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Originally published In Press as doi:10.1074/jbc.M605387200 on October 29, 2006

J. Biol. Chem., Vol. 281, Issue 52, 40379-40388, December 29, 2006
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Disruption of RhoGDI and RhoA Regulation by a Rac1 Specificity Switch Mutant*

Ka-Wing Wong{ddagger}, Sina Mohammadi{ddagger}, and Ralph R. Isberg{ddagger}§1

From the §Howard Hughes Medical Institute, {ddagger}Department of Molecular Biology and Microbiology, Tufts University Medical School, Boston, Massachusetts 02111

Received for publication, June 5, 2006 , and in revised form, October 6, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rho family GTPases are important regulators of the actin cytoskeleton. Activation of these proteins can be promoted by guanine nucleotide exchange factors containing Dbl and Pleckstrin homology domains resulting in membrane insertion of a Rho family member, whereas the inactive GDP-bound form is sequestered primarily in the cytoplasm, bound to the guanosine dissociation inhibitor RhoGDI. Dominant interfering variants of Rac1, but not Cdc42, inhibit beta1 integrin-promoted uptake of Yersinia pseudo tuberculosis. Unexpectedly, we found that the Rac1(W56F) guanine nucleotide exchange factors specificity switch mutant blocked invasin-promoted uptake as well as Cdc42-dependent uptake of enteropathogenic Escherichia coli. Fluorescence resonance energy transfer experiments demonstrated that Rac1(W56F) retained the ability to be loaded with GTP, bind a downstream effector, and interact with RhoGDI. Mutational analyses of intragenic suppressors and coexpression studies demonstrated that binding of the Rac1(W56F) mutant to RhoGDI appeared to play a role in the inhibition of uptake. As RhoGDI inhibits RhoA, overactivation of RhoA may account for the uptake interference caused by Rac1(W56F). Consistent with this model, a dominant interfering form of RhoA restored significant uptake in the presence of the Rac1(W56F) mutant but had no effect on another interfering Rac1 form. Furthermore, the cellular GTP-RhoA level was elevated by the presence of Rac1(W56F) mutant protein. These data are consistent with the proposition that Rac1(W56F) blocks invasin-promoted uptake by preventing RhoGDI from inactivating RhoA. We conclude that RhoGDI allows cross-talk between Rho family members that promote potentially antagonistic processes, and disruption of this cross-talk can interfere with invasin-promoted uptake.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Successful pathogens often associate with host cells prior to initiating disease. This association results in either adhesion to the extracellular surface of target cells or internalization of the pathogen into the host cell (1). In the case of the Gram-negative enteropathogenic Yersinia species, disease is initiated by internalization of the bacteria into M cells located on the surface of the patches of the Peyer within the small intestine, eventually leading to microbial growth within lymph nodes in an extracellular locale (2-5). For Yersinia pseudotuberculosis, uptake of the bacterium into cultured cells is the result of binding by the outer membrane protein invasin to multiple host beta1 chain integrin receptors (6, 7). The engagement of integrins by invasin in many ways mimics the binding of natural substrates to this receptor, including the recognition of a site on the integrin that appears to overlap with that recognized by fibronectin (8). The one distinguishing feature of invasin is that it binds integrins with a unique high affinity, and this characteristic appears key to ensuring successful uptake of the bacterium (8, 9).

Among the properties shared with other integrin substrates is the ability of invasin to promote localized activation of the small GTPase Rac1 after integrin engagement at the site of bacterial adhesion (10, 11). Rac1 belongs to the large family of Rho GTPases that regulates most known actin rearrangements in host cells and includes the members Cdc42 and RhoA (12). As is true of many Rho family members, most of the Rac1 in resting cells is in an inactive state, associated with RhoGDI in a soluble cytoplasmic complex (13). Upon cell stimulation by either growth factors or adhesion receptor engagement, guanine nucleotide exchange factors (GEFs)2 become activated and associate with the membrane, whereas the Rac1·RhoGDI complex translocates to the plasma membrane and dissociates (14, 15). The GDI-free Rac1, in turn, assumes the active GTP-bound state, and the membrane-inserted GTPase drives actin rearrangements. Inactivation of Rac1 occurs by GTP hydrolysis stimulated by GTPase-activating proteins (GAP), returning inactive Rac1 to its cytoplasmic complex with RhoGDI (16).

It is not well understood how beta1 integrin receptor engagement by invasin leads to Rac1 activation or which GEFs transmit signals to Rho family members after integrin clustering. One model posits that receptor engagement leads to a phosphotyrosine signal that in turn may activate GEFs (17-20). In support of this model, proteins involved in phosphotyrosine cascades downstream from integrin engagement, such as focal adhesion kinase (FAK) and CrK-associated substrate, are required for invasin-mediated uptake of bacteria (21-23). Although a specific GEF that responds to such a cell surface generated signal by invasin has not been identified, the GEF that is functionally important for invasin-promoted uptake is likely to show specificity toward Rac1. The dominant negative Rac1(N17) mutant blocks invasin-mediated bacterial uptake, whereas the dominant negative Cdc42(N17) does not (6, 24). Furthermore, after Y. pseudotuberculosis adhesion, Cdc42 is not activated as efficiently as a Cdc42 mutant that responds to Rac1-specific GEFs. Therefore, manipulation of Rac1 function appears central to invasin-promoted uptake.

Although RhoGDI is normally thought to be an inhibitor of Rho family function by maintaining its binding partners in an inactive state within a cytoplasmic locale, there is potential for it playing a role in positively promoting specific cytoskeletal rearrangements (14, 25, 26). Regulated GTPase function requires that the Rho family member be recruited to the appropriate site and that activation be limited to this region in the cell. RhoGDI maintains a readily available pool of the GTPase that can respond to such signals rapidly, perhaps shuttling Rho family members directly to this site (25). Furthermore, some Rho family members can have antagonistic effects on specific cytoskeletal rearrangements, and RhoGDI may dampen the activity of these proteins. For instance, activated RhoA has been demonstrated to inhibit neural cell migration as well as interfere with invasin-promoted bacterial uptake and phagocytosis of charged beads (6, 27-29). In these cases, it is thought that stimulation of actin stress fiber formation by RhoA interferes with localized membrane movement promoted by cytoskeletal rearrangements.

The fact that only a subset of Rho family members can play a positive role in promoting specific cytoskeletal events means that upstream signals must discriminate between different GTPases. Much of this discrimination appears to be provided by the RhoGEFs, which activate only a subset of GTPases (15). Many but not all of these GEFs have Dbl and pleckstrin homology domains (30). Structural analysis of side chain contacts between one of these Dbl and pleckstrin homology-GEFs, Tiam1, and its target Rac1 protein has identified potential residues important for substrate specificity (31). Remarkably, a single side chain difference in Cdc42 and Rac1 appears sufficient to confer substrate specificity (32, 33).

In this report we studied the consequences of a Rac1 specificity switch mutant on integrin-mediated bacterial uptake. Surprisingly, the mutant was shown to be a potent inhibitor of invasin-mediated uptake that behaved very differently from classic Rac1 dominant interfering mutants. The results of this analysis indicate that the activity of RhoA needs to be tightly regulated to prevent it from interfering with invasin-mediated uptake.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, DNA Constructs, and Transfections—COS-1 and HeLa cells were cultured and transfected as described previously (6) in Dulbecco's modified Eagle's medium with low glucose (Invitrogen) containing 10% fetal bovine serum (Hyclone). Transfection of cell lines was performed using Lipofectamine PLUS or Lipofectamine 2000 reagents (Invitrogen) according to manufacturer's protocols on coverslips placed in 24-well dishes. DNA for transient transfection was prepared by using the Endofree Maxiprep kit (Qiagen). pCGT-Rac1(WT), pCGT-Rac1(G12V), and pCGT-Rac1(T17N) were kind gifts of Dr. Jim Bliska (The State University of New York (SUNY), Stony Brook, NY). Monomeric fluorescence protein-tagged expression plasmids pmCFP-Rac1 and pmYFP-PBD are described elsewhere (6, 10). The mammalian expression vectors that express N-terminal Myc-tagged RhoA and Myc-tagged Cdc42 were provided by Dr. Kit Wong (University of California, San Francisco, CA) and Rac1(6Q) by Ulla Knaus (Scripps Research Institute, San Diego, CA). The vector expressing RhoGDI-mYFP was generated by cloning the open reading frame from the cDNA of the RhoGDI{alpha} isoform (Guthrie Research Institute, Sayre, PA) into pmYFP-N1 to obtain pRhoGDI-mYFP. The Rac1(T35L), (Y40C), (W56F), (R66A), (C189S) mutations and the RhoGDI(D185A) mutation were introduced into pCGT-Rac1 or pRhoGDI-mYFP, respectively, by site-directed mutagenesis using the QuikChange protocol (Stratagene, La Jolla, CA). All plasmids were verified by DNA sequencing. The properties of each of the mutant derivatives are described in Table 1.


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  		TABLE 1
Predicted properties of mutants used in this study

 
Bacterial Strains and Assay for Bacterial Uptake into Cultured Cells—The Y. pseudotuberculosis YPIII(P-) strain is cured of the virulence plasmid, lacks the YadA adhesin, and expresses no antiphagocytic proteins (35). The enteropathogenic Escherichia coli (EPEC) strain E2348/69 was provided by John Leong (University of Massachusetts, Worcester, MA) (36). Y. pseudotuberculosis YPIII(P-) was grown on LB plates at 26 °C for 48 h. To perform uptake assays, a single colony was inoculated into Luria-Bertani broth and allowed to grow with shaking at 26 °C overnight. Bacteria were then subcultured into fresh medium and grown for ~3 h until A600 = 0.7, as described (10). The EPEC strain was grown as described (37). Transfected COS-1 cells adherent to coverslips were typically incubated with Y. pseudotuberculosis YPIII(P-) for 20 min at a multiplicity of infection = 50 at 37 °C, whereas transfected HeLa cells were challenged with EPEC for 3 h according to Jepson et al. (37). After the incubation with bacteria, the adherent cells were processed for immunofluorescence staining to visualize epitope-tagged Rac1 or RhoA and to distinguish extracellular or internalized bacteria exactly according to the procedure we described previously (6). Briefly, the monolayers were fixed in 3% paraformaldehyde and probed with primary antibody directed against either Y. pseudotuberculosis or EPEC followed by a fluorescent secondary antibody (anti-IgG conjugated to either Alexa Fluor 594 or Cascade Blue) to detect extracellular bacteria. The cells were then permeabilized (6) and probed with a combination of antibodies directed against the bacteria and Rho family member to allow detection of both intracellular and extracellular bacteria. The coverslips were then probed with appropriate secondary antibodies. Secondary antibodies used to detect the Rho family members were conjugated to either Cascade Blue or Alexa Fluor 488. All secondary antibodies were obtained from Molecular Probes, Inc.

Fluorescence Resonance Energy Transfer Analysis of Rac1-RhoGDI Interaction in Single Cells—Localized activation of Rac1 derivatives was determined using a fluorescence resonance energy transfer (FRET) assay described previously, measuring the interaction of mCFP-Rac1 with mYFP-PBD (10). In this assay, a FRET signal occurred when mCFP-Rac1 was loaded with GTP, allowing binding of the fluorescent protein to the p21 binding domain (PBD) moiety. A similar strategy was used to quantitate interaction of RhoGDI with Rac1 in which binding of mCFP-Rac1 to mYFP-RhoGDI was measured by FRET (10). The set up of the microscope and the filters used for FRET excitation and emission were identical to those used previously (10). Briefly, to measure FRET, images from filter sets dedicated for YFP, CFP, and FRET fluors were first captured, and then two random regions of interest, each about 10% of the area of a whole cell located within the cytoplasmic region, were chosen for analysis. FRET from these regions was calculated as sensitized FRET by subtracting the bleed through signal of CFP into the YFP channel and cross-excitation of YFP by the CFP channel, using exactly the same procedure as described previously (10).

Measurement of RhoA Activation—HeLa cells were transfected in 6-well dishes with pCGT-Rac1(WT), pCGT-Rac1(W56F), or mYFP-RhoGDI{alpha}(D185A). To determine the quantity of GTP-loaded RhoA 48 h post-transfection, the cells were washed twice with ice-cold phosphate-buffered saline and resuspended in 300 µl of lysis buffer (20 mM Tris, pH 7.5, 125 mM NaCl, 1% Nonidet P-40, and complete protease inhibitor mixture (Roche)). Lysates were incubated at 4 °C with agitation for 5 min and then cleared by centrifugation at 8000 rpm for 5 min at 4 °C. 250 µl of cleared lysates were then incubated with 20 µl of Rhotekin-RBD GST beads (Cytoskeleton Inc., Denver, CO) for 30 min at 4 °C with agitation. Beads were pelleted in a microfuge, then washed four times (25 mM Tris, pH 7.5, 30 mM MgCl2, 40 mM NaCl, 0.1% Nonidet P-40) by successive centrifugation. Bound Rho proteins and 30 µl of the input lysate were detected by Western blotting using a monoclonal antibody against RhoA (Santa Cruz Biotechnology) followed by densitometry analysis using Kodak Image Station 440CF (Kodak).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Rac1(W56F) Variant Having Altered GEF Specificity Does Not Reverse the Effects of Rac1(T17N)—Invasin-promoted uptake of Y. pseudotuberculosis into nonphagocytic cells is dependent on the function of Rac1 and occurs in the absence of Cdc42 signaling, based on the fact that bacterial uptake is inhibited by the dominant negative Rac1(T17N) mutant but not by Cdc42(T17N) (6). The Rac1(T17N) mutation is thought to be dominant negative because it forms a tight complex with a GEF specific for Rac1, preventing the GEF from activating endogenous Rac1 protein (Table 1). To test the model that titration of Rac1-specific GEF causes uptake inhibition, whereas titration of a Cdc42-specific GEF has no effect, we analyzed a Rac1 mutation that switches the specificity of GEF recognition to see whether this suppresses the dominant inhibitory properties of Rac1(T17N).

The tryptophan side chain at position 56 (Trp-56) in Rac1 confers substrate specificity, allowing preferential recognition of Rac1-specific Dbl-containing GEFs (32, 33). In turn, the phenylalanine residue at position 56 (Phe-56) in Cdc42 allows recognition of Cdc42-specific Dbl-GEF proteins (32, 33). GEF specificity can be simply inverted by performing either the Trp -> Phe conversion in Rac1 or the Phe -> Trp change in Cdc42, allowing Cdc42(F56W) to recognize some Rac1-specific GEFs such as Tiam1 and allowing Rac1(W56F) to recognize the Cdc42-specific GEF intersectin (32, 33). The prediction from this model is that the point mutation W56F should relieve potential titration of a Rac1 GEF by the Rac1(T17N) mutant and reverse the effects of the dominant inhibitory mutant. Therefore, we tested whether the Rac1(T17N/W56F) double mutant could reverse the inhibitory effects of the T17N mutation. To this end, transfected COS-1 cells producing either Rac1(WT), Rac1(T17N), or the double mutant Rac1(T17N/W56F) were challenged with Y. pseudotuberculosis, and the efficiency of bacterial uptake was determined. Surprisingly, the double mutant Rac1(T17N/W56F) was as strong an inhibitor of invasin-promoted uptake as the parental Rac1(T17N) (Fig. 1A). Either the W56F residue change did not cause altered specificity of Rac1(T17N) or the Rac1(W56F) variant itself was a dominant inhibitory mutant.


Figure 1
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  		FIGURE 1.
Rac1(W56F) is an inhibitor of invasin-mediated Yersinia uptake and EPEC uptake. A, COS-1 cells transfected with plasmids encoding T7-tagged Rac1 with noted residue changes or Myc-tagged Cdc42(T17N) were challenged with Y. pseudotuberculosis (YPIII(P-)) for 20 min. B, HeLa cells transfected as noted were challenged with EPEC for 60 min. Monolayers were fixed and processed for the immunofluorescence protection assay using rabbit polyclonal anti-Yersinia or chicken polyclonal anti-EPEC antibodies and mouse monoclonal anti-Rac1 or mouse monoclonal anti-Myc as detection reagents (see "Materials and Methods"). Untransfected cells were included as control. The percentage of cell-associated bacteria that were internalized was determined from 50 cells of each coverslips (see "Materials and Methods"). Data for four coverslips from each group are displayed as mean ± S.E.

 
To test the model that the W56F mutation produces an inhibitory Rac1 molecule, COS-1 cells transfected with the single mutant Rac1(W56F) were challenged with Y. pseudotuberculosis. The Rac1(W56F) mutant impaired uptake as severely as did Rac1(T17N) (Fig. 1A). Therefore it appears that altering the GEF specificity of Rac1 renders it an inhibitor of invasin-promoted uptake.

Rac1(W56F) Interferes with Cdc42-dependent Uptake of EPEC into HeLa Cells—As the Rac1(W56F) mutation is predicted to bind Cdc42-specific GEFs, and it has unexpected effects on Rac1-dependent uptake, we considered that Cdc42-dependent events also may be antagonized by this mutant. One such Cdc42-dependent event is uptake of EPEC by HeLa cells. Uptake of this organism is inhibited by Cdc42(T17N), but not by Rac1(T17N) (37), and is a good model to determine the effects of Rac1(W56F) on events that are Cdc42-dependent and Rac1-independent. Rac1(W56F) efficiently interfered with EPEC uptake (Fig. 1B). The inhibition of uptake observed was as strong as that for Cdc42(T17N), whereas Rac1(T17N) had no effect (Fig. 1B). Therefore, Rac1(W56F) could interfere with signaling events promoted by multiple small GTPases.

Rac1(W56F) Binds Effectors and RhoGDI—One of the key characteristics of the Rac1(T17N) dominant negative mutant is that it assumes an inactive conformation that fails to bind to downstream effectors (38). We sought to determine whether Rac1(W56F) had a similar mechanism of interference of Y. pseudotuberculosis uptake and is unable to bind effectors. We measured the binding of Rac1 derivatives to the PBD of PAK1 by FRET, both throughout the uninfected cells and in the region around phagosomes after incubation with Y. pseudotuberculosis. In the case of wild type Rac1, the activated conformation that results from GTP loading allows binding to PBD, which is measured by quantifying the amount of FRET that occurs in cells transfected simultaneously with mCFP-Rac1 variants and mYFP-PBD (10). COS-1 cells transfected with CFP-Rac1(WT) resulted in a robust FRET readout throughout the cell when normalized to the amount of CFP-Rac1 that was expressed (Fig. 2A). The mCFP-Rac1(T17N) derivative, however, was unable to generate any detectable FRET with mYFP-PBD, as predicted by its inability to assume an active conformation, nor was there detectable FRET using the effector binding domain mutant Rac1(Y40C) (Fig. 2A). Under the same conditions, the interfering Rac1(W56F) variant behaved very differently from other defective Rac1 forms. The mCFP-Rac1(W56F) derivative produced levels of FRET that were as high as Rac1(WT) when normalized to the mCFP-Rac1 concentration in the cell and approached the levels of binding observed for the constitutively active Rac1(G12V) mutant (Fig. 2A). This result was independent of the expression levels of Rac1 in the cell. If the amount of sensitized FRET was displayed as a function of the level of mCFP-Rac1 in individual cells, the binding of the Rac1(W56F) mutant to the PBD activation readout was at least as efficient as that observed for Rac1(WT) at all expression levels (Fig. 2B). This result is similar to that observed previously, using an affinity precipitation approach to quantitate binding of Rac1(W56F) to bead-immobilized PBD (32, 33). In addition, in areas where nascent phagosomes formed around bound Y. pseudotuberculosis, the recruited mCFP-Rac1(W56F) was able to generate an enhanced FRET readout with PBD as well (Fig. 2C). No such concentration of FRET signal was observed with the dominant interfering Rac1(T17N) mutant in this study or in our previous work (Fig. 2A; 10).


Figure 2
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  		FIGURE 2.
W56F does not reduce GTP loading of Rac1 in cultured cells. A, Rac1(W56F) form shows activation levels similar to Rac1(WT). COS-1 cells were transfected with mYFP-PBD in combination with either mCFP-Rac1(WT) (control) or mCFP-Rac1 derivatives having noted residue changes. The transfectants were then subjected to FRET analysis, and normalized FRET was calculated, expressed as amount PBD relative to the amount of Rac1 found in each cell (see "Materials and Methods"). Data for normalized FRET relative to Rac1 concentration was determined from 16 regions of interest, which represented two cytoplasmic regions from each of 8 cells, quantitated as mean ± S.E. B, activation of Rac1(W56F) is observed over a large range of mCFP-Rac1 concentration. FRET from 20 regions of interest, which represent two cytoplasmic regions from each of 10 cells, were plotted as a function of the intensity of the mCFP-Rac1 donor. Displayed are wild type and mutant forms of mCFP-Rac1 cotransfected with YFP-PBD. C, Rac1(W56F) localized about incoming bacteria can be activated. Displayed are COS-1 cells transfected with the mYFP-PBD and mCFP-Rac1 plasmids having noted residue changes and allowed to incubate with Y. pseudotuberculosis YPIII(P-) for 30 min. The cells were fixed and probed with anti-Y. pseudotuberculosis, revealed with anti-rabbit IgG-Alexa Fluor 594, and subjected to image analysis. Images were captured in the CFP, YFP, and FRET channels followed by capture of Alexa Fluor 594 (see "Materials and Methods"). Displayed are the noted channels. sFRET is sensitized FRET, using corrections as described under "Materials and Methods." Arrows point to phagosomes having recruited Rac1.

 
The second readily assayable property of the classic Rac1(T17N) dominant negative mutant is its inability to bind RhoGDI. Both the GTP loaded or GDP loaded forms of geranylgeranylated Rac1 or Cdc42 bind to RhoGDI, which inhibits Rho family members from interacting with GEFs at the membrane (11, 39). We developed a FRET-based system, similar to the one for Rac1-PBD, to monitor Rac1-RhoGDI interaction (Fig. 3A). In this assay, mCFP-Rac1 derivatives acted as fluorescence emission donors with RhoGDI-mYFP acting as the excitation acceptor (Fig. 3A). RhoGDI-YFP was able to generate FRET when coexpressed with Rac1(WT), whereas the Rac1(R66A) derivative, which is defective for RhoGDI binding, failed to produce FRET with RhoGDI-YFP (Fig. 3, B and C). No FRET signal was detected with mCFP-Rac1(T17N), consistent with the earlier report that Rac1(T17N) fails to coimmunoprepitate with RhoGDI, further confirming the fidelity of this readout (Fig. 3D) (13). In contrast to the results with Rac1(T17N), the Rac1(W56F)-RhoGDI pair produced FRET as efficiently as Rac1(WT) (Fig. 3D). These data indicate that Rac1(W56F) behaved distinctly from Rac1(T17N) in terms of its ability to bind RhoGDI or effectors. Moreover, the data also indicate that Rac1(W56F) can localize in the cytosol and bind RhoGDI in contrast to the exclusive plasma membrane localization reported for Rac1(T17N) (13). Therefore, by several criteria, the physical properties of Rac1(W56F) are distinctly different from Rac1(T17N), and the mechanisms of dominant interference of uptake by the two proteins are likely to be different.

We also used the FRET-based assay to analyze the Rac1(6Q) mutant that had been shown previously to be defective for binding RhoGDI, based on coimmunoprecipitation experiments (40). This mutant, which replaces the carboxyl terminal polybasic region with six Gln residues, is predicted to affect a variety of other functions of Rac1, including phosphoinositol-4-phosphate-5-kinase binding, Rac1 self-association, and plasma membrane localization (Table 1). We found that the defect in binding of RhoGDI by this mutant was not absolute, as there was considerable residual binding by the mutant (Fig. 3D, 6Q). Furthermore, the addition of the W56F lesion to 6Q (Rac1(W56F6Q)) did not cause further decay in binding, consistent with the proposition that Rac1(W56F) was fully capable of interacting with RhoGDI in the cell (Fig. 3D). As had been observed with the FRET analysis of PBD binding, the results regarding binding of the Rac1 derivatives to RhoGDI were independent of expression levels in the cell. When the amount of sensitized FRET found in individual cells was plotted as a function of the expression levels of mCFP-Rac1 (Fig. 3E), the conclusions were identical to those obtained when the FRET readings were normalized to expression levels (Fig. 3D).


Figure 3
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  		FIGURE 3.
Rac1(W56F) retains binding to RhoGDI. A, schematic representation of intermolecular FRET from mCFP-Rac1 to RhoGDI-mYFP. Upper panel, in a cytoplasmic locale, the pool of mCFP-Rac1 GTP binds to RhoGDI-mYFP. This allows mCFP emission to excite RhoGDI-mYFP, resulting in emission at 527 nm, detected by using the FRET filter set (see "Materials and Methods"). Lower panel, the predicted result of the presence of the R66A mutation is that mCFP-Rac1(R66A) should be unable to bind mYFP-PBD and should not generate a FRET signal. B and C, FRET between mCFP-Rac1 and RhoGDI-mYFP is dependent on intact interaction surface between Rac1 and RhoGDI. COS-1 cells were cotransfected with RhoGDI-mYFP and either mCFP-Rac1(WT) or mCFP-Rac1(R66A) followed by determination of sensitized FRET and normalized FRET (see "Materials and Methods"). Displayed are the channels for CFP and YFP emissions, the relative Rac1 activation throughout the cell as determined by sensitized FRET, as well as the Rac1 activation normalized to the Rac1 concentration. Both the intensities of sensitized FRET and normalized FRET were displayed according to the color-gradient scales indicated below panels. C, the Rac1(R66A) mutation eliminates FRET between mCFP-Rac1 and RhoGDI-YFP. Displayed are mean FRET values normalized to the Rac1 concentration in the cell (see "Materials and Methods"). D, the Rac1(6Q) retains residual RhoGDI binding. Displayed are denoted Rac1 variants subjected to FRET analysis as determined by the interaction between CFP-Rac1 derivatives and RhoGDI-YFP. FRET values are normalized to mCFP-Rac1 concentrations. For all data, sensitized FRET values were normalized against Rac1 concentration from 16 regions of interest, representing two cytoplasmic regions from each of 8 cells, displayed as mean ± S.E. (C and D). E, Rac1(W56F) retains binding to RhoGDI over a range of expression levels in host cells. FRET was determined as in panels B and D, except FRET values were not normalized to mCFP-Rac1 concentrations. Instead, FRET intensities from 20 regions of interest, which represent two cytoplasmic regions from each of 10 cells, were plotted as a function of the intensity of the mCFP-Rac1 donor. Displayed are FRET values for noted mutations in mCFP-Rac1 derivatives.

 
Rac1 Mutations Affecting RhoGDI Binding Suppress Dominant Inhibition of Uptake by Rac1(W56F)—To explain the reason for dominant inhibition of uptake by Rac1(W56F), mutations that affected binding of Rac1 to various proteins were introduced into Rac1(W56F) with the hope of identifying lesions that reversed the effects of the inhibitory W56F mutation. The rationale behind this approach is that if the inhibition of uptake by Rac1(W56F) is because of titration of a particular protein, then a second mutation should prevent the titration and suppress the effects of the W56F variant. Residue changed blocking effector signaling (Y40C) (Table 1, Fig. 4A), and membrane ruffling (T35L) (data not shown) had no effect on the inhibitory features of Rac1(W56F) or Rac1(T17N). In addition, mutations in the polybasic domain of Rac1 (Rac1(6Q)) did not reduce inhibition of uptake by Rac1(W56F) or Rac1(T17N) (Fig. 4B, Rac1(6Q); Table 1).

To analyze the role of RhoGDI binding in mediating the inhibitory effects of the Rac1(W56F) mutant, we focused on the Rac1(R66A) and C189S mutations that are totally defective for RhoGDI binding (41) (Table 1; Fig. 3C and data not shown). When introduced onto Rac1(W56F), it was found that R66A significantly reversed the effect of Rac1(W56F) on uptake inhibition without altering the interference properties of Rac1(T17N) (Fig. 4C). The C189S mutation, which prevents C-terminal prenylation and is essential for RhoGDI binding and plasma membrane localization, was particularly effective in reversing inhibition of uptake by Rac1(W56F) (Fig. 4D). In contrast, C189S failed to reduce interference of uptake by the classic inhibitors Rac1(T17N) and Rac1(D118A) (Fig. 4D). We therefore conclude that the ability of Rac1(W56F) to interact with RhoGDI is central to the inhibitory effect of Rac1(W56F) on invasin-promoted uptake. This lends further support to the model that Rac1(W56F) exerts its effects in a fashion distinct from other characterized dominant inhibitors, and that RhoGDI may play a positive role in the uptake process.

Overexpression of a Noninhibitory RhoGDI Mutant Suppresses the Effects of Rac1(W56F)—The above data suggest that Rac1(W56F) inhibits Rac1 signaling by modulating RhoGDI activity in some fashion. To test this, we determined whether overexpression of RhoGDI could increase the amount of invasin-promoted uptake in Rac1(W56F) transfected cells. Unfortunately, RhoGDI is well documented to have negative effects on Rac1 signaling by extracting Rac1 from plasma membrane (13), and predictably, overexpression of RhoGDI inhibited invasin-promoted uptake (data not shown). Therefore, we analyzed the recently described RhoGDI(D185A) mutant that is unable to extract Cdc42 from membranes but nevertheless still retains some ability to associate with Cdc42 (14)(Table 1). The RhoGDI(D185A) mutant was shown to be ~50% less efficient than the wild type RhoGDI at binding Rac1, as measured by FRET analysis, indicating that the mutation results in lowered affinity when Rac1 is the substrate (Fig. 5A). Consistent with reduced affinity, transfection of the RhoGDI(D185A) mutant did not interfere with invasin-promoted uptake (Fig. 5B). When RhoGDI(D185A) was cotransfected with Rac1(W56F), there was significantly more uptake than in cells expressing only Rac1(W56F) (Fig. 5B, Rac1(W56F) + RhoGDI(D185A)). This suppression, although not complete, is specific for Rac1(W56F) because the RhoGDI(D185A) mutant did not reverse the effects of Rac1(T17N) (Fig. 5C). Therefore, it appears that at least some of the effects of Rac1(W56F) are mediated by its manipulation of RhoGDI function.


Figure 4
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  		FIGURE 4.
Rac1(W56F) is suppressed by mutations that interfere with RhoGDI binding. The mechanism of dominant inhibition of uptake by Rac1(W56F) is different from that of Rac1(N17). COS-1 cells overexpressing various T7-tagged Rac1 variants were challenged with Y. pseudotuberculosis (YPIII(P-)) for 20 min, fixed, and processed for the immunofluorescence protection assay using rabbit polyclonal anti-Yersinia and mouse monoclonal anti-HA antibodies (see "Materials and Methods"). In some cases, untransfected cells were included as control. The percentage of cell-associated bacteria that were internalized was determined from 50 cells on each coverslip. Data for four coverslips from each group were analyzed and displayed as mean uptake on each coverslip ± S.E. (A and B). Y40C and the 6Q mutations do not suppress the ability of Rac1(W56F) or Rac1(T17N) to block invasinmediated uptake. C and D, the blockade of invasin-mediated uptake by Rac1(W56F) can be suppressed by the R66A and C189S mutations.

 
Inhibition of RhoA Activity Restores Invasin-promoted Uptake in the Presence of Rac1(W56F)—By binding Rho family members and maintaining them in a cytoplasmic locale, the primary consequence of RhoGDI binding is to inhibit the function of a Rho family GTPase. This implies that the Rac1(W56F) mutant alters the ability of RhoGDI to inhibit a small GTPase. By this model, the presence of the Rac1(W56F) mutant results in excess activity of one of the GTPase binding partners of RhoGDI. There are two primary candidates that may be misregulated because of the loss of RhoGDI function: Cdc42 and Rho. Dominant negative Cdc42(T17N) had no effect on invasin-mediated uptake in the presence of Rac1(W56F), indicating that increased Cdc42 activity probably was not responsible for the phenotype of Rac1(W56F) (Fig. 5D). The other major candidate is RhoA. The constitutively active RhoA(G14V) formed severely attenuated invasin-promoted uptake, reproducing previous results (27) (Fig. 5E). In contrast, we found that dominant negative RhoA(T19N) could enhance invasin-mediated uptake (Fig. 5E). RhoA activity, therefore, appeared antagonistic to uptake in this system. We therefore examined the possibility that sequestration of RhoGDI by Rac1(W56F) prevented RhoGDI from interfering with RhoA activity. Overexpression of RhoA(T19N) stimulated invasin-mediated uptake in the presence of Rac1(W56F) to the same level as RhoGDI(D185A), suggesting that sequestration of RhoGDI away from RhoA was responsible for Rac1(W56F)-mediated inhibition of invasin-mediated uptake (Fig. 5B). This suppression of Rac1(W56F) by RhoA(T19N) was specific because RhoA(T19N) could not elevate uptake in the presence of Rac1(T17N) (Fig. 5C). Based on these results, the enhanced RhoA activity that results from binding of the Rac1(W56F) mutant to RhoGDI contributes to inhibition of invasin-mediated uptake by the Rac1 mutant.

Cells that Express Rac1(W56F) Have a Higher Cellular GTP-RhoA Levels than Cells Expressing Rac1(WT)—The above results indicate that overactivation of RhoA, perhaps by titration of RhoGDI, was the reason that the Rac1(W56F) mutant interfered with bacterial uptake. To test this directly, cells transfected with plasmids overexpressing either Rac1(WT) or Rac1(W56F) were assayed for their cellular GTP-RhoA levels by affinity precipitation, using Rhotekin-agarose beads to detect GTP loading on RhoA. The amount of activated RhoA in cells transfected with Rac1(W56F) was 3-4 times higher than that observed in either cells transfected with Rac1(WT) or empty plasmid control (Fig. 5F). Consistent with the model that the RhoGDI(D185A) derivative reverses the effects of Rac1(W56F) by reducing RhoA activation in the cell, cotransfection of Rac1(W56F) and RhoGDI(D185A) resulted in levels of RhoA activation that were similar to those observed in untransfected cells (Fig. 5F). Therefore, excess RhoA activity is associated with both the presence of the Rac1(W56F) mutation and a corresponding reduction in invasin-mediated uptake in this system.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Rac1(W56F) was initially characterized as a Rac1 variant that switches its GEF recognition specificity (32, 33) such that it is able to associate in vitro with the Cdc42 Dbl-GEF intersectin but is unable to bind a number of Rac1 Dbl-GEFs. The protein, however, still retains the ability to be recognized by the unconventional GEF DOCK180 so that it is able to receive a subset of upstream signals that are recognized by Rac1(WT) (42). Furthermore, the mutant appears to behave identically to Rac1(WT) in terms of GTP loading and effector binding with no reported inhibitory effects on Rac1 signaling (32, 33). Surprisingly, we found that the Rac1(W56F) variant itself interfered with integrin-mediated uptake of Y. pseudotuberculosis.


Figure 5
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  		FIGURE 5.
Defective uptake caused by the Rac1(W56F) variant is associated with misregulation of RhoA. A, noninhibitory RhoGDI(D185A) mutant has depressed binding efficiency to Rac1. COS-1 cells cotransfected with mCFP-Rac1(WT) and RhoGDI-mYFP or RhoGDI-D185A-mYFP were analyzed by FRET, as described in the legend to Fig. 3. B, invasin-mediated uptake in the presence of Rac1(W56F) is restored by overproduction RhoGDI(D185A) or dominant negative RhoAT19N mutants. The percentages of cell-associated bacteria that were internalized were determined from 50 cells on each coverslip and displayed as means from four coverslips ± S.E. C, Rac1(T17N)-mediated blockade of invasin-mediated uptake is insensitive to the presence of RhoGDI(D185A) or RhoA-T19N. COS-1 cells overexpressing T7-tagged Rac1(T17N) alone or together with either RhoGDI(D185A) or RhoAT19N were analyzed. D, Cdc42(T17N) cannot suppress the inhibition of Rac1(W56F) on invasin-mediated uptake. COS-1 cells were analyzed that expressed T7-Rac1(W56F) alone or together with Myc-tagged Cdc42(T17N). E, RhoA activity negatively regulates invasin-mediated uptake. Cells were transfected with plasmids overexpressing Myc-tagged dominant negative RhoA-T19N or constitutive active RhoA-G14V. F, increased RhoA activation in cells containing Rac1(W56F) is blocked by overexpression of RhoGDI(D185A). HeLa cells transfected with the noted combinations of pCGT-Rac1(WT), pCGT-Rac1(W56F), or pCGT-RhoGDI(D185A) were subjected to affinity precipitation of cellular GTP-Rho. The amount of RhoA in the precipitate as well as in the input cell lysate was determined by Western blotting using a monoclonal RhoA antibody followed by densitometry analysis (see "Materials and Methods"). Graph displays the mean of three determinations from a typical experiment. % active RhoA represents mean ± S.E. of RhoA found in affinity precipitate relative to the total RhoA in the cell. Displayed below graph is immunoblot probed with anti-RhoA, showing one of the sets of samples used to obtain data.

 
Several lines of evidence indicate that the interference with bacterial uptake caused by Rac1(W56F) was distinct from that of the well characterized dominant negative mutant Rac1(T17N). First, Rac1(W56F) inhibited not only the Rac1-dependent pathway for invasin-mediated uptake but also blocked the Cdc42-dependent entry of EPEC (Fig. 1), which is resistant to the presence of Rac1(T17N) (37). Second, unlike Rac1(T17N), Rac1(W56F) appeared to be able to assume an active conformation after GTP loading (Fig. 2) and was able to bind to RhoGDI (Fig. 3). Third, we were able to identify intragenic suppressor mutations in Rac1 that specifically reversed the effects of Rac1(W56F) without affecting interference caused by Rac1(T17N) (Fig. 4). Both intragenic suppressor mutations affected RhoGDI binding by Rac1, indicating that the Rac1(W56F) variant may modulate normal RhoGDI function as part of its mechanism of interference with invasin-promoted uptake. Fourth, overexpression of either a lowered affinity RhoGDI mutant or the dominant negative RhoA(T19N) enhanced invasinpromoted uptake in the presence of Rac1(W56F), whereas the same strategies had no effect on interference caused by Rac1(T17N). Finally, cellular RhoA activity was elevated in the presence of Rac1(W56F), a consequence probably central to its ability to inhibit uptake (Fig. 5) and consistent with the fact that the dominant inhibitory RhoA(T19N) could selectively reverse the effects of Rac1(W56F). Taken together, it appears that the primary effect of the Rac1(W56F) mutant is that it is unable to inhibit the activity of RhoA, which in turn interferes with invasin-promoted uptake (Fig. 6).

Rac1 can exert negative regulation of RhoA at multiple levels, although not all the molecular details are known regarding this modulation. The best appreciated regulatory strategy is the ability of activated Rac1 to relieve inhibition of p190RhoGAP via a process that involves both reactive oxygen species and inactivation of the low molecular weight protein tyrosine phosphatase (44). The resulting activation of p190RhoGAP causes Rho to undergo GTP hydrolysis, with consequent loss of Rho from the membrane, sequestering it in a cytoplasmic locale bound to RhoGDI. A second model proposed is that Rac1 can inactivate at least one Rho-specific GEF by stimulating PAK kinase activity (46). Although the Rac1(W56F) variant may be unable to down-regulate RhoA, we think it unlikely that this is the result of a defect in its ability to inactivate a RhoGEF via PAK. This is because the mutant still retains the ability to bind the PBD of PAK1, and furthermore, this mechanism cannot explain the dominant inhibitory properties of Rac1(W56F) (Fig. 2). Rather, it seems more likely that the effects are exerted via RhoGDI, RhoGAPs, or perhaps both. We have evidence that interference with uptake by this mutant requires structural information that is required for recognition of RhoGDI (Fig. 6), although this only partially explains interference by Rac1(W56F). This is because a residue change that eliminates detectable RhoGDI binding to Rac1(W56F) does not completely suppress the uptake defect, and similar intermediate effects were observed after overproduction of RhoGDI(D185A) (Fig. 5).


Figure 6
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  		FIGURE 6.
A model for RhoGDI-mediated inactivation of RhoA during invasin-mediated uptake. Left, events involved in allowing efficient bacterial uptake. After engagement of beta1 integrin by invasin, inactive Rac1 bound to RhoGDI was recruited to a site on the membrane where it was activated and released from RhoGDI, creating a pool of free RhoGDI. The free cytoplasmic pool of RhoGDI is proposed to inhibit the activity of RhoA, resulting in a local environment that is compatible with Rac1-promoted events necessary for efficient bacterial uptake. Right, events resulting in depressed bacterial uptake. In the presence of overexpressed Rac1(W56F), RhoGDI remains in association with Rac1(W56F) and fails to inhibit RhoA activity. As a result, the failure of RhoGDI to depress RhoA activity leads to inhibition of invasin-promoted uptake. Alternatively, a factor other than RhoGDI may be responsible for the RhoA inhibition, and overexpression of the RhoGDI mutant would simply displace this hypothetical factor from Rac1, freeing it to directly inhibit RhoA.

 
RhoA function cannot be efficiently regulated in the presence of Rac1(W56F), opening up the possibility that the mutant can also interfere with cytoskeletal processes not normally associated with Rac1 activity. This may explain why there was severe depression of EPEC uptake into HeLa cells, even though this process is not inhibited by the classic Rac1(T17N) mutation (38). That the Rac1(W56F) variant had previously appeared to be nothing more than a simple switch of specificity mutant, without confounding complications, can be explained by the fact that the assays used to analyze the variant were not inhibited by enhanced RhoA activity. For instance, the mutant is used to study the Rac1-dependent NADPH oxidase production of superoxide, a process that probably requires RhoA (43). In addition, Rac1(W56F) has been analyzed in cells in which there is either an additional mutation conferring constitutive activation of Rac1 or there is an overproduced copy of a GEF (32, 33). In either case, the strong global signaling that occurs under these types of experimental conditions may obscure any inhibitory activities that are more readily observable when analyzing localized membrane changes such as those involved in bacterial uptake.

That inhibition of RhoA activity could significantly overcome the block in uptake caused by Rac1(W56F) is reminiscent of several reports demonstrating an antagonistic relationship between activation of Rho and Rac1. Successful completion of cytoskeletal events controlled by Rac1 often involves preventing activated Rho from interfering with these rearrangements (28, 45). For instance, Rho interferes with Rac1-activated neural cell migration, perhaps by stabilizing an adhesion event that favors cell immobilization rather than movement (28). More relevant to the work reported here, both uptake of charged beads (29) and invasin-promoted internalization of Y. pseudotuberculosis (6, 27) are inhibited by activation of RhoA, indicating that Rac1-promoted phagocytic events are antagonized by RhoA. Although there are number of possible explanations for this interfering relationship, there are two properties of the GTPases that are relevant to uptake. First, continued RhoA activation results in Rac1-promoted membrane ruffling events that have aberrant morphologies (28). This deranged ruffling may interfere with phagocytosis, causing undirected ruffling events that cannot be coordinated to successfully complete the uptake process. Second, the activity of Rho proteins leads to the formation of actin stress fibers that potentially immobilize the mammalian cell surface (12). Such a rigid cell surface may interfere with membrane movement around an incoming particle. In either case, a successful phagocytic event clearly requires tight negative control of Rho.

Our characterization of Rac1(W56F) provides an unappreciated role for RhoGDI in potentially dampening the activity of antagonistic Rho family members (Fig. 6). After beta1 integrin receptor engagement by invasin, a GEF must be activated to facilitate the guanine nucleotide exchange of Rac1-GDP into Rac1-GTP and to allow release of RhoGDI from Rac1 (48). Free Rac1-GTP then goes on to activate downstream pathways of actin polymerization events for bacterial uptake. RhoGDI, however, may have some other role in the uptake process than simply negatively regulating Rac1. The ability of RhoGDI to negatively regulate interfering Rho activity allows another level of control of bacterial uptake that is distinct from the demonstrated positive role of RhoGDI in directing Rac1 to be delivered to membrane sites (11). Instead, RhoGDI released from Rac1 is proposed here to have the potential to exert an indirect positive effect by interfering with unwanted RhoA activity (Fig. 6). In this way, RhoGDI could potentially mediate cross-talk between Rac1 and RhoA signaling and amplify the Rac1 signaling by antagonizing the opposing activity. Future work should help uncover other positive roles RhoGDI may play in Rac1 signaling and the nature of cytoskeletal rearrangements that interfere with invasin-promoted uptake.


   FOOTNOTES
 
* This work was supported by Howard Hughes Medical Institute (HHMI), Award R37AI23538 from NIAID, National Institutes of Health, and program project Grant P30DK34928 from NIDDK, National Institutes of Health. 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 An investigator of Howard Hughes Medical Institute (HHMI). To whom correspondence should be addressed: Dept. of Molecular Biology and Microbiology and Howard Hughes Medical Inst., Tufts University School of Medicine, 150 Harrison Ave., Boston, MA 02111. Tel.: 617-636-1392; Fax: 617-636-0337; E-mail: Ralph.Isberg{at}tufts.edu.

2 The abbreviations used are: GEF, guanine nucleotide exchange factor; GDI, guanine nucleotide dissociation inhibitor; EPEC, enteropathogenic Escherichia coli; FRET, fluorescence resonance energy transfer; PBD, p21 binding domain; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Matt Heidtman, Matthias Machner, Molly Bergman, Vicki Auerbuch, and Marion Dorer for review of the text and Drs. Jim Bliska, Ulla Knaus, and Kit Wong for supplying plasmids.



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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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T. T. G. Ho, S. D. Merajver, C. M. Lapiere, B. V. Nusgens, and C. F. Deroanne
RhoA-GDP Regulates RhoB Protein Stability: POTENTIAL INVOLVEMENT OF RhoGDI{alpha}
J. Biol. Chem., August 1, 2008; 283(31): 21588 - 21598.
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A. A. Birukova, E. Alekseeva, A. Mikaelyan, and K. G. Birukov
HGF attenuates thrombin-induced endothelial permeability by Tiam1-mediated activation of the Rac pathway and by Tiam1/Rac-dependent inhibition of the Rho pathway
FASEB J, September 1, 2007; 21(11): 2776 - 2786.
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