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INTRODUCTION |
Arp2/3-dependent actin polymerization plays a critical
role in controlling motile structures including membrane ruffling
(1-7). Two small G-proteins, Rac and Cdc42, regulate actin
polymerization and are responsible for the assembly of membrane ruffles
and filopodia, respectively (8). Recent work has elucidated the
essential role of the Arp2/3 complex in the Rac- or Cdc42-mediated
actin polymerization (6). Actin alone polymerizes rather slowly because of the rate-limiting process of nucleation. The Arp2/3 complex greatly
accelerates the nucleation process in a Rac- or
Cdc42-dependent way; Rac or Cdc42 activates the effector
proteins of WASP family proteins including N-WASP and WAVE (SCAR).
Together they activate the nucleation activity of the Arp2/3 complex
and induce rapid actin polymerization. The Arp2/3 complex also binds to
the side of actin filaments and initiates branched actin
polymerization, resulting in the formation of a dendritic actin network.
The appearance and extent of membrane ruffling vary widely depending on
cell types, as well as cellular conditions and external circumstances.
For example, fibroblasts generally show more vigorous ruffling than do
epithelial cells. Membrane ruffling is regulated by a variety of
external signals including serum or growth factors, extracellular
matrix, and cell-cell contacts (9-12). On the other hand, intrinsic
signals appear to control constitutive membrane ruffling of motile
cells. It is thus conceivable that, in addition to the small
G-proteins, other proteins may modulate Arp2/3-dependent actin polymerization. Candidates with such functions are actin-binding proteins present in membrane ruffles. For example, cortactin activates Arp2/3-dependent actin polymerization and stabilizes
branched actin polymerization in vitro (13-15).
Tropomyosin, on the other hand, has been shown to reduce
Arp2/3-dependent branching and branched nucleation (16).
Most recently, coronin has been reported to bind to the Arp2/3/VCA
complex and inhibit the de novo nucleation activity of
Arp2/3 (17).
Caldesmon is an actin-binding protein that is localized in stress
fibers, as well as in membrane ruffles (18, 19). In vitro,
caldesmon inhibits actin-activated myosin ATPase (20), and caldesmon
together with tropomyosin protects actin filaments from severing
activities of gelsolin and anneals gelsolin-severed actin filaments
(21, 22). These two activities are likely to be involved in regulating
the actomyosin contractility and stability of stress fibers,
respectively (23-28). It is not clear, however, what functions
caldesmon plays in membrane ruffles. We have examined the effects of
caldesmon on Arp2/3-dependent actin polymerization and
found that caldesmon inhibits the Arp2/3-dependent actin
nucleation process.
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MATERIALS AND METHODS |
Protein Purification--
Skeletal muscle G-actin was prepared
as described (29), and pyrene dye was conjugated as described (30). The
Arp2/3 complex was purified from HeLa cells using the method described
in Ref. 31, except that the incubation at 37 °C was omitted. The
GST1-tagged C-terminal region
of N-WASP (GST-VCA) was cloned by PCR using the N-WASP-expressing
baculovirus (a kind gift from Drs. Takenawa and Miki, Tokyo University)
as the template, expressed in bacteria, and purified as described (31).
Rat nonmuscle caldesmon was expressed in bacteria and purified as
described (32). Smooth muscle caldesmon was purified from chicken
gizzard by the method described in Ref. 33 with slight modification as
described (34). Cdc2 kinase was prepared as described previously (35).
Calmodulin and
-phosphatase were purchased from Sigma and New
England Biolabs (Beverly, MA), respectively.
Polymerization Assay--
Actin polymerization was measured by
pyrene fluorescence using a Perkin Elmer spectrofluorometer, LS-50B.
Mg-ATP G-actin (2 µM 5% pyrene-labeled actin) was mixed
with various proteins in polymerization buffer (final condition: 10 mM imidazole buffer, pH 7, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM DTT) at time 0. When we examined the effects of
Ca2+/calmodulin, 0.4 mM CaCl2 was
added instead of EGTA. The high concentration of DTT was included to
prevent the dimerization of caldesmon (36).
Phosphorylation of Caldesmon with cdc2 Kinase--
Smooth muscle
caldesmon was phosphorylated by cdc2 kinase as described (35). The
level of phosphorylation (determined by 32P incorporation)
was found to be 4.5 ± 0.5 mol/mol. In some experiments, phosphorylated caldesmon was dephosphorylated by the incubation with
-phosphatase at 30 °C for 30 min, which resulted in the decrease
of the incorporation to 0.8 ± 0.4 mol/mol.
-Phosphatase was
denatured by heat treatment, and dephosphorylated caldesmon was
recovered in a heat-stable fraction.
Actin Binding Assay--
F-actin was polymerized from Mg-ATP
G-actin (final concentration, 2 µM) in the polymerization
buffer (10 mM imidazole buffer, pH 7, 50 mM
KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM DTT, 4 µM phalloidin) for 40 min. After
polymerization, caldesmon (final concentration, 0.5 µM)
was added and incubated for 10 min, and then varying concentrations (5-20 nM) of Arp2/3 and GST-VCA (50 nM) were
added. The samples were incubated for 20 min at room temperature and
centrifuged in a Beckman Airfuge (26 p.s.i. × 20 min). Both pellets
and supernatants were suspended in SDS sample buffer and analyzed by
the Western blot method using mouse monoclonal antibody against Arp3
(BD Biosciences Pharmingen, San Diego, CA). A standard curve was made
by immunoblotting known concentrations of Arp2/3 complex with the same
antibody and used to quantitatively determine the levels of Arp3 using Kodak one-dimensional Image Analysis Software (Eastman Kodak Co., Rochester, NY).
Electron Microscopy--
The average length of actin filaments
at the equilibrium state (after pyrene fluorescence reached a plateau)
was measured by electron microscopy using negative staining technique
(36). More than 1000 actin filaments were measured for each
polymerization experiment.
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RESULTS |
Caldesmon Effectively Inhibits
Arp2/3-dependent Actin Polymerization--
We
first examined effects of caldesmon on Arp2/3-mediated actin
polymerization. Actin (2 µM), Arp2/3 (15 nM),
and VCA (50 nM) were mixed with varying concentrations
(0-2 µM) of either smooth muscle or nonmuscle caldesmon,
and polymerization was initiated by the addition of salt at time 0. As
Fig. 1 (A and B)
shows, the higher the concentration of caldesmon, the longer the delay in actin polymerization. To quantitate the effects of caldesmon, we
measured the delay times required to reach one-fifth of the final
fluorescence, as well as slopes at the half-maximal fluorescence (proportional to the number of barbed ends, assuming that caldesmon did
not change the elongation rate). As Fig.
2 shows, the delay time is increased from
100 to 200 s for smooth muscle caldesmon and to 210 s for
nonmuscle caldesmon. The delay is saturated at 0.25 µM
for smooth muscle caldesmon and at 0.5-1 µM for
nonmuscle caldesmon. The inhibition of the polymerization rates is
saturated at approximately 1 µM of either type of
caldesmon. It is noteworthy that caldesmon at a very low concentration
(50 nM) has more effect on the delay time than on the slope
of polymerization. The inhibition by caldesmon is at least as effective
as that reported for tropomyosin (16).

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Fig. 1.
Caldesmon delays Arp2/3-induced actin
polymerization. The effects of caldesmon on Arp2/3-induced actin
polymerization were examined by monitoring pyrene fluorescence. The
conditions were: 2 µM actin (5% pyrene labeled actin);
50 nM VCA; 15 nM Arp2/3 complex in 10 mM imidazole buffer, pH 7, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM DTT, and 0-2 µM caldesmon. Representative
data from three independent experiments are shown. A,
nonmuscle caldesmon. 0 µM, open circles; 0.05 µM, filled squares; 0.1 µM,
open triangles; 0.25 µM,
multiplication signs; 0.5 µM,
filled circles; 1 µM, open
squares; 2 µM, filled triangles.
B, smooth muscle caldesmon. 0 µM, open
circles; 0.05 µM, filled squares; 0.1 µM, open triangles; 0.25 µM,
multiplication signs; 0.5 µM, filled
circles; 1 µM, open squares; 2 µM, filled triangles. C, effects of
smooth muscle caldesmon (0.5 µM) on actin polymerization
in the absence of Arp2/3 or VCA. Filled squares, actin
alone; open squares, actin plus caldesmon. The other
conditions are the same as in A or B.
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Fig. 2.
Effects of caldesmon on maximum rates of
actin polymerization and the retardation time. The maximum
rates of polymerization (filled squares) were measured
by the slopes at half-maximum fluorescence obtained from the
experiments shown in Fig. 1. The retardation (open circles)
was measured by times required to reach at one-fifth of the final
fluorescence obtained from the experiments shown in Fig. 1.
A, nonmuscle caldesmon. B, smooth muscle
caldesmon.
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Without Arp2/3, the effect of caldesmon on actin polymerization was
minimal. The delay time was changed from 165 to 180 s when 0.5 µM of caldesmon was added in the absence of Arp2/3 (Fig. 1C). The difference was only 9%. In contrast, the same
concentration (0.5 µM) of caldesmon delayed
Arp2/3-dependent polymerization to a much greater extent
(from 99.4 to 193.2 s, corresponding to a 94% increase; see Fig.
2B). The polymerization rate (slopes at the half-maximal
fluorescence) also showed little change in the absence of Arp2/3;
caldesmon decreased the rate by merely 5%. In contrast, the same
concentration of caldesmon decreased the rate by 62% in the presence
of Arp2/3 (Fig. 2B). These results suggest that caldesmon
affects Arp2/3-dependent processes of actin polymerization.
The C terminus of caldesmon is indispensable for the inhibitory effect.
Although the N terminus of caldesmon contains a major myosin-binding
site, the C terminus has the actin-binding domains in addition to the
domains that bind to tropomyosin or calmodulin (37). We found that the
C-terminal half (Glu235-Val531 of rat
nonmuscle caldesmon) was able to show inhibitory effects on
Arp2/3-dependent actin polymerization similar to the
full-length molecule, whereas the N terminus showed no effects on actin
polymerization (results not shown). This result suggests that actin
binding ability is important for the inhibition.
Caldesmon Does Not Largely Alter Elongation Rates--
The
retardation of Arp2/3-dependent actin polymerization could
be caused by the reduction of the elongation rate from barbed ends, by
the inhibition of actin nucleation, or by both. We first examined
whether caldesmon slows the elongation from barbed ends of
Arp2/3-capped actin seeds. To make seeds, actin (2 µM,
without pyrene label) was polymerized in the presence of the Arp2/3
complex (20 nM) and VCA (50 nM). The seeds were
then diluted 10-fold and incubated with 0.3 µM
pyrene-labeled G-actin with or without caldesmon to monitor barbed end
polymerization. We conclude from the following reasons that this
assay reasonably tests the effects of caldesmon on elongation from
barbed ends. First, the concentration of G-actin is below the critical
concentration of pointed ends so that barbed end polymerization was
monitored (final concentrations: 0.2 µM F-actin seeds,
0.3 µM G-actin, with or without 0.5 µM
caldesmon). Second, caldesmon did not increase the annealing of
Arp2/3-capped actin filaments in this condition, and thus the number of
barbed ends was not changed with or without caldesmon. When
Arp2/3-capped actin filaments with two different colors were mixed with
or without caldesmon, the percentage of annealed actin filaments
(visualized by combined actin filaments with two colors) was the same,
indicating that caldesmon did not dissociate Arp2/3 complexes from
pointed ends. Third, no nucleation occurred under this condition. It
was possible that Arp2/3 and VCA used for making seeds could induce de novo nucleation. However, no actin polymerization was
observed within 200 s (Fig.
3A, filled circles)
when 0.5 µM G-actin was mixed with 2 nM
Arp2/3 and 5 nM VCA (the same concentrations used in this
assay). Fourth, caldesmon binding is faster than actin elongation at
the barbed ends. Because the binding of caldesmon to F-actin is rather
a fast reaction (k+ = 1 × 107
M
1 s
1) (38), most actin
filaments would be saturated with caldesmon before any significant
actin polymerization could occur at the free barbed ends; half of
caldesmon binds to actin in 0.1 s in this condition, whereas only
0.2% of actin seems to polymerize at the ends at the same time.

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Fig. 3.
Caldesmon does not significantly reduce the
rate of actin elongation. A, actin polymerization from
F-actin seeds in the presence or absence of caldesmon was monitored.
F-actin was first polymerized from 2 µM G-actin in the
presence of 20 nM Arp2/3 and 50 nM VCA for 15 min and used as F-actin seeds. Then the seeds were diluted 10 times
with pyrene labeled G-actin (final concentration of pyrene actin, 0.3 µM; F-actin seeds, 0.2 µM) with or without
caldesmon (final concentration of caldesmon, 0.5 µM).
Elongation from the seeds was immediately measured by an increase in
pyrene fluorescence. Open squares, actin alone; open
triangles, actin plus smooth muscle caldesmon; open
circles, actin plus nonmuscle caldesmon; closed
circles, control (actin polymerization without seeds; G-actin of
0.5 µM was incubated with 2 nM Arp2/3
and 5 nM VCA). Virtually no actin polymerization
occurred. The buffer conditions were the same as those of Fig. 1.
B, the initial elongation rates were calculated from the
slopes in A. The values were normalized as "actin alone"
to 100%.
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As Fig. 3B shows, caldesmon inhibits the elongation rates to
a small extent. The rates were decreased 20% by smooth muscle caldesmon and 10% by nonmuscle caldesmon. This small extent of inhibition, however, does not account for the large decreases in the
rates of actin polymerization by caldesmon as shown in Figs. 1 and 2 in
which the slope at half-maximum fluorescence was decreased by 62% by
0.5 µM caldesmon.
Caldesmon Reduces Nucleation Rates--
We then examined how
caldesmon affects the apparent nucleation rate, k*, and the
apparent elongation rate, k+. According to the
theory of actin polymerization devised by Oosawa and Asakura (39), the
nucleation rate k* is proportional to the inverse of
<i>*t, where <i> is the average
length of actin filaments at the equilibrium state, and t is
the time when a certain amount of monomers has polymerized. On the
other hand, the elongation rate k+ is
proportional to <i>/t.
Fig. 4 shows the time courses of actin
polymerization in the absence (panel A) or presence
(panel B) of caldesmon when the concentrations of the Arp2/3
complex were changed from 0 to 20 nM. At the equilibrium,
the lengths of more than 1000 actin filaments were measured by electron
microscopy, and the length distribution is shown as the histogram in
Fig. 5. It is clear that filament length
becomes longer when caldesmon is present. Table
I shows the average lengths,
<i>, of actin filaments, as well as times (t1/5) at which 20% of F-actin was polymerized
relative to the final level of F-actin. Both average lengths and
t1/5 become longer in the presence of caldesmon than
in its absence. We have then calculated relative changes in
k* and k+ by normalizing the rates,
k* and k+, of polymerization of actin
alone to 1.0. Fig. 6 shows the plots of
relative values of k* and k+
versus Arp2/3 concentrations.

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Fig. 4.
Effects of caldesmon on the kinetics of
Arp2/3-dependent actin polymerization. Actin
polymerization was measured in the presence (B) or absence
(A) of smooth muscle caldesmon (1 µM) at
varying concentrations of the Arp2/3 complex (0-20 nM).
Buffer conditions were the same as in Fig. 1. The Arp2/3 concentrations
were: 0 nM (open squares), 5 nM
(open triangles), 10 nM (open
circles), and 20 nM (multiplication signs).
The data are representative of three independent experiments.
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Fig. 5.
Histogram of length distribution of actin
filaments polymerized with or without caldesmon. After actin
polymerization reached equilibrium in the experiment shown in Fig. 4,
the lengths of actin filaments were determined by electron microscopy,
and distribution is shown as histograms. Open bars, without
caldesmon; filled bars, with caldesmon. Note that the
filament lengths become longer in the presence of caldesmon.
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Fig. 6.
Caldesmon reduces nucleation rates of
Arp2/3-dependent actin polymerization but not the
elongation rates. A, the relative nucleation rate
constants (k*) are calculated from Table I according to the
theory of Oosawa and Asakura on protein polymerization (39) and plotted
versus Arp2/3 concentrations. The rate constants are
normalized as actin alone to 1. Inset, the
Arp2/3-dependent increases in the nucleation rates
(k* 1) are proportional to the square of Arp2/3
concentrations. Closed squares, without caldesmon;
open squares, with caldesmon. B, the relative
elongation rate constants (k+) calculated from
Table I are plotted versus Arp2/3 concentrations. The
constants are normalized as in A. Closed squares,
without caldesmon; open squares, with caldesmon.
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These plots indicate that caldesmon greatly decreases the nucleation
rate, k* (Fig. 6A), but not the elongation rate,
k+ (Fig. 6B). As expected,
k* increased as the concentration of Arp2/3 increases (Fig.
6A). Importantly, k* in the presence of caldesmon
(open squares) is decreased to one-third of that in the
absence of caldesmon (filled squares). On the other hand, k+ (Fig. 6B) seems to be the same
whether caldesmon is present (open squares) or absent
(filled squares), although k+
decreases when the Arp2/3 concentration becomes high. The reason for
the decrease is not clear at this time. It is interesting to note that
the Arp2/3-dependent increase in k* is
proportional to the second power of Arp2/3 concentrations (Fig.
6A, inset). This relationship may be reflected by
the fact that Arp2/3 nucleation activity is greatly increased when the
Arp2/3 complex binds to the side of an actin filament (29, 40).
Caldesmon Does Not Interact with the Arp2/3
Complex--
The kinetic analyses shown above (Figs. 4-6) strongly
suggest that caldesmon inhibits the nucleation activity of Arp2/3
complexes. One possibility was that caldesmon might directly associate
with Arp2/3 complex or VCA and interfere the nucleation activity.
However, we were unable to detect the binding of caldesmon to the
Arp2/3 complex, VCA, or a VCA-Arp2/3 complex when the association was examined by a pull-down assay using affinity column chromatography or
by the surface plasmon resonance method (data not shown). This suggests
that caldesmon may indirectly inhibit the nucleation activity of Arp2/3 complex.
Caldesmon Inhibits Branched Nucleation of Arp2/3 by
Reducing Actin Binding of Arp2/3--
How does caldesmon
indirectly reduce the nucleation activity of Arp2/3? Arp2/3 is able to
nucleate actin in two ways. One is de novo nucleation
(without polymerized actin), and the other is branched nucleation via
binding of Arp2/3 to the side of F-actin filaments. Branched nucleation
has been reported to be far more effective than de novo
nucleation (29, 40). Because caldesmon binds to the side of actin
filaments, it is possible that caldesmon inhibits the latter nucleation
by inhibiting Arp2/3-F-actin binding.
To test this possibility, we performed the following two experiments.
We first asked whether caldesmon reduces the ability of F-actin seeds
as the secondary activator of Arp2/3 nucleation. F-actin seeds (final
concentration, 0.3 µM) were first mixed with or without
0.5 µM caldesmon, and then Arp2/3, VCA, and
pyrene-labeled G-actin were added to initiate polymerization. As
Fig. 7 shows, actin polymerization in the
presence of Arp2/3 occurred very rapidly with almost no lag time when
F-actin seeds without caldesmon were added (Fig. 7, filled
squares; compare the actin polymerization without F-actin seeds
shown in Fig. 1). The addition of caldesmon (Fig. 7A,
nonmuscle caldesmon; Fig. 7B, smooth muscle caldesmon) to
F-actin seeds, however, greatly reduces the ability of F-actin seeds (compare open squares with filled squares).
The effect is specific to Arp2/3-dependent actin
polymerization because, in the absence of Arp2/3, the addition of
either nonmuscle (Fig. 7A) or smooth muscle (Fig.
7B) caldesmon showed only small (for nonmuscle caldesmon) or
no (for smooth muscle caldesmon) effects on actin polymerization
(compare filled and open circles). These results
suggest that caldesmon inhibits branched nucleation mediated by the
binding of Arp2/3 to F-actin seeds. However, because there was an
excess amount of caldesmon in these assays, it is possible that free
caldesmon may also inhibit de novo nucleation of Arp2/3.

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Fig. 7.
Actin filaments with caldesmon are less
effective activator of Arp2/3. Actin polymerization using F-actin
seeds with or without nonmuscle (A) or smooth muscle
(B) caldesmon is shown. F-actin seeds were made by
polymerizing 6 µM Mg-ATP G-actin (without pyrene) in the
same buffer conditions as described in Fig. 1 with 12 µM
phalloidin for 30 min and used for seeds. For F-actin seeds with
caldesmon, 10 µM caldesmon was added after actin
polymerization and incubated for 10 min. F-actin seeds (final
concentration, 0.3 µM) with (open symbols) or
without (filled symbols) caldesmon (final concentration, 0.5 µM) were mixed with pyrene labeled G-actin (final
concentration, 2 µM G-actin, 5% pyrene-labeled) to start
actin polymerization. Squares, with Arp2/3 and GST-VCA;
circles, without Arp2/3 and GST-VCA. Filled
squares, F-actin seeds without caldesmon, with 15 nM
Arp2/3 and 50 nM GST-VCA. Open squares, F-actin
seeds with caldesmon, with 15 nM Arp2/3, and 50 nM GST-VCA. Filled circles, F-actin seeds
without caldesmon, without Arp2/3 or GST-VCA. Open circles,
F-actin seeds with caldesmon, without Arp2/3 or GST-VCA.
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We next examined whether caldesmon inhibits binding of Arp2/3 to
F-actin, because actin binding of Arp2/3 is required for branched
nucleation. Using co-sedimentation assays, we examined whether
caldesmon affects actin binding of Arp2/3. F-actin (2 µM)
with or without caldesmon (0.5 µM) was incubated with
varying concentrations of Arp2/3 (5-20 nM), and the levels
of Arp2/3 in F-actin pellets were determined by quantitative Western
blot method using the monoclonal antibody against Arp3. As Fig.
8 shows, the levels of bound Arp2/3 are
reduced to 30-60% of the controls, when actin was preincubated with
either nonmuscle or smooth muscle caldesmon. These results indicate
that caldesmon inhibits Arp2/3 binding to F-actin.

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Fig. 8.
Caldesmon reduces the affinity of Arp2/3 to
F-actin. A, a representative Western blot showing the
levels of F-actin-bound Arp3 in the presence or absence of caldesmon.
F-actin (2 µM) with or without caldesmon was incubated
with 5-20 nM of Arp2/3 and GST-VCA (50 nM) for
20 min. Then the mixtures were centrifuged to pellet F-actin. The
levels of Arp3 in F-actin pellets were detected by immunoblotting with
monoclonal antibody against Arp3. Control, without
caldesmon; +NM CaD, with 0.5 µM nonmuscle
caldesmon; +SM CaD, with 0.5 µM smooth muscle
caldesmon. The total amounts of Arp2/3 added to F-actin are indicated
below each lane. B, the levels of F-actin-bound
Arp2/3 were determined by scanning the Western blot shown in
A. A standard curve was made by blotting the series of known
concentrations of Arp2/3 on the same membranes and used to calculate
the levels of Arp2/3. The total amounts of Arp2/3 added to F-actin were
indicated below each lane. The error bars
represent the variations of three independent experiments.
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Reversal of the Inhibition of Caldesmon by
Ca2+/Calmodulin or by Phosphorylation with cdc2
Kinase--
The interaction of caldesmon with actin is regulated by
Ca2+/calmodulin or by phosphorylation of caldesmon with a
variety of kinases including cdc2 kinase (35, 41). Because actin
binding of caldesmon is important for its effects on Arp2/3, we
examined whether these agents regulate the effects of caldesmon on
Arp2/3 nucleation. As Fig. 9 shows,
Ca2+/calmodulin negated the effects of the inhibition of
caldesmon. Although caldesmon alone (filled triangles)
retarded Arp2/3-dependent actin polymerization greatly
(compare with filled squares), the addition of
Ca2+/calmodulin (open triangles) to caldesmon
almost completely reversed the inhibitory effect of caldesmon. As an
additional control, we examined the effect of calmodulin alone on
Arp2/3-dependent actin polymerization and found no
significant effect (open squares).

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Fig. 9.
Ca2+/calmodulin reverses the
effect of caldesmon on the Arp2/3-induced actin polymerization.
The effects of Ca2+/calmodulin on the inhibition of
caldesmon of the Arp2/3 actin polymerization were monitored by pyrene
fluorescence. The conditions were: 2 µM actin (5%
pyrene-labeled actin); 50 nM VCA; 15 nM Arp2/3
complex in 10 mM imidazole buffer, pH 7, 50 mM
KCl, 1 mM MgCl2, 0.4 mM
CaCl2, 10 mM DTT, and 0.5 µM
caldesmon. A, nonmuscle caldesmon; B, smooth
muscle caldesmon. Filled squares, Arp2/3-induced
polymerization; open squares, with Arp2/3 and calmodulin (2 µM) alone; open triangles, Arp2/3-induced
actin polymerization with caldesmon and Ca2+/calmodulin (2 µM); filled triangles, Arp2/3-induced
polymerization with caldesmon but without calmodulin.
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We also found that phosphorylation of caldesmon with cdc2 kinase
completely negates the inhibition of caldesmon (Fig.
10). Although unphosphorylated
caldesmon (Fig. 10, ×) greatly inhibited Arp2/3-dependent
actin polymerization, caldesmon phosphorylated by cdc2 kinase to 4-5
mol/mol showed no inhibition on Arp2/3-dependent actin
polymerization. The time course without caldesmon (filled squares) was identical to that with phosphorylated caldesmon
(open circles). The release of inhibition was
phosphorylation-specific; when caldesmon was dephosphorylated with
-phosphatase, caldesmon regained its inhibitory effect on
Arp2/3-dependent actin polymerization (filled
triangles). Dephosphorylation was not complete (phosphorylation level was 0.8 ± 0.4 mol/mol), which appears to explain the fact that dephosphorylated caldesmon showed less inhibition than
unphosphorylated caldesmon.

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Fig. 10.
Phosphorylation by cdc2 kinase inhibits the
effect of caldesmon on the Arp2/3-induced actin polymerization.
The effects of cdc2 phosphorylation on the inhibition of caldesmon of
the Arp2/3 actin polymerization were monitored by pyrene fluorescence.
The conditions were: 2 µM actin (5% pyrene labeled
actin); 50 nM VCA; 15 nM Arp2/3 complex in 10 mM imidazole buffer, pH 7, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM DTT. Filled squares, Arp2/3 only without
caldesmon; multiplication signs, with 0.5 µM
unphosphorylated smooth muscle caldesmon; open circles, 0.5 µM smooth muscle caldesmon phosphorylated with cdc2
(Pi incorporation was 4.5 ± 0.5 mol/mol.);
filled triangles, 0.5 µM -phosphatase
treated smooth muscle caldesmon. This caldesmon was first
phosphorylated with cdc2 then dephosphorylated by treatment with
-phosphatase (Pi incorporation was decreased to 0.8 ± 0.4 mol/mol).
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DISCUSSION |
Our data indicate that caldesmon reduces the nucleation rate of
the Arp2/3-dependent actin polymerization by inhibiting
actin binding of Arp2/3. Three lines of evidence support this
conclusion. First, kinetic analyses showed that caldesmon reduced the
nucleation rate, whereas it did not alter the elongation rate largely
(Fig. 6). Second, F-actin seeds bound with caldesmon were much less effective in activating nucleation by Arp2/3 than were F-actin seeds
without caldesmon (Fig. 7). Finally, caldesmon inhibited actin binding
of Arp2/3 (Fig. 8). These results strongly suggest that caldesmon
reduces the nucleation rate by inhibiting branched nucleation,
which is similar to the way of the inhibition reported for
tropomyosin (16). The inhibition of branched nucleation is probably
related to the way of actin binding of these two proteins; both
tropomyosin and caldesmon bind to the side of filaments. Although it
has not been shown that tropomyosin inhibits actin binding of Arp2/3,
side binding of tropomyosin is likely to reduce the actin binding
affinity of Arp2/3 as did caldesmon. Another possibility for the
inhibition of branching would be that caldesmon stimulates the
conversion of ATP-actin to ADP-actin. However, we did not detect any
stimulation of actin ATPase by caldesmon (data not shown).
We attempted to observe directly, by fluorescent microscopy, whether
caldesmon inhibits branched F-actin formations. However, we found that
this assay had the following problems, which made results unreliable.
First, the binding of caldesmon made F-actin filaments more rigid and
resistant to shearing force than F-actin without caldesmon. During
microscopic observation, we observed that branched F-actin without
caldesmon was frequently broken at its branch sites by shearing force,
whereas branched F-actin with caldesmon was preserved better under the
same conditions. Second, caldesmon tends to form a dimer in the absence
of reducing agents, and dimer caldesmon can cross-link actin filaments
(36). Although a high concentration of DTT reduced dimer formation of most but not all caldesmon, residual caldesmon dimers appear to generate loosely cross-linked actin filaments. Such loosely
cross-linked actin filaments were quite similar to branched filaments
under a microscope, again making accurate quantitation of branching quite difficult.
The inhibition of Arp2/3-induced actin polymerization by caldesmon is
likely to have physiological significance. First, caldesmon and Arp2/3
are co-localized in membrane ruffles. Second, the intracellular concentration of caldesmon appears high enough to affect
Arp2/3-dependent actin polymerization. For example,
fibroblasts contain ~2-8 µM caldesmon (32). Because
total actin concentration is approximately 100-200 µM,
the molar ratio of caldesmon to actin could be between 1:100 and 1:12,
in which range caldesmon was shown to affect actin polymerization in
this work (Figs. 1 and 2). Fourth, changes in caldesmon concentrations
appear to alter the motile activity of the peripheral membranes. For
example, caldesmon has been reported to be down-regulated in many
transformed cells (19, 26). This down-regulation is well correlated to
the increases of membrane ruffling upon cell transformation.
Furthermore, in our preliminary experiments, we found that
microinjection of caldesmon into normal rat kidney cells reduced
membrane ruffling movement considerably (data not shown).
Caldesmon may be a key molecule that could confer
phosphorylation-dependent or
Ca2+/calmodulin-dependent regulation of
Arp2/3-mediated actin polymerization. We have demonstrated that the
inhibition of caldesmon of Arp2/3-dependent actin
polymerization is released by phosphorylation with cdc2 kinase (Fig.
10) and by Ca2+/calmodulin (Fig. 9). This release is likely
to be caused by the inhibition of actin binding of caldesmon by these
agents. Although tropomyosin has been reported to inhibit
Arp2/3-dependent actin polymerization (16), it is mainly
localized in stress fibers, and the actin binding of tropomyosin is not
regulated like caldesmon. Because other kinases including MAPK (43) and
protein kinase C (44) were also reported to regulate the actin binding
of caldesmon, caldesmon may play a role in motile phenomena in a
variety of signal transduction pathways.
For example, growth factors like PDGF are known to induce membrane
ruffles. PDGF also causes caldesmon phosphorylation by MAPK. Because
the phosphorylation sites of caldesmon by MAPK partially overlap with
those by cdc2 kinase, phosphorylation with MAPK is likely to reverse
the inhibition of caldesmon on Arp2/3 nucleation. Indeed, our
preliminary data showed that caldesmon phosphorylated by MAPK lost its
inhibition of Arp2/3-dependent actin polymerization (data
not shown). Thus, although growth factors like PDGF activate small
G-proteins such as Cdc42 and Rac, which in turn initiate membrane
ruffles, phosphorylation of caldesmon by MAPK would enhance Arp2/3-induced actin polymerization, leading to more vigorous membrane
ruffling and cell movement during growth factor treatment (45, 46).
This notion appeared to be supported by a report showing that caldesmon
phosphorylation by MAPK is involved in PDGF-stimulated cell migration
of smooth muscle cells (46).
Caldesmon phosphorylation by cdc2 kinase may also have physiological
significance. It has been recently reported that Arp2/3 plays an
important role in the completion of cytokinesis in yeast (47) as well
as Drosophila (42). We have shown here that phosphorylation of caldesmon by cdc2 kinase completely abolished the inhibition of
caldesmon on Arp2/3 (Fig. 10). Because our previous study has shown
that caldesmon is phosphorylated by cdc2 kinase during mitosis (35,
41), it is possible that the release of the inhibition of caldesmon may
be involved in Arp2/3-mediated assembly of contractile rings. Future
studies should be conducted to define how the regulation of
actin-caldesmon binding controls actin polymerization and cell motility
during signal transduction.