Department of Cell Biology, The Tokyo Metropolitan Institute of Medical
Science, Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan.
Present address: Medical & Biological Laboratories Co., Ltd., 1063-103,
Ohara, Terasawaoka, Ina-city, 396-0002, Japan
* Author for correspondence (e-mail: moriyama{at}rinshoken.or.jp )
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Summary |
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Key words: Actin, Adenylyl cyclase-associated protein, Cofilin/ADF, Cell motility, Actin-interacting protein 1
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Introduction |
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CAP/Srv2 was first identified in S. cerevisiae as a binding
partner of adenylyl cyclase, Cyr1, which is activated by a small G-protein,
Ras (Field et al., 1990;
Fedor-Chaiken et al., 1990
).
CAP has three domains. The N-terminal domain binds to Cyr1 and is involved in
its Ras-responsiveness (Gerst et al.,
1991
; Mintzer and Field,
1994
; Nishida et al.,
1998
). The C-terminal domain binds to G-actin and strongly
inhibits actin polymerization (Freeman et
al., 1995
). The precise function of the internal, proline-rich
domain of CAP is still unclear. Mammals have at least two CAP homologues (CAP1
and CAP2). Both CAP1 and CAP2, like CAP/Srv2, have been reported to bind to
G-actin at their C-terminal domains and not at their N-terminal domains
(Hubberstey et al., 1996
).
Recently, a CAP homologue of Drosophila has been reported to play a
role in developmental morphogenesis, probably through its effect on the actin
cytoskeleton (Benlali et al.,
2000
; Baum et al.,
2000
).
We found two proteins, p65 and p55, in relative abundance in the
actin-cofilin complex of a human cell extract, and we determined p55 to be
CAP1. Partial sequencing of the p65 peptides revealed that it is a homologue
of actin-interacting protein 1, Aip1 (K.M. and I.Y., unpublished). Several
lines of evidence indicate that Aip1 homologues assist cofilin through lateral
association with F-actin (Iida and Yahara,
1999; Okada et al.,
1999
; Aizawa et al.,
1999
; Rodal et al.,
1999
). Thus, CAP1 may also support cofilin function. Here, we have
characterized human CAP1, and its N- and C-terminal domains, in terms of its
ability to regulate actin dynamics and co-operate with cofilin. Our novel
findings demonstrate that CAP1 effectively recycles actin and cofilin, thereby
allowing a rapid turnover of actin filaments, which is an essential driving
force behind cell motility.
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Materials and Methods |
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pQE63H was constructed by replacing the NcoI/Bpu1102 I-portion of pQEH-cof with a polylinker sequence: 5'-CCATGGCTAGCCATCATCACCACCATCACGGCtctagaGTCGACCTGCAGGCATGCAAGCTTCGACCTCGAGGGGGGGCCCGGTACCCGGGGATCCAGATCAGCTTAATTAGCTGAGC-3', in which the italicized sequence encodes for a His-tag and the lower case letters represent a XbaI site. This operation eliminated any cofilin-encoding sequences. Another vector, pHSE63, was made by replacing the NcoI/XbaI portion of pQE63H with the S-tag-encoding NcoI/EcoRI-170 bp of pBAC-2cp (Novagen, Madison, WI), in which GAATtctaga was the junction between the EcoRI- and XbaI sites.
I.M.A.G.E. Consortium human EST clones zr28g06 (encoding CAP1) and au45b04
(encoding CAP2) were obtained from Genome Systems (St. Louis, MO). The 690 bp
cDNA fragment encoding the N-terminal domain of CAP1 (CAP1-NT) was substituted
for the NheI/BamHI portion of pQE-cof.His. This product,
pQE-CAP1-N229. His, directed the efficient expression of 229 CAP1 N-terminal
residues in E. coli. The 670 bp fragment encoding the C-terminal
domain of CAP1 (CAP1-CT) was transferred into XbaI/HindIII
sites of pHSE63, thereby generating pHSE-CAP1254, which directed
moderate expression of 221 CAP1 C-terminal residues in E. coli.
pUSR-cof.HA and pSRA7-cof have been used to express HA-tagged cofilin in
mammalian cells (Moriyama et al.,
1996). In the present study, the 690 bp fragment encoding CAP1-NT
was substituted for the cofilin-coding region of pUSR-cof.HA in order to make
pUSR-CAP1-N229.HA. pUSR-CAP2-N228.HA was constructed in a similar manner.
pSRA-CAP1
254 was made by replacing the cofilin-coding region of
pSRA7-cof with the XbaI/BamHI 670 bp fragment of
pHSE-CAP1
254. pUSH-CAP1 was generated from pUSH-cof, then used for the
production of His-tagged CAP1 in HEK293 cells.
cDNAs for human p57WD (coronin), fascin, EF1 and
CAP1 were obtained from human U937 mRNA by RT-PCR. Plasmids for mammalian
expression of their HA-tagged forms were created in a similar manner to
pSRA-CAP1
254 and used for the experiment shown in
Fig. 1B.
|
Preparation of cofilin-associated proteins
HEK293 cells were transfected by lipofectamine (Gibco BRL, Grand Island,
NY) using a plasmid that drives the expression of Cof-His6, a
porcine cofilin carrying a His-tag at its C-terminus. After 2 days, confluent
cells were rinsed with Hepes-buffered saline and lysed in lysis solution (20
mM Tris-HCl, pH 7.5, 0.5% Triton X-100, 0.1 mM DTT, 1 mM PMSF, 2 µg/ml
aprotinin, 10 µg/ml leupeptin). The crude lysate was clarified by
centrifugation, quantified for protein concentration using Bio-Rad protein
assay reagent and adjusted to 6 mg/ml by diluting with the lysis solution.
Then, 0.5 ml of the lysate was incubated with 30 µl of
Ni2+-resin to adsorb Cof-His6 and associated molecules.
The resin was washed three times with the lysis solution and bound material
was eluted by exposure to increasing concentrations of NaCl.
Protein expression and purification
The N- and C-terminal domains of CAP1 were expressed in E. coli
BL21(Rep4) carrying pQE CAP1-N229.His and pHSE-CAP1254, respectively.
CAP1-CT contained both a His-tag and an S-tag at its N-terminus, whereas
CAP1-NT possessed only a C-terminal His-tag. The domains produced were
purified using a Ni2+ column, as previously described for
His-tagged cofilin (Moriyama et al.,
1996
). Full-length CAP1 was prepared from HEK293 cells with a
His-tag at its N-terminus. Cells (50 dishes) were transfected with pUSH-CAP1.
After 2 days, lysate was prepared and applied to a Ni2+ column. The
column was washed thoroughly with a 0-800 mM NaCl gradient in 20 mM Tris-HCl,
0.1 mM ATP, 0.05 mM DTT, 0.5 mM PMSF (pH 7.5). Following this, the protein was
eluted using a 10-500 mM imidazole gradient in the same buffer. Peak fractions
were dialysed and fractionated with a SP-Sepharose column and a 0-700 mM
gradient of NaCl. Peak fractions were concentrated and processed in two
different columns. One aliquot was gel-filtrated through a Superdex 200pg
column (HR 16/60), and the other was reapplied onto a Ni2+ column.
The latter column was washed with 4 M urea, 20 mM Tris-HCl (pH 8) in order to
liberate actin that was tightly bound to His6-CAP1. Then, pure
His6-CAP1 was eluted with a 0-500 mM gradient of imidazole.
Purified CAP1 and its domains were dialysed in 20 mM Tris-HCl, 50 mM KCl, 0.2
mM DTT (pH 7.5). The protein concentration was determined by the method of
Gill and von Hippel (Gill and von Hippel,
1989
), because CAP1 domains exhibited only a little difference
(within 5.9%) in absorbance at 280 nm between native and denatured states.
Antibodies
HSE-CAP1254 (CAP1-CT) was cleaved with enterokinase (Novagen),
leading to the release of an N-terminal extra peptide (containing His- and
S-tags), which was removed by adsorption to a Ni2+ column. Rat
antiserum to CAP1-CT was made by Sawady Technology (Tokyo, Japan) and
affinity-purified with a CAP1-CT-conjugated Sepharose column.
Alkaline-phosphatase-labeled antibody to rat IgG was purchased from ICN
Biomedicals (Aurora, OH). Cy3-conjugated anti-rat IgG was obtained from
Jackson (West Grove, PA). Rabbit antibodies to actin, cofilin and a mouse
antibody to an HA-tag (12CA5) were previously described
(Iida et al., 1992
;
Moriyama et al., 1996
).
Subunit exchange assay
Preparation of N-(1-pyrene) iodoacetamide-labeled actin
(pyreneactin) and the method of fluorescence measurement were described
previously (Moriyama and Yahara,
1999). 6 µM Mg-actin was polymerized in the presence or absence
of 18 nM gelsolin. 40 µl of fully polymerized actin was removed and added
to 60 µl of 17 mM Tris-HCl, 10 mM Hepes-KOH, 2 mM MgCl2, 100 mM
KCl, 0.5 mM EGTA, 0.1 mM DTT (pH 7.2) containing the CAP1-domains and/or
cofilin. Then it was immediately mixed with 20 µl of 1.2 µM pyreneactin
monomer (50%-labeled), which had been converted to its Mg-ATP-bound form just
before being added. The incorporation of pyrene-actin into unlabeled F-actin
was monitored by a Perkin-Elmer LS-50B Luminescence spectrophotometer.
Dilution-induced depolymerization assay
10 µM Mg-actin (10% pyrene-labeled) was polymerized in the presence or
absence of 40 nM gelsolin over the course of 4 hours. Depolymerization was
initiated by a 20-fold dilution with 20 mM Tris-HCl (pH 7.5), 2 mM
MgCl2, 100 mM KCl, 0.5 mM EGTA, 0.1 mM DTT containing the
CAP1-domains and/or cofilin. The decline in fluorescence was immediately
monitored. The dilution solution for the gelsolin-capped filaments was
supplemented with 2 nM gelsolinactin complex to cap the barbed ends.
Observation of actin turnover kinetics
The kinetics of actin filament turnover (treadmill) were observed using
1,N6-etheno ATP (ATP)-labeled F-actin, which was
prepared according to the procedure of Didry et al.
(Didry et al., 1998
). 6 µM
ATP-actin was converted to its Mg-bound form and was polymerized in the
presence or absence of the CAP1 domains and/or cofilin in 10 mM Tris-HCl, 7 mM
Hepes-KOH, 2 mM MgCl2, 100 mM KCl, 20 µM
ATP, 0.5 mM EGTA,
0.1 mM DTT (pH 7.1). After a steady state (apparent equilibrium) was reached,
ATP was added at a final concentration of 0.25 mM, and the fluorescence was
recorded by measuring emission at 410 nm and excitation at 360 nm.
Nucleotide exchange reaction on G-actin
15 µM of Ca-actin was incubated in 80 µM MgCl2 with 0.2 mM
EGTA in G-buffer (2 mM Tris-HCl, 0.2 mM ATP, 0.1 mM CaCl2, 0.1 mM
DTT, pH 7.8) for 5 minutes and then reacted with 20 units/ml of hexokinase
(Sigma, St. Louis, MO) and 0.3 mM D-glucose. After 2 hours at 4°C, free
nucleotides were removed with a Dowex1-X8 ion exchange resin. The remaining 12
µM of Mg-ADP-actin was supplemented with 36 µM ADP. 10 µl of
Mg-ADP-actin monomer was mixed with an equal volume of 18 µM cofilin or its
solvent for 3 minutes. Then, 100 µl of 60 µM ATP solution
containing the CAP1 domains was added, and the increase in fluorescence was
recorded. The reaction buffer contained 20 mM Hepes-KOH, 8 mM Tris-HCl, 2 mM
MgCl2, 80 mM KCl, 0.1 mM EGTA and 0.1 mM DTT (pH 6.9).
Other methods
Co-sedimentation with F-actin, His-tag pull-down assay and gel densitometry
were performed as described by Moriyama and Yahara
(Moriyama and Yahara, 1999).
The methods used for western blotting and immunostaining of fibroblasts were
previously described by Moriyama et al.
(Moriyama et al., 1996
).
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Results |
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CAP1 dramatically enhances the effects of cofilin on actin
dynamics
We expressed His-tagged CAP1 in HEK293 cells and purified it by column
chromatography (Fig. 2). Gel
filtration through a Superdex 200 column suggested that CAP1 exists in a large
complex (possibly an oligomer) because it eluted between the elution positions
of ferritin (440 kDa) and thyroglobulin (669 kDa). Some of the CAP1 molecules
were tightly associated with actin at this step
(Fig. 2, lane 5). At the second
round of chromatography on a Ni2+ column, the bound actin was
eliminated by washing with urea, and pure His6-CAP1 was obtained
after renaturation (Fig. 2,
lane 6). When the purified His6-CAP1 was gel filtered as above, it
also eluted between ferritin and thyroglobulin even in the absence of actin,
further suggesting that CAP1 is oligomeric (data not shown).
|
The effect of His6-CAP1 on cofilin-induced changes in actin
dynamics was examined in the following experiments
(Fig. 3A-C). One of the unique
functions of cofilin is the remarkable acceleration of the turnover (or
treadmilling) of actin filaments (Carlier
et al., 1997; Rosenblatt,
1997
; Lappalainen, 1997). In the presence of cofilin, the
steady-state turnover of F-actin was dramatically accelerated by CAP1
(Fig. 3A). The faster turnover
of F-actin with cofilin is primarily brought about by its activity to speed up
depolymerization at the pointed end of actin filament
(Carlier et al., 1997
;
Moriyama and Yahara, 1999
).
CAP1 moderately stimulated the depolymerization of gelsolin-capped actin
filaments both in the presence and absence of cofilin
(Fig. 3B). 0.5 µM of CAP1
increased the apparent rate constant for depolymerization by 1.7-fold in the
absence of cofilin and 1.9-fold in its presence
(Fig. 3B). In addition to such
stimulation of subunit release at the pointed end, we found that CAP1 promoted
the cofilin-enhanced turnover of F-actin by facilitating actin assembly at the
barbed end of actin filament. CAP1 increased the maximal rate of incorporation
of actin monomers into pre-existing actin filaments in the presence of cofilin
(Fig. 3C;
Fig. 5E). These results clearly
contrast with those of a number of studies concerning CAP1 or CAP homologues
(Gieselmann and Mann, 1992
;
Freeman et al., 1995
;
Gottwald et al., 1996
),
because they have reported that CAP homologues are strong inhibitors of actin
polymerization. The cause of this discrepancy is implicated, in part, in the
results shown in Fig. 3C. We
noticed that CAP1 had a biphasic effect on the rate of subunit incorporation
into F-actin as a function of concentration in the presence of cofilin, that
is, a dosedependent increase was observed up to 1 µM, but higher
concentrations of CAP1 were less effective
(Fig. 3C; Fig. 5E). Furthermore, such a
biphasic pattern of stimulation was observed even in the absence of cofilin
when the amount of CAP1 was decreased. The optimal concentration of CAP1 was
lower (
0.2 µM) than that observed in the presence of cofilin
(Fig. 3C; Fig. 5E). Thus, high
concentrations of CAP1 inhibit subunit incorporation into F-actin. This may
account for some of discrepancy between our results and those of previous
studies (Gieselmann and Mann,
1992
; Freeman et al.,
1995
). CAP1 did not facilitate subunit incorporation into
gelsolincapped filaments and rather prevented it in the absence of cofilin
(data not shown), indicating that CAP1-facilitated assembly of actin occurred
solely at the barbed end of actin filament and that CAP1 does not sever actin
filament. These properties of CAP1 are qualitatively similar to those of
profilin, as discussed later.
|
|
The N-terminal domain of CAP1 mediates its cofilin-dependent
interaction with actin
According to the available literature, CAP homologues exert their effects
on actin exclusively by their C-terminal domains. We expressed both domains of
CAP1 in E. coli, purified each one and examined their activity. A
simple binding assay verified an association between the C-terminal domain of
CAP1 (CAP1-CT) and actin (Fig.
4). The complex of actin and CAP1-CT bound tightly to DNase I
(data not shown), indicating that the bound actin was G-actin, consistent with
previous reports. Neither the cofilin-actin complex nor actin-free cofilin
were, however, bound by CAP1-CT, although p55 was originally found within a
complex containing both cofilin and actin
(Fig. 1). This suggests that
another region of CAP1 might mediate binding with the complex. The N-terminal
domain (CAP1-NT) did not bind G-actin efficiently in the absence of cofilin,
whereas the CAP1-CT did (Fig.
4A). Surprisingly, when cofilin was present, CAP1-NT bound both
actin and cofilin effectively, whereas CAP1-CT bound only cofilin-free G-actin
under physiological ionic condition (Fig.
4A, lanes 1-6). Cofilin did not directly associate with CAP1-NT in
the absence of actin, which suggests that cofilin strengthens the otherwise
weak association between unpolymerized actin and CAP1-NT.
|
The association of CAP1 domains with F-actin was assessed by examining their co-sedimentation with polymerized actin (Fig. 4B, C). In the absence of cofilin, both N- and C-terminal domains of CAP1 slightly sedimented with F-actin. However, co-sedimentation of CAP1-NT and CAP1-CT with F-actin was almost non-existent following the inclusion of cofilin (Fig. 4B, C). The possible oligomeric nature of the full-length CAP1 made it difficult to evaluate its lateral association with F-actin (the CAP1-actin complex obtained from the Superdex column step was partially sedimented by analogous centrifugation). CAP1-CT efficiently decreased the amount of sedimentable F-actin both in the presence or absence of cofilin (Fig. 4B). Interestingly, CAP1-NT decreased the amount of sedimentable F-actin in the presence of cofilin but not in its absence (Fig. 4C).
Novel roles for the N- and C-terminal domains of CAP1 in stimulating
cofilin-enhanced actin dynamics
The effect of each CAP1 domain on actin dynamics was examined. As well as
full-length CAP1, both CAP1-NT and CAP1-CT enhanced cofilin-induced
acceleration of F-actin turnover, whereas their effect was only marginally
visible in the absence of cofilin (Fig.
5A). It was noted that either CAP1-NT or CAP1-CT exhibited weaker
enhancement of cofilin activity than full-length CAP1, but the simultaneous
addition of both domains produced activity almost equal to that of full-length
CAP1 (Fig. 5A). CAP1-NT also
increased the depolymerization rate of gelsolin-capped actin filaments, which
was faster in the presence of cofilin and/or CAP1-CT
(Fig. 5B). This effect was
significant even in the absence of cofilin
(Fig. 5B), suggesting a direct
interaction of CAP1-NT with the pointed end of actin filament.
We found another novel activity of CAP1. It dramatically accelerated
nucleotide exchange on G-actin, a process to regenerate readily polymerizable
ATP-actin (Fig. 5C,D). The
C-terminal domain of CAP1 had a similar effect
(Fig. 5C,D). The opposite
effect was reported for cofilin/ADF
(Nishida, 1985;
Blanchoin and Pollard, 1998
).
In fact, cofilin remarkably delayed the nucleotide exchange
(Fig. 5C). This property of
cofilin must pose a serious obstacle to the rapid turnover of actin filaments.
With regard to cofilin-bound G-actin, CAP1-CT showed weaker nucleotide
exchange activity than CAP1 (Fig.
5C,D). CAP1-NT did not affect the rate of nucleotide exchange in
the absence of CAP1-CT, even when cofilin was present
(Fig. 5C). In addition, it did
not show any effect on the acceleration observed with CAP1-CT in the absence
of cofilin (data not shown). When G-actin was complexed with cofilin, however,
CAP1-NT enhanced the stimulative effect of CAP1-CT and reproduced almost
similar effects to those obtained with full-length CAP1
(Fig. 5C,D). Therefore, CAP1
promotes actin dynamics not only by enhancing the stimulatory effects of
cofilin with regard to actin turnover but also by reducing its inhibitory
effect.
CAP1 facilitated the assembly of actin monomers onto the barbed end of pre-existing actin filaments (Fig. 3C). The same experiment was carried out with the CAP1 domains, and the maximum rates of subunit incorporation were derived (Fig. 5E). 0.2-0.5 µM of CAP1-CT increased the maximal rate of incorporation of actin monomers into pre-existing actin filaments in the absence of cofilin. In the presence of cofilin, a larger increase was observed, up to 1.8 µM of CAP1-CT. The biphasic profile of this effect with CAP1-CT resembled that obtained with full-length CAP1 (Fig. 5E). 1 µM of CAP1-NT did not obviously affect this activity of CAP1-CT (Fig. 5E), but a much larger amount of CAP1-NT inhibited the subunit incorporation into F-actin either in the presence or absence of CAP1-CT and/or cofilin (data not shown). Thus, CAP1-NT cannot enhance cofilin-induced fragmentation of actin filaments nor facilitate elongation of them.
CAP1 colocalizes with cofilin and actin in dynamic regions of
spreading cells
Immunofluorescent staining of spreading fibroblasts showed colocalization
of CAP1, cofilin and actin in dynamic regions of the cells
(Fig. 6A,B). CAP1 was present
with actin in the lamellipodia, but little was observed on the stress fibers
(Fig. 6A). It also existed with
cofilin in the lamellipodia and in the dorsal ruffles
(Fig. 6B). CAP1 was also
diffusely present in the inner cytoplasm, as was cofilin.
|
Next, we expressed CAP1-NT or -CT carrying an HA1-epitope tag in fibroblasts and examined the localization of each domain (Fig. 6Ca-f). CAP1-NT clearly colocalized with actin and cofilin in the lamellipodia (Fig. 6Ca-d). In contrast, CAP1-CT was primarily distributed diffusely throughout the cytoplasm and only weaker staining was visible even in the actin-rich area of lamellipodia (Fig. 6Ce,f; arrow). Thus, the N-terminal domain is primarily responsible for accumulating CAP1 with cofilin and actin in dynamic peripheral regions of spreading cells.
In addition to CAP1, another isoform, CAP2, exists in mammals
(Yu et al., 1994,
Swiston et al., 1995
). We
examined the localization of human CAP2-NT and found that it also colocalized
with cofilin in the lamellipodia (Fig.
6Cg,h). Our recent biochemical study has also verified that the
properties of CAP2-NT are very similar to those of CAP1-NT, with particular
reference to the association with the cofilin-actin complex (K.M. and I.Y.,
unpublished).
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Discussion |
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This is the first report to reveal the association between actin and the
N-terminal domain of CAP. Interestingly, this interaction is dramatically
enhanced by cofilin and is responsible for CAP1 binding to the cofilin-actin
complex. Previous studies have failed to observe the association between actin
and the N-terminal domain of CAP1 or CAP homologues because of the
cofilin-dependent nature of the interaction. Cofilin prevents several
actin-binding molecules, such as tropomyosin and phalloidin, from associating
with F-actin by altering the twist of the actin filaments
(McGough et al., 1997). In
contrast, Aip1 homologues prefer cofilin-bound F-actin as its target
(Iida and Yahara, 1999
;
Okada et al., 1999
;
Aizawa et al., 1999
;
Rodal et al., 1999
). The
actin-binding property of CAP1 is different from other known actin-binding
molecules. Cofilin strengthens the association between the CAP1-NT and
unpolymerized actin, while reducing the basal weak association of CAP1 domains
with F-actin (Fig. 4). It may
also be possible that the observed co-sedimentation of CAP1 domains with
F-actin was due to non-specific trapping by the F-actin pellet. Our results
strongly argue that CAP1-NT associates primarily with the binary actin-cofilin
complex, but it remains to be determined whether CAP1-NT associates with other
forms of the complex containing three or more molecules of actin and/or
cofilin.
The intracellular localization of CAP1 was consistent with its biochemical
properties. It is present in dynamic regions of the cell periphery, which are
also enriched with both actin and cofilin. Interestingly, CAP1-NT is primarily
responsible for the colocalization of CAP1 with actin and cofilin. Thus, it is
possible that association of the N-terminal domain of CAP1 with the
actin-cofilin complex determines CAP1 localization because the cofilin-actin
complex was recognized specifically by the CAP1-NT but not by the CAP1-CT
(Fig. 4A). Further analysis of
CAP2-NT showed that the actin-modulating function of the N-terminal domain is
conserved between human CAP1 and CAP2 (K.M. and I.Y., unpublished). Noegel et
al. (Noegel et al., 1999)
reported that the enriched localization of a Dictyostelium CAP
homologue at anterior and posterior edges of cells required its N-terminal
domain but not its C-terminal one. Thus, interaction of CAP-NT with actin may
occur in this organism, too, and possibly be conserved in different
species.
Synergistic effects of CAP1 and cofilin in accelerating actin
dynamics
A rapid turnover of actin filaments is essential for actin-based cell
motility, and cofilin-ADF accelerates this turnover
(Carlier et al., 1997;
Rosenblatt et al., 1997
;
Lappalainen et al., 1997). Our novel findings illustrate several mechanisms
behind the rapid turnover observed in the presence of both CAP1 and cofilin.
CAP1 greatly enhances the effect cofilin has on the turnover rate of actin
filaments (Figs 3,
5). Our detailed dissection of
this effect revealed that it is brought about by four distinct activities, as
illustrated in Fig. 7. First,
CAP1 facilitates the addition of ATP-actin monomers onto the barbed end of
actin filament, an effect that is more pronounced in the presence of cofilin.
This effect is biphasic and is a function of the concentration of CAP1 and
mediated by its C-terminal domain (Fig.
5E). As discussed above, the effective concentration range for
this biphasic trend is significantly extended in the presence of cofilin.
However, this property of CAP1-CT has another side to it that is, a
much larger amount of it greatly accelerates the disassembly of actin filament
exclusively at the barbed end (K.M. and I.Y., unpublished). This effect argues
for the presence of an interaction between CAP1-CT and the barbed end. Second,
CAP1 increases the rate of subunit release at the pointed end of the actin
filament and enhances the more potent, analogous effect of cofilin. The
N-terminal domain primarily contributes to this activity of CAP1
(Fig. 5B), and the C-terminal
domain augments it, probably by recycling cofilin and/or the N-terminal domain
of CAP1. Third, CAP1 accelerates nucleotide exchange on G-actin to regenerate
readily polymerizable ATP-actin (Fig.
5C,D). CAP1-CT is essentially responsible for this activity.
CAP1-NT was found to play an interesting role in this activity. Although it
had no apparent effect on nucleotide exchange in the absence of cofilin, it
was found to relieve the inhibitory effect of cofilin on nucleotide exchange
only when CAP1-CT was present together. This finding sheds light on the
physiological significance of the reported interaction between the N- and
C-terminal domains within CAP1 and CAP2 molecules
(Hubberstey et al., 1996
). The
pronounced effect of CAP1 on cofilin-induced acceleration of actin turnover
would be achieved through the integration of the four activities of CAP1,
which involves a coordinated interplay between its N- and C-terminal
domains.
|
Fragmentation of actin filaments affects actin dynamics. Our analysis refutes the possibility that CAP1 or its domains sever actin filaments in the absence of cofilin. At this time, however, we cannot completely exclude the possibility that CAP1 enhances severing by cofilin. If such enhancement does indeed occur, we think it is merely an indirect effect of CAP1. After released from the pointed end of the filament, the ADP-actin-cofilin complex should dissociate much faster in the presence of CAP1. As a result, released cofilin may make a second attack on F-actin to sever filaments again.
As mentioned above, cofilin strongly inhibits the nucleotide exchange of
ADP-actin. This property of cofilin probably impedes the turnover of actin
filaments. Any factor that stimulates dissociation of the ADP-actin monomer
from cofilin should promote actin turnover. Higher eukaryotes possess cofilin
kinases (including LIM kinases), which inhibit actin-binding by cofilin
through phosphorylation (Arber et al.,
1998; Yang et al.,
1998
; Smertenko et al.,
1998
; Lian et al.,
2000
; Aizawa et al.,
2001
). Phosphoinositides, which are present even in lower
eukaryotes, also prevent cofilin from binding to actin
(Yonezawa et al., 1990
;
Iida et al., 1993
;
Aizawa et al., 1995
). It is
thought that cofilin-kinases and/or phosphoinositides might promote the
dissociation of the cofilin-actin complex. Our study identified CAP1 as a more
promising factor to have such a property. CAP1 is perhaps better suited for
this role because it relieves the inhibitory effect of cofilin on nucleotide
exchange on G-actin via the interplay of its N- and C-terminal domains. In
addition, its C-terminal domain is capable of stimulating nucleotide exchange
on its own. Therefore, CAP1 effectively recycles both actin and cofilin for
rapid actin turnover.
Freeman et al. (Freeman et al., 2000) recently reported that the microinjection of CAP1 into fibroblasts stimulated the formation of actin filaments in the cytoplasm and proposed a possible role of CAP1 in promoting actin assembly in vivo. Although they did not uncover how CAP1 could promote the actin assembly in cells, our present study will help interpret their observation.
Functional similarity and difference with profilin
In S. cerevisiae, loss of function of the C-terminal domain of CAP
is compensated by profilin, a small actin-binding protein, and the cells
lacking profilin are phenotypically similar to those lacking CAP-CT
(Vojtek et al., 1991). In
addition, expression of both mRNA and protein of CAP is remarkably upregulated
in profilin-minus cells of Dictyostelium
(Gottwald et al., 1996
). The
role of profilin in actin turnover has been studied in detail
(Pring et al., 1992
;
Pantaloni and Carlier, 1993
;
Perelroizen et al., 1996
;
Selden et al., 1999
;
Wolven et al., 2000
). Profilin
accelerates nucleotide exchange on G-actin and facilitates subunit addition
onto the barbed ends. Thus, profilin promotes the turnover of actin filaments
in a similar manner to CAP1 and exhibits synergy with cofilin as well
(Didry et al., 1998
). In spite
of these studies, CAP homologues have long been regarded as mere inhibitors of
actin polymerization. CAP1-CT actually resembles profilin in its activity as
revealed by our study. In reality, this similarity probably underlies the
observed suppression of
CAP-CT by profilin in yeast. In clear
contrast to CAP1, profilin does not associate with the cofilin-actin complex
and thus cannot directly dissociate the complex. In other words, profilin does
not possess CAP1-NT-like activity. Hence, CAP1 is unique in having a
cofilin-dependent activity in its N-terminal domain.
Possible cellular regulators of CAP1 function
Internal proline-rich regions of CAP homologues have been reported to
interact with several proteins implicated in cytoskeletal regulation
(Freeman et al., 1996;
Lila and Drubin, 1997
). In
addition, CAP homologues contain a sequence related to a verprolin homology
domain, which is part of the WASP-family of actin-binding proteins
(Baum et al., 2000
). These
regions may also affect the modulation of actin dynamics by CAP homologues or
cofilin in living cells.
Moreover, CAP is a component of the Ras signalling pathway in S.
cerevisiae. In response to glucose, Ras activates Cyr1, an adenylyl
cyclase, to stimulate cAMP synthesis and promote cell growth. CAP exists as a
complex with Cyr1 and aids Ras signalling
(Field et al., 1990;
Fedor-Chaiken et al., 1990
).
The N-terminal region of CAP mediates its binding to Cyr1, and this
association generates a second binding site for Ras
(Shima et al., 2000
). To date,
relevant mammalian targets of CAP1-NT or CAP2-NT have not been identified,
although human CAP1 may bind to yeast adenylyl cyclase in fission yeast
(Yu et al., 1994
). This is the
first study to identify a physiologically relevant mammalian target: the
cofilin-actin complex. However, it is likely that the interaction between CAP1
and actin-cofilin may be regulated by an unidentified partner of CAP1-NT,
possibly a Ras/Cyr1-like signalling molecule.
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