Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9040
Gelsolin and CapG are actin regulatory proteins that remodel the cytoskeleton in response to
phosphatidylinositol 4,5-bisphosphate (PIP2) and Ca2+
during agonist stimulation. A physiologically relevant
rise in Ca2+ increases their affinity for PIP2 and can
promote significant interactions with PIP2 in activated
cells. This may impact divergent PIP2- dependent signaling processes at the level of substrate availability.
We found that CapG overexpression enhances PDGF-stimulated phospholipase C (PLC
) activity (Sun, H.-q.,
K. Kwiatkowska, D.C. Wooten, and H.L. Yin. 1995. J. Cell Biol. 129:147-156). In this paper, we examined the
ability of gelsolin and CapG to compete with another
PLC for PIP2 in live cells, in semiintact cells, and in
vitro. We found that CapG and gelsolin overexpression
profoundly inhibited bradykinin-stimulated PLC
. Inhibition occurred at or after the G protein activation
step because overexpression also reduced the response
to direct G protein activation with NaF. Bradykinin responsiveness was restored after cytosolic proteins, including gelsolin, leaked out of the overexpressing cells.
Conversely, exogenous gelsolin added to permeabilized
cells inhibited response in a dose-dependent manner.
The washout and addback experiments clearly establish
that excess gelsolin is the primary cause of PLC inhibition in cells. In vitro experiments showed that gelsolin and CapG stimulated as well as inhibited PLC
, and
only gelsolin domains containing PIP2-binding sites
were effective. Inhibition was mitigated by increasing
PIP2 concentration in a manner consistent with competition between gelsolin and PLC
for PIP2. Gelsolin and
CapG also had biphasic effects on tyrosine kinase-
phosphorylated PLC
, although they inhibited PLC
less than PLC
. Our findings indicate that as PIP2 level
and availability change during signaling, cross talk between PIP2-regulated proteins provides a selective
mechanism for positive as well as negative regulation of
the signal transduction cascade.
GELSOLIN (33) AND CAPG (36) ARE MEMBERS OF AN ACTIN FILAMENT SEVERING AND CAPPING PROTEIN FAMILY.
THEY ARE ACTIVATED BY Ca2+ and inhibited by polyphosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate (PIP2)1 (15, 33, 36). It is postulated that agonist-induced changes in Ca2+ and plasma membrane PIP2
levels alter the partitioning of gelsolin between the plasma
membrane, cytosol, and actin filament ends (2, 3, 7, 8, 12,
19). These changes initiate actin filament severing and capping to remodel the cytoskeleton (14).
There is also emerging evidence to suggest that actin
regulatory protein binding to PIP2 may have implications
beyond a direct effect on the cytoskeleton. In vitro, gelsolin modulates the activity of several important signaling
enzymes through an effect on their interactions with PIP2.
It alters the activity of phosphoinositide-specific phospholipase C (PLC) (1), phospholipase D (30), and phosphoinositide 3-OH kinase (30; Lu, P., A. Hsu, D. Wong, H. Yun, H.L. Yin, and C. Chen, manuscript submitted for
publication). Therefore, gelsolin and CapG may be components of a signaling complex that transduces external
stimuli to the cytoskeleton. The possibility of cross talk between divergent PIP2-binding proteins through regulation of substrate availability is particularly relevant as more
PIP2-binding proteins are identified. Many pleckstrin homology domain proteins with important signaling functions (27) bind PIP2 with affinity comparable to that of the
gelsolin family (18). We recently demonstrated that gelsolin and CapG affinity for PIP2 is increased six- to eightfold
by micromolar amounts of Ca2+ (18). They can therefore
compete effectively for PIP2, particularly when cytosolic
[Ca2+] increases and PIP2 level drops in agonist-stimulated
cells. We reported that NIH3T3 clones expressing slightly
more CapG than control clones have increased PDGF-stimulated PLC In this paper, we use a variety of approaches to determine how overexpression affects another PLC, PLC Cell Lines
NIH3T3 cell lines stably overexpressing human gelsolin were gifts of Drs.
D.J. Kwiatkowski and C. Cunningham (Brigham and Women's Hospital,
Boston, MA). They were obtained by transfection of a human cytoplasmic
gelsolin expression vector and clonally selected with neomycin (9). Early
passage cells that had been directly quantitated for gelsolin content were
used, and their gelsolin expression was reconfirmed in our laboratory (see
below). CapG overexpression cells were obtained as described in reference 31. Control (Ctrl) cells were transfected with the vector without a
cDNA insert. Cells were maintained in DME with 10% FCS.
For inositol 1,4,5-trisphosphate (IP3) and intracellular Ca2+ measurements, cell monolayers were starved in DME/0.2% serum for 24 h. They
were switched to a completely defined 1:1 DME/F12 medium supplemented with 20 mM Hepes, pH 7.4, 0.5 mg/ml BSA, 1 µg/ml insulin, and 1 µg/ml transferrin (Q-medium) for an additional 18 h before agonist application. In some cases, cells were treated with 100 ng/ml pertussis toxin
(List Biological Lab., Inc., Campbell, CA) overnight. Bradykinin and
PDGF (AB chains) were from Sigma Chemical Co. (St. Louis, MO) and
Upstate Biotechnology, Inc. (Lake Placid, NY), respectively.
Streptolysin O Permeabilization
Cells were trypsinized and resuspended in Chelex 100-treated buffer containing 100 mM KCl, 20 mM NaCl, 20 mM Hepes, pH 7.4. 5 × 106 cells
were permeabilized in 0.4-0.6 U/ml streptolysin O (SLO) (Murex Diagnostics Limited, Dartford, England) with 1 mg/ml creatinine phosphate,
0.1 mg/ml creatinine phosphokinase, 5 mM MgCl2, and 3 mM ATP (21).
Cells were usually permeabilized for 4 min at 37°C before agonist application.
Gelsolin PIP2-binding Domains
Gelsolin NH2-terminal half domains containing zero to three PIP2-binding
sites were expressed in Escherichia coli and isolated as described (35, 37).
The final products were >99% pure, based on Coomassie blue staining of
protein bands in sodium dodecyl sulfate polyacrylamide gels. Protein concentrations were determined by the method of Bradford (5). P2, a 22-mer
synthetic peptide encompassing gelsolin residues 150-169 (CKHVVPNEVVVQRLFQVKGRR, amino-terminal cysteine added) (16), was synthesized by solid phase methods. CapG and thymosin Ca2+ Measurements
Serum-deprived cells were washed and incubated with a buffer containing
Fura 2 (2.5 µM), 1 mg/ml BSA, and 2 mg/ml glucose for 1 h at room temperature (31). Cells were trypsinized briefly and resuspended in a buffer
containing BSA and glucose and warmed to 37°C. Fluorescence measurements were performed with 2 × 106 cells in 1.5 ml with slow stirring at
37°C, at excitation and emission wavelengths of 340 and 500 nm, respectively. [Ca2+] was calibrated by adding 25 µg/ml digitonin and 0.25 mM
MnCl2 to obtain Fmax and Fmin, respectively. A KdCa2+ of 224 nM was used
for calculation.
IP3 Content
Cell monolayers starved in Q-medium were stimulated with 5 µM bradykinin for 7 or 15 s, and the reaction was stopped by adding an equal volume of a 5% perchloric acid solution containing 10 mM EDTA and 1 µM
ATP. The soluble extract was neutralized with 1:1 freon/octylamine, and
IP3 content was assayed by competition with exogenous [3H]IP3 to bind
calf cerebellum microsomes (31). IP3 standards were extracted and assayed under identical conditions. The IP3 content of permeabilized cells
was determined with cells in suspension under conditions identical to
those used for Ca2+ measurements.
Immunoblotting
Cells were lysed in cold RIPA buffer containing a protease inhibitor cocktail as described previously (31). Lysate protein concentrations were determined by the micro-BCA method (Pierce Chemical Co., Rockford, IL).
Proteins were electrophoresed on 5-10% polyacrylamide, discontinuous
pH slab gels in the presence of SDS and transferred to nitrocellulose for
Western blotting. Blots were probed with rabbit anti-mouse gelsolin,
which recognizes the endogenous gelsolin (31), and/or a monoclonal anti-
human gelsolin (clone 2C4), which recognizes human but not mouse
gelsolin (6). Endogenous mouse and overexpressed human gelsolin were quantitated by comparing the intensity of gelsolin bands in cell lysates
with that of purified mouse and human plasma gelsolins, respectively, as
described in reference 9. In leak-out experiments, ctrl cells were permeabilized with streptolysin O at 37°C and centrifuged at 1,500 rpm in an Eppendorf microcentrifuge for 1 min at room temperature. Samples were
blotted with rabbit anti-mouse gelsolin, rabbit anti-PLC PLC Assays
Recombinant PLC Phospholipase C Ca2+ Signaling in CapG Overexpressing Cells
We showed previously that CapG overexpression increased Ca2+ and IP3 response to PDGF (31). These results suggest that CapG enhanced PLC
Table I.
Effects of Overexpression on Ca2+ Signals
To explore the basis for the opposite effects of CapG
overexpression on PDGF- vs. bradykinin-activated PLC,
we felt that studying cells with a wider range of overexpression and comparing cells overexpressing related proteins would be important. In spite of repeated attempts,
we were not able to isolate stable CapG clones with higher
overexpression. We therefore directed our effort to gelsolin-overexpressing clones, which have a wider range of
overexpression (9).
Effects of Gelsolin Overexpression on PIP2 Hydrolysis
Western blotting with human gelsolin-specific antibody,
which recognizes the transfected human gelsolin but not
endogenous mouse gelsolin, confirmed that the clones had
a range of gelsolin overexpression (Fig. 2, bottom), as described previously (9). C2 was not positive, indicating that
it had lost the expression vector, and was not studied further. Blotting with anti-mouse gelsolin, which cross-reacts
poorly with human gelsolin, showed that the endogenous
gelsolin level in overexpressing cells was comparable to
that of the ctrl cells (Fig. 2, top). Therefore, there was no
compensatory change in endogenous gelsolin expression. Total gelsolin level, determined by quantitative Western
blotting using purified human and mouse gelsolin standards, confirmed that the levels of expression were similar
to that shown previously (9) (Table I).
Gelsolin overexpression modified the Ca2+ response to
bradykinin. C3 that has 1.3 times as much gelsolin as ctrl
was slightly more responsive to bradykinin and PDGF
(Table I). In contrast, C6, C1, C4, and C5 that have higher
overexpression had a striking decrease in bradykinin-
induced Ca2+ signal (Fig. 3, Table I). The PDGF response
was also reduced, but to a lesser extent (Table I). Since inhibition was observed with multiple independently isolated clones, the phenotype was attributed to gelsolin
overexpression and was not an artifact of clonal selection.
The stimulation we observed with the lowest overexpressing clone, C3, suggests that gelsolin may also increase signaling. However, because only one such clone was available, ruling out clonal variations unrelated to gelsolin was
not possible.
The effect of gelsolin on PLC Characterization of the Gelsolin Effect on the
Bradykinin Response
Since the bradykinin response was strikingly inhibited, we
will focus on this aspect in the rest of the paper. Bradykinin stimulation of Ca2+ release is a multistep process that
involves bradykinin binding to receptors, activation of heterotrimeric G proteins, PLC Addition of Exogenous Gelsolin to Streptolysin
O-permeabilized Ctrl Cells
To determine if the change in PLC
Exogenous CapG also inhibited IP3 rise, while thymosin
Regeneration of Bradykinin Response after Cytosol
Leakage from Semiintact Cells
Our data showed that gelsolin, either overexpressed in intact cells or added to semiintact cells, reduced bradykinin
responsiveness. Removal of gelsolin should restore the
bradykinin response. Cells were permeabilized for progressively longer intervals before bradykinin stimulation
to allow gelsolin and other cytosolic proteins to leak out.
After 3 min, gelsolin and actin were detected in the medium (Fig. 5 A). More proteins leaked out with longer
treatment. In contrast, PLC
The effect of cytosol leakage on IP3 generation was determined by stimulating cells permeabilized for different
intervals with bradykinin (Fig. 5 B). Both intact and permeabilized ctrl cells increased IP3 after bradykinin stimulation. The semiintact cell's response was more robust,
suggesting that soluble inhibitors had leaked out or that
other clamping mechanisms were disrupted. As shown
above, intact C5 had a much lower IP3 response than ctrl cells. At 2 min after SLO treatment, C5 was still inhibited
relative to ctrl. Between 2 to 5 min, C5 response increased
to a level comparable to that of ctrl. With longer permeabilization, both ctrl and C5 were less active, probably due
to loss of membrane integrity and depletion of soluble cofactors such as GTP. The convergence of the IP3 response
between ctrl and C5 was not due to differential leakout of
PLC Effects of Gelsolin on Phospholipase C In Vitro
It has been reported that gelsolin inhibits several PLC
isozymes in vitro (1). In light of our in vivo results, we reexamined this issue to determine if gelsolin and CapG inhibit PLC
Gelsolin inhibited Ca2+-activated PLC The inhibitory effect of GS1-406 was overcome by increasing PIP2 concentration. As expected, PLC Experiments using gelsolin domains with and without
PIP2-binding sites confirmed that gelsolin altered PLC activity through PIP2 binding. GS1-406, encompassing gelsolin segments 1-3, contains at least two (and possibly three)
PIP2-binding sites (Fig. 7 A). The S1 and S2 PIP2-binding
sites have been mapped precisely by deletion analyses (37)
and peptide analog studies (16, 37). The existence of a
third site is inferred from previous experiments after deleting the S2 site and is predicted to be in S3 (37). GS1-406, which binds PIP2 with an estimated Kd of 2.9 µM and
a stoichiometry of ~3 (18), had the most activating and inhibitory effects (Fig. 7 B). GS150-406 (S2-3), which binds
with a similar Kd but with a stoichiometry of ~2, was slightly
less effective. GS150-267 that contains a single PIP2-binding
site was markedly less effective, while GS171-267 with no
PIP2-binding site had no effect. These results showed that
gelsolin altered PLC activity through PIP2 binding, and
maximal effect was observed with the participation of two
to three PIP2-binding sites. Binding to multiple PIP2 may
form a more stable complex that is less likely to be displaced
by PLC, and the larger polypeptides are likely to be more
effective in sterically hindering PLC access to its substrate.
P2, the PIP2-binding peptide derived from gelsolin S2
(16), had no effect at low concentration and stimulated
PLC In this paper, we examined the ability of gelsolin and
CapG to compete with PLC for PIP2 in live cells, in semiintact cells, and in vitro. The various approaches help to
define the mechanism by which gelsolin and CapG modulate PLC activity in two independent signaling pathways.
Our results suggest that these cytoskeletal proteins may
participate in signaling by controlling the availability of
PIP2 substrates.
The CapG and gelsolin overexpressing cell lines were
isolated independently by two different laboratories, using
the same parent NIH3T3 cell line and expression vector.
Their remarkably similar effects on PLC Another feature that emerges from these studies is that
there is a difference in the sensitivity of PLC Inhibition of PLC is most likely due to competition of
gelsolin and CapG with PLC for PIP2. Steric hindrance
may also be a contributing factor. The mechanism by
which PLC is enhanced by a small increase in CapG or
gelsolin is not known. Since only gelsolin domains with
PIP2-binding sites stimulate, this effect depends on PIP2
binding. The simplest hypothesis is that gelsolin and CapG
improve the presentation of PIP2 to PLC by altering the phospholipid conformation. Among the gelsolin domains
tested, P2, the small synthetic peptide containing a single
PIP2-binding site, is most stimulatory in vitro. However, it
does not have any effect on PLC in semiintact cells. This
peptide has been used by others to examine the relation
between gelsolin and PIP2 in cells (8, 13). P2 added to
semiintact platelets blocks Rac1-induced uncapping of actin filament + ends, and this effect is overcome by adding
PIP2 (13). Overexpression of P2 in a permanently transfected cell line reduces gelsolin association with the
plasma membrane (8). Both experiments indicate that P2
competes with gelsolin for PIP2, although effects on other
PIP2 -requiring processes have not been ruled out. Our result suggests that PLC was not affected by increasing P2
levels in cells. Additional experiments will be required to
determine if other parameters are altered by P2.
In conclusion, we show that gelsolin and CapG interact
with PIP2 in vivo, and a modest increase in concentration
has profound effects on two PIP2 signaling pathways.
Overexpression is used as a tool to probe intracellular
function. Our overexpression results have physiological
relevance. While total gelsolin and CapG content do not
change per se, cytosolic concentration and membrane
binding are expected to fluctuate during signaling. Gelsolin and CapG bind to and are released from filament ends
and the membrane PIP2 content changes due to hydrolysis
and resynthesis. Ca2+ increases gelsolin and CapG affinity
for PIP2 (18), enhancing their ability to compete with
other PIP2-binding proteins at a time when membrane
PIP2 content decreases. CapG and gelsolin can provide
positive and negative inputs on PLC signaling pathways, and these pathways are modulated selectively. The data
presented here indicate that there is significant cross talk
between components of the transmembrane signal machinery and actin cytoskeleton at multiple levels, including
that of the generation of important second messengers.
activity (31), supporting this possibility.
However, we did not anticipate a stimulatory effect since
previous in vitro studies indicate that gelsolin and profilin,
another PIP2-regulated actin-binding protein, inhibit PLC
(1, 10).
, and
the basis for interaction in vivo. We also compared the effects of CapG and gelsolin overexpression. Gelsolin and
CapG are present in similar concentration (~0.7 µM) in
NIH3T3 cells (9, 31). The gelsolin clones have a wider range
of overexpression (9), and much more is known about
gelsolin PIP2-binding sites (16, 34, 37). They have the same
motility phenotype as CapG-overexpressing cells (9, 31),
but their phosphoinositide metabolism has not been examined.
Materials and Methods
4 were expressed
in E. coli and purified as described previously (35, 38).
3 (gift of P. Sternweis, University of Texas Southwestern Medical Center, Dallas, TX)
(11), and monoclonal antiactin (clone C4; Boehringer Mannheim Corp.,
Indianapolis, IN).
1 expressed in SF9 cells and purified to homogeneity
was a gift of Dr. E. Ross (University of Texas Southwestern Medical Center) (4). Vesicles containing 100 µM phosphatidylethanolamine, 10 µM
phosphatidylserine, 10 µM PIP2, and 0.028 µM [3H] PIP2 were made by
sonicating in the PLC buffer (25 mM Hepes, 80 mM KCl, 3 mM EGTA,
0.5 mM DTT, pH 7.0). Vesicles were preincubated with gelsolin domains
in the presence of 11 µM free [Ca2+] at 4°C for 10 min, and enzyme reaction was initiated by adding 4 ng (per 60 µl) PLC
1 and warming to 37°C.
Reactions were terminated after 1-4 min by adding TCA and 3 mg/ml
BSA. Precipitated proteins were removed by centrifugation, and radioactive IP3 in the TCA-soluble supernatant was determined by scintillation
counting. Triplicate samples were assayed per condition. PIP2 hydrolysis was linear for 15 min under the conditions used. In some experiments, PIP2, ranging from 5-200 µM, was presented as micelles.
1, purified from bovine brain (26), was a gift of Dr.
S.G. Rhee (National Institute of Health, Bethesda, MD). A preparation
enriched in EGF receptor tyrosine kinase was prepared from A431
plasma membrane by Triton X-100 extraction and passaged through a
wheat germ agglutinin column with N-acetyl-D glucosamine (32). The
eluted receptor preparation was dialyzed and used at 3 µg/ml to phosphorylate 0.6 µg/ml PLC
1 , in the presence of 20 µM ATP and 3 µM EGF at
4°C for 1 h (22). Its activity was assessed by incubating PLC
1 with
[32P]ATP to detect EGF- dependent 32P incorporation. PLC
1 activity was
assayed by using 6-12.5 ng PLC
1/60 µl reaction mixture, and with vesicles
containing 10 µM PIP2/0.02 µM [3H]PIP2/100 µM PE. The buffer conditions were as described above. The reaction was linear for at least 15 min
at 37°C. Unphosphorylated PLC
1 control was incubated with the kinase
preparation in the absence of ATP and EGF.
Results
in vivo, although
gelsolin (1) and CapG (see below) are predominantly inhibitory in vitro. We therefore examined the effect of
overexpression on PLC
, which is activated through the
bradykinin/heterotrimeric G protein pathway. 5 µM bradykinin stimulated ctrl cells to increase cytosolic Ca2+
in a sharp peak (Fig. 1 A). CapG clones had a reduced
Ca2+ response (Fig. 1 A and Table I). Likewise, the IP3 response was also attenuated. Bradykinin-stimulated ctrl
cells generated IP3 to a maximal level at 7 s (Fig. 1 B). The
CapG clones generated less IP3 at 7 and 15 s. Ca2+ signal
and IP3 response were inhibited to a similar extent, suggesting that CapG inhibited PLC
and Ca2+ signaling by a
similar mechanism (see Fig. 3 B).
Fig. 1.
Effect of CapG
overexpression on response
to bradykinin. Serum-
deprived fibroblasts were
stimulated with 5 µM bradykinin. (A) Ca2+ tracings from
a representative experiment
were shown. Fura 2 was used
as an indicator. Top, ctrl; bottom, gk15. Arrows indicate time of bradykinin addition.
(B) IP3 generation as a function of time after stimulation.
Cells were extracted with
PCA, and IP3 level was determined by a receptor binding assay. Results shown
were from a single representative experiment performed
in triplicate. Values were
mean ± SEM.
[View Larger Versions of these Images (10 + 18K GIF file)]
Fig. 3.
Effect of gelsolin and
CapG overexpression. (A) Ca2+
tracings. Cells were stimulated
with 5 µM bradykinin or 20 mM
NaF. Tracings were from a representative experiment. Arrows
indicate time of agonist addition. Ctrl, mock-transfected
cells; C1, gelsolin-overexpressing cells. (B) Comparing the effects of gelsolin and CapG overexpression. Cells were stimulated
with 5 µM bradykinin or 20 mM
NaF. Ca2+ spike and IP3 generation were measured. The peak
response of overexpressing cells
was expressed as a percent of
ctrl cells exposed to the same
stimulus. Results shown were
the average of two experiments.
Filled circles, Ca2+ signal after
bradykinin stimulation; open circles, IP3 generated in response
to bradykinin; triangles, Ca2+
signal after NaF treatment.
[View Larger Versions of these Images (6 + 18K GIF file)]
Fig. 2.
Western blotting of gelsolin-overexpressing clones.
(Top) Anti-mouse gelsolin (anti-mG), detecting endogenous
gelsolin. (Bottom) Anti-human gelsolin (anti-hG), detecting the
expression of human gelsolin in human gelsolin cDNA-transfected cells. ctrl, control-transfected NIH3T3 cells; C, gelsolin
overexpressing clones.
[View Larger Version of this Image (27K GIF file)]
activity per se was confirmed by quantitating IP3. Ctrl and overexpressing clones
had similar basal IP3 levels (~10 pmol/mg cell protein).
Gelsolin clones had reduced IP3 response (Fig. 3 B), and
the extent of inhibition correlated with that of the Ca2+ response. Overall, our results are consistent with the hypothesis that the gelsolin- and CapG-induced change in Ca2+
signal was due to PLC
inhibition.
hydrolysis of PIP2, and IP3-induced release of Ca2+ from intracellular stores. Ctrl cells
responded to as low as 0.1 µM bradykinin, and Ca2+ release was maximally stimulated at 1 µM (data not shown).
C5 had a very small Ca2+ peak, even at 50 µM bradykinin,
suggesting that the problem was unlikely to be due to decreased receptor affinity. To identify the step compromised by gelsolin overexpression, NaF was used to activate G proteins directly, bypassing receptor involvement. NaF induced a slow Ca2+ rise (Fig. 3 A), which had a smaller
amplitude than the bradykinin peak (320 ± 50 nM).
Gelsolin overexpression reduced the NaF response, suggesting that it acted at or downstream of the heterotrimeric G protein activation step (Fig. 3 A). However, gelsolin did not inhibit the NaF response to as great an extent as
the bradykinin response (Fig. 3 B). The Ca2+ peak for C4
was reduced to 1.8 and 47.8% of ctrl after bradykinin and
NaF stimulation, respectively. The difference may be due
to activation of different PLCs. NaF activates all heterotrimeric G proteins, while bradykinin activates the alpha
subunit (G
q/11) (11, 29). This was confirmed with pertussis toxin that inhibits Gi but not G
q/11. In ctrl cells, pertussis toxin reduced the NaF response by 45.7% but had minimal effect on the bradykinin response (data not shown).
Our result suggested that G
q/11-activated PLCs were more
sensitive to gelsolin inhibition than G
i-activated enzymes.
activity is directly attributable to gelsolin, a gelsolin NH2-terminal half polypeptide (45 kD, spanning gelsolin residues 1-406 [GS1-406])
that contains at least two PIP2-binding sites (17, 35) was
added to SLO-permeabilized ctrl cells. Under the permeabilizing conditions used, cells retained agonist responsiveness, as demonstrated previously for another type of cell
(21). Fig. 4 showed the result of a representative experiment. The mean IP3 response in the presence of 4 and 32 µM GS1-406 were 46.5 ± 4.0% (n = 8) and 6.4 ± 1.1%
(n = 3) of control, respectively. Thus, exogenous gelsolin
added to permeabilized cells mimicked the inhibitory effects of gelsolin overexpression.
Fig. 4.
Effects of exogenous gelsolin on the IP3 response of
permeabilized ctrl cells. Ctrl cells were permeabilized with SLO
for 4 min, incubated with gelsolin GS1-406 for 3 min, and stimulated with 10 µM bradykinin for 7 s. IP3 release was assayed.
Data shown were from a representative experiment, and each
point was performed in triplicate.
[View Larger Version of this Image (11K GIF file)]
4, an actin-binding protein that does not interact with
PIP2 (38), had no effect at 10 µM. (IP3 response was 91.7 and 101.8% of control after bradykinin stimulation in two
experiments.) These results suggested that inhibition of
IP3 generation in cells was linked to an increase in PIP2-binding proteins.
3, the predominant PLC in
NIH3T3 cells, did not leak out to a significant extent, as
would be consistent with its stable plasma membrane association. There was no difference in the leakage of PLC
3
between ctrl and C5.
Fig. 5.
Restoration of bradykinin response to gelsolin-overexpressing cells after SLO permeabilization. (A) Time course of leakage of cytosolic proteins. Ctrl cells in suspension were permeabilized at 37°C with SLO and centrifuged for 1 min at room temperature. Pellets and supernatants were electrophoresed on SDS-polyacrylamide gels and blotted with anti-mouse gelsolin, antiactin, or anti-PLC3. (B). IP3 generation after bradykinin stimulation. Ctrl cells and C5 were exposed to SLO for the amount of time indicated and then stimulated with bradykinin. The C5 Ca2+ peak was expressed as a percent of ctrl under identical conditions. Data shown were the means of
two separate experiments.
[View Larger Versions of these Images (13 + 46K GIF file)]
3 (Fig. 5 A). Recovery of bradykinin-stimulated PLC
activity indicates that reduced bradykinin responsiveness
in intact overexpressing cells is unlikely to be due to
bradykinin receptor defects. A similar conclusion was
reached using NaF to stimulate G proteins directly (Fig. 3
A). Therefore, we conclude that gelsolin depresses PLC
activity in intact C5, and reducing gelsolin concentration
relieves the inhibition.
and PLC
differentially, and whether inhibition
is due to PIP2 binding. Gelsolin GS1-406 that contains two
identified PIP2-binding sites increased Ca2+-activated PLC
at 1 µM and inhibited at higher concentrations (Fig. 6 A).
A biphasic effect has been reported previously (1), although the stimulatory phase was ignored. CapG stimulated and inhibited with a comparable dose response, consistent with its similar affinity as gelsolin for PIP2 (18).
Gelsolin also had a biphasic effect on PLC
phosphorylated with EGF receptor kinase. In contrast, gelsolin did
not increase the activity of nonphosphorylated PLC
(Fig.
6 B) and inhibited it more strongly. The differential effects
on phosphorylated and unphosphorylated PLC
, including
activation of the phosphorylated enzyme at low concentration, were similar to that reported previously for profilin
(10). A direct comparison between gelsolin and profilin
showed that gelsolin was a stronger inhibitor (data not shown), consistent with their different affinity for PIP2 (18, 20). Although gelsolin was reported to bind PLC
(1), we
were not able to detect such an interaction either with purified proteins or in cell lysates (data not shown). Therefore, gelsolin is not likely to act by binding PLC directly.
Fig. 6.
Effect of gelsolin
on PLC activity in vitro. (A)
PLC1 was activated with 11 µM Ca2+, and IP3 release
from PIP2/PE/PS vesicles after 1 min was determined. The specific activity was 5.48 µmol/mg/min. Enzyme activity without added gelsolin
was expressed as 100%, after
background subtraction. (Background was less than
10% without added Ca2+.)
(B) PLC
with and without
EGF receptor (EGFR) tyrosine kinase activation. PLC
was incubated with EGF receptor kinase in the presence
and absence of ATP and
EGF. The activity of the phosphorylated and unphosphorylated enzymes were assayed in the presence of increasing amounts of gelsolin.
IP3 release from PIP2/PE/PS vesicles after 4 min was expressed as percent of control
(in the absence of gelsolin).
The specific activity after
background subtraction was
3.74 µmol/mg/min. (C) Effect of increasing PIP2 micelle concentration on the
ability of gelsolin to inhibit
PLC
. IP3 released into the
supernatant after 15 min was
plotted as a function of PIP2
concentration.
[View Larger Versions of these Images (14 + 17K GIF file)]
more than phosphorylated PLC
. 2 µM gelsolin stimulated phosphorylated PLC
but was located on the inhibitory arm of the
PLC
dose-response curve (Fig. 6, A and B). Stimulation
of phosphorylated PLC
may contribute to the increased
PDGF response in clones overexpressing low levels of
CapG and in the single low-level gelsolin-overexpressing clone (C3). The higher sensitivity of PLC
to inhibition may explain why PLC
and PLC
were affected in opposite directions in CapG clones and why the gelsolin clones
showed more inhibition of PLC
than PLC
. Thus, the differential inhibitory effects of gelsolin on PLC
and PLC
in vitro recapitulated the in vivo effects of overexpression.
generated
more IP3 as the substrate concentration increased. 5 µM
GS1-406 completely inhibited PLC
activity at 5 and 10 µM PIP2 but was progressively less effective at higher PIP2
concentration (Fig. 6 C). No inhibition was observed at
PIP2 concentrations above 120 µM. The simplest interpretation was that gelsolin inhibited PLC
by substrate competition, as suggested for PLC
in an earlier study (1).
Fig. 7.
Gelsolin-modulated PLC1 activity through PIP2 binding. (A) Gelsolin NH2-terminal domains (GS1-406) containing
none or between one to three PIP2-binding sites. Gelsolin segments 1, 2 and 3 are as defined in reference 17. *PIP2-binding
sites that have been localized. ?, suspected PIP2-binding site that has not yet been located precisely. (B) Effects of gelsolin domains on PLC
1. (C) Effect of P2 on PLC
1. In B and C, enzyme activity was assayed for 1 min and expressed as percent of control in the absence of exogenous protein.
[View Larger Versions of these Images (16 + 15K GIF file)]
by more than threefold at 53 µM (Fig.7 C). We were
not able to use more P2 to determine if higher concentrations were inhibitory. P2 may stimulate by organizing PIP2
in a more favorable conformation for PLC hydrolysis. It
may be more stimulatory than the parent S2 domain because its positive effects are not neutralized by steric hindrance to block PLC access. However, in contrast to the
marked stimulation in vitro, P2 had minimal effect on
PLC
activity in permeabilized cells. Semiintact cells treated
with 100 µM P2 showed 72.5 ± 6.7% (n = 9) of the bradykinin-induced IP3 response of untreated cells. No stimulation was observed between 5-100 µM (data not shown).
We cannot explain why P2 had divergent effects in vitro
and in vivo.
Discussion
strongly support
the conclusion that gelsolin and CapG bind PIP2 in vivo to
inhibit PLC
. There are, however, differences with respect
to PLC
. At an apparently similar level of overexpression,
CapG clone gK8 and gelsolin clone C6 had increased and
decreased response to PDGF, respectively. The divergent
effects of gelsolin and CapG may be due to multiple factors. CapG has one PIP2-binding site, while gelsolin has
two (37). CapG is half the size of gelsolin (36). Thus,
CapG may be more readily displaced by activated PLC
and may not sterically block PLC
access to PIP2 as effectively. CapG is phosphorylated (23), and phospho-CapG
may have a different affinity for PIP2 (Lu, P., A. Hsu, D. Wong, H. Yan, H.L. Yin, and C. Chen, manuscript submitted for publication). Another possibility may be different
intracellular localization. CapG is a nuclear and cytosolic
protein, while gelsolin is excluded from the nucleus (24).
There is emerging evidence for phosphoinositide signaling pathways in nuclei (Lu, P., A. Hsu, D. Wong, H. Yan, H.L.
Yin, and C. Chen, manuscript submitted for publication),
and CapG may therefore act in the nucleus.
and PLC
towards gelsolin and CapG. This may be explained in several ways. Phosphorylated PLC
may have a higher affinity for PIP2 than PLC
and is therefore better able to compete with gelsolin for PIP2. Differential sensitivity may
account for the paradoxical finding that CapG-overexpressing cells have increased PLC
(31) but reduced PLC
activity. Another possibility is that PLC isozymes may use
different pools of PIP2, and gelsolin/CapG have differential access to these pools. PIP2 pools with distinct turnover
characteristics have been identified (25), although their relation to the different PLC isozymes, gelsolin and CapG, has not been examined.
Received for publication 3 October 1996 and in revised form 19 June 1997.
Address all correspondence to Helen L. Yin, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9040. Tel.: (214) 648-7967. Fax: (214) 648-8685. e-mail: YIN01@UTSW. SWMED.EDUWe are grateful to David Kwiatkowksi and Casey Cunningham for the
gelsolin overexpressing cell lines, Sue Goo Rhee for PLC1 and Paul
Sternweis for anti-PLC
3. We thank Elliot Ross and Gloria Biddlecome
(University of Texas Southwestern Medical Center) for initiating the
PLC
1 assays and providing for recombinant PLC
1.
This work was supported by the National Institutes of Health grant GM51112.
C, gelsolin-overexpressing lines; ctrl, control clones; IP3, inositol 1,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; SLO, streptolysin O.
1. |
Banno, Y.,
T. Nakahima,
T. Kumada,
K. Ebisawa,
Y. Nonomura, and
Y. Nozawa.
1992.
Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.
J. Biol. Chem.
267:
6488-6494
|
2. | Barkalow, K., W. Witke, D.J. Kwiatkowski, and J.H. Hartwig. 1996. Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein. J. Cell Biol. 134: 389-399 [Abstract]. |
3. | Barkalow, K., W. Witke, D.J. Kwiatkowski, and J.H. Hartwig. 1996. Coordinated regulation of platelet actin filament barbed ends by gelsolin and capping protein. J. Cell Biol. 134: 389-399 [Abstract]. |
4. |
Biddlecome, G.H.,
G. Berstein, and
E.M. Ross.
1996.
Regulation of phospholipase C-![]() |
5. | Bradford, M.M.. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Annu. Rev. Biochem. 72: 248-254 . |
6. | Chaponnier, C., H.L. Yin, and T.P. Stossel. 1987. Reversibility of gelsolin/ actin interaction in macrophages. Evidence of Ca2+-independent pathways. J. Exp. Med. 165: 97-106 [Abstract]. |
7. | Chaponnier, C., H.L. Yin, and T.P. Stossel. 1987. Reversibility of gelsolin/ actin interaction in macrophages. Evidence of Ca2+-dependent and Ca2+-independent pathways. J. Exp. Med. 165: 97-106 [Abstract]. |
8. | Chen, P., J.E. Murphy-Ullrich, and A. Wells. 1996. A role for gelsolin in actuating epidermal growth factor receptor-mediated cell motility. J. Cell Biol. 134: 689-698 [Abstract]. |
9. | Cunningham, C.C., T.P. Stossel, and D.J. Kwiatkowski. 1991. Enhanced motility in NIH 3T3 fibroblasts that overexpress gelsolin. Science (Wash. DC). 251: 1233-1236 |
10. | Goldschmidt-Clermont, P.J., J.W. Kim, L.M. Machesky, S.G. Rhee, and T.D. Pollard. 1991. Regulation of phospholipase C-y1 by profilin and tyrosine phosphorylation. Science (Wash. DC). 251: 1231-1233 |
11. |
Gutowski, S.,
A. Smrcka,
L. Nowak,
D. Wu,
M. Simon, and
P.C. Sternweiss.
1991.
Antibodies to the ![]() |
12. | Hartwig, J.H., K.A. Chambers, and T.P. Stossel. 1989. Association of gelsolin with actin filaments and cell membranes of macrophages and platelets. J. Cell Biol. 108: 467-479 [Abstract]. |
13. | Hartwig, J.H., G.M. Bokoch, C.L. Carpenter, P.A. Janmey, L.A. Taylor, A. Toker, and T.P. Stossel. 1995. Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell. 82: 643-653 |
14. | Janmey, P.A.. 1994. Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly. Annu. Rev. Physiol. 56: 169-191 |
15. |
Janmey, P.A., and
T.P. Stossel.
1989.
Gelsolin-polyphosphoinositide interaction.
J. Biol. Chem.
264:
4825-4831
|
16. |
Janmey, P.A.,
J. Lamb,
P.G. Allen, and
P.T. Matsudaira.
1992.
Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin.
J. Biol. Chem.
267:
11818-11823
|
17. | Kwiatkowski, D.P., T.P. Stossel, S.H. Orkin, J.E. Mole, H.R. Colten, and H.L. Yin. 1986. Plasma and cytoplasmic gelsolins contain a duplicated actin-binding domain. Nature (Lond.). 323: 455-458 |
18. | Lin, K., E. Wenegieme, P. Lu, C. Chen, and H.L. Yin. 1997. Gelsolin binding to phosphatidylinositol 4,5 bisphosphate is modulated by calcium and pH. J. Biol Chem. In press. |
19. | Lind, S.E., P.A. Janmey, C. Chaponnier, T.-J. Herbert, and T.P. Stossel. 1987. Reversible binding of actin to gelsolin and profilin in human platelet extracts. J. Cell Biol. 105: 833-842 [Abstract]. |
20. | Lu, P., W. Shieh, S.G. Rhee, H.L. Yin, and C. Chen. 1996. Lipid products of phosphoinositide 3-kinase bind human profilin with high affinity. Biochemistry. 35: 14027-14034 |
21. | Muallem, S., K. Kwiatkowska, X. Xu, and H.L. Yin. 1995. Actin filament disassembly is a sufficient final trigger for exocytosis in nonexcitable cells. J. Cell Biol. 128: 589-598 [Abstract]. |
22. |
Nishibe, S.,
M.I. Wahl,
S.M.T. Hernandez-Sotomayor,
N.K. Tonks,
S.G. Rhee, and
G. Carpenter.
1990.
Increase of the catalytic activity of phospholipase C-![]() |
23. |
Onoda, K., and
H.L. Yin.
1993.
gCap39 is phosphorylated. Stimulation by
okadaic acid and preferential association with nuclei.
J. Biol. Chem.
268:
4106-4112
|
24. | Onoda, K., F.-X. Yu, and H.L. Yin. 1993. gCap39 is a nuclear and cytoplasmic protein. Cell Motil. Cytoskel. 26: 227-238 |
25. |
Pike, L.J., and
L. Casey.
1996.
Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains.
J.
Biol. Chem.
271:
26453-26456
|
26. | Rhee, S.G., S.H. Ryu, K.Y. Lee, and K.S. Cho. 1991. Assays of phosphoinositide-specific phospholipase C and purification of isozymes from bovine brains. Methods Enzymol. 197: 502-511 |
27. | Shaw, G.. 1996. The pleckstrin homology proteins: an intriguing multifunctional protein module. BioEssays. 18: 35-46 |
28. | Singh, S.S., A. Chauhan, N. Murakami, and V.P. Chauhan. 1996. Profilin and gelsolin stimulate phosphatidylinositol 3-kinase activity. Biochemistry. 35: 16544-16549 |
29. | Smrcka, A.V., J.R. Hepler, K.O. Brown, and P.C. Sternweis. 1991. Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq. Science (Wash. DC). 251: 804-807 |
30. | Steed, P.M., S. Nagar, and L.P. Wennogle. 1996. Phospholipase D regulation by a physical interaction with the actin-binding protein gelsolin. Biochemistry. 35: 5229-5237 |
31. | Sun, H.-Q., K. Kwiatkowska, D.C. Wooten, and H.L. Yin. 1995. Effects of CapG overexpression on agonist-induced motility and second messenger generation. J. Cell Biol. 129: 147-156 [Abstract]. |
32. |
Wahl, M.I.,
G.A. Jones,
S. Nishibe,
S.G. Rhee, and
G. Carpenter.
1992.
Growth factor stimulation of phospholipase C-y1 activity. Comparative
properties of control and activated enzymes.
J. Biol. Chem.
267:
10447-10456
|
33. | Yin, H.L., and T.P. Stossel. 1980. Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent protein. Nature (Lond.). 218: 583-586 . |
34. | Yin, H.L., K. Iida, and P.A. Janmey. 1988. Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the side of actin filaments. J. Cell Biol. 106: 805-812 [Abstract]. |
35. |
Yu, F.,
D. Zhou, and
H.L. Yin.
1991.
Chimeric and truncated gCap39 elucidate the requirements for actin filament severing and end capping by the
gelsolin family of proteins.
J. Biol. Chem.
266:
19269-19275
|
36. | Yu, F.-X., P.A. Johnston, T.C. Sudhof, and H.L. Yin. 1990. gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein. Science (Wash. DC). 250: 1413-1415 |
37. |
Yu, F.-X.,
H.-Q. Sun,
P.A. Janmey, and
H.L. Yin.
1992.
Identification of a
polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin.
J. Biol. Chem.
267:
14616-14621
|
38. |
Yu, F.-X.,
S.-C. Lin,
M. Morrison-Bogorad,
M.A.L. Atkinson, and
H.L. Yin.
1993.
Thymosin ![]() ![]() |