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Vol. 17, Issue 3, 1096-1109, March 2006
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* Department of Biochemistry, Institute for Cancer Research, Faculty Division The Norwegian Radium Hospital, 0310 Oslo, Norway;
Department of Molecular Biosciences, University of Oslo, 0316 Oslo, Norway
Submitted August 16, 2005;
Revised November 30, 2005;
Accepted December 5, 2005
Monitoring Editor: Suzanne Pfeffer
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Sandvig et al., 2002). The toxin is composed of two moieties: the enzymatically active A-chain that is noncovalently attached to a stable pentamer of B-chains that binds to Gb3 and mediates the endocytic uptake of the toxin. In some cells, Stx is endocytosed mainly by clathrin-dependent endocytosis (Sandvig, 2001), although other mechanisms exist (Nichols et al., 2001; Lauvrak et al., 2004; Saint-Pol et al., 2004). Importantly, the toxin can induce its own transport to clathrin-coated pits (Sandvig et al., 1989). When Stx is added to HeLa cells at 0°C, the toxin becomes evenly distributed at the cell surface. However, after a short incubation at 37°C, the toxin moves to clathrin-coated pits and is internalized. In toxin-sensitive cells a larger fraction of the toxin seems to be internalized from clathrin-coated pits at the cell surface, and clathrin is required for endosome-to-Golgi transport of the toxin as well (Lauvrak et al., 2004; Saint-Pol et al., 2004). Because it has been shown by different groups that the fatty acid of Gb3 is important for efficient Golgi transport (Sandvig et al., 1994; Lingwood, 1999), the composition of Gb3 also may play a role for the endocytic pathway used. A raft localization of StxB was recently found to be required for efficient retrograde transport (Falguieres et al., 2001).
In some hematopoetic cells, Stx has been shown to trigger a rapid signaling cascade that might lead to apoptosis (for review, see Cherla et al., 2003). In a number of other cell types induction of signaling leading to apoptosis seems to be mediated by the ribocytotoxic stress induced by the A-fragment after entry into the cytosol (Foster and Tesh, 2002; Smith et al., 2003). Thus, in the latter cases it takes time before the reported signaling takes place.
Because Stx seems to affect its own endocytosis by inducing its transport to clathrin-coated pits (Sandvig et al., 1989), we decided to investigate whether this might occur as a response to a rapid toxin-induced signaling, similarly to what has been reported for endocytic uptake of other ligands. The ability of extracellular ligands to trigger signaling cascades has been well characterized (for review, see Hunter, 2000). The most striking effect is a shift in phospho-proteins, mediated by kinases and phosphatases able to modify the phosphorylation status of cellular proteins (Hunter, 2000). Stimulation of cells by, for example, growth factors needs to be restricted to a spatiotemporal window. To this end, cells have developed a battery of processes capable of attenuating the effect of the stimulating ligand. One of these is the endocytosis and subsequent lysosomal degradation of a ligand in complex with its receptor (for review, see Sorkin and Von Zastrow, 2002). This degradation is tightly regulated, and, interestingly, the signaling cascade triggered by the receptor is also regulating the degradation of the ligand. Thus ligands are able to affect their own intracellular trafficking (Di Fiore and De Camilli, 2001; Gonzalez-Gaitan and Stenmark, 2003; Teis and Huber, 2003; Bache et al., 2004). One of the most studied families of endocytosed receptors is the antibody receptors FcR (Daeron, 1997). These receptors do not possess any enzymatic activity, thus they need to recruit kinases to trigger a signaling cascade within the cell and also to regulate their internalization. Src family kinases (SFKs) and Syk kinase seem to be crucial partners in this process (van Oers and Weiss, 1995; Bolen and Brugge, 1997). Sequential activation of SFKs and Syk is necessary for both the uptake of the receptor into the cell (Park and Schreiber, 1995; Majeed et al., 2001; Strzelecka-Kiliszek et al., 2002) and for the trafficking to lysosomes (Bonnerot et al., 1998). Similarly, Stx in human B-cells activates at least two main tyrosine kinases, Lyn (a SFK) and Syk (Mori et al., 2000), and this activation occurs very fast. Also, in ACHN renal cells Stx activates the SFK Yes (Katagiri et al., 1999). In this paper, the authors were able to show that this tyrosine kinase is recruited to Gb3-enriched structures and occurs in the same fraction as Stx in a sucrose gradient.
In the present study, we have asked the following question: Is Stx able to regulate and increase the efficiency of its entry and intracellular transport by such signaling? We have investigated the signaling occurring upon binding of Stx to HeLa cells, a highly toxin-sensitive cell line previously found to internalize Stx at least partly through clathrin-coated pits. In this paper, we provide evidence for the involvement of a tyrosine kinase in uptake and intracellular transport of Stx. As demonstrated, the toxin induces phosphorylation of clathrin by a Syk-dependent pathway that involves the formation of a complex containing both clathrin heavy chain (CHC) and Syk. Depletion of Syk by small interfering RNA (siRNA), overexpression of a kinase dead mutant of Syk (Syk KD), or inhibition of Syk activity by addition of piceatannol or the more general tyrosine kinase inhibitor genistein, all reduced the uptake of Stx, indicating that toxin signaling is important for its entry. Furthermore, toxin-induced tyrosine phosphorylation of CHC is inhibited in cells treated with siRNA against Syk, or overexpressing Syk KD, but increased when Syk wild-type (WT) is overexpressed. Finally, we here show that Golgi transport of Stx also is reduced under all conditions were Syk activity is abolished, reflecting the fact that inhibition of signaling decreases the entry of the toxin.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-33P]ATP and [3H]leucine were from Amersham Biosciences (Little Chalfont, Buckinghamshire, United Kingdom). Rabbit anti-Stx antibody was obtained by standard immunization. Rabbit polyclonal anti-Syk antibody (N-19) and mouse monoclonal anti-Syk (4D10) were from Santa Cruz Biotechnology (Santa Cruz, CA). 4G10 mouse monoclonal anti-phosphotyrosine antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-CHC antibodies are mouse monoclonal X-22 (Abcam, Cambridge, United Kingdom) and mouse monoclonal N-terminal fragment (Research Diagnostics, Flanders, NJ). Genistein (BIOMOL Research laboratories, Plymouth Meeting, PA), piceatannol (Calbiochem, San Diego, CA), and SU6656 (Calbiochem) were resuspended in dimethyl sulfoxide (DMSO), and frozen aliquots were stored at –20°C. When used in experiments, the DMSO concentration never exceeded 0.2%. The StxB plasmid encoding the B-chain-Sulf2 was a kind gift from Dr. B. Goud (Institut Curie, Paris, France).
Cells
HeLa cells were grown under 5% CO2 in DMEM with 10% fetal calf serum (FCS). Both media were supplemented with penicillin at 100,000 U/l, streptomycin at 100,000 U/l, and L-glutamine at 2 mM.
Cloning, Mutagenesis, and Transfection
An expressed sequence tag containing the complete coding sequence of Syk (clone BC011399
[GenBank]
; Invitrogen, Carlsbad, CA) was amplified by Pfx polymerase (Invitrogen) using specific primers (5'-ATA GAA TTC GCA TGG CCA GCA GCG GCA-3' and 5'-GCT CTA GAT TAGT TCA CCA CGT CAT-3'). The amplicon was digested with EcoRI and XbaI (underlined) and cloned into a pcDNA4 vector (Invitrogen). Kinase dead mutant (Syk KD) was generated using specific primers designed to change K402 to an arginine (5'-AAA CCG TGG CTG TGA gAA TAC TGA AAA ACG-3' and 5'-CGT TTT TCA GTA TTc TCA CAG CCA CGG TTT-3') and the human Syk cDNA as a template, the position changed is shown in lowercase letters. The mutagenesis was performed using the QuikChange kit (Stratagene, La Jolla, CA) following the manufacturer's procedure. The constructs were totally sequenced.
Cells were seeded out into 5-cm dishes (2 x 105 cells/dish) or into a 24-well plate (2 x 104 cells/well) and grown overnight. Then, the cells were transfected with Syk WT or Syk KD using FuGENE-6 (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's manual. The cells were left for 24 h before they were used for experiments.
For siRNA transfection studies, cells were seeded out in the absence of penicillin and streptomycin. After 24 h, the cells were transfected with either SMARTpool Syk siRNA, containing four designed siRNAs against Syk pooled (Upstate Biotechnology) (which we have termed Syk siRNA 1), Syk siRNA 2 (5'-GGAUGCUGGUUAUGGAGAU-3'), or a nonspecific control pool (Upstate Biotechnology) by using Oligofectamine (Invitrogen) according to the procedure given by the company. Four hours after transfection, 10% FCS, penicillin at 100,000 U/l, and streptomycin at 100,000 U/l were added to the cells, and the cells were further left for 
40 h before they were used for experiments. To verify knockdown of Syk, cells were harvested for real-time reverse transcription-PCR 2 d after transfection. RNA was isolated using the TRIzol reagent (Invitrogen), and cDNA was synthesized using the iScriptcDNA synthesis kit from Bio-Rad (Hercules, CA), following the manufacturer's instructions. PCR amplifications were conducted using the iQSYBR Green Supermix (Bio-Rad). The PCR reaction was performed in an iCycler thermal cycler with an iCycler iQ real-time PCR detection system (Bio-Rad) under the following conditions: 3 min at 95°C to activate the enzyme followed by 50 cycles at 95°C denaturation for 10 s and 60°C annealing and extension for 35 s. Finally, a melt-curve analysis was added to exclude nonspecific amplification. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as an internal control in all experiments. Primer sets used in the PCR reactions were as follows: for Syk, 5'-TGGAGATGGCAGAACTTGGT-3' and 5'-TTCTTATCCTTGACATGTCTGT-3' and for GAPDH, 5'-GGAAGGTGAAGGTCGGAGTC-3' and 5'-GGAAGATGGTGATGG GATTTC-3'. The amount of Syk mRNA was quantified to be almost completely removed (with 80–90%) in our experiments after 2 d of transfection with 200 nM Syk siRNA 1. Syk siRNA 2 was slightly less efficient; the same time and concentration reduced the Syk mRNA level by 70–80%.
Immunoprecipitation and Immunoblotting
Clathrin was immunoprecipitated from the sonicated 2-(N-morpholino)ethanesulfonic acid (MES)-buffer cell lysates by using the mouse monoclonal clathrin heavy chain antibody (X22) (Abcam) prebound to protein G-Sepharose beads (overnight; 4°C). The immunoprecipitates was washed (3 times) with Tris-buffered saline (20 mM Tris-Cl, pH 7.6, and 0.5 M NaCl) and solubilized in SDS-PAGE sample buffer. The immunoprecipitated clathrin was run on a 7.5% SDS-PAGE, and the proteins were blotted onto a polyvinylidene diflouride (PVDF) membrane. The presence of phosphorylated clathrin was detected using an anti-phosphotyrosine horseradish peroxidase-conjugate (BD Transduction Laboratories, Lexington, KY) and the enhanced signal chemiluminescence reagent (Pierce Chemical, Rockford, IL).
Tyrosine-phosphorylated clathrin was immunoprecipitated by using the slurry of an anti-phosphotyrosine column (Upstate Biotechnology) in batch (Walchli et al., 2004). Briefly, 40 µl of the slurry was incubated with 106 cells previously lysed and sonicated in MES buffer (0.1 M MES, pH 6.5, 0.5 mM MgCl2, 1 mM EGTA, 1 mM orthovanadate, and a Complete protease inhibitor cocktail [Roche Diagnostics]) overnight at 4°C. After several washes of the slurry with MES buffer, the tyrosine-phosphorylated proteins were eluted with 65 µl of MES buffer containing phenylphosphate. Then, 20 µl of this eluate was run on a 7% SDS-PAGE. After transfer of proteins onto a PVDF membrane, the presence of clathrin was detected by using a mouse monoclonal antibody (Research Diagnostics).
Syk detection was performed as follows. After transfection, cells were grown in complete medium for 24 h. They were then starved in HEPES medium for 4 h and treated with Stx for the indicated time. If inhibitors were used, the cells were preincubated with these compounds for 30 min before the addition of the toxin. After the incubation times indicated, lysates were prepared in NP-40.buffer (50 mM Tris, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, and a tablet of Complete protease inhibitor cocktail [Roche Diagnostics]). Then, the lysates were passed trough a 21-gauge syringe, centrifuged for 5 min at 5000 rpm in an Eppendorf centrifuge, and sonicated three times for 10 s. The cleared supernatant was used for immunoprecipitation (IP) with rabbit polyclonal anti-Syk antibody (N-19) (Santa Cruz Biotechnology) precoated on protein A/G beads overnight at 4°C. The IP was then washed twice in phosphate-buffered saline (PBS)/NP-40 lysis buffer (1:1), and the adsorbed material was analyzed by 7.5% SDS-PAGE under reducing conditions and transferred onto a PVDF membrane. The presence of Syk kinase was detected using mouse anti-Syk antibody (4D10) (Santa Cruz Biotechnology), and the phosphorylation state of Syk was analyzed using a mouse anti-phosphotyrosine antibody. Coimmunoprecipitation (CoIP) of clathrin was analyzed using a mouse anti-CHC antibody. Signal intensities of the bands were quantified using the ImageQuant 5.0 software (Amersham Biosciences).
Preparation of STxB-Sulf2
The modified version of Shiga toxin B-chain containing a tandem of sulfation sites (STxB-Sulf2) was produced in Escherichia coli BL21 (DE3) cells essentially as described previously (Su et al., 1992). Briefly, a 10-ml overnight bacterial culture grown at 37°C was inoculated in 500 ml of LB medium and grown further to an OD600 of 0.6. The culture was heat induced for 4 h at 42°C, and the cells were harvested by centrifugation. The pellet was washed twice with 10 mM Tris-HCl, pH 8; resuspended in 25% sucrose, 1 mM Na2EDTA, and 10 mM Tris-HCl, pH 8.0; and gently shaken at 30°C for 10 min. Cells were harvested by centrifugation and resuspended in ice-cold distilled water. After centrifugation, the supernatant was dialyzed overnight against 20 mM Tris-HCl, pH 7.5, loaded on a Resource Q column (Amersham Biosciences), and eluted with a 0–600 mM NaCl gradient in 20 mM Tris-HCl, pH 7.5.
Sulfation of STxB-Sulf2
HeLa cells were washed twice with sulfate-free medium (MEM 12–126; Cambrex Bio Science, Verviers, Belgium) before incubation with 0.2 mCi/ml Na235SO4 for 3 h at 37°C in the same medium. When indicated, different inhibitors were added to the medium during the last 30 min of this incubation. Then STxB-Sulf2 was added to the medium, and the incubation was continued for 1 h. The cells were then washed twice with cold PBS before they were lysed (0.1 M NaCl, 10 mM Na2HPO4, pH 7.4, 1 mM EDTA, 1% Triton X-100, and 60 mM n-octyl-glucopyranoside, supplemented with a mixture of Complete protease inhibitors [Roche Diagnsotics]). The lysate was centrifuged for 10 min at 5000 rpm in an Eppendorf centrifuge, and the cleared lysate was immunoprecipitated with rabbit anti-Stx antibody immobilized on protein A-Sepharose beads (Amersham Biosciences) overnight at 4°C. The beads were then washed twice with PBS containing 0.35% Triton X-100, and the adsorbed material was analyzed by 12% SDS-PAGE under reducing conditions and transferred onto a PVDF membrane. The membrane was then exposed to a PhosphoImager screen (Amersham Biosciences), and signal intensities of the bands were quantified using the ImageQuant 5.0 software.
Preparation of TAG- and Biotin-labeled Stx and Transferrin
Stx and transferrin were labeled with N-hydroxysuccinimide ester-activated Tris(bipyridine) chelated ruthenium(II) TAG (BioVeris, Gaithersburg, MD) according to the manufacturer's procedure and simultaneously biotinylated with the reducible ImmunoPure NHS-SS-Biotin (Pierce Chemical).
Endocytosis Assays
Endocytosis of Stx and transferrin was measured using a BioVeris detection system, which uses electrochemiluminescence detection, as described previously (Lauvrak et al., 2004). Briefly, the cells were washed with HEPES medium and incubated with TAG- and biotin-labeled Stx (25 ng/ml) in HEPES medium containing 2 mg/ml bovine serum albumin (BSA) for 20 min at 37°C or with TAG- and biotin-labeled transferrin (50 ng/ml) for 5 min at 37°C. When indicated, the cells were preincubated with different inhibitors for 30 min before incubation with Stx. The cells were subsequently washed twice with cold buffer (0.14 M NaCl, 2 mM CaCl2, and 20 mM HEPES, pH 8.6). Then, to remove the SS-linked biotin on cell surface-bound protein, half of the cells were treated with 0.1 M MESNa in the same buffer but supplemented with 2 mg/ml BSA for 20 min at 0°C. The other half was incubated with BSA-containing buffer alone. Subsequently, the cells were washed twice in cold buffer (0.14 M NaCl, 2 mM CaCl2, and 20 mM HEPES, pH 7.4) before they were lysed (0.1 M NaCl, 5 mM MgCl2, 50 mM HEPES, pH 7.0, 1% Triton X-100, and 60 mM n-octyl-glucopyranoside). The lysates were centrifuged for 5 min at 14,000 rpm in an Eppendorf centrifuge, and the amount of TAG- and biotin-labeled Stx or transferrin in the cleared lysates were measured using streptavidin beads (Dynal Biotech, Oslo, Norway) and the BioVeris detection system instrument. In the cell lysate, only protein that is TAG-labeled and still biotinylated is detected via binding to the streptavidin beads; thus, MESNa-treated cells represent the amount of endocytosed protein, whereas untreated cells represent the total amount of protein associated with the cells. Endocytosis of iodinated ricin and transferrin was monitored as in Rodal et al. (1999).
Immunofluorescence
Cells grown on coverslips were transfected with Syk WT or Syk KD 24 h before the experiment. After two washes with HEPES medium, the cells were incubated with Shiga toxin (250 ng/ml) for 45 min at 37°C. The cells were then wash twice with HEPES medium and incubated at 37°C for the indicated time. The cells were subsequently fixed with 3% paraformaldehyde in PBS, permeabilized with 0.2% Triton X-100 for 7 min, and blocked with 5% FCS. Transfected cells were identified using rabbit anti-Syk antibodies (Santa Cruz Biotechnology) followed by Cy3-labeled donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA), and Stx was revealed using the mouse anti-Stx (VT1; Toxin Technology, Sarasota, FL) and a Cy2-labeled donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories). The fixed cells were analyzed using a LSM 510 Meta confocal microscope (Carl Zeiss, Jena, Germany). Pictures were taken of thin single plane sections.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) and during internalization of the B-cell receptor (BCR) from clathrin-coated pits (Stoddart et al., 2002). Similarly to EGF and the BCR, Stx seems to be internalized from clathrin-coated pits in HeLa cells by a toxin-induced mechanism (Sandvig et al., 1989). We therefore investigated whether Stx, although it binds a glycolipid receptor, can affect clathrin phosphorylation. It has previously been shown that Stx is able to induce a stimulation of tyrosine phosphorylation that peaks after 5 min in lymphocytes (Foster et al., 2000; Mori et al., 2000). Similarly, we found that also in HeLa cells Stx induced tyrosine phosphorylation of a variety of proteins even after 5 min of incubation (our unpublished data). To investigate whether clathrin was one of these phosphorylated proteins, the pool of tyrosine-phosphorylated proteins was immunoprecipitated by using anti-phosphotyrosine (anti-PY) antibodies (see Materials and Methods), and CHC was visualized by Western blot analysis. As shown in Figure 1A (left), CHC rapidly gets phosphorylated upon Stx binding. The amount of clathrin in the whole cell lysates (WCLs) was the same. The Stx-induced CHC phosphorylation was dependent on the amount of toxin added; it increased upon incubation with increasing amounts of Stx (Figure 1A, right). It should be noted that even as little as 10 ng/ml Stx gave a strong increase in CHC phosphorylation. To confirm this Stx-dependent clathrin phosphorylation, we also performed IP of clathrin and then revealed the presence of phosphorylated CHC with anti-PY antibodies (Figure 1B). An increase in phosphorylated clathrin after stimulation with Stx (Figure 1B, left) as well as the B-subunit alone (Figure 1B, right) was also observed by this approach. The figure further shows that CHC is constitutively phosphorylated in HeLa cells. Quantification of the amount of phosphorylated clathrin from 8 experiments (n = 1–5 for the different time points) revealed that there was an increase already after 5 min and that the peak was seen after 15 min with an increase to 170% for both Stx and StxB (Figure 1B, graph). From these data, we can conclude that Stx activates a signaling cascade involving tyrosine kinases that increase the phosphorylation status of CHC.
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), we decided to investigate whether Syk is activated during Stx entry into HeLa cells. It is known that Syk is present at a low concentration in HeLa cells (Renedo et al., 2001; our unpublished data) as well as in many other nonlymphoid cell lines (Yanagi et al., 2001). To facilitate our studies, we therefore overexpressed human Syk in HeLa cells and monitored toxin-induced tyrosine phosphorylation of Syk after immunoprecipitation of the kinase. Indeed, Syk autophosphorylation is accepted as a hallmark of its activation state (Rowley et al., 1995). As shown in Figure 2A, Stx holotoxin (as well as the purified B-chain; Figure 4) triggers an early signaling reflected by fast phosphorylation of Syk. Quantification revealed that the amount of phosphorylated Syk was increased to 180% already after 2.5 min, and after 7.5 min the amount was doubled compared with nonstimulated cells (Figure 2A, graph). Furthermore, as little as 10 ng/ml toxin is sufficient to activate the kinase (Figure 2B). However, a higher toxin concentration (250 ng/ml) gave a stronger signal, and in this situation there was a more than twofold increase in the amount of phosphorylated Syk (Figure 2B, graph). Importantly, even at the highest concentration of Stx, the Syk inhibitor piceatannol inhibited Syk phosphorylation (Figure 2B), demonstrating and confirming that the signal observed on anti-PY Western blot is due to a proper Syk kinase activity. Parallel experiments with Syk KD demonstrated that this mutant did not become phosphorylated (Figure 2C), confirming that Stx triggers a Syk activation resulting in its autophosphorylation.
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Complex Formation between Syk and Clathrin
The Syk kinase has two tandem SH2 domains, a linker domain, and at its C terminus, a catalytic domain. The SH2 domains of Syk are involved in binding to tyrosine-phosphorylated receptors via the so-called immunoreceptor tyrosine-based activation motif (ITAM), and this binding leads to tyrosine kinase activation of Syk (van Oers and Weiss, 1995). Even in HeLa cells that were not incubated with Stx, there is a low level of CHC tyrosine phosphorylation (Figure 1). We decided to investigate whether Syk is able to form a complex with clathrin. By transfecting HeLa cells with Syk wild type, we were able to demonstrate that a stable complex of clathrin and Syk was already present in cells that had not been incubated with Stx but that after addition of Stx or the B-subunit, there seems to be more clathrin present in a complex with Syk (Figure 4). Control experiments on untransfected cells showed that the Syk antibody alone was not able to coimmunoprecipitate CHC (our unpublished data). However, future studies are required to investigate this complex.
Importance of Syk for Endocytosis of Stx
To investigate whether the Stx-induced signaling described above is important for the uptake of the toxin, we investigated the effect of depleting Syk by siRNA on Stx endocytosis. Stx is a highly stable protein, and very little toxin is degraded. Also recycling of Stx plays a minor role during the incubation times used (our unpublished data). As shown in Figure 5A, depletion of Syk by siRNA 1 resulted in a strong inhibition of Stx endocytosis; the effect was greatest at early time points (40% reduction after 5 and 10 min compared with 20% after 20 min). Quantification of the degree of endocytosis (as percentage of total cell-associated toxin) from two independent experiments (each done in triplicate) after Stx endocytosis for 10 min is also shown (Figure 5A, columns). Also, by using Syk siRNA 2, we were able to observe a decrease in Stx endocytosis, although the effect was slightly less than for Syk siRNA 1 (30% reduction after 10 min) (Figure 5A, columns). This might be explained by a slightly lower reduction in Syk mRNA level after transfection with Syk siRNA 2 (70–80%) compared with Syk siRNA 1 (80–90%) (see Materials & Methods), which is not surprising because Syk siRNA 1 is a mixture of four different siRNA oligos against Syk. To strengthen the suggestion that Syk is implicated in Stx endocytosis, we also analyzed the effect of the general tyrosine inhibitor genistein as well as the Syk inhibitor piceatannol, on the toxin uptake. As shown in Figure 5B, both genistein (25 µg/ml) and piceatannol (50 µM) reduced toxin entry during a 20-min period (with 40 and 30%, respectively), supporting the idea that Syk is indeed important for Stx endocytosis. Lower concentrations of piceatannol had a smaller inhibitory effect. As expected, the combination of piceatannol and genistein did not give any further reduction than genistein alone (our unpublished data). We also examined Stx endocytosis after shorter times, and incubation with Stx for 10 min after treatment with genistein or piceatannol gave a similar inhibition (40 and 20%, respectively) (our unpublished data). In contrast, SU6656, a highly specific SFK inhibitor, had essentially no effect on Stx uptake (10%, even at high concentration) (Figure 5B). Also, PP1 and PP2, two other SFK inhibitors, had no effect on the toxin uptake (our unpublished data). Thus, in contrast to the results reported for BCR (Stoddart et al., 2002), T-cell receptor (TCR) (Crotzer et al., 2004) and EGF receptor (EGFR) (Wilde et al., 1999), Src or a related kinase does not seem to be involved in regulation of Stx endocytosis. The fact that endocytosis of Stx can be reduced by piceatannol to almost the same extent as observed in the presence of genistein suggests that the main tyrosine kinase involved in Stx uptake in HeLa cells is Syk. Furthermore, overexpression of Syk KD inhibited Stx endocytosis by 25% compared with transfection with the vector alone (Figure 5C), which is comparable with the reduction observed in cells treated with piceatannol because the estimated transfection efficiency was 60–70% in these experiments. Interestingly, overexpression of Syk WT did not increase the uptake of Stx (Figure 5C). This suggests that the amount of endogenous Syk is sufficient to give optimal activity on endocytosis. To further confirm these biochemical data, we have also monitored Stx entry after transfection with Syk (WT or KD) by using confocal microscopy. In this assay, we observed that 70% of the cells transfected with Syk KD showed a decrease in Stx entry after 5 min of incubation with the toxin compared with untransfected cells (our unpublished data). In cells overexpressing Syk WT, we could not observe any significant changes.
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We then tested whether Stx only affects its own endocytosis or whether uptake of other ligands also was changed. To this end, we have measured the uptake of transferrin, which enters via a constitutive clathrin-dependent mechanism, and ricin, which is taken in via both clathrin-dependent and -independent mechanisms. However, neither the uptake of transferrin nor ricin endocytosis was affected by piceatannol or genistein treatment (Figure 5D). We have also performed endocytosis experiments with cells transfected with Syk (WT or KD), and again neither ricin nor transferrin was affected by the kinase (our unpublished data). These data clearly indicate that this Syk-regulated uptake is specific for Stx.
To confirm that the Syk-regulated uptake of Stx occurred via a clathrin-dependent mechanism, we have investigated the effect of genistein on Stx endocytosis in our antisense CHC cells (Iversen et al., 2003). As shown in Figure 6A, genistein inhibits the uptake of Stx only when CHC is expressed (+tet), but in the absence of functional CHC (–tet) genistein had essentially no effect (<10%). To verify that the clathrin-dependent endocytosis was blocked in the absence of tetracycline in our experiments, transferrin endocytosis was analyzed in parallel (Figure 6B). From these data, we can conclude that because a general tyrosine kinase inhibitor is able to selectively affect Stx uptake when functional clathrin is present, CHC-dependent uptake is the only tyrosine kinase-regulated route for Stx. This is also in agreement with the 40% reduction in Stx endocytosis that we observe in Syk siRNA 1 transfected cells—the other 60% most likely representing clathrin-independent endocytosis.
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Involvement of Tyrosine Kinases in the Transport of Stx to the Golgi Apparatus
To study the involvement of tyrosine kinases in intracellular transport of Stx, we have used a genetically modified Stx B-chain that can be sulfated (Johannes et al., 1997), a modification that occurs only in the trans-Golgi network (TGN). By using radioactive sulfate, one can quantify transport of StxB to the TGN. As shown in Figure 7A, both the general tyrosine kinase inhibitor genistein and the Syk inhibitor piceatannol reduced the sulfation of StxB. A quantification of bands from three independent experiments (Figure 7A, graph) revealed that Stx B-chain sulfation was reduced to 30% of the control level by genistein, whereas piceatannol reduced sulfation with 50%. Importantly, we have also examined the effect of StxB sulfation in cells depleted for Syk by siRNA (Figure 7B). Quantification from two independent experiments revealed a 45% decrease in Stx transport to the Golgi apparatus in Syk-depleted cells compared with cells transfected with nonspecific siRNA (Figure 7B, graph), which fits well with the reduction observed with piceatannol. This reduction is also comparable with the one observed in the endocytosis measurements, suggesting that the decreased transport to the TGN is mainly because of reduced toxin endocytosis. Because genistein has a stronger inhibitory effect on StxB sulfation than piceatannol, other tyrosine kinases than Syk might regulate endosome to TGN transport of StxB. The sulfation data support the view that the measurements of toxin endocytosis actually reflect toxin on its way to the Golgi apparatus, and they support the notion that Syk is important for endocytosis.
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Notably, the conclusion that Syk is important for endocytosis and therefore also for Golgi transport also can be demonstrated by confocal microscopy. Cells overexpressing the mutant kinase Syk KD showed a clear reduction in Stx content in the Golgi area as well as in vesicles compared with untransfected cells (Figure 8). We could not observe any difference in cells transfected with Syk WT compared with untransfected cells (our unpublished data), supporting the proposition that overexpression of Syk does not affect the toxin uptake. A reduced transport to the Golgi apparatus was also observed in cells transfected with Syk siRNA 1 compared with cells transfected with nonspecific siRNA, and quantification from two independent experiments revealed that depletion of Syk reduced the colocalization between Stx and TGN with 62% (our unpublished data).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Furthermore, the fact that Syk inhibition gives the same reduction in sulfation of StxB as the inhibition seen in endocytosis suggests that Syk-induced phosphorylation of clathrin might be important for endocytosis of Stx only, and not also for endosome-to-Golgi transport of Stx, although this process also is dependent on clathrin in toxin-sensitive cells (Lauvrak et al., 2004; Saint-Pol et al., 2004). The results also suggest that clathrin-dependent uptake of Stx is important for transport to the Golgi apparatus. There seems to be several pathways between endosomes and the Golgi apparatus, and in the case of Stx the toxin can at high concentrations reach the Golgi apparatus even in the absence of functional clathrin (Lauvrak et al., 2004; Saint-Pol et al., 2004). Toxin transport to the ER could in principle occur without the toxin passing the TGN; however, we do not have any evidence for such a pathway so far when it comes to Stx transport.
Tyrosine phosphorylation of CHC has been reported also under several other circumstances. Tyrosine phosphorylation of CHC occurs upon binding of EGF (Wilde et al., 1999) and nerve growth factor (NGF) (Beattie et al., 2000), during BCR internalization (Stoddart et al., 2002), TCR signaling, and internalization (Crotzer et al., 2004) and during oxidative stress (Ihara et al., 2002), but in these cases phosphorylation is caused by Src kinases, because inhibitors of this group of enzymes were shown to interfere with this process. However, in a recent article by Sorkina et al. (2002), it was reported that although the commonly used Src inhibitor PP2 inhibited EGF uptake, a similar inhibition was not observed with a more specific inhibitor of Src (SU6656), raising the question of whether another kinase was involved in EGF-induced endocytosis. Interestingly, similarly to our data, a basal level of clathrin tyrosine phosphorylation was observed also in Daudi and Ramos cells (Stoddart et al., 2002) and in chicken embryo fibroblasts transformed by Rous Sarcoma virus (Martin-Perez et al., 1989). In vitro assays performed on tyrosine kinases have shown that Syk and Src have different amino acid requirements for substrate specificity (Bewarder et al., 1996; Schmitz et al., 1996; Ruzza et al., 2003). It is therefore likely that Syk induces phosphorylation of different tyrosine residue(s) than Src in clathrin. Indeed, we are currently working on the binding of Syk to CHC, and we have obtained convincing evidence that the phosphorylation site(s) of Syk on CHC is not the same as the one used by Src kinase (our unpublished data). To which extent this has different effects on clathrin and its interacting partner proteins is not known, but it might very well be the case. After H2O2-induced oxidative stress, there is a redistribution of clathrin from the membrane to the cytosol, leading to a reduction of transferrin endocytosis. In contrast, NGF binding increased the amount of clathrin at the cell surface and the uptake of transferrin, and similarly, EGF increased the number of clathrin-coated pits at the cell surface (Wilde et al., 1999). In our studies, we could not measure any difference in transferrin uptake after addition of Stx (our unpublished data), suggesting that the cellular events differ. If clathrin is recruited to the site of Stx binding, then phosphorylated and incorporated into a clathrin-coated pit, these pits do not necessarily contain transferrin receptors to the same extent as those already present, and one might therefore not see an increase in transferrin uptake. In agreement with such an idea are the following results from other laboratories: Cbl mutants affected EGF internalization without inhibiting transferrin uptake (Jiang and Sorkin, 2003) and a dominant negative Eps15 mutant inhibited EGFR endocytosis by retaining the receptors in clathrin-coated pits without affecting endocytosis of transferrin receptors (Confalonieri et al., 2000). Another possibility is that clathrin-coated pits with Stx also contain transferrin, but that these pits just lead to a dilution of transferrin in all clathrin-coated pits present at the membrane. Importantly, as suggested for the BCR receptor (Stoddart et al., 2002), clathrin phosphorylation might be a negative signal for clathrin-coated pit formation. It might actually delay the process, providing the ligand to be internalized with more time to locate to clathrin-coated domains. In our study, we have shown that Stx binding is followed by CHC phosphorylation. However, the increase in phosphorylation caused by the overexpression of Syk does not improve the toxin uptake. Thus, the level of the endogenous enzyme seems to be sufficient to facilitate maximum uptake of Stx. It should be noted that although genistein selectively may inhibit pinching off of caveolae and not clathrin coated pits with prelocalized transferrin receptors (for review, see Marks and Pagano, 2002), inhibition of tyrosine phosphorylation might still inhibit relocalization of a given receptor to clathrin-coated pits. This has been shown to be the case for the EGF receptor (Lamaze et al., 1993) and seems to be the case also for cholera toxin, which can enter from clathrin-coated pits in some cell types (Torgersen et al., 2001).
An important question without any answer so far is: How is Stx able to activate Syk? It is interesting in this connection that also antibodies to Gb3 are reported to cause signaling in lymphocytes, but this signaling is presumably of a different type than what occurs after addition of toxin (Tetaud et al., 2003). Thus, cross-linking of Gb3 may have different consequences for the cell, depending on what is bound. Therefore, it is tempting to suggest that the glycosphingolipid receptor for Stx, Gb3, and the toxin itself or the binding moiety of the toxin (the B-chains) interact with a transmembrane structure able to mediate a signal to the cytosolic side of the membrane. It has been reported that the toxin can interact with other proteins at the cell surface (Shimizu et al., 2003), and the possibility exists that these might be important for signaling. Also, aggregation of Gb3 caused by binding of the pentameric B-moiety, perhaps in lipid rafts, might play a role. In the case of BCR, the binding of antigen to the receptor is associated with transfer of BCR to lipid rafts and then there is an induction of clathrin phosphorylation (Stoddart et al., 2002). Interestingly, there is a larger fraction of clathrin that is phosphorylated in rafts in these cells compared with the rest of the membrane. Similarly, it has been shown that Stx which is internalized in toxin-sensitive cells is associated with lipid rafts, and it is known that receptor internalization and transport to the Golgi apparatus are dependent on the length of the fatty acid (Sandvig et al., 1994; Lingwood, 1999). It is possible that this part of the receptor is important for the signaling described in the present article. Whether the activity of Syk in the case of Stx internalization is dependent on raft structures and the type of lipids that make up Gb3 in different cells would be a question for future studies.
Stx is not unique when it, as a ligand, uses Syk activation as a strategy to facilitate entry. Similarly, antibody receptors (FcR) in macrophages, although they do not have enzymatic activity, are able to activate Syk after cross-linking by antibodies. On antibody binding, an SFK first phosphorylates the receptor on ITAM, and then these phosphorylated motifs are recognized by the SH2 domains of Syk. The binding of Syk to these motifs leads to activation of Syk and signaling. However, in contrast to Stx entry, the FcR and the antibody is then internalized via phagocytosis. The different internalization strategies requiring Syk might be a consequence of the scaffolding proteins involved. Thus, signaling can induce uptake by different endocytic pathways. It should be noted that Syk can be activated by different mechanisms. Interestingly, Syk can be activated through interaction with integrin 
cytoplasmic domains by a mechanism that is independent of phosphorylation (Woodside et al., 2001, 2002).
As shown in the present article, Stx entry seems to be regulated by stimulation of a signaling cascade. Endocytosis of the toxin seems to be the first step triggered by Syk tyrosine kinase, whereas other tyrosine kinases might be involved in the regulation of later steps of the toxin journey. Importantly, as shown here, the toxin is an active player in its transport.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: Stx, Shiga toxin; TGN, trans-Golgi network.
These authors contributed equally to this work. 
Address correspondence to: Kirsten Sandvig (kirsten.sandvig{at}labmed.uio.no).
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Together, these p values indicate that the increase in CHC phosphorylation is significant.
