated by 12% SDS-PAGE, followed by staining with Coomassie Brilliant Blue (CBB) or by autoradiography (AutoRad) of a dried gel. The migration positions of molecular mass markers are shown on the left.

 



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  		FIGURE 3.
Type III-delivered ExoT(R149K) ADP-ribosylates GFP-Crk-I, but not GFP-Crk-I(R20K), in HeLa cells. A, HeLa cells were transfected with pGFP-Crk-I and then infected with P. aeruginosa {Delta}exoU exoT::Tc transformed with pUCP-ExoT(R149K) at an m.o.i. of 8:1 bacteria/cells. At the indicted times post-infection, cells were permeabilized with tetanolysin and incubated with [32P]NAD as described under "Materials and Methods." Cells were boiled in SDS sample buffer and separated by 12% SDS-PAGE, followed by autoradiography (AutoRad) or by immunoblotting using anti-GFP antibody as the primary antibody, followed by enhanced chemiluminescence. The migration positions of molecular mass markers are shown on the left. B, HeLa cells were transfected (TF) with pGFP, pGFP-Crk-I (wild-type (WT)), or pGFP-Crk-I(R20K), followed by infection (IF) with P. aeruginosa {Delta}exoU exoT::Tc transformed with pUCP-ExoT(R149K,E383D,E383D) (ExoT-KDD) or pUCP-ExoT(R149K) (ExoT-R149K). Four h post-infection (inf.), cells were permeabilized with tetanolysin and incubated with [32P]NAD as described for A, boiled in SDS sample buffer, and separated by 12% SDS-PAGE, followed by autoradiography (AutoRad) or by immunoblotting using anti-GFP, anti-HA, or anti-actin as the primary antibody. Asterisks indicate the migration positions of auto-ADP-ribosylated ExoT(R149K), GFP-Crk-I, and endogenous Crk-I.

 



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  		FIGURE 4.
ADP-ribosylation of Crk-I blocks Crk-I/p130cas/paxillin interaction. HeLa cells were incubated for 3 h in 1 mM sodium orthovanadate. Cells were harvested and lysed in Triton X-100 lysate buffer. The Triton X-100-soluble fraction was used in a GST pull-down assay with wild-type GST-Crk-I (WT), GST-Crk-I-ADPr, or GST-Crk-I(R20K) as described under "Materials and Methods." Proteins, lysate (input), GST pull-down supernatant (S), and GST pull-down (PD) were subjected to 8% SDS-PAGE, transferred to polyvinylidene difluoride, and probed with the indicated primary antibody (4G10, anti-GST, anti-paxillin, or anti-p130cas) as indicated. Bound antibodies were visualized with the appropriate horseradish peroxidase-conjugated secondary antibody and by enhanced chemiluminescence. The migration positions of molecular mass markers are shown on the left.

 
Neither ADP-ribosylated Crk-I nor Crk-I(R20K) Associates with the Focal Adhesion Proteins p130cas and Paxillin—Arg20 is located within the SH2 domain of Crk and participates in the binding of Crk to phosphotyrosine in binding partners. Phosphorylated p130cas and paxillin are focal adhesion proteins that directly bind the Crk SH2 domain (20, 23, 27–31) and were used to determine whether the ADP-ribosylation of Crk at Arg20 interferes with the association of Crk and binding partners.

Initial experiments used a GST pull-down assay to determine whether ADP-ribosylation or the R20K mutation interferes with the ability of Crk to bind phosphorylated p130cas and paxillin in HeLa cell lysate. Under identical conditions, Crk-I coprecipitated both paxillin and p130cas, whereas neither Crk-I-ADPr nor Crk-I(R20K) coprecipitated either of these focal adhesion proteins (Fig. 4). In addition to the preferred binding of GST-Crk-I to these specific focal adhesion proteins, analysis of the antibody 4G10 (anti-phosphotyrosine)-probed blot showed that GST-Crk-I coprecipitated more phosphorylated proteins than either Crk-I-ADPr or Crk-I(R20K) (Fig. 4). This suggests that either the ADP-ribosylation of Crk-I at Arg20 or the introduction of R20K reduced the general phosphotyrosine-binding capacity of Crk-I. Measurement of the GST fusion component showed the presence of comparable amounts of GST-Crk-I, GST-Crk-I-ADPr, and GST-Crk-I(R20K) in the coprecipitate. A control with GST beads alone did not coprecipitate detectable amounts of the phosphoproteins paxillin and p130cas.

A far Western analysis was used to determine whether disruption of the interaction by ADP-ribosylation with focal adhesion proteins could be attributed to direct inhibition of Crk binding to the focal adhesion proteins. p130cas proteins were enriched from HeLa cell lysate by immunoprecipitation and then phosphorylated with Src kinase, which directly phosphorylates p130cas proteins (27, 30, 32). Consistent with a previous report (22), p130cas proteins showed an apparent increase of ~20 kDa upon Src kinase treatment, indicating hyperphosphorylation (Fig. 5A, left panel). The bands in Fig. 5A that migrated below the 116-kDa marker were neither phosphorylated nor reactive with anti-rabbit antibody and may represent a degradation product of p130cas or a crossreactive protein recognized by polyclonal anti-p130cas antibody.



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  		FIGURE 5.
Phosphorylated p130cas binds directly to GST-Crk-I, but not GST-Crk-I-ADPr or GST-Crk-I(R20K). HeLa cells were lysed in Triton X-100 lysate buffer and immunoprecipitated (IP) with protein A-conjugated anti-p130cas antibody. Immunoprecipitates were washed with Src reaction buffer and incubated with (+) or without (–) Src kinase and ATP. Reactions were then boiled in SDS sample buffer, separated by 8% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. The membranes were either probed with antibody 4G10 (anti-phosphotyrosine) or anti-p130cas antibody as the primary antibody (A) or subjected to a far Western assay with wild-type GST-Crk-I (WT), GST-Crk-I-ADPr, GST-Crk-I(R20K), or GST (B) as described under "Materials and Methods." The arrows on the right indicate the migration positions of phosphorylated p130cas (P-Cas) and p130cas (Cas). The migration positions of molecular mass markers are shown on the left.

 



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  		FIGURE 6.
Overexpression of GFP-Crk-I protects HeLa cells from rounding by ExoT. HeLa cells were transfected overnight with 300 ng of pGFP, pGFP-Crk-I, or pGFP-Crk-I(R20K), followed by infection with P. aeruginosa {Delta}exoU exoT::Tc transformed with pUCP-ExoT(R149K,E383D,E383D) (ExoT-KDD) or pUCP-ExoT(R149K) (ExoT-R149K) at an m.o.i. of 8:1. A, 4 h post-infection, cells were fixed in 1% paraformaldehyde and visualized by fluorescent microscopy (magnification x20). B, shown are the results from the quantitation of cell rounding in transfected cells. Data are the means ± S.E. of three independent experiments.

 
Tyrosine phosphorylation of p130cas was confirmed by probing with antibody 4G10 (Fig. 5A, right panel). Direct binding of phosphorylated p130cas to the indicated GST-Crk-I constructs was tested by far Western analysis. Although the direct binding of GST-Crk-I to phosphorylated p130cas was observed, neither GST-Crk-I-ADPr nor GST-Crk-I(R20K) bound phosphorylated p130cas directly (Fig. 5B).

Overexpression of Crk-I Rescues HeLa Cells from ExoT-mediated Cell Rounding—The effect of overexpression of GFP-Crk-I and GFP-Crk-I(R20K) on ExoT-mediated cell rounding was investigated next. It was not possible to determine the expression of GFP-Crk-I relative to endogenous Crk because anti-Crk antibody failed to detect endogenous Crk-I. Controls showed that, in HeLa cells overexpressing GFP, infection with ExoT(R149K,E383D,E383D) resulted in ~20% cell rounding, whereas infection with ExoT(R149K) resulted in ~60% cell rounding. In contrast, overexpression of wild-type GFP-Crk-I reduced cell rounding by ExoT(R149K) by ~50%, indicating that ExoT(R149K)-induced cell rounding involves the Crk pathway. In cells that overexpressed GFP-Crk-I(R20K), cells infected with P. aeruginosa expressing ExoT(R149K) were rounded to a greater extent compared with cells overexpressing GFP-Crk-I (Fig. 6). This is consistent with the in vitro interaction data and suggests that Crk-I(R20K) is defective in interacting with Crk SH2 domain-binding proteins. The reason for the enhanced cell rounding may be related to the presence of a functional SH3 domain within Crk-I(R20K) to sequester downstream molecules, which made cells more sensitive to ExoT-induced cell rounding. Overexpression of Crk-I and Crk-I(R20K) did not affect nonspecific cell rounding elicited by ExoT(R149K,E383D,E383D), which lacks RhoGAP and ADP-ribosyltransferase activities.

Expression of Dominant-active Rac1 Rescues ExoT ADP-ribosyltransferase-induced Cell Rounding—Because Crk proteins are cell signaling adaptors that modulate the actin cytoskeleton through the downstream effector Rac1 (18, 33, 34), the ability of dominant-active (DA) Rho GTPases to rescue ExoT-induced cell rounding was determined. DA-RhoA, DA-Rac1, and DA-Cdc42 were expressed in HeLa cells to test their ability to rescue HeLa cells from ExoT-induced cell rounding.



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  		FIGURE 7.
Expression of DA-Rac1 protects HeLa cells from rounding by ExoT. HeLa cells were transfected overnight with 200 ng of plasmid encoding dominant-active small Rho GTPases (pGFP-RhoA(Q61E), pGFP-Rac1(Q63E), or pGFP-Cdc42(Q63E)), followed by infection with P. aeruginosa {Delta}exoU exoT::Tc transformed with pUCP-ExoT(R149K,E383D,E383D) (ExoT-KDD) or pUCP-ExoT(R149K) (ExoT-R149K) at an m.o.i. of 8:1 A, 4 h post-infection, cells were fixed in 1% paraformaldehyde and visualized by fluorescent microscopy (magnification x20). B, shown are the results from the quantitation of cell rounding in transfected cells. Data are the means ± S.E. of three independent experiments. C, HeLa cells were transfected overnight with 300 ng of pGFP-Crk or pGFP-Crk(R20K) with or without 200 ng of DA-Rac1, followed by infection with P. aeruginosa {Delta}exoU exoT::Tc transformed with pExoT(R149K) (ExoT-R149K) at an m.o.i. of 8:1. Four h post-infection, cells were fixed in 1% paraformaldehyde, and cell rounding in transfected cells was quantitated. Data are the means ± S.E. of three independent experiments.

 
Expression of GFP-DA-RhoA, GFP-DA-Rac1, and GFP-DA-Cdc42 in HeLa cells was similar as detected by Western blotting (data not shown). Relative to GFP-transfected cells, expression of GFP-DA-Rac1 reduced the ability of ExoT(R149K) to induce cell rounding (Fig. 7). Similarly, GFP-DA-Cdc42 reduced the ability of ExoT(R149K) to induce cell rounding, but with a lower efficiency compared with GFP-DA-Rac1. The intermediate protection elicited by Cdc42 relative to Rac1 may be related to the ability of Cdc42 to activate Rac1, which has been shown in fibroblasts (35). Expression of GFP-DA-RhoA did not affect the intrinsic cell rounding by ExoT(R149K,E383D,E383D) or the ability of ExoT(R149K) to induce cell rounding. Controls showed that expression of GFP did not affect the ability of ExoT(R149K) to induce cell rounding (Fig. 6).

DA-Rac1 was used to determine the relationship between the Crk-I (Arg20) and Rac1 signaling. Although overexpression of Crk-I(R20K) did not affect the actin cytoskeleton, Crk-I(R20K)-enhanced cell rounding by ExoT was reduced by DA-Rac1 (Fig. 7C). This is consistent with the ability of DA-Rac1 to reverse ExoT ADP-ribosyltransferase activity and supports a model in which Rac1 functions downstream of Crk-I and the modulation of the actin cytoskeleton by ExoT is through Crk and involves Rac1 signaling.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
ExoT ADP-ribosylates Crk at Arg20, a conserved residue in Crk family proteins. The NMR structure of the SH2 domain in complex with a phosphotyrosine-containing peptide indicates that Arg20 forms hydrogen bonds with the phosphate group of phosphotyrosine (36). In this study, we observed that even a conserved substitution (Arg to Lys) disrupted SH2 domain interactions. A similar phenotype was reported for Arg38, which also forms hydrogen bonds with the phosphate group of phosphotyrosine. Thus, both Arg20 and Arg38 are complementary in action, stabilizing the phosphopeptide. R38K is deficient in SH2 domain-mediated signaling and is used as a dominant-negative Crk protein (37–40).



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  		FIGURE 8.
ADP-ribosylation of Crk proteins by ExoT. Integrin receptors are activated by extracellular stimuli, bringing Src kinase and focal adhesion kinase (FAK), to phosphorylate p130cas and paxillin, which form a focal adhesion complex. Phosphorylated focal adhesion complex proteins recruit Crk to the membrane through interactions with the Crk SH2 domain. The SH3 domains of Crk proteins bring the downstream Rac guanine nucleotide exchange factor (DOCK180) and the Rap guanine nucleotide exchange factor (C3G) to the membrane. Activated Rac1 stimulates downstream signal transduction, leading to phagocytosis and wound healing, whereas activated Rap1 stimulates cell proliferation. ExoT ADP-ribosylates Crk proteins, directly blocking the upstream interaction between the focal adhesion complex and the Crk SH2 domain to uncouple Crk-mediated signal transduction. The model was adapted from Ref. 5.

 
Functional SH2 and SH3 domains are required for Crk to function as an adaptor protein where ADP-ribosylation of Crk protein by ExoT directly disrupts Crk signal transduction pathways (focal adhesion protein-Crk-DOCK180-Rac1). In this model, integrin receptors are activated by extracellular stimuli (undefined) and recruit Src kinase and focal adhesion kinase to phosphorylate p130cas and paxillin, which subsequently form a focal adhesion complex on the membrane. The phosphorylated (active) focal adhesion complex recruits Crk through interactions with the Crk SH2 domain. ADP-ribosylated Crk fails to bind phosphorylated focal adhesion proteins (Fig. 8; modified from Ref. 5). Crk-I is a splice variant of Crk-II; both proteins have identical SH2 domains that are ADP-ribosylated by ExoT (12), which disrupts Crk-I and Crk-II function. Fig. 4 shows that other Crk SH2 domain/protein interactions were also disrupted by ADP-ribosylation or the R20K mutation of Crk, which implicates a more global effect of the ADP-ribosylation of Crk beyond paxillin and p130cas. The cellular and biochemical function of the second SH3 domain in Crk-II is not clear. Crk-II can also be regulated by phosphorylation at Tyr221 within the Crk-II spacer region by the Abl kinases (41), which can lead to the dissociation of Crk SH3 domain-binding protein (42–48). The relationship between phosphorylation and ADP-ribosylation of Crk is not clear. Crk-I is not regulated by phosphorylation.

Crk family adaptors play multiple roles in cell physiology, including phagocytosis, focal adhesion, and cell proliferation and migration (33, 49, 50). Crk proteins activate several different effector proteins such as guanine nucleotide exchange factors (C3G and DOCK180) for small GTPases (Rap1 and Rac1, respectively). Rac1 controls actin dynamics, integrin-mediated cell adhesion and cell shape changes, and engulfment of dead cell bodies. Crk-DOCK180-Rac1 is a conserved pathway mediating phagocytosis (51, 52), whereas Crk-C3G-Rap1 is not well understood, but appears to be involved in integrin-matrix adhesion and cell proliferation (33). Other Crk effector proteins include the Abl family non-receptor tyrosine kinases (47, 48, 53), HPK1 and KHS serine/threonine kinases (54–57), and phosphatidylinositol 3-kinase (58–60). ADP-ribosylation of Crk proteins in different cell types may disrupt different cellular functions. ExoT ADP-ribosyltransferase-induced cell rounding was rescued by DA-Rac1, but not DA-RhoA (Fig. 7), suggesting that Rac1 signaling is the primary contributor for the cell rounding phenotype. Although overexpression of Crk-I(R20K) did not have an apparent affect on cell morphology, overexpression of Crk-I(R20K) made cells sensitive to ExoT-induced cell rounding upon infection. This sensitivity to Crk-I(R20K) was reversed by DA-Rac1 (Fig. 7C), supporting the model in which ExoT causes cell rounding through the Crk pathway and Rac1 is the primary Rho GTPase involved in this regulation. ExoT ADP-ribosyltransferase activity did not have global effects on the nucleotide state of Rac1, measured as p21-activated kinase pull-down (61) or the movement of Rac1 from the membrane to cytosol (62).4 This indicates that only a fraction of Rac1 is linked to the Crk signaling pathway or that the ExoT ADP-ribosyltransferase domain needs to shift the nucleotide state of only a fraction of Rac1 to elicit a physiological effect.

Crk-mediated phagocytosis and focal adhesion appear to be a common pathway modulated by several bacterial pathogens. Like P. aeruginosa, the Yersinia are extracellular pathogens. YopH, a type III effector of Yersinia, is a phosphatase that targets focal adhesion complex proteins such as focal adhesion kinase and p130cas to down-regulate Crk-mediated phagocytosis (63, 64). In contrast, Shigella flexneri, an intracellular pathogen, stimulates the Crk-mediated pathway to facilitate uptake. Abl family tyrosine kinases (Abl and Arg) accumulate at the site of Shigella entry and phosphorylate Crk-II at Arg221, which activates Rac1, a step required for Shigella internalization (65). In addition to modification of the Crk pathway by bacterial pathogens, a recent study showed that epidermal growth factor stimulation activates breast tumor kinase, which phosphorylates paxillin at Tyr31 and Tyr118 to activate Rac1 via Crk-II, which contributes to tumor migration and invasion (66).

ExoT and ExoS are bifunctional type III cytotoxins (5) and share 76% primary amino acid homology. Although sharing common RhoGAP activity, the ADP-ribosyltransferase substrates for ExoS and ExoT are unique. ExoS is polysubstrate-specific and ADP-ribosylates a broad range of substrates, eliciting a cytotoxic phenotype in cultured cells. In contrast, ExoT ADP-ribosylates a restricted set of substrates, primarily Crk-I and Crk-II, and is not cytotoxic for cultured cells (12). ExoT is found in most clinical isolates of P. aeruginosa (67), suggesting that ExoT has a fundamental function that is required for bacterial survival. The RhoGAP activity and ADP-ribosylation of ExoT are complementary for P. aeruginosa to express anti-internalization activity. The RhoGAP domain of ExoT inactivates the three major subsets of Rho GTPases (RhoA, Rac1, and Cdc42) (61, 68). The significance of utilizing two distinct but complementary mechanisms is not clear, but may provide P. aeruginosa an opportunity to neutralize the phagocytic properties of epithelial cells as well as macrophages and neutrophils. ExoT ADP-ribosyltransferase activity disrupts the p130cas-Crk-DOCK180 pathway, a conserved phagocytosis pathway, and explains the previous observation of the ability of ADP-ribosylation by ExoT to inactivate the actin cytoskeleton (7).


   FOOTNOTES
 
* This work was supported by United States Public Health Service Grant RO1 AI30162 from the National Institutes of Health (to J. T. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115. Back

2 To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-8412; Fax: 414-567-6535; E-mail: jtb01{at}mcw.edu.

3 The abbreviations used are: Exo, exoenzyme; RhoGAP, Rho GTPase-activating protein; SH, Src homology; GST, glutathione S-transferase; HA, hemagglutinin; IEF, isoelectric focusing; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Crk-I-ADPr, ADP-ribosylated Crk-I; LC, liquid chromatography; MS/MS, tandem mass spectrometry; GFP, green fluorescent protein; m.o.i., multiplicity of infection; DA, dominant-active; PIPES, 1,4-piperazinediethanesulfonic acid. Back

4 Q. Deng and J. T. Barbieri, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Liam B. Moran (Thermo Electron Corp., Waltham, MA) for assistance with mass spectrometry.



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