The Small GTP-binding Protein Rac Promotes the Dissociation of Gelsolin from Actin Filaments in Neutrophils*

Alexandre ArcaroDagger

From the Institute of Biochemistry, University of Fribourg, CH-1700 Fribourg, Switzerland

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Gelsolin is an actin filament-capping protein that has been shown to play a key role in cell migration. Here we have studied the involvement of phosphoinositide 3-kinase (PI 3-kinase) and GTP-binding proteins (G-proteins) in the regulation of gelsolin-actin interactions in neutrophils. Inhibition of PI 3-kinase activity in vivo by wortmannin did not affect the dissociation of actin-gelsolin (1:1) complexes induced by neutrophil stimulation with N-formyl-Met-Leu-Phe. Guanosine 5'-[gamma -thio]triphosphate (GTPgamma S) indirectly promoted the dissociation of actin-gelsolin complexes in a cell-free system using neutrophil cytosol, and this effect was blocked by the GDP dissociation inhibitor for Rho (Rho-GDI). The GTPgamma S-loaded ialpha 2 and the beta 1gamma 2 subunits of heterotrimeric G-proteins (Gialpha 2 and Gbeta 1gamma 2) also triggered actin-gelsolin dissociation in a Rho-GDI-sensitive manner. GTP-loaded activated Rac, but not activated Rho, induced the dissociation of cytosolic actin-gelsolin complexes. The guanine nucleotide exchange on Rac was increased by addition of GTPgamma S-loaded Gialpha 2 or Gbeta 1gamma 2 to neutrophil cytosol. These findings suggest that activation of Rac by G-protein-coupled receptors in neutrophils triggers uncapping of actin filaments, independently of PI 3-kinase.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Gelsolin is a Ca2+- and polyphosphoinositide-regulated actin-binding protein involved in the signaling cascade activated by chemotactic agonists such as fMLP1 that controls actin polymerization in neutrophils (1, 2), a response thought to be essential for cell motility (3-5). In vitro and in the presence of Ca2+, gelsolin rapidly disassembles actin filaments by noncovalent severing and remains attached to their barbed ends (6, 7), a function that can be inhibited by the phosphoinositides PtdIns(4)P and PtdIns(4,5)P2 (8-10). This results in actin oligomers blocked at their fast growing barbed ends. EGTA causes the dissociation of one of the two actin monomers bound to gelsolin, generating an EGTA-resistant actin-gelsolin (1:1) complex (6, 7, 11), which can, in vitro, be dissociated by anionic phospholipids (PtdIns(4,5)P2 and PtdIns(4)P) (10). EGTA-resistant actin-gelsolin (1:1) complexes have been reported to be present in various cell extracts (12, 13), including neutrophils, and the levels of these complexes decrease in response to cell stimulation by fMLP (14). This response parallels an increase in the nucleating activity at the barbed ends of actin filaments (14, 15) and an increase in the cellular F-actin content, suggesting that reversal of gelsolin binding to actin filaments barbed ends can initiate actin polymerization in response to chemoattractants.

Dissociation of actin-gelsolin (1:1) complexes in neutrophils has been proposed to occur independently of the increase in cytosolic Ca2+ that is triggered by fMLP (14) and to involve phosphoinositide resynthesis, leading to accumulation of PtdIns(4)P and PtdIns(4,5)P2 (2). Actin polymerization and actin-gelsolin dissociation are, however, maximal at a time (10-15 s after fMLP stimulation) when PtdIns(4,5)P2 levels drop (16, 17), implying that PtdIns(4,5)P2 synthesis does not correlate with these responses in vivo (18, 19).

PtdIns(3,4,5)P3 has thus been proposed to control actin-gelsolin dissociation (19-22) because, in contrast to PtdIns(4,5)P2, the cellular levels of this phospholipid rise upon fMLP stimulation, with a time course similar to both actin polymerization and actin-gelsolin dissociation. Moreover, PtdIns(3,4,5)P3 production is Ca2+-independent (19, 22). PtdIns(3,4,5)P3 is produced from PtdIns(4,5)P2 by the action of PI 3-kinase (17) and has been suggested to be a novel second messenger mediating cytoskeletal rearrangements and respiratory burst activation in response to chemoattractants (19-24).

There is now compelling evidence that cell surface receptor-activated actin rearrangements are controlled by small G-proteins of the Rho family in various motile cells. In fibroblasts, platelet-derived growth factor has been shown to control the formation of lamellipodia by activating Rac (25), whereas the assembly of stress fibers and focal contacts was mediated by Rho (26). In neutrophils, ADP-ribosylation of Rho by the C3 exoenzyme impaired chemotaxis (27), whereas in HL-60 cells inactivation of Rho by Clostridium limosum toxin inhibited basal but not fMLP-stimulated actin polymerization (28). Studies in permeabilized neutrophils showed that activation of G-proteins with GTPgamma S could trigger actin polymerization (29) by increasing the number of free barbed ends (30). The GTPgamma S-induced actin polymerization was not dependent on the presence of ATP, suggesting that this response is not dependent on protein or lipid phosphorylations (29). A recent report showed that GTPgamma S also induced actin polymerization in a cell-free system by activating Cdc42 and that this response did not correlate with PtdIns(4,5)P2 synthesis (31).

Here we assess the importance of PtdIns(3,4,5)P3 production in the fMLP-stimulated actin-gelsolin dissociation in neutrophils. We also provide evidence for the existence of a phosphoinositide-independent signaling pathway involving Rac that can trigger actin-gelsolin dissociation in neutrophil cytosol.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- BAPTA-AM, Pluronic F-127, N-(1-pyrene)iodoacetamide, and fluorescein-phalloidin were from Molecular Probes; fMLP, PtdIns(4,5)P2, PtdIns(4)P, and PtdIns were from Sigma; [alpha -32P]GTP (3000 Ci/mmol) was from Hartmann Analytic; wortmannin was a generous gift of T. G. Payne, Sandoz, Basel; 2C4 hybridoma producing anti-gelsolin monoclonal antibody was donated by C. Chaponnier, Geneva; PtdIns(3,4,5)P3 (di-palmitoyl) was from R. Gigg, London. The pGEX2T plasmid constructs for the expression of Rho-GDI, Gly-12 Rac1, Val-12 Rac1, Val-14 RhoA, Val-12 Ala-35 Rac1 were the gift of A. J. Ridley and A. Hall, London. C3 transferase was donated by L. A. Feig, Boston. The pQE6 plasmids for the expression of Gialpha 2, the pBB131 plasmid for the expression of N-myristoyltransferase, and the baculoviruses for the expression of His6-Gialpha 1, Gbeta 1, and Ggamma 2 were the gifts of T. Kozasa and A. G. Gilman, Dallas. The antiserum AS 269/1 against Gialpha 2 was the gift of K. Spicher, Berlin. A PtdIns(4,5)P2-binding peptide (32) was a gift of D. J. Kwiatkowski, Harvard University. An activated version of p65PAK was a gift of A. Abo, Onyx Pharmaceuticals. Other reagents were obtained from sources described in Ref. 24.

Recombinant Protein Production-- Myristoylated recombinant Gialpha 2 was prepared as described (33, 34). The fractions of each purification step were assayed for Gialpha 2 by Western blotting using the specific rabbit antiserum AS 269/1. The pooled active fractions of the Bio-Gel hydroxylapatite were dialyzed against HED (33, 34), concentrated to 1 mg/ml, and stored at -80 °C in the presence of 50 µM GDP. For GTPgamma S loading, recombinant Gialpha 2 was incubated with 1 mM GTPgamma S in the presence of MgCl2 20 mM MgCl2 for 1 h at 37 °C.

Recombinant Gbeta 1gamma 2 was prepared as described (35). Purified recombinant Gbeta 1gamma 2 was extensively dialyzed against buffer D (35), reconcentrated, and stored at -80 °C.

Rho-GDI, Val-12 Rac1, Val-12 Ala-35 Rac1, Val-14 RhoA, and C3 transferase were expressed in Escherichia coli as GST fusion proteins and subsequently purified on glutathione-Sepharose CL-4B beads (25, 26). The recombinant G-proteins were cleaved from the GST by thrombin digestion, treated with p-aminobenzamidine-agarose, and stored at -80 °C. Alternatively, GST-Rac1 was eluted from the glutathione beads by competition with free glutathione (36), dialyzed against 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM MgCl2, 1 mM dithiothreitol and stored at -80 °C. GDP- or GTP-bound forms of the small G-proteins were prepared by incubation with 10 mM EDTA at room temperature for 10 min in the presence of the respective guanine nucleotide. 20 mM MgCl2 was then added, and the samples were placed on ice.

The purity and concentration of recombinant proteins were determined by SDS-PAGE and Coomassie Blue staining. Moreover, the biological activity of Rac1 and of Rho-GDI was tested in a cell-free NADPH-oxidase assay (data not shown).

Dissociation of Pure Actin-Gelsolin Complexes by Polyphosphoinositides in Vitro-- This assay was essentially performed as in Ref. 8. Rabbit skeletal muscle ATP-actin was purified as described (37). ADP-actin was prepared as described (38). Plasma gelsolin was purified from bovine plasma as described (39). Gelsolin (0.375 µM) was mixed with freshly gel-filtered (Sephadex G-150 column (37)) monomeric ATP-actin (0.1875 µM) in buffer A (8) and PtdIns(3,4,5)P3, PtdIns(4,5)P2, PtdIns(4)P, or PtdIns sonicated in buffer A at various concentrations for 5 min at 25 °C. EGTA-resistant actin-gelsolin (1:1) complexes were immunoprecipitated using purified anti-gelsolin monoclonal antibody (clone 2C4) coupled to CNBr-activated Sepharose CL-4B. The immunoprecipitates were washed once with buffer A and analyzed by SDS-PAGE. After Coomassie Blue staining, the gels were scanned on a Bio-Rad GS-670 flatbed imaging densitometer. The molar ratios of gelsolin and actin were calculated as in Ref. 14, assuming that the actin/gelsolin molar ratio is equal to the ratio of the measured absorbances of the 93-kDa (plasma gelsolin) and 42-kDa (actin) protein bands multiplied by 2.21 (see Ref. 14).

Assay of the Severing Activity of Gelsolin in Vitro-- This assay was performed exactly as described (8). Purified F-actin was coupled to N-(1-pyrene)iodoacetamide (40-42). Bovine plasma gelsolin was diluted to 25 nM in buffer B (8) and incubated for 5 min at 25 °C with PtdIns(3,4,5)P3, PtdIns(4,5)P2, PtdIns(4)P, or PtdIns sonicated in water at various concentrations. Pyrene-labeled F-actin was then added to a final concentration of 200 nM, and fluorescence at 407 nm (excitation 370 nm) was recorded on a Perkin-Elmer Luminescence Spectrometer LS 50 B. The slope of the fluorescence decrease between 0 and 40 s was used to calculate the relative severing activity of gelsolin.

Neutrophil Isolation-- Neutrophils were isolated from acid citrate dextrose decoagulated buffy coats (provided by the Swiss Red Cross Laboratory, Fribourg). Remaining erythrocytes in the sediments of Lymphoprep gradients were lysed by isotonic ammonium chloride treatment, as described in detail elsewhere (43).

Subcellular Fractionation-- Isolated neutrophils were resuspended at 108 cells/ml in HEPES buffer (20 mM HEPES (pH 7.4), 138 mM NaCl, 4.6 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM diisopropyl fluorophosphate, 20 µM leupeptin, 18 µM pepstatin, 1 mM EDTA, 12.5 units/ml Benzon nuclease, 5% glycerol (w/v)) and placed in a nitrogen bomb for 60 min at 35 bars on ice. After homogenization with a Potter-Elvehjem, the lysate was centrifuged 10 min at 400 × g at 4 °C; the supernatant was centrifuged 20 min at 9000 × g at 4 °C, and the supernatant was centrifuged 45 min at 121,500 × g at 4 °C. The supernatant ("cytosol") was collected and stored at -80 °C until use.

Ca2+ Depletion of Neutrophils-- For Ca2+ depletion, neutrophils were resuspended in phosphate-buffered saline supplemented with 2 mM EGTA and incubated with 25 µM BAPTA-AM (in Me2SO containing 20% (w/v) Pluronic F-127) for 30 min at room temperature. The cells were then pelleted by centrifugation and resuspended at the desired concentration in the respective assay buffer without Ca2+ and containing 2 mM EGTA. The effectiveness of Ca2+ depletion was checked by measuring changes in intracellular Ca2+ levels in response to fMLP. For this purpose, cells were loaded with 5 µM Fura 2-AM and were stimulated with 100 nM fMLP. Two wavelength excitation was performed at 340 and 380 nm, and the intracellular [Ca2+] ([Ca2+]i) was calculated from the emission at 510 nm. Cells that had been treated with BAPTA-AM did not show any increase in [Ca2+]i in response to fMLP.

Measurement of the Quantity of Gelsolin Bound to Actin in EGTA-resistant (1:1) Complex in Neutrophils-- These assays were essentially carried out as described (14). Neutrophils were resuspended at 1.5 × 108/ml in HBSSG2+ (14) and preincubated with Me2SO (control) or wortmannin (dissolved in Me2SO) for 10 min at 37 °C. 100 µl of cells were then added to an equal volume of HBSSG2+ containing Me2SO (control) or 200 nM fMLP for 15 s at 37 °C. The stimulation was stopped by the addition of 200 µl of lysing buffer (14). After centrifugation, actin-gelsolin (1:1) complexes were immunoprecipitated from the supernatants using immobilized purified anti-gelsolin monoclonal antibody and washed as described (14). After SDS-PAGE and Coomassie Blue staining, the gels were scanned as above. The molar ratios of gelsolin and actin were calculated (14) assuming that the actin/gelsolin molar ratio is equal to the ratio of the measured absorbances of the 90-kDa (cytoplasmic gelsolin) and the 42-kDa (actin) protein bands on the gel multiplied by 2.14. The differences in molecular mass between plasma and neutrophil gelsolin are due to an N-terminal extension of 25 amino acids on plasma gelsolin (44).

Quantification of Barbed End Nucleating Activity-- Pyrene-labeled F-actin prepared as above was depolymerized and gel-filtered through a Sephadex G-150 column (37). Barbed end nucleating activity was analyzed (15). Neutrophils were resuspended at 5 × 106/ml in suspension buffer (15) and preincubated for 10 min at 37 °C with Me2SO (control) or wortmannin, prior to stimulation with 100 nM fMLP for 15 s. The cells were then diluted to 3.33 × 105/ml in suspension buffer containing 0.2% (w/v) Triton X-100 (15), and 1 µM monomeric pyrene-actin (gel-filtered through Sephadex G-150) was added. The slope of the fluorescence increase at 407 nm between 100 and 200 s was used as an indicator of barbed end nucleating activity.

Determination of Cellular Filamentous Actin-- Filamentous actin was stained with fluorescein-labeled phalloidin according to Ref. 45 with modifications described elsewhere (46, 47). Neutrophils were resuspended at 107/ml in HBSSG2+ and preincubated with wortmannin or Me2SO (control) for 10 min at 37 °C. The cells were then stimulated with 100 nM fMLP or Me2SO for 15 s, and 100 µl was added to an equal volume of ice-cold staining solution (8% formaldehyde, 200 µg of lysophosphatidylcholine/ml, and 0.33 µM fluorescein-phalloidin in phosphate-buffered saline). Samples were assayed for bound fluorescence at 520 nm (excitation 495 nm) after overnight incubation at 4 °C.

EGTA-resistant Actin-Gelsolin (1:1) Complexes in Neutrophil Cytosol-- Neutrophil cytosol (1.0 × 107 cell eq) was treated with EGTA 10 mM with or without GTPgamma S or ATPgamma S for 5 min at room temperature, followed by 2 mM MgCl2 prior to incubation for 10 min at 37 °C. As a control, complexes of purified gelsolin and ATP- or ADP-actin prepared as above, but in HEPES buffer, were incubated for 10 min at 37 °C with or without GTPgamma S or ATPgamma S. EGTA-resistant actin-gelsolin (1:1) complexes were immunoprecipitated and analyzed by SDS-PAGE as described above. For experiments with recombinant GTPgamma S-loaded Gialpha 2, controls were treated with the corresponding volume of HED treated with 1 mM GTPgamma S and 20 mM MgCl2. For experiments with recombinant Gbeta 1gamma 2, controls were treated with a corresponding volume of buffer D, and all samples were supplemented with 0.1 µM GTPgamma S. When small G-proteins were tested, controls were incubated with the corresponding volume of buffer treated with 100 µM GTP or GDP, 10 mM EDTA, and 20 mM MgCl2.

Measurement of GDP-GTP Exchange on GST-Rac1 in Neutrophil Cytosol-- Purified recombinant GST-Rac1 was preloaded with GDP and added (10 µM final concentration) to neutrophil cytosol containing 10 mM EGTA, 12.5 µM GTP, and 93.8 µCi/ml [alpha -32P]GTP in the presence of 2.5 µM recombinant GTPgamma S-loaded Gialpha 2, HED pretreated with GTPgamma S and MgCl2 (control), 2.5 µM recombinant Gbeta 1gamma 2 or buffer D (control). The samples were incubated for 10 min at 37 °C and placed on ice, and glutathione-Sepharose CL-4B (40 µl of a 1:1 suspension in HEPES buffer) was added. After incubation for 30 min at 4 °C, the beads were collected by centrifugation and washed three times with Wash buffer (48). The samples were split in two. One-half was assayed for bound radioactivity by scintillation counting, and the other was analyzed for protein content by SDS-PAGE and Coomassie Blue staining. The ratio of bound radioactivity (cpm) to protein content (µg) was used as an indicator of the rate of exchange of GDP to GTP on GST-Rac1.

    RESULTS
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References

Modulation of Gelsolin/Actin Interactions by Phosphoinositides in Vitro-- It has been shown in previous reports that increased phosphorylations of the inositol head group of phosphoinositides enhance their ability to dissociate actin-gelsolin complexes (PtdIns(4,5)P2 > PtdIns(4)p > PtdIns) (8-10). We have now evaluated the capacity of PtdIns(3,4,5)P3 to dissociate EGTA-resistant equimolar actin-gelsolin complexes and to inhibit the actin filament severing activity of gelsolin in vitro. When purified actin-gelsolin complexes were exposed to phosphoinositides prior to immunoprecipitation, PtdIns(3,4,5)P3 was found to be equally potent to PtdIns(4,5)P2 in dissociating actin from gelsolin (EC50 = 19 µM, Fig. 1A). The actin filament severing activity of gelsolin was measured by the relative rate of pyrene-labeled F-actin depolymerization. In these experiments, PtdIns(3,4,5)P3 was somewhat more efficient than PtdIns(4,5)P2 in inhibiting the gelsolin-mediated severing of actin filaments (EC50 of 4 µM versus 10 µM, Fig. 1B).


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Fig. 1.   Dose-dependent dissociation of actin-gelsolin complexes by phosphoinositides (A) and inhibition of the severing activity of gelsolin by increasing amounts of phosphoinositides (B). A, purified gelsolin was premixed with purified monomeric actin and subsequently incubated with PtdIns(3,4,5)P3 (circles), PtdIns(4,5)P2 (squares), PtdIns(4)P (diamonds), and PtdIns (triangles) at indicated concentrations, in the presence of EGTA. Equimolar actin-gelsolin complexes were immunoprecipitated and analyzed by SDS-PAGE and densitometry. Values are means from four experiments ± S.E. for PtdIns(3,4,5)P3 and PtdIns(4,5)P2, single points for for PtdIns(4)P and PtdIns. B, purified gelsolin was incubated with PdtIns(3,4,5)P3 (circles), PtdIns(4,5)P2 (squares), PtdIns(4)P (diamonds), and PtdIns (triangles) at the indicated concentrations. Pyrene-labeled F-actin was added, and the gelsolin-dependent severing activity was measured. Mean of four experiments with PtdIns(3,4,5)P3 or PtdIns(4,5)P2 ± S.E., single values for PtdIns(4)P and PtdIns.

Effect of PI 3-Kinase Inhibition and Ca2+ Depletion on Agonist-induced Actin-Gelsolin Dissociation and Actin Polymerization in Neutrophils-- While in resting neutrophils a large fraction of gelsolin is associated with actin, and exposure of the cells to fMLP leads to the rapid release of actin from gelsolin (14). This is paralleled by an increase in free barbed ends (14, 15) and a doubling of the cellular F-actin content (45, 49, 50). At a time point when these three transient responses reach their maximum, the effects of the PI 3-kinase inhibitor wortmannin and of Ca2+ depletion were studied. As shown in Fig. 2A, 100 nM fMLP caused a decrease of the amount of actin associated with gelsolin to 1/7 of the levels in unstimulated cells within 15 s, which is in agreement with previous reports (14).


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Fig. 2.  

Effect of wortmannin and Ca2+ depletion on the fMLP-stimulated decrease in actin-gelsolin complexes (A), on the fMLP-stimulated increase in barbed end nucleating activity (B), and on the fMLP-stimulated actin polymerization (C) in neutrophils. A, normal (filled boxes) or Ca2+-depleted (open boxes) neutrophils were preincubated with Me2SO (control) or wortmannin at indicated concentration prior to stimulation with fMLP (100 nM for 15 s, where indicated). Actin-gelsolin (1:1) complexes were immunoprecipitated and analyzed by SDS-PAGE and densitometry. Mean of three experiments ± S.E. B, isolated untreated (filled boxes), Pluronic F-127-treated, or Ca2+-depleted neutrophils (open boxes) were preincubated with Me2SO (control) or wortmannin at indicated concentration prior to stimulation with 100 nM fMLP for 15 s. The cells were then lysed, and barbed end nucleating activity was determined as described under "Experimental Procedures." Mean of three experiments ± S.E. C, untreated (filled boxes) or Ca2+-depleted (open boxes) neutrophils were preincubated with Me2SO (control) or wortmannin 1 µM, prior to stimulation with 100 nM fMLP for 15 s. The cells were then fixed, permeabilized, and stained for F-actin content using fluorescein-phalloidin. The cellular F-actin content is expressed as % of the untreated control. Mean of three different experiments ± S.E.

Pretreatment of neutrophils with 0.1-10 µM wortmannin prior to stimulation did not significantly affect the fMLP-induced breakdown of actin-gelsolin complexes as compared with control cells (Fig. 2A). While wortmannin at 0.1 µM completely inhibits PI 3-kinase in a specific manner (24, 51), the substance also affects myosin light chain kinase activity at micromolar concentrations (52). Since the signal controlling actin-gelsolin dissociation has been proposed to be Ca2+-independent (14), we analyzed the levels of complexes in Ca2+-depleted cells. Ca2+ depletion resulted in a dramatic decrease in the basal levels of actin-gelsolin complexes (actin/gelsolin ratio = 0.07, Fig. 2A). Stimulation of Ca2+-depleted neutrophils lowered the actin/gelsolin ratio further from 0.07 to 0.02. The effect of wortmannin in Ca2+-depleted cells was pronounced, as it inhibited the agonist-mediated dissociation of actin and gelsolin completely at 1 µM.

The observation that depletion of intracellular calcium pools with BAPTA strongly lowered the basal levels of complexes is probably due to the fact that there is a turnover of gelsolin molecules at the barbed ends of actin filaments in the cell and that the reassociation of gelsolin to actin is blocked in the absence of calcium (11, 12) (as well as the severing activity of gelsolin that could also produce actin oligomers with capped barbed ends). This decrease in basal levels of complexes is probably not due to increased cellular levels of PtdIns(4,5)P2 or PtdIns(4)P (possibly caused by inhibition of Ca2+-dependent phospholipase C), as in 32PO4-labeled neutrophils the basal levels of [32P]PtdIns(4,5)P2 and [32P]PtdIns(4)P were not significantly affected by Ca2+ depletion (data not shown). Ca2+ depletion totally abolished the fMLP-stimulated breakdown of [32P]PtdIns(4,5)P2 but not the production of [32P]PtdIns(3,4,5)P3, as previously reported (data not shown) (22).

We tested various pharmacological agents for their ability to inhibit the fMLP-mediated breakdown of actin-gelsolin complexes in neutrophils. Inhibitors of protein kinases like staurosporine, genistein, and erbstatin did not inhibit the response, which was also insensitive to preincubation with 0.5% ethanol (an inhibitor of fMLP-induced phosphatidic acid production), but required metabolic energy, as it was inhibited by incubating the cells with 2-deoxyglucose and antimycin A. Stimulation of heterotrimeric G-proteins with AlF4 (50 µM, 5 mM) could mimic the effect of fMLP on the complexes in the intact cell (data not shown).

When neutrophils were stimulated with 100 nM fMLP for 15 s and subsequently treated with detergent for a barbed end nucleating activity assay, the stimulation elevated the relative rate of actin polymerization by a factor of 2 (Fig. 2B), in accordance with previous reports (15). Wortmannin showed no significant effect at 100 nM, although doses higher than 1 µM produced a 50% inhibition of the fMLP-induced rise in nucleating activity (Fig. 2B). The same amounts of inhibitor produced no significant effect on the barbed end nucleating activity in resting cells (data not shown). Ca2+ depletion per se induced an increase in free barbed ends (Fig. 2B). The stimulation of Ca2+-depleted cells with 100 nM fMLP caused a further (1.7-fold) increase in barbed end nucleating activity, which is 80% of the response in normal cells (see also Ref. 15). The effect of wortmannin in Ca2+-depleted cells was similar to normal cells.

The basal levels of cellular F-actin content were lowered by wortmannin (1 µM) pretreatment (by 23%) or Ca2+ depletion (by 13%). On the other hand, both treatments left the fMLP-mediated actin polymerization intact (Fig. 2C), as also reported under different conditions previously (45-47).

Effect of GTPgamma S on Cytosolic Actin-Gelsolin Complexes-- To study the regulation of actin-gelsolin dissociation by G-proteins, we developed a cell-free system using cytosolic fractions of neutrophils obtained by nitrogen cavitation. The cytosolic preparations displayed levels of EGTA-resistant actin-gelsolin (1:1) complexes ranging from 0.5 to 1 (mol/mol), thus reflecting the levels of complexes in the soluble fraction of Triton X-100 extracts of resting neutrophils.

When neutrophil cytosol was exposed to GTPgamma S, the actin/gelsolin ratio in subsequently obtained anti-gelsolin immunoprecipitates was significantly reduced to less than 60% (Fig. 3A). The maximal effect of GTPgamma S occurred already at 1 µM. Increasing concentrations of GTPgamma S up to 100 µM did not further affect the level of complexes in the cytosol (Fig. 3B). 100 µM GDP did not dissociate the complexes (data not shown). ATPgamma S up to 100 µM was less effective than GTPgamma S in dissociating actin from gelsolin, indicating that GTP is not utilized by a diphosphonucleotide kinase to generate ATP and that the latter is not involved in the dissociation process (Fig. 3, A and B). When actin-gelsolin complexes were preformed from purified ATP- or ADP-actin and gelsolin, both GTPgamma S and ATPgamma S did not dissociate the 1:1 complex (Fig. 3A). This indicates that GTPgamma S does not dissociate cytosolic actin-gelsolin complexes directly but that it activates additional factors in the cytosol that mediate its effect on the complexes. These observations are confirmed by the finding that actin-gelsolin (1:1) complexes purified from neutrophil cytosol by immunoprecipitation could not be dissociated directly by 100 µM GTPgamma S and ATPgamma S (data not shown).


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Fig. 3.   Dissociation of cytosolic actin-gelsolin (1:1) complexes by GTPgamma S and ATPgamma S (A) and dose-dependence of GTPgamma S and ATPgamma S in the dissociation of cytosolic actin-gelsolin (1:1) complexes (B). A, neutrophil cytosol (filled boxes) or actin-gelsolin complexes prepared from purified gelsolin and purified ADP- or ATP-actin (open boxes) were incubated with 100 µM GTPgamma S or 100 µM ATPgamma S, prior to immunoprecipitation of actin-gelsolin (1:1) complexes and SDS-PAGE analysis. Data are means ± S.E. from six different experiments and are plotted as % of untreated control. The control value of the actin/gelsolin molar ratio is 0.8399 for neutrophil cytosol, 1.0065 for ATP-actin/gelsolin, and 0.9723 for ADP-actin/gelsolin. * indicates a significant difference as compared with control (Student's t test, p < 0.05). B, neutrophil cytosol was incubated with GTPgamma S (circles) or ATPgamma S (squares) at indicated concentrations. Actin-gelsolin (1:1) complexes were immunoprecipitated and analyzed by SDS-PAGE. Values are means from two different experiments ± S.E. presented as % of untreated controls.

It has been shown previously that Rho-GDI forms tight complexes with members of the Rho family of small G-proteins and that the complex is inactive in various processes (53, 54). When neutrophil cytosol was supplemented with 2.8 µM recombinant Rho-GDI, the protein reversed the action of subsequently added GTPgamma S but did not affect the level of actin-gelsolin complexes in untreated cytosol (Fig. 4A). Incubation of the cytosol with recombinant C3 transferase, ribosylating specifically Rho proteins (55-57), did not inhibit the GTPgamma S-mediated dissociation of actin and gelsolin, whereas 1 µM wortmannin did not inhibit the response either (data not shown).


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Fig. 4.  

Reversal of the GTPgamma S-stimulated actin-gelsolin dissociation by Rho-GDI (A) and activation of actin-gelsolin dissociation in neutrophil cytosol by recombinant Gialpha 2 and Gbeta 1gamma 2 (B and C). A, neutrophil cytosol was pretreated with buffer (control) or 2.8 µM Rho-GDI and incubated with or without (control) 100 µM GTPgamma S. B, EGTA-treated neutrophil cytosol was incubated with HED buffer supplemented with GDP or GTPgamma S (control) or with purified recombinant Gialpha 2 (2.5 µM) preloaded with GDP or GTPgamma S. Alternatively, the cytosol was incubated with buffer D or recombinant Gbeta 1gamma 2 (2.5 µM). C, neutrophil cytosol was preincubated with buffer (control) or Rho-GDI (2.8 µM) and subsequently incubated with GTPgamma S-loaded Gialpha 2 or Gbeta 1gamma 2 (2.5 µM). Actin-gelsolin (1:1) complexes were immunoprecipitated and analyzed by SDS-PAGE. A and B, data are means ± S.E. from six different experiments and are plotted as % of control. C, data are means ± S.E. from six different experiments and are plotted as % of buffer-treated controls. * indicates a significant difference as compared with control (Student's t test, p < 0.05).

The Action of GTP-binding Proteins on Cytosolic Actin-Gelsolin Complexes-- Myristoylated recombinant Gialpha 2 at 2.5 µM bound to GTPgamma S (but not to GDP) induced a significant decrease of about 40% in the levels of cytosolic actin-gelsolin (1:1) complexes, as compared with the control treated with HED buffer containing GTPgamma S and MgCl2 (Fig. 4B). The presence of GTPgamma S in the HED buffer used to preload the recombinant Gialpha 2 (10 µM final concentration) induced by itself a significant decrease in the levels of the cytosolic complexes (Fig. 4B). The effect of recombinant Gialpha 2 was indirect, since it was not observed on purified actin-gelsolin complexes. Recombinant Gbeta 1gamma 2 at 2.5 µM also induced a significant decrease of about 50% in cytosolic actin-gelsolin (1:1) complexes, when the cytosol was supplemented with 0.1 µM GTPgamma S (Fig. 4B). The effect of GTPgamma S-loaded Gialpha 2 and of Gbeta 1gamma 2 on cytosolic complexes could be blocked by Rho-GDI (Fig. 4C), suggesting that heterotrimeric G-proteins can activate proteins of the Rho family in neutrophil cytosol, which in turn trigger actin-gelsolin dissociation. This is confirmed by the observation that the dissociation of actin-gelsolin in neutrophil cytosol could be induced by the addition of GTP-loaded, constitutively activated Val-12 Rac1 at 5 µM (Fig. 5A). Its dissociating potential was specific as GDP-loaded Val-12 Rac1, inactive GTP-Val-12 Ala-35 Rac1 or GTP-Val-14 RhoA did not cause dissociation of cytosolic actin-gelsolin complexes at the same concentration (Fig. 5A). The effect of GTP-loaded Val-12 Rac1 occurred in a concentration-dependent fashion with a 50% dissociation at around 10 µM (Fig. 5B). Preformed complexes of purified actin and gelsolin, however, could not be dissociated even in the presence of 25 µM recombinant GTP-loaded Val-12 Rac1 (data not shown), which suggests that a downstream target of Rac, but not the small G-protein itself, interacts with the actin-gelsolin complex. The decrease in cytosolic actin-gelsolin complexes induced by GTP-loaded Val-12 Rac1 was not inhibited by C3 transferase, confirming that Rho is not involved in the process (data not shown).


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Fig. 5.  

The influence of small G-proteins on actin-gelsolin complexes in neutrophil cytosol (A and B) and lack of effect of a PI(4,5)P2-binding peptide on the response to Val-12 Rac1 (V12rac1) (C). A, purified recombinant small G-proteins were preloaded with GDP or GTP and were added at a final concentration of 4.8 µM to neutrophil cytosol. Actin-gelsolin (1:1) complexes were immunoprecipitated and quantitated by SDS-PAGE and densitometry. Values are plotted as % of the controls (without recombinant protein, but with the respective guanine nucleotide) ± S.E., n = 6. B, EGTA-treated neutrophil cytosol was incubated with GTP-bound V12Rac1 at indicated concentration. Actin-gelsolin (1:1) complexes were analyzed as above. Data are means ± S.E. from two different experiments and are plotted as % of controls treated with a corresponding volume of buffer and GTP. C, neutrophil cytosol was supplemented with a PI(4,5)P2-binding peptide (50 µM final concentration) or buffer (control). GTP-loaded Val-12 Rac1 (4.8 µM) or buffer supplemented with GTP (control) was then added, and actin-gelsolin complexes were analyzed as in A. Values are plotted as % of the buffer-treated control (100%) ± S.E., n = 4. * indicates a significant difference as compared with control (Student's t test, p < 0.05).

The effect of GTPgamma S in the cytosol was not caused by an increase in the polyphosphoinositide concentration of the cytosol, since we failed to detect the corresponding amount of PtdIns(3,4,5)P3, PtdIns(4,5)P2, or PtdIns(4)P that would have accounted for the decrease in the levels of complexes in the GTPgamma S-treated cytosol (data not shown). GTPgamma S did not induce an incorporation of 32P into phospholipids when [gamma -32P]ATP was included in the cytosol (data not shown).

The effects of GTPgamma S-loaded Gialpha 2, Gbeta 1gamma 2, and GTP-loaded Val-12 Rac1 on cytosolic actin-gelsolin (1:1) complexes were not significantly affected by the presence of neomycin (10 µM) or wortmannin (1 µM) in the cytosol (data not shown), excluding the involvement of phosphoinositides such as PtdIns(3,4,5)P3 or PtdIns(4,5)P2. Furthermore, a gelsolin-derived peptide that has been shown to bind specifically to PtdIns(4,5)P2 (32) did not inhibit the response to GTP-bound Val-12 Rac1 (Fig. 5C) and to GTPgamma S-loaded Gialpha 2 and Gbeta 1gamma 2 (data not shown), at a concentration of 50 µM. This confirms that the action of the GTP-binding proteins on actin-gelsolin complexes is not dependent on phosphoinositides.

Activation of Nucleotide Exchange on GST-Rac1 by Heterotrimeric G-proteins in Neutrophil Cytosol-- To gain further insight in the mechanism of activation of Rac by heterotrimeric G-proteins, an assay to measure the exchange rate of GDP to GTP on Rac in neutrophil cytosol was developed. GST-Rac1 was preloaded with GDP and added to neutrophil cytosol containing [alpha -32P]GTP, in the presence or absence of heterotrimeric G-proteins. GST-Rac1 was then immobilized on glutathione beads, and the amount of bound nucleotide was quantified by scintillation counting. GST-Rac1 was able to bind specifically [alpha -32P]GTP in neutrophil cytosol, since samples containing immobilized GST-Rac1 bound about 10 times more radioactive nucleotide than samples containing immobilized GST alone (data not shown). The identity of the nucleotide bound to GST-Rac1 was determined after denaturation of the protein and analysis of the eluted nucleotide by ion-exchange thin layer chromatography, using a standard protocol (48). This showed that immobilized GST-Rac1 was mainly bound to GDP under the condition used, with a ratio of GDP/GTP of about 10 (data not shown), which can be explained by hydrolysis of the bound [alpha -32P]GTP by the intrinsic GTPase activity of Rac. Addition of recombinant GTPgamma S-loaded Gialpha 2 (2.5 µM) (but not GDP-loaded Gialpha 2) induced a significant 2-fold increase in the amount of radioactive nucleotide bound to GST-Rac1, as compared with control samples treated with HED supplemented with GTPgamma S and MgCl2 (Fig. 6). Recombinant Gbeta 1gamma 2 (2.5 µM) was also able to significantly increase the exchange of GDP to GTP on GST-Rac1, although to a lesser extent (1.5-fold) (Fig. 6). When GST-Rac1 was incubated with recombinant GTPgamma S-loaded Gialpha 2 or Gbeta 1gamma 2 in HEPES buffer and in the absence of cytosol, no significant change in the amount of radioactive nucleotide bound to GST-Rac1 could be observed (data not shown).


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Fig. 6.   Activation of the exchange of GDP to GTP on GST-Rac1 by recombinant Gialpha 2 and Gbeta 1gamma 2. Purified recombinant GST-Rac1 was preloaded with GDP and added to EGTA-treated neutrophil cytosol containing [alpha -32P]GTP in the presence of recombinant GDP- or GTPgamma S loaded Gialpha 2 or Gbeta 1gamma 2 (2.5 µM). GST-Rac1 was subsequently precipitated with glutathione beads, and the samples were assayed for bound radioactivity and protein content. Data are means ± S.E. from three independent experiments and are expressed in % controls treated with buffer and nucleotides. * indicates a significant difference as compared with control (Student's t test p < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The results presented in this article show that gelsolin is not specifically regulated by PtdIns(3,4,5)P3 in vitro, since PtdIns(3,4,5)P3 was able to dissociate actin-gelsolin complexes with a potency similar to PtdIns(4,5)P2. Considering that, upon neutrophil stimulation with fMLP, the maximal concentration of PtdIns(3,4,5)P3 does not exceed 10% of the concentration of PtdIns(4,5)P2 (17), it can be speculated that PtdIns(3,4,5)P3 production is probably not the signal triggering actin-gelsolin dissociation, assuming a uniform distribution of the polyphosphoinositides in the inner leaflet of the cell membrane. The observed lack of selectivity of the severing activity of gelsolin in vitro for PtdIns(3,4,5)P3 as compared with PtdIns(4,5)P2 supports this idea. The fMLP-stimulated breakdown of actin-gelsolin (1:1) complexes in neutrophils and the increase in barbed end nucleating activity were not significantly affected by wortmannin at a concentration of 0.1 µM, which shows that activation of PI 3-kinase is not responsible for these responses and supports the conclusion of the in vitro studies.

Since Ca2+ depletion caused a strong decrease in the basal levels of actin-gelsolin complexes and also increased the basal barbed end nucleating activity of neutrophil lysates, it has to be concluded that increasing the number of free barbed ends in the cell is not sufficient to induce actin polymerization, since the F-actin levels in Ca2+-depleted cells were not increased in comparison to normal cells. A possible explanation for this is the fact that actin monomers are sequestered in the resting cell (58), so that they cannot add to the freed barbed ends. This hypothesis is supported by experiments in other cell types (59) and suggests that fMLP induces actin polymerization in neutrophils both by increasing the number of free barbed ends and by monomer desequestration (2).

The neutrophil cytosol appears to contain a GTPgamma S-stimulable factor that can trigger the dissociation of endogenous actin-gelsolin complexes. Considering the evidence that the response of the cytosol to GTPgamma S is blocked by Rho-GDI, it must be assumed that a member of the Rho family of small G-proteins is involved in this response. As the effect of GTPgamma S on actin-gelsolin complexes was mimicked by GTP-loaded Val-12 Rac1 and not Val-14 RhoA, it can be proposed that cytosolic Rac is the GTPgamma S-stimulable factor responsible for actin-gelsolin dissociation. The insensitivity of this response to C3 transferase confirms that Rho is not involved in the pathway leading to actin-gelsolin dissociation.

Gialpha 2 and Gbeta 1gamma 2 were both found to be able to trigger actin-gelsolin dissociation, which is in agreement with previous studies reporting effector activation both by Galpha and Gbeta gamma subunits (reviewed in Ref. 60). Since the Gialpha 2- and Gbeta 1gamma 2-stimulated dissociation of cytosolic actin-gelsolin was Rho-GDI-sensitive, heterotrimeric G-proteins may be able to trigger Rac activation in the neutrophil cytosol, a model supported by other studies (61, 62). However, the GTPgamma S-stimulated dissociation of the cytosolic actin-gelsolin complexes is probably due to a direct activation of endogenous Rac proteins, since we detected only a very small amount of Gialpha 2 in neutrophil cytosol, representing only a few percent of the protein present in membrane preparations (data not shown). This cytosolic pool of Gialpha 2 does not represent an amount of protein equivalent to the amount of recombinant Gialpha 2 that has to be added to the cytosol to trigger dissociation of actin-gelsolin complexes. It has been shown that Rac is complexed with Rho-GDI in neutrophil cytosol (63) and that GTPgamma S does not dissociate the Rac-Rho-GDI complex directly (62). It must thus be proposed that a fraction of the Rac present in the cytosolic fraction used in our studies is not bound to Rho-GDI and is activated by the addition of GTPgamma S. This hypothesis is consistent with the observation that preincubation of the cytosol with Rho-GDI inhibits the effect of subsequently added GTPgamma S. Dissociation of Rac from Rho-GDI may occur during the preparation of the cytosolic fractions and exposure of the Rac-Rho-GDI complex to anionic phospholipids (64), during the cavitation of the cellular membranes. Our results demonstrate, however, that PI 3-kinase is not responsible for the fMLP-stimulated dissociation of actin-gelsolin complexes, implying that an anionic phospholipid different from PtdIns(3,4,5)P3 is responsible for the Rac-Rho-GDI dissociation in this signaling pathway in vivo.

The evidence that both GTPgamma S-loaded Gialpha 2 and Gbeta 1gamma 2 were able to significantly increase the exchange rate of GDP to GTP on GST-Rac1 in neutrophil cytosol suggests the existence of a cytosolic nucleotide exchange factor for Rac that can be activated by heterotrimeric G-proteins. The identity of the exchange factor is not known at present, but several guanine nucleotide exchange factors specific for Rac have been described and are possibly involved in this pathway (reviewed in Ref. 65). PI 3-kinase has been reported to be involved in the activation of Rac downstream of growth factor receptors (66), which is in contrast with the results presented here, since wortmannin did not inhibit the heterotrimeric G-protein-induced activation of nucleotide exchange on GST-Rac1 and dissociation of actin-gelsolin complexes. A possible explanation for this discrepancy is that serpentine receptors, like the neutrophil fMLP receptor, activate an alternative signaling cascade controlling Rac activation, which involves a G-protein-activated exchange factor for Rac. This hypothesis is consistent with the observation that bombesin-stimulated membrane ruffling, which requires Rac, is not blocked by wortmannin in Swiss 3T3 cells (67).

The cytosolic component downstream of Rac mediating its effect on actin-gelsolin complexes is unknown. Two targets of Rac1 have been described in neutrophils (68) as follows: p67Phox and a 68-kDa kinase analogous to p65PAK (69). We tested both purified recombinant p67Phox and an activated version of p65PAK, which failed to induce cytosolic actin-gelsolin dissociation in the assay described here. These two proteins are probably not the only downstream targets of Rac, since it has been shown that Rac interacts with numerous proteins, some of which contain a consensus Rac-binding motif (CRIB) (70). POR-1, a Rac-binding protein that is involved in membrane ruffling (71), and IQGAP, which binds Rac and is localized to membrane ruffles (72), are two potential mediators of the Rac-stimulated dissociation of actin-gelsolin dissociation described in this report.

In platelets, activated Rac induced an increase in barbed end nucleating activity, which was explained by an increase in the cellular levels of PtdIns(4,5)P2, since this response was blocked by a PtdIns(4,5)P2-binding peptide derived from gelsolin (32). When tested in our assay, the peptide did not inhibit the actin-gelsolin dissociation induced by GTP-loaded Val-12 Rac1, GTPgamma S-loaded Gialpha 2, and Gbeta 1gamma 2, which is in agreement with the finding that these responses are insensitive to neomycin treatment. It must thus be concluded that the G-protein-activated decrease in cytosolic actin-gelsolin complexes we observe is not dependent on phosphoinositides. The increase in barbed end nucleating activity induced by Rac in platelets (32) may be due to the dissociation of a capping protein distinct from gelsolin. Alternatively, it can be argued that the Rac-induced actin polymerization observed in permeabilized platelets (32) involves the membrane-associated cytoskeleton, which is absent from the cytosolic extracts used in the present study. Actin-gelsolin dissociation could thus be regulated differentially, depending on the cellular compartmentalization, membrane-associated complexes being a target for polyphosphoinositides, whereas the complexes found in the cytosol may be regulated by protein-protein interactions and/or protein phosphorylation.

GTPgamma S and Cdc42 were shown to induce actin polymerization in neutrophil lysates, independently of PtdIns(4,5)P2 synthesis (31). By using a similar experimental approach, we show that Rac can trigger the dissociation of gelsolin from the barbed ends of cytosolic actin oligomers, a response that is not mediated by PtdIns(3,4,5)P3 or PtdIns(4,5)P2. The discrepancy between the inability of Rac to induce actin polymerization (31) and its effect on actin-gelsolin complexes can be explained by the fact that dissociation of the complexes may not be sufficient to trigger actin polymerization in the cytosol, which is supported by the results presented above on intact, calcium-depleted neutrophils. Assuming that Cdc42 acts upstream of Rac in controlling cytoskeletal rearrangements (73, 74), it can be speculated that Cdc42 increases barbed end availability via Rac and actin monomer desequestration by a Rac-independent pathway. Since the ability of Cdc42 to induce actin polymerization was attributed to an interaction with PAK1 (31), an activated version of which (p65PAK) was inactive in the actin-gelsolin dissociation assay, an attractive hypothesis would be that Cdc42-mediated activation of PAK1 controls actin monomer availability.

The involvement of cascades of G-proteins in the control of agonist-induced actin rearrangements in fibroblasts (25, 26, 73, 74), mast cells (61), and neutrophils (27-31) is now well established. We provide evidence that activation of Rac by G-protein-coupled receptors in neutrophils controls the release of gelsolin from the barbed ends of actin filaments, independently of PI 3-kinase.

    ACKNOWLEDGEMENTS

I am grateful to Prof. Dr. Andreas Conzelmann and Dr. Matthias P. Wymann (Institute of Biochemistry, University of Fribourg, Switzerland) for their support and advice. I thank Prof. M. D. Waterfield (Ludwig Institute for Cancer Research, University College London) for support and Dr. Anne J. Ridley for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by a grant from the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Ludwig Institute for Cancer Research, University College London, 91 Riding House St., London W1P 8BT, UK. Tel.: 00441718784128; Fax: 00441718784040; Email arcaro{at}ludwig.ucl.ac.uk.

1 The abbreviations used are: fMLP, N-formyl-Met-Leu-Phe; ATPgamma S, adenosine 5'-[gamma -thio]triphosphate; Me2SO, dimethyl sulfoxide; F-actin, filamentous actin; G-protein, GTP-binding protein; Gialpha 2 and Gbeta 1gamma 2 ialpha 2 and beta 1gamma 2 subunits of heterotrimeric G-proteins; GTPgamma S, guanosine 5'-[gamma -thio]triphosphate; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-triphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(4)P, phosphatidylinositol 4-monophosphate; PtdIns, phosphatidylinositol; PI 3-kinase, phosphoinositide 3-kinase; Rho-GDI, GDP dissociation inhibitor for Rho; PAGE, polyacrylamide gel electrophoresis; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; GST, glutathione S-transferase.

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Abstract
Introduction
Procedures
Results
Discussion
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