From the Institute of Biochemistry, University of Fribourg, CH-1700 Fribourg, Switzerland
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ABSTRACT |
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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-[
-thio]triphosphate
(GTP
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 GTP
S-loaded i
2 and the
1
2 subunits of heterotrimeric
G-proteins (Gi
2 and
G
1
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 GTP
S-loaded Gi
2
or G
1
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.
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INTRODUCTION |
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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 GTPS could trigger actin polymerization (29) by
increasing the number of free barbed ends (30). The GTP
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 GTP
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.
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EXPERIMENTAL PROCEDURES |
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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; [-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 Gi
2, the pBB131 plasmid for the expression
of N-myristoyltransferase, and the baculoviruses for the
expression of His6-Gi
1,
G
1, and G
2 were the gifts of T. Kozasa
and A. G. Gilman, Dallas. The antiserum AS 269/1 against
Gi
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
Gi2 was prepared as described (33, 34). The
fractions of each purification step were assayed for
Gi
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 GTP
S loading, recombinant
Gi
2 was incubated with 1 mM
GTP
S in the presence of MgCl2 20 mM
MgCl2 for 1 h at 37 °C.
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 GTPS or
ATP
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 GTP
S or ATP
S. EGTA-resistant actin-gelsolin (1:1)
complexes were immunoprecipitated and analyzed by SDS-PAGE as described
above. For experiments with recombinant GTP
S-loaded Gi
2, controls were treated with the
corresponding volume of HED treated with 1 mM GTP
S and
20 mM MgCl2. For experiments with recombinant
G
1
2, controls were treated with a
corresponding volume of buffer D, and all samples were supplemented
with 0.1 µM GTP
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 [-32P]GTP in the presence of 2.5 µM recombinant GTP
S-loaded
Gi
2, HED pretreated with GTP
S and
MgCl2 (control), 2.5 µM recombinant G
1
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.
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RESULTS |
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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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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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Effect of GTPS 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.
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The Action of GTP-binding Proteins on Cytosolic Actin-Gelsolin
Complexes--
Myristoylated recombinant
Gi2 at 2.5 µM bound to GTP
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 GTP
S and MgCl2 (Fig. 4B). The presence of GTP
S in the
HED buffer used to preload the recombinant
Gi
2 (10 µM final
concentration) induced by itself a significant decrease in the levels
of the cytosolic complexes (Fig. 4B). The effect of
recombinant Gi
2 was indirect, since it was
not observed on purified actin-gelsolin complexes. Recombinant
G
1
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
GTP
S (Fig. 4B). The effect of GTP
S-loaded
Gi
2 and of
G
1
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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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 [-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
[
-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
[
-32P]GTP by the intrinsic GTPase activity of Rac.
Addition of recombinant GTP
S-loaded Gi
2
(2.5 µM) (but not GDP-loaded
Gi
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 GTP
S and
MgCl2 (Fig. 6). Recombinant
G
1
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 GTP
S-loaded Gi
2 or G
1
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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DISCUSSION |
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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 GTPS-stimulable factor
that can trigger the dissociation of endogenous actin-gelsolin complexes. Considering the evidence that the response of the cytosol to
GTP
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 GTP
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 GTP
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.
Gi2 and G
1
2
were both found to be able to trigger actin-gelsolin dissociation,
which is in agreement with previous studies reporting effector
activation both by G
and G
subunits (reviewed in Ref. 60).
Since the Gi
2- and
G
1
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 GTP
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 Gi
2 in
neutrophil cytosol, representing only a few percent of the protein
present in membrane preparations (data not shown). This cytosolic pool
of Gi
2 does not represent an amount of
protein equivalent to the amount of recombinant
Gi
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
GTP
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 GTP
S. This hypothesis is consistent
with the observation that preincubation of the cytosol with Rho-GDI
inhibits the effect of subsequently added GTP
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 GTPS-loaded Gi
2
and G
1
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,
GTPS-loaded Gi
2, and
G
1
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.
GTPS 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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; ATPS, adenosine
5
-[
-thio]triphosphate; Me2SO, dimethyl sulfoxide; F-actin, filamentous actin; G-protein, GTP-binding protein;
Gi
2 and G
1
2
i
2 and
1
2 subunits of
heterotrimeric G-proteins; GTP
S, guanosine
5
-[
-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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REFERENCES |
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