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Vol. 15, Issue 3, 1124-1133, March 2004
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B Activation by Conveying the SCF Complex and IkB
to the Ruffling Membranes
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* Institut National de la Santé et de la Recherche Médicale U452, IFR50, Faculté de Médecine, 06107 Nice, France;
Laboratory of Ultrastructures, Istituto Superiore di Sanità, 00161 Rome, Italy; and
Consiglio Nazionale delle Ricerche Anaerobies, Institut Pasteur, 75724 Paris, France
Submitted May 15, 2003;
Revised August 13, 2003;
Accepted October 24, 2003
Monitoring Editor: Mark Ginsberg
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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B (NF-B) is a ubiquitously expressed transcription factor that plays a central role in directing a vast range of cellular functions. Its activation is controlled by the Rac GTPase and relies on the coordinated cooperation of the E3–ligase complex SCF
TrCP, composed by Skp-1/Cullin-1, Rbx/Roc1, and the -TrCP proteins. Recently, Cullin-1 has been reported to form a complex with the activated Rac GTPase. Here, we show that the specific activation of the Rac GTPase, besides directing its own positioning, induces the relocalization of the SCF component Cullin-1 to the ruffling membranes. This occurred only if the ruffles were stimulated by the Rac GTPase and was accompanied by the repositioning to the same intracellular compartment of the SCF protein Skp-1 and the ubiquitin-like molecule Nedd-8. The SCF substrate IkB
was also directed to the ruffling membranes in a Rac-dependent way. The novelty of these findings is in respect to the demonstration that the correct positioning at the ruffling membranes is crucial for the subsequent series of events that leads to IkB proteasomal degradation and the resultant activation of NF-B. Consequently, this points to the role of Rac as a docking molecule in NF-B activation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). To date, the mammalian Rho family comprises at least 16 members, including Rho, Rac, and Cdc42. Besides the first recognized ability of regulating the actin cytoskeleton (Hall, 1998), Rho GTPases also play a pivotal role in guiding a variety of other cellular processes, such as cell proliferation, transformation, and gene transcription. In fact, they participate in the activation of nuclear factor-B (NF-B) (Perona et al., 1997; Montaner et al., 1998), a group of structurally related and evolutionarily conserved transcription factors involved in regulating the expression of genes required for inflammatory responses, cell growth, and suppression of apoptosis (reviewed in Ghosh et al., 1998). In mammals, NF-B consists of five members, namely, Rel (c-Rel), RelA (p65), RelB, NF-kB1 (p50 and its precursor p105), and NF-kB2 (p52 and its precursor p100). They form both homo- and heterodimers, of which the best characterized form of NF-B is a heterodimer composed of p50 and p65 subunits. This heterodimer, in resting cells, is sequestered in the cytoplasm by association with the inhibitory subunit IkB (Beg et al., 1992). Signals leading to NF-B activation trigger a transduction pathway responsible for IkB phosphorylation at two specific serine residues. Phosphorylation allows IkB polyubiquitination on specific lysine residues and subsequent proteolytic degradation by the 26S proteasome. After degradation of IkB, NF-B heterodimer is translocated into the nucleus for gene trans-activation (Baldwin, 1996).
The ubiquitination process, although involving several steps, always results in the recognition and modification of the target protein via the E3-ubiquitin-protein-ligases (Ciechanover and Schwartz, 1998). Several classes of E3s have been described and, among them, the SCF (Skp1/Cul-1/F-box protein complex) complexes that recognize phosphorylated substrates. These tetrameric complexes are composed by Skp-1/Cullin-1 and Rbx/Roc1, which are common to all SCF, and a variable F-box protein (Deshaies, 1999). Ubiquitination of IkB, as does -catenin, is achieved upon its association with an SCF complex that contains the -TrCP (SCF-TrCP) as F-box protein (Karin and Ben-Neriah, 2000). In vitro, the capacity of SCF-TrCP to bind IkB is enhanced upon Cullin-1 posttranslational neddylation, a modification consisting of the covalent addition of a Nedd-8 ubiquitin-like molecule to lysine 720 (Read et al., 2000). The Cullin-1 K720R mutant, impaired in its neddylation capacity, was localized in the nucleus, suggesting that Nedd-8 modification of Cullin-1 is required for its exit from the nucleus (Furukawa et al., 2000). Interestingly, the E3 ubiquitination ligase subunit Cullin-1 was noted to form a complex with activated Rho GTPases, and in particular with Rac (Senadheera et al., 2001), raising the hypothesis that Rac GTPase-Cullin-1 interaction could serve to regulate E3 subcellular localization.
To verify such a hypothesis, we undertook a study using an Escherichia coli protein toxin that specifically activates the Rho GTPases, named cytotoxic necrotizing factor 1 (CNF1). It acts by deamidating a pivotal glutamine residue of RhoA, Rac, and Cdc42 (glutamine 63 of Rho or glutamine 61 of Rac and Cdc42) into glutamic acid (Flatau et al., 1997; Schmidt et al., 1997; Lerm et al., 1999). Recent findings have established that CNF1-induced permanent activation of RhoA, Rac, and Cdc42 (Doye et al., 2002; Lerm et al., 2002) sensitizes them to ubiquitination and proteasome degradation. Interestingly, some carcinoma cell lines, including HEp-2 cells, respond to CNF1 with a lower efficiency of Rac ubiquitination and degradation that results in a sustained Rac activation (Doye et al., 2002). This identifies CNF1 as a preferential tool for studying cellular activities linked to Rac activation in HEp-2 epithelial cells.
In this article, we provide evidence that protein complexes involved in the activation of the transcription factor NF-B are spatially relocalized at the level of membrane ruffles, after Rac activation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-actin [clone AC-74]); Upstate Biotechnology (Lake Placid, NY) (anti-IkB); and Santa Cruz Biotechnology (San Diego, CA) (anti-Nedd-8 and anti-p65). If indicated, cells were treated with 10-9 M CNF1, either wild type or catalytic inactive mutant C866S purified as described previously (Flatau et al., 1997).
Inhibition Experiments
For proteasome inhibition, HEp-2 cells were preincubated for 1 h with 10 µM MG132 (Sigma-Aldrich) before being challenged with CNF1 for different time points. For experiments aimed at perturbing lipid raft organization, 2 µg/ml filipin (Sigma-Aldrich) were added after 1 h of incubation with CNF1. Toxin exposure was prolonged for an additional 3 h. To verify whether cholesterol addition would interfere with filipin activity, cells treated with CNF1 for 1 h were exposed to 60 µg/ml cholesterol together with 2 µg/ml filipin until the end of toxin treatment. Inhibition of Rho GTPases was achieved by pretreating HEp-2 cells for 2 h with 1 µg/ml of the chimeric toxin C3B (Aullo et al., 1993), LT82, or LT9048 from Clostridium sordellii (Popoff et al., 1996; El Hadj et al., 1999) before being challenged with CNF1 for 4 h.
Transfections of HEp-2 Cells
HEp-2 cells were transfected with 2 µg of pcDNA3-myc-IkB-mutant (S32A and S36A) (Traenckner et al., 1995), pcDNA3-HA-Cullin-1 (Lisztwan et al., 1998), pcDNA3-HA-Cullin-1 K720R (Kawakami et al., 2001), pHm200-HA-Skp-1 (Winston et al., 1999), and p(6KappaB)Luc, controlled by a minimal thymidine kinase promoter and six reiterated B sites (Bottero et al., 2001). The construction for GST-RacL61 and GST-RacN17 expressions were obtained by subcloning in the plef-vector (Rudert et al., 1996) RacL61 and RacN17 from pXJ-HA-Rac mutants (Doye et al., 2002). PEGFP-ARF6 was a gift from Philip Chavrier (Derrien et al., 2002). Exgen500 (Euromedex, France) was used to transfect cells according to the manufacturer's instructions. Eighteen hours after transfection, CNF1 was added and maintained for different incubation times.
Immunofluorescence and Confocal Microscopy
Control and CNF1-treated HEp-2 cells, either transfected or not, maintained in minimal Eagle's medium supplemented with 10% fetal calf serum were stained as follows: for p65 immunostaining, cells were fixed in acetone: methanol, 1:1 (vol/vol) for 10 min at room temperature and air dried. After 1 h of preincubation with phosphate-buffered saline (PBS) containing 10% of AB human serum, cells were incubated with the p65 antibody for 1 h at room temperature. After three washes in PBS, cells were incubated for 1 h at room temperature with fluorescein isothiocyanate-labeled anti-rabbit antibody. After three additional washings in PBS, cells were mounted on glass coverslips and analyzed with a Nikon Microphot fluorescence microscope. For immunolabeling with anti-caveolin-1 and anti-IkB, cells were fixed in 4% paraformaldehyde and permeabilized with 1% Triton X-100. For immunolabeling with anti--actin, anti-hemagglutinin (HA), anti-GST, anti-Nedd-8, anti-Rac, and anti-IkB, cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) and permeabilized with 0.05% saponin (Sigma-Aldrich). Cells were processed as described previously (Doye et al., 2002) and analyzed with a TCS-SP confocal microscope (Leica, Heidelberg, Germany).
Luciferase Assay
HEp-2 cells seeded at 105 per well in a 24-well plate were transfected as described above, with both 2 µg of a luciferase reporter gene and 0.5 µg of pSV40-galactosidase control vector (Promega, Madison, WI) per well. Eighteen hours after transfection, cells were intoxicated with 10-9 M CNF1 and lysed in 50 µl of lysis buffer (Promega) at different times. Soluble extracts were assessed for both luciferase activity and -galactosidase activity, as recommended by the manufacturer. Results were expressed as arbitrary values corresponding to the value of the luciferase activity normalized to the -galactosidase activity or fold activation.
Western Blots and GST-PAK Pull-Down Assay
To test the activity of Rac1 in HEp-2 cells after different times of incubation with CNF1, pull-down assay was performed by using 0.5 mg of cell lysates and 40 µg of GST-PAK70-106. Proteins were resolved on 12% SDS-PAGE followed by transfer on polyvinylidene difluoride membranes (Amersham Biosciences, Saclay, France). Rac1 binding to GST-PAK70-106, total Rac as well as IkB protein levels were determined by Western blot analysis as described previously (Doye et al., 2002).
Statistical Analysis
The values reported in graphs are the means ± SD from three separate experiments performed in duplicate. The Student's t test was used for analysis of statistical significance. A p value <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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B, and the SFC complex, we took advantage of the recent demonstration that, in HEp-2 cells, CNF1 produces a sustained activation of Rac that is due to the very low Rac ubiquitination activity in this cell type (Doye et al., 2002). To verify this finding in our experimental system, we performed a pull-down experiment by using the Rac effector PAK linked to GST protein. We found, in accordance to what was reported previously (Doye et al., 2002), that exposure to CNF1 activated Rac in a time-dependent manner, and this resulted in the activation being sustained for at least 24 h (Figure 1A). The next step was to view the localization of the components of the SCF complex after Rac activation by using confocal microscopy. We transfected HEp-2 cells with the recombinant protein HA-Cullin-1- and we found that although in control cells Cullin-1 was dispersed throughout the cytoplasm, in cells exposed to CNF1 the Cullin-1 relocalized to the actin-rich membrane ruffles (Figure 1B). Transfecting cells with the dominant positive mutant GST-RacL61 or dominant negative mutant GST-RacN17 demonstrated the specific involvement of Rac in such a phenomenon. In fact, RacL61 was capable of mimicking the displacement of Cullin-1 to ruffles promoted by CNF1 (Figure 2A), an ability that was impaired by the dominant negative mutant RacN17 (Figure 2B). As a control, we transfected cells with the ADP-ribosylation factor 6 (ARF6), a small GTPase involved in the aspects of endocytosis, exocytosis, membrane recycling, and also membrane ruffling (Hunzicker-Dunn et al., 2002). As shown in Figure 2C, ARF6-promoted ruffles were devoid of Cullin-1 signal, demonstrating that the presence of Cullin-1 at the ruffles level is specifically governed by Rac. Because it is known that to be active, Cullin-1 must be linked to the ubiquitin-like molecule Nedd-8 (Wu et al., 2000), we stained cells with an anti-Nedd-8 antibody, and we found that in control cells Nedd-8 was dispersed throughout the cytoplasm, whereas in CNF1-treated cells, it was displaced to the ruffles (Figure 3A), in the same way as that of Cullin-1. Consistent with this, the neddylation-impaired mutant Cullin-1 K720R failed in moving to the ruffling membranes after CNF1 stimulation (Figure 3B), but rather seemed localized in the nucleus, as reported previously by another group (Wu et al., 2000). To verify the presence of Skp-1, a further component of the SCF complex, at the level of membrane ruffles raised by Rac activation, HEp-2 cells were transfected with HA-Skp-1 and stained with an antibody against the tag-HA. Skp-1, a cytoplasmic/nuclear glycoprotein associated with polyubiquitination (West, 2003) was dispersed into the cell cytoplasm of control cells and relocalized to the ruffling membranes, similarly to what occurs for Cullin-1, in CNF1-treated cells (Figure 3C). Considered together, these results permit the assumption that the SCF complex is relocalized to ruffling membrane upon CNF1 exposure.
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Rac Activation Produces IkB Recruitment into Membrane Ruffles and Proteasome Degradation
From our results on Rac-dependent SCF relocalization, we hypothesized the recruitment to the ruffles of one of the main substrates of SCF activity, that is, IkB. As viewed by confocal microscopy, IkB was diffusely spread throughout the cytoplasm of control cells, whereas upon exposure to CNF1, it could also be detected at the level of ruffling membranes, colocalized with Rac (Figure 4A). This phenomenon was already discernible after 2 h of toxin exposure (our unpublished data) but became particularly evident starting from 4 h of challenge with CNF1 (Figure 4A). Because these results suggested a spatial relationship between activated Rac and IkB, we then verified whether there was an eventual dependence on Rac activation of IkB recruitment to ruffling membranes. Hence, we transfected HEp-2 cells with the dominant positive mutant HA-RacL61 or the dominant negative mutant HA-RacN17. As shown in Figure 4B, RacL61 was capable of promoting the recruitment of IkB to the ruffles, whereas RacN17, the dominant negative mutant, blocked the CNF1-induced relocalization of IkB to the ruffling membranes. These data suggest the possibility that the IkB displacement to ruffles is necessary for the subsequent SCF-dependent IkB ubiquitination. In support of this hypothesis, we found that ubiquitin was partially redistributed to ruffling membranes after CNF1 exposure (our unpublished data). Hence, we contemplated whether the Rac-induced relocalization of IkB, together with the SCF complex and ubiquitin, could result in its degradation. To address this question, we measured by Western blotting the amount of IkB in CNF1-treated cells. As shown in Figure 5A, IkB depletion had already occurred after 2 h of toxin exposure and was followed by IkB reappearance within 24 h of challenge with CNF1 (our unpublished data). This was a proteasome-dependent phenomenon because the Rac-induced IkB degradation was totally prevented by the addition of 10 µM proteasome inhibitor MG132 (Figure 5B). It is worth noting that the CNF1 C866S, which represents the catalytic mutant defective in the ability to activate the Rho GTPases (Schmidt et al., 1998), did not induce the proteolytic degradation of IkB (Figure 5A), thus proving the necessity of the catalytic activity of the toxin for IkB depletion.
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Rac Activation Induces the Nuclear Translocation of the Transcription Factor NF-B
Because IkB degradation is a crucial step for NF-B activation, we investigated whether the CNF1-induced Rac activation could lead to its translocation and activation. First, we analyzed by fluorescence microscopy the ability of the NF-B p65 subunit to translocate into the nucleus by using an antibody against p65 (Figure 6A). Whereas nuclei in control cells were completely devoid of signal, CNF1 promoted the nuclear translocation of p65, as evidenced by positively stained nuclei (Figure 6A). This was a time-dependent phenomenon that started 3 h after CNF1 exposure and lasted for several hours (Figure 6B). Because CNF1-induced Rac activation lasted for at least 24 h (Fig. 1A) and in contrast, p65 translocation seemed to decrease after 24 h of CNF1 treatment, we also verified in the time frame between 24 and 48 h whether CNF1 was able to induce p65 translocation. We found that, after the peak obtained between 4 and 10 h (p < 0.05), in the time frame ranging between 24 and 48 h, a minimal level of p65 translocation was observed, always lower than that observed after the initial times of CNF1 exposure. In fact, in none of the cases this was higher than 12% (our unpublished data), thus suggesting a steady state activation of the NF-B transcription factor. NF-B translocation correlated with its gene transactivation activity, as viewed by transfecting HEp-2 cells with a plasmid corresponding to a luciferase reporter gene under the control of six-repeated B-binding sites. The challenge with CNF1 led to a significant increase (p < 0.05) in the luciferase activity that started 6 h after addition of the toxin (Figure 6C). The apparent discrepancy between data on p65 nuclear staining and luciferase activity (Figure 6, B and C) can be ascribed to the lack of a linear correlation between them.
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Although Rac was reported to be the principal Rho GTPase activated by CNF1 in HEp-2 cells (Doye et al., 2002), still the toxin also activates Rho and Cdc42 (Flatau et al., 1997; Schmidt et al., 1997). Furthermore, NF-B activation has been reported to be regulated by all the three Rho GTPases (Perona et al., 1997; Cammarano and Minden, 2001). Hence, to verify that p65 translocation to the nucleus was dependent only on Rac, HEp-2 cells were for 2 h preexposed to 1 µg/ml Rho-inhibiting toxins, before being challenged with CNF1 for 4 h. We used, in particular, the chimeric toxin C3B, that specifically inhibits Rho A, B, and C (Aullo et al., 1993), Clostridium sordellii LT82 or LT9048, which inhibit Rac but not Rho and Cdc42 (Popoff et al., 1996) and Rac plus Cdc42 (El Hadj et al., 1999), respectively. C3B failed in inhibiting the CNF1-induced p65 nuclear translocation, whereas both LT toxins did have a blocking effect (Figure 6D) (LT82+CNF1 and LT9048+CNF1 versus CNF1 alone; p < 0.05). These results indicate Rac as inducer of NF-B activation, supporting the master role of this GTPase in the positioning and function of the NF-B–activating complex.
Is Rac-induced Protein Recruitment to Ruffling Membranes Having a Role in NF-B Activation?
All in all, our results indicate that CNF1 induced 1) the relocalization to the ruffles of Rac together with the SCF complex and IkB, 2) IkB proteasomal degradation, and 3) NF-B activation. Hence, we contemplated whether the observed protein complex relocalization to the ruffles could be an essential step for the occurrence of the subsequent events. IkB-SA 32-36, the classical dominant negative mutant with its inability to be phosphorylated (Bottero et al., 2001), was cotransfected with the luciferase reporter gene described above. Despite the addition of CNF1, the luciferase activity resulted in being nearly eliminated by the presence of IkB-SA 32-36 (Figure 7A). Interestingly, when CNF1 was added to cells transfected with IkB-SA 32-36, it failed in dislocating the mutant to the ruffling membranes (Figure 7B), indicating that IkB positioning at the level of ruffles is a prerequisite for NF-B activation. Furthermore, because lipid rafts represents a typical domain of a membrane involved in signal transduction, we challenged HEp-2 cells with filipin, a compound known to sequester cholesterol and thus used to disrupt the organization of lipid rafts (Orlandi and Fishman, 1998). Interestingly, filipin significantly reduced the percentage of p65-positive nuclei (p < 0.05) induced by toxin treatment (Figure 8A). This was not due to alterations in the rate of CNF1-induced Rac activation, as demonstrated by a pull-down assay (our unpublished data) and in accordance to what was reported previously (Contamin et al., 2000). As a control, to verify whether the impairment of CNF1-induced p65 translocation could be due to sequestration of cholesterol by filipin, 60 µg/ml cholesterol and 2 µg/ml filipin were simultaneously added to CNF1-treated cells. What we found was that cholesterol prevented the inhibiting effect of filipin (our unpublished data), suggesting the involvement of cholesterol-rich domain in the pathway leading to NF-B activation. Hence, we verified whether IkB could colocalize with caveolin-1, a typical marker of lipid rafts (Harris et al., 2002), by performing a double staining for caveolin-1 and IkB, both in control and in CNF1-treated HEp-2 cells. Whereas in control cells both proteins were dispersed throughout the cytoplasm, in HEp-2 cells challenged with the toxin caveolin-1 and IkB colocalized at the level of ruffling membranes (Figure 8B). All in all, these findings reinforce our hypothesis indicating lipid rafts as pivotal in the CNF1-induced NF-B activation.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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B activation, that is, the SCF complex components Cullin-1 and Skp-1, and the NF-B inhibitor IkB. More importantly, this occurred only if the ruffles were raised by the Rac GTPase. The novelty of these findings resides in the demonstration that the right positioning at the ruffling membranes, and more precisely in cholesterol-rich lipid rafts, is mandatory for the accomplishment of the subsequent series of events that leads to IkB proteasomal degradation and the resultant activation of NF-B. A scheme that summarizes our results is drawn in Figure 9.
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Interestingly, it has recently been reported that activated Rac family members bind to Cullin-1, and it has been suggested that this interaction may serve to regulate the E3 ligase subcellular localization (Senadheera et al., 2001). Starting from this hypothesis, we herein report the first evidence that ruffling membrane dislocation of activated Rac (Doye et al., 2002) is accompanied by the repositioning to the same intracellular compartment of Cullin-1 and Skp-1. Presumably, thus the whole SCF apparatus deputed to IkB ubiquitination that consequently signals for IkB degradation was at the ruffle level. Notably, Read et al. (2000) reported that phosphorylated IkB (that is, the form targeted for ubiquitination) exclusively associates with a form of Cullin-1 that is singly modified by the ubiquitin-like protein Nedd-8. We found that not only Cullin-1 but also Nedd-8 was relocalized to the Rac-induced ruffling membranes. Because Cullins are, up to now, the only proteins reported to be modified by Nedd-8 and because Nedd-8 itself is localized primarily in the nucleus (Kamitani et al., 1997), our results suggest that the Nedd-8 protein detected at the level of the Rac-induced ruffles is part of the active Nedd-8 modified Cullin-1. The presence in the same compartment of endogenous IkB and ubiquitin strongly supported this hypothesis. This suggests also the possibility that the activated Rac GTPase recruits to ruffles both the p65/p50/IkB complex and the SCF complex by functioning as a docking molecule. We would like to point out that Cullin-1 was reported to be primarily localized in the nucleus and, only in a small percentage of cells, in the cytosol (Furukawa et al., 2000). Thus, this is the first report showing a Rac-dependent displacement of Cullin-1 to the ruffling compartment. All this is in accordance with the general assumption that certain proteins, after a proper activatory stimulus, need to be precisely relocalized to exert their function. Proteins of the Rho family are targeted to specific subcellular compartments, thus playing an essential role once the right positioning is achieved (Kraynov et al., 2000). In particular, the Rac GTPase targeted to the membrane has been reported to recruit the cytosolic p67phox protein for the assembly of the NADPH-oxidase complex (Gorzalczany et al., 2000). On the other hand, activation of Rac leads to a simultaneous membrane recruitment and activation of kinases involved in the production of phosphoinositides, thus regulating the membrane ruffling as well as the endocytosis and exocytosis (Wei et al., 2002). In our case, the Rac-dependent grouping of all SCF, IkB, and ubiquitin to ruffles is a crucial newly described prerequisite for the activation of the transcription factor NF-B.
It is worth noting that ruffles serve in numerous cellular processes and must consequently recruit all the components required for their functions. On the other hand, it is well-established that proteins involved in cell signaling can partition into subdomains of the plasma membrane, namely, lipid rafts that contain high concentrations of cholesterol and glycosphingolipids and where the cell signaling is controlled through different mechanisms (Pike, 2003). The demonstration that CNF1-treated cells require the cholesterol availability for an efficient NF-B activation and the finding that IkB colocalizes with caveolin-1 at the level of CNF1-induced ruffling membranes define the lipidic subdomains of recruitment that are present in the ruffling membranes. Therefore, in our case, lipid rafts can be viewed as signaling platforms where the requested components for NF-B activation convey and colocalize. This conceivably serves to facilitate their interaction that would occur rapidly and efficiently because of the spatial proximity of the interacting components.
NF-B plays a central role in regulating the expression of several genes involved in immune, inflammatory, and apoptotic processes (Gosh et al., 1998). Intriguingly, NF-B also provides a link between toxins hijacking the Rho GTPases and apoptosis in the bacterial world (Fiorentini et al., 2003). Hence, it is not surprising that such a pivotal transcription factor can somehow be governed by the central switching molecule Rac. From the point of view of bacteria, challenging Rac with a protein toxin can represent, together with the others, a strategy to handle this transcription factor and to avoid their own elimination. On the other hand, the immune system has evolved a variety of receptors to recognize invading bacteria, including the so-called Toll-like receptors that are able to convey signals to transcription factors that orchestrate the inflammatory response (Zhang and Ghosh, 2001). It is worth noting that NF-B activation can be mediated by Toll-like receptors and is controlled by Rac (Arbibe et al., 2000) and, although it involves different molecules, is dependent on the assembly of a multiprotein complex in the same manner as the one herein described. Of interest, epithelial cells have a family of cytosolic proteins, one of which, Nod1 (nucleotide-binding oligomerization domain) also mediates NF-B activation in response to invasive bacteria (Girardin et al., 2001). Finally, the Rac-induced activation of the transcription factor NF-B by the bacterial toxin CNF1 can furnish a molecular framework to our previous observations showing a protective effect of the toxin to apoptotic stimuli (Fiorentini et al., 1998).
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Present address: Department of Drug Research and Evaluation,
Istituto Superiore di Sanità, 00161 Rome, Italy. 
|| C.F. and A.F. are principal investigators.
¶ Corresponding author. E-mail address: alessia.fabbri{at}iss.it.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, control cells; 
, C3B-treated cells; 
, LT82-treated cells; and 
, LT9048-treated cells).
