Inorganic phosphate regulates the binding of cofilin to
actin filaments
Andras Muhlrad
1
, Dmitry Pavlov
2
, Y. Michael Peyser
1
and Emil Reisler
2
1 Institute of Dental Sciences, School of Dental Medicine, Hebrew University of Jerusalem, Israel
2 Department of Chemistry and Biochemistry and the Molecular Biology Institute, University of California, USA
Actin dynamics, the polymerization and depolymeriza-
tion of actin filaments and formation of ordered actin
assemblies, is critical to many events of cell motility,
including the movement of whole cells, cell division,
vesicular transport and exo- and endocytosis. The
essential processes of actin dynamics are closely regula-
ted in the cell by a large number of actin binding pro-
teins and small molecules. The large family of actin
depolymerizing factor (ADF) ⁄ cofilin (AC) proteins [1]
has a central role in regulating actin dynamics. These
small proteins, which are ubiquitous in all eukaryotic
cells, increase the depolymerization and nucleation of
actin filaments and accelerate their treadmilling [2].
AC proteins accelerate the turnover of actin filaments
by severing them [3–6], thereby increasing the number
of the free pointed and barbed ends, or by increasing
monomer dissociation from the pointed end of fila-
ments [7], and ⁄ or by both processes [8–10]. The action
of these proteins on actin is promoted in most cases by

(Received 3 January 2006, accepted 8
February 2006)
doi:10.1111/j.1742-4658.2006.05169.x
Inorganic phosphate (Pi) and cofilin ⁄ actin depolymerizing factor proteins
have opposite effects on actin filament structure and dynamics. Pi stabilizes
the subdomain 2 in F-actin and decreases the critical concentration for
actin polymerization. Conversely, cofilin enhances disorder in subdomain 2,
increases the critical concentration, and accelerates actin treadmilling. Here,
we report that Pi inhibits the rate, but not the extent of cofilin binding to
actin filaments. This inhibition is also significant at physiological concen-
trations of Pi, and more pronounced at low pH. Cofilin prevents conforma-
tional changes in F-actin induced by Pi, even at high Pi concentrations,
probably because allosteric changes in the nucleotide cleft decrease the
affinity of Pi to F-actin. Cofilin induced allosteric changes in the nucleotide
cleft of F-actin are also indicated by an increase in fluorescence emission
and a decrease in the accessibility of etheno-ADP to collisional quenchers.
These changes transform the nucleotide cleft of F-actin to G-actin-like.
Pi regulation of cofilin binding and the cofilin regulation of Pi binding to
F-actin can be important aspects of actin based cell motility.
Abbreviations
AC, ADF ⁄ cofilin; ADF, actin depolymerizing factor; D-loop, DNase I binding loop; K
sv
,K
sv
¼ [(F
0
⁄ F))1]Æquencher M
)1
, where F
0

mers contain ADP and there is no cap at the barbed
end. ADP–F-actin is less stable and has a high critical
concentration for polymerization (reviewed in [24]). Pi
from the medium can bind stoichiometrically to the
nucleotide cleft of ADP–F-actin protomers and ADP–
G-actin if the Pi concentration in the solution is high
enough. The K
d
for Pi in ADP–F-actin protomers is
1.5 mm at pH 7.0 [23], while in G-ADP-actin the K
d
for
Pi is an order of magnitude higher [25]. Because K
d
increases with increasing pH [23], it was concluded that
the actin bound Pi species is H
2
PO
4
–
[23]. Pi lowers sig-
nificantly the critical concentration for polymerization
of ADP-actin [26] by decreasing the rate of protomer
dissociation at both filament ends, and in particular at
the barbed end [23]. On the other hand, Pi has a negli-
gible effect on the critical concentration for polymeriza-
tion of F-actin in the presence of ATP, because of the
protective ATP or ADP–Pi cap at the barbed end of the
filament. However, Pi stabilizes F-actin structure both
in the absence (ADP–F-actin) and presence of ATP

and slows, whilst BeFx completely prevents, the bind-
ing of actophorin to actin filaments. In addition, acto-
phorin was reported to promote the dissociation of Pi
from freshly polymerized F-actin [34]. However, no
attempt was made to study the effect of physiological
phosphate concentrations on cofilin binding to F-actin
at various pH values relevant to the regulation of
AC proteins.
In view of the important and antagonistic effects of
ADF ⁄ cofilin and phosphate on actin dynamics, we
examined here the binding of cofilin to F-actin and its
effect on F-actin structure in the presence of various
concentrations of Pi at pH 8.0 and 6.5. We also studied
the effect of cofilin on the nucleotide cleft of F-actin. We
used yeast cofilin because in its presence the critical con-
centration for actin polymerization is relatively low
(0.7 lm) even at pH 7.8 [7]. Thus, at high enough
F-actin concentrations, its depolymerization does not
significantly influence data analysis. To monitor cofilin
binding, we used fluorescence and proteolysis methods.
TRC-labeled F-actin shows a large fluorescence inten-
sity decrease upon cofilin binding [21], while subdo-
main 2 cleavage by subtilisin and trypsin is greatly
accelerated by cofilin, independently of the increased fil-
ament treadmilling [21]. We found here that the rate of
cofilin binding is strongly inhibited by Pi even at physio-
logical Pi concentrations. Cofilin greatly reduces the
affinity of Pi to the nucleotide cleft because of conform-
ational changes, which render the cleft of F-actin pro-
tomers G-actin like. The mutual regulation of cofilin

binding was pH independent between pH 6.5 and 8.0
(Table 1). We also checked the effect of phosphate on
the fluorescence emission spectrum of TRC–F-actin
(data not shown). We found that except for a very
small red shift, Pi essentially did not affect the spec-
trum of the TRC moiety on the D-loop of F-actin.
The incubation time (1–24 h) of TRC–F-actin with Pi
had no effect on cofilin binding (data not shown).
Effect of cofilin on Pi binding to F-actin and on
the conformation of the nucleotide binding cleft
Although Pi inhibited the rate of cofilin binding to
F-actin, we could not detect any displacement of cofilin
by even 30 mm Pi, using either TRC–F-actin fluores-
cence or subtilisin digestion assays (data not shown).
This indicates very low affinity of Pi to cofilin-occupied
F-actin. It is plausible that cofilin induced changes in the
nucleotide binding cleft are responsible for a reduction
of Pi affinity to F-actin. To test whether such changes
indeed occur in F-actin, we examined the fluorescence
emission of etheno-ADP (e-ADP) on actin, with and
without the bound cofilin. Figure 2A shows a 54%
fluorescence increase upon binding of cofilin to F-actin,
confirming nucleotide cleft perturbation. Addition of
cofilin to G-actin increased slightly (6.3%) the fluores-
0.1
0.2
0.3
0.4
0.5
01020304050

strength was equalized in all solutions with NaCl or KCl at pH 8.0
and 6.5, respectively. Cofilin (5.0 l
M) was added and the time
course of fluorescence intensity change was monitored in a
stopped-flow instrument at 20 °C, as described in Experimental
procedures. (A), pH 8.0; (B), pH 6.5.
Table 1. Effect of Pi on the rate constants of cofilin binding to
TRC–F-actin at pH 8.0 and 6.5. Data were obtained by fitting the
binding curves in Fig. 1 to mono-exponential expression. All rates
were normalized to the rate determined in the absence of Pi at the
same pH.
pH
Pi concentration
(m
M)
Rate constants of
cofilin binding (s
)1
)
Normalized
rates (%)
8.0 0 0.1924 100
2 0.1261 65.5
30 0.0444 23.0
6.5 0 0.8676 100
2 0.1283 14.8
30 0.0438 5.0
Pi regulates cofilin binding to F-actin A. Muhlrad et al.
1490 FEBS Journal 273 (2006) 1488–1496 ª 2006 The Authors Journal compilation ª 2006 FEBS
cence of e-ATP in the nucleotide cleft (data not shown).

F-actin (10 lm) was digested with subtilisin in the
presence and absence of 12 lm cofilin and 30 mm Pi
(Fig. 4). The digestion was started 30 s after the
0
100000
200000
300000
A
380 420 460
500
Wavelength (nm)
Fluorescence (A.U.)
8
µ
M cofilin
no cofilin
B
01020304050
0.95
1.00
1.05
1.10
1.15
1.20
Nitromethane (m
M)
F
o
/


at pH 6.5 the acceleration of the cleavage was lar-
ger in the absence of Pi than in its presence. We
also studied the effect of incubation time of
Pi–F-actin with cofilin on the rate of subtilisin diges-
tion at pH 8.0 and 6.5 (Fig. 5). At pH 8.0, after
3 min incubation with cofilin, the cleavage rate of
Pi–F-actin became as fast as that of F-actin without
Pi, and 20 s incubation with cofilin was enough to
almost completely activate the subtilisin digestion
(Fig. 5A). Similar results were obtained by trypsin
digestion of Pi–F-actin in the presence of cofilin at
pH 8.0 (data not shown). On the other hand, at
pH 6.5 the subtilisin cleavage of Pi–F-actin was still
inhibited after 20 s, and even 180 s, incubation
with cofilin (Fig. 5B). The rate of cofilin-activated
subtilisin cut of Pi–F-actin is faster at pH 8.0 than
at pH 6.5, while the binding rates of cofilin at
these pH values, as monitored by TRC fluorescence,
are equal.
Discussion
Cofilin and phosphate are important regulators of
actin dynamics in the cell. Their effects on the struc-
ture of actin filaments are antagonistic to each other.
Cofilin, and AC proteins in general, disorder the
Fig. 4. Effect of cofilin on the subtilisin digestion of Pi-TRC–F-actin
at pH 8.0 and 6.5. TRC–F-actin (10 l
M) was incubated with 30 mM
NaPi or KPi in pH 8.0 or pH 6.5 F-buffer, respectively, for 1 h on
ice. The ionic strength was equalized in all solutions with NaCl or
KCl at pH 8.0 and 6.5, respectively. After 30 s incubation with

dissociation of Pi from actin filaments in the presence
of Acanthamoeba actophorin and inhibited binding of
this AC protein at high, nonphysiological Pi concen-
tration [34]. Here, we found that the rate but not the
extent of yeast cofilin binding to F-actin is inhibited
even at physiological concentrations of Pi at pH values
8.0 and 6.5, which promote and inhibit AC protein
induced actin depolymerization, respectively [4].
In agreement with Ressad et al. [37], we observed
that in the absence of Pi the binding of cofilin is faster
at low pH than at high pH. This is in contrast to cofi-
lin’s depolymerizing effect, which is stronger at high
pH. However, according to our observations, the rate
of cofilin binding to F-actin in the presence of
2–30 mm Pi is the same at pH 6.5 and 8.0. The influ-
ence of Pi on the relative rates of cofilin binding at
these two pH values can be explained by the finding
that the Pi species that binds to F-actin is H
2
PO
4
–
[23].
At pH 8.0 the main species of Pi is HPO
4
2–
, while at
pH 6.5 the H
2
PO

detected via TRC fluorescence data, because these
are unchanged by Pi (data not shown) and monitor
only cofilin binding to F-actin. The difference in
subtilisin digestion of F-actin at the two pH values
probably derives from the stronger binding of Pi at
pH 6.5 than at pH 8.0 (see above). It is also possible
that the effect of Pi (in the nucleotide cleft) on
F-actin structure is cooperative, as is the case for
the BeFx analog of Pi [31], and that the Pi coopera-
tivity is higher at pH 6.5 than at pH 8.0. It is more
difficult to detect any significant effect of Pi on the
preformed F-actin–cofilin complexes. High concentra-
tions of Pi (30 mm) cannot displace cofilin from
F-actin, probably because of further weakening of
the intrinsically weak binding of Pi to F-actin. This
may be due to cofilin induced allosteric changes in
the nucleotide binding cleft of actin. The cofilin
induced increase in the fluorescence and a decrease
in quenching of e-ADP (Fig. 2) also indicate nucleo-
tide cleft perturbation in F-actin. The latter indicates
that in the presence of cofilin the bound nucleotide
in F-actin becomes less accessible and probably more
buried in the F-actin structure. Notably, cofilin bind-
ing has an opposite effect on ADP and Pi in the
nucleotide binding cleft, i.e., it buries ADP and faci-
litates Pi release from the cleft. We may speculate
that the Pi release is promoted by opening of the
‘back door’ [38], while ADP becomes less accessible
through the closing of the ‘main door’ on the top of
the protomer or by some other mechanism. Similarly

hand, cofilin accelerates Pi dissociation from F-actin
[34] and prevents conformational changes that accom-
pany Pi binding. Thus, Pi, cofilin, and pH can intri-
cately regulate actin dynamics with a profound impact
on the actin based cell motility.
Experimental procedures
Reagents
TRC and e-ATP were obtained from Molecular Probes
(Eugene, OR). ATP, trypsin, soybean trypsin inhibitor, sub-
tilisin (Carlsberg), phenylmethylsulfonyl fluoride were pur-
chased from Sigma Chemical Co. (St Louis, MO). Bacterial
transglutaminase was a generous gift from K Seguro
(Ajimoto Co., Inc., Kawasaki, Japan).
Proteins
G-actin was prepared from back and leg muscles of rabbit
by the method of Spudich & Watt [42] and stored in G-buf-
fer containing 5.0 mm Tris ⁄ HCl, 0.2 mm CaCl
2
, 0.2 mm
ATP, 0.5 mm dithiotreitol, pH 8.0. F-actin was prepared
from G-actin by polymerizing it with 2.0 mm MgCl
2
. Yeast
cofilin was prepared as described previously [17]. The con-
centrations of cofilin and unlabeled skeletal muscle a-actin
were determined spectrophotometrically by using the extinc-
tion coefficients E
1%
280
¼ 9.2 and E

TRC and 0.18 mgÆmL
)1
bacterial transglutaminase in
G-buffer pH 8.0, at 22 °C for 2 h. Reagent excess was
removed on PD-10 filtration column (Amersham Pharmacia
Biotech Inc., Piscataway, NJ) equilibrated with G-buffer.
Preparation of e-ADP–F-actin
ATP in skeletal muscle G-actin was substituted with
e-ATP as follows. G-actin was passed through a desalting
column (Amersham, PD10) of Sephadex G-25 equilibrated
with ATP-free G-buffer. The eluted actin was supplemen-
ted with 20-fold molar excess of e-ATP and was incubated
for 1 h on ice. Excess e-ATP was removed from G-actin
by passing it through another PD10 column. Actin was
polymerized by addition of 2.0 mm MgCl
2
and during the
polymerization the actin-bound e-ATP was hydrolyzed to
e-ADP.
Fluorescence measurements
Fluorescence emission spectra were recorded in a PTI
spectrofluorometer (Photon Technology Industries, South
Brunswick, NJ), in G-buffer for G-actin, and in G-buffer
containing 2.0 mm MgCl
2
for F-actin. The excitation wave-
length for TRC and e-ADP was set at 544 and 350 nm,
respectively. For quenching of e-ADP and time course of
TRC fluorescence change the emission wavelength was set
at 420 and 583 nm, respectively. The time course of TRC

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1496 FEBS Journal 273 (2006) 1488–1496 ª 2006 The Authors Journal compilation ª 2006 FEBS