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INTRODUCTION |
The cortical region of eukaryotic cells is made up of the plasma
membrane and the underlying protein meshwork of the cytoskeleton. The
cortical cytoskeleton plays a role in phenomenon such as cellular morphology, membrane domain specialization, and cell-cell and cell-substratum interactions (1, 2). In addition, the cortical cytoskeleton is involved in signal transduction events that regulate membrane trafficking, cell migration, and growth regulation (3-7). A
major portion of the cortical cytoskeleton is made up actin filaments
whose physiological state is governed by actin-binding proteins. These
actin-binding proteins control actin filament polymerization,
filament-filament interaction, and filament-plasma membrane interaction
(reviewed in Ref. 8).
The proteins that link the actin cytoskeleton with the plasma membrane
are particularly diverse in their actions. An example is the
ezrin-radixin-moesin (ERM)1
family of proteins (9). These proteins are known to be involved in
cytoskeletal-membrane events such as cell adhesion (10-12), Rho- and
Rac-mediated cell morphology (13-16) as well as Akt-mediated cell survival (17). Further discovery and characterization of cortical
actin-binding proteins will be important in elucidating the in
vivo roles that the eukaryotic cell cortex and its binding partners play within the cell.
Annexin II is a member of the annexin family of proteins that are well
characterized by their ability to bind to acidic phospholipids in a
Ca2+-dependent manner (18). In addition to
their phospholipid binding activity, most family members discovered to
date bind F-actin in a Ca2+-dependent manner
(19). Annexin II is unique among the annexins in that it exists as both
a monomer and a tetramer within the cell. Annexin II tetramer (AIIt)
consists of two copies of annexin II bound to a dimer of p11, a member
of the S-100 family of Ca2+-binding proteins. Within the
cell, it has been established that annexin II is localized throughout
the cell, whereas AIIt is localized to the plasma membrane-actin
cytoskeleton interface (20-23). It has been suggested that
intracellular AIIt acts as a link between the cytoskeleton and the
plasma membrane, although the physiological consequences of this
proposed interaction are unknown (24).
Although both annexin II and AIIt bind F-actin in vitro,
only AIIt bundles F-actin (25). The bundling of F-actin by AIIt was
rapid and reversible (t0.5 = 6 s) and of
moderate affinity (apparent Kd (AIIt) of 0.18 µM). This process was dependent on the presence of
Ca2+, with half-maximal values obtained near 2 µM Ca2+. The region of annexin II that
contributes to the bundling activity of AIIt bundling activity was a
region of
-helix in the fourth domain of annexin II that exhibited
homology to an actin-binding site of myosin (26). Although this region
of annexin II was critical for the F-actin bundling activity of AIIt,
it did not participate in F-actin binding. In contrast to the F-actin
bundling domain of AIIt, the F-actin binding domain of annexin II has
remained elusive.
Here we present evidence establishing that the C terminus of the
annexin II subunit of AIIt comprises an F-actin binding domain. Interestingly, the last 9 amino acid residues of the C terminus of the
annexin II subunit appear to be entirely responsible for the F-actin
binding activity of AIIt.
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EXPERIMENTAL PROCEDURES |
Mutagenesis of Annexin II--
A bacterial expression vector
(pAED4.91) containing the wild-type sequence for annexin II was mutated
using the QuikChange site-directed mutagenesis kit (Stratagene).
Briefly, mutagenic primers were synthesized to introduce premature stop
codons immediately following Ala329, Asp325,
and Thr322 in the annexin II cDNA. The resultant
plasmids encoded for a series of C-terminally truncated annexin II
proteins, which we have named CT
9, CT
13, and CT
16,
respectively. These plasmids were then transformed into
Escherichia coli BL21(DE3) and grown as previously described
(27).
Purification of Wild-type and C-terminal Truncated Annexin
II--
After 4 h of induction with
isopropyl-1-thio-
-D-galactopyranoside, bacteria were
collected by low speed centrifugation. They were subsequently sonicated
in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 1 mM DTT) containing protease inhibitors, and following centrifugation, the annexin II mutants were found to be
insoluble, contrary to wild-type annexin II. The mutant annexin II
proteins were solubilized using 6 M guanidinium
hydrochloride followed by dialysis against several changes of buffer A. After solubilization, the mutant annexin II proteins were purified in the same manner as wild-type annexin II via hydroxylapatite,
heparin-Sepharose affinity, and gel permeation chromatography as
reported previously (27). The elution profiles of the recombinant
wild-type and mutant annexin II on hydroxylapatite, heparin affinity,
and gel permeation chromatography were indistinguishable. In addition, the circular dichroism spectra of each of the proteins were very similar, indicating little secondary structure perturbation.
Purification of Recombinant p11--
The production of the
recombinant p11 construct and the subsequent protein purification
scheme have been previously reported (27, 28). Briefly, bacteria were
grown and induced with isopropyl-1-thio-
-D-galactopyranoside as described above for annexin II. The
resultant cell lysate supernatant was then applied to a Fast Q
ion-exchange column, after which Fast S ion-exchange chromatography was
performed on the unbound flowthrough fraction, followed by gel
permeation chromatography.
Purification of Recombinant Annexin II Tetramer--
Equimolar
amounts of recombinant annexin II (WT, CT
9, CT
13, or CT
16) and
recombinant p11 were incubated at 4 °C for 30 min and then subjected
to gel permeation chromatography to separate the monomeric from the
tetrameric forms of annexin II. The elution profiles of the recombinant
WT and CT
9, CT
13, and CT
16 AIIt were identical, suggesting
that a gross change in the conformation of the mutant AIIt had not
occurred as a result of the truncations. Proteins were subsequently
stored in buffer A at
80 °C until use.
Phospholipid Vesicle Aggregation Assay--
In a 12- × 75-mm
culture tube, 50 µl each of 20 mg/ml phosphatidylserine,
phosphatidylethanolamine, and cholesterol (dissolved in chloroform)
were shelled by N2 gas. The resultant residue of lipids was
resuspended in 1 ml of phospholipid aggregation buffer (30 mM HEPES, pH 7.5, 50 mM KCl) and sonicated at
75 watts for three 10-s bursts with a Braun probe sonicator to generate
phospholipid vesicles. This stock lipid solution, at a concentration of
1 mg/ml, was prepared fresh daily. Phospholipid vesicle aggregation was assayed in an ELx-808 spectrophotometric plate reader
(Bio-Tek Instruments) at 450 nm in a final volume of 200 µl.
Initially, phospholipid vesicles, either 200 µM
Ca2+ or 3 mM EGTA, and phospholipid aggregation
buffer were combined resulting in a final phospholipid vesicle
concentration of 0.05 mg/ml. To initiate the reaction, 10 µg of
annexin II (WT, CT
9, CT
13, or CT
16) was added to the above
mixture. Absorbance readings were then recorded for an additional 15 min as described previously (27).
Phospholipid Vesicle Binding Assay--
Phospholipid vesicle
binding was performed as described above for vesicle aggregation. The
reaction was initiated by the addition of varying amounts of annexin II
and incubated at room temperature for 15 min. After the incubation
period, the reaction mixture was centrifuged at 14,000 × g in a desktop centrifuge. The pelleted phospholipid
vesicles were resuspended in 30 µl of SDS sample buffer and boiled
for 3-5 min (29). The phospholipid vesicle pellet was then resolved by
SDS-PAGE and stained with Coomassie Blue, and densitometric analysis of
the annexin II band was performed using a Hewlett Packard ScanJet 4c
flatbed scanner and ImageQuaNT software (Molecular Dynamics). The
densitometric intensity of annexin II centrifuged in phospholipid
aggregation buffer without phospholipid vesicles was subtracted from
all readings. Unless otherwise stated, the results are expressed as the
percentage maximum of phospholipid binding by either WT annexin II or
AIIt.
F-actin Bundling Assay--
F-actin was prepared from rabbit
skeletal muscle as outlined previously (30). F-actin bundling was
measured by the light scattering intensity perpendicular to the
incident light in a PerkinElmer Life Sciences LS-50B luminescence
spectrophotometer as described previously (25). The excitation and
emission wavelengths were both set to 400 nm with slit widths of 10 nm
and 5 nm, respectively. F-actin (0.60 µM) was incubated
in bundling buffer (25 mM MOPS, pH 7.0, 0.5 mM
DTT, 0.33 mM ATP, 500 µM CaCl2)
to a final volume of 600 µl. F-actin bundling was initiated by the
addition of varying amounts of WT, CT
9, CT
13, or CT
16 AIIt.
After a 15-min incubation at room temperature, light scattering
intensity was recorded in at least triplicate. The light scattering
intensity of F-actin alone in bundling buffer was subtracted from all readings.
F-actin Binding Assay--
F-actin binding was performed as
described above for bundling, except that the reaction volume was
adjusted to 200 µl. After a 15-min incubation period, the sample was
centrifuged at 400,000 × g for 30 min in a Beckman
Optima TLX Ultracentrifuge. The pellet was solubilized at room
temperature for 1 h in 25 µl of 100 mM KCl and SDS
sample buffer, followed by boiling for 3-5 min. To quantitate the
protein remaining in the supernatant after centrifugation, the
supernatants were concentrated using chloroform/methanol precipitation as described previously (31). The precipitated protein was treated with
SDS sample buffer as described above for the pelleted fraction. The
pellet fractions or the concentrated supernatant fractions were then
resolved by SDS-PAGE, followed by densitometric analysis as described
above for the phospholipid binding assay. The densitometric intensity
of annexin II centrifuged in bundling buffer without F-actin was
subtracted from all readings. The results, unless otherwise stated, are
expressed as the percentage maximum of actin binding by either WT
annexin II or AIIt.
Miscellaneous Techniques--
Protein concentrations were
determined using Coomassie Brilliant Blue, and standardizing the
concentrations to BSA, as described by Bradford (32). All reagents used
were of analytical grade purity. Data was analyzed using Sigma Plot
(Jandel Scientific).
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RESULTS |
C-terminal Actin-binding Motifs--
The C terminus of many
actin-binding proteins contains the F-actin binding domain. Examples
include the villin superfamily (33), the 34-kDa
Dictyostelium discoideum bundling protein (34), vinculin
(35), human erythroid dematin (36), and the ERM proteins (37,
38). Interestingly, many F-actin-binding proteins, such as the ERM
family and AIIt, are thought to form a link between the actin
cytoskeleton and the plasma membrane. Furthermore, the ERM proteins and
AIIt form F-actin bundles as a consequence of their F-actin binding
activity (18, 25, 26, 39-42). We therefore investigated the
possibility that the F-actin-binding site of AIIt was also contained
within its C terminus.
Initially, we constructed a mutant AIIt composed of native p11 and
mutant annexin II subunits. The mutant annexin II subunit was
constructed such that the 16 amino acids of the C terminus were
truncated (CT
16 AIIt). As shown in Fig.
1, analysis of the F-actin bundling
activity of CT
16 AIIt by light scattering revealed that the F-actin
bundling activity of this mutant AIIt was severely diminished compared
with WT AIIt. To confirm that the decrease in light scattering was due
to a loss in the formation of supramolecular structures (bundles) of
F-actin, we took advantage of the fact that F-actin does not sediment
at 14,000 × g but that F-actin bundles can be
harvested at this centrifugal force. The lack of bundling activity by
the CT
16 AIIt was thus shown to be due to a reduced interaction
between F-actin and the mutant AIIt (Fig. 1, inset). This
suggested that the C-terminal region of annexin II was important in
mediating the binding to F-actin.

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Fig. 1.
F-actin bundling kinetics of WT and
CT 16 AIIt. F-actin (0.6 µM)
was incubated at 20 °C in a buffer containing 25 mM
MOPS, pH 7.0, 50 mM KCl, 0.33 mM ATP, 0.5 mM DTT, and 500 µM CaCl2 (600 µl of total volume). WT (open circles) or CT 16
(filled triangles) AIIt was added at a concentration of 0.14 µM to initiate the bundling reaction (buffer A was also
added in a separate reaction as a negative control (filled
circles)). Immediately after addition of AIIt, the reactions were
analyzed for F-actin bundling activity by light scattering.
Inset, immediately following the bundling reactions
described above, the mixtures were centrifuged at 14,000 × g. The pelleted material was then resolved on SDS-PAGE and
stained with Coomassie Blue to assess the amount of F-actin
bundle-associated AIIt (lane 1, buffer A control; lane
2, WT AIIt; lane 3, CT 16 AIIt). The upper
band is actin (42 kDa), and the lower band is the
annexin II band (36 kDa) from AIIt. The results are representative of
three independent experiments.
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To further characterize this domain, we truncated the C terminus of
annexin II by 9 (CT
9) and 13 (CT
13) amino acid residues and
examined the F-actin binding activity of these proteins.
F-actin Binding by Mutant Annexin II--
It has been previously
shown that annexin II binds F-actin in a
Ca2+-dependent manner in vitro (19,
42). We assessed the ability of the C-terminal truncated annexin II
mutants (CT
9, CT
13, and CT
16) to bind to F-actin using high
speed centrifugation (400,000 × g). Under these
experimental conditions, F-actin-binding proteins will cosediment with
F-actin. As shown in Fig. 2A,
at submaximal annexin II concentrations the truncated proteins have
decreased F-actin binding, together with a shift in the
K0.5 (annexin II). Furthermore, addition of 1 mg/ml BSA did not affect this binding phenomenon (data not shown),
suggesting that this interaction was specific. Shown in Fig.
2A, inset, is a comparison of the supernatants
and pellets of each of the mutants at submaximal annexin II
concentration (1.4 µM). The amount of CT
9, CT
13, and CT
16 annexin II that cosedimented with F-actin was ~45, 50, and 60% that of WT annexin II, respectively.

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Fig. 2.
Annexin II-mediated F-actin binding.
A, concentration dependence of F-actin binding by WT,
CT 9, CT 13, and CT 16 annexin II. F-actin (0.6 µM)
was incubated at 20 °C in a buffer containing 25 mM
MOPS, pH 7.0, 50 mM KCl, 0.33 mM ATP, 0.5 mM DTT, and 500 µM CaCl2 (200 µl of total volume). WT (filled circles), CT 9
(open circles), CT 13 (filled triangles), or
CT 16 (open triangles) annexin II was added at the
indicated concentrations, and the reaction mixture was incubated at
20 °C for 10 min. After the incubation period, the samples were
centrifuged at 400,000 × g for 30 min and then
resolved by SDS-PAGE. The annexin II bound to F-actin was quantitated
by densitometric analysis of the Coomassie Blue-stained gel.
Inset, effect of annexin II C-terminal deletion on F-actin
binding. F-actin binding was performed as described above with a fixed
annexin II concentration of 1.4 µM. The annexin II bound
to F-actin (P) was compared with the unbound fraction of
annexin II (S) as described under "Experimental
Procedures." Results shown are representative of three independent
experiments. B, Ca2+ dependence of F-actin
binding by WT, CT 9, CT 13, and CT 16 annexin II. F-actin binding
was performed as described above, except CaCl2 was added at
the indicated concentrations. WT (filled circles), CT 9
(open circles), CT 13 (filled triangles), or
CT 16 (open triangles) was added at a concentration of 1.4 µM. Results shown are expressed as the percentage of
maximal binding displayed by WT annexin II and are representative of
three independent experiments.
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We next measured the ability of both WT and mutant annexin II to
interact with F-actin at different concentrations of Ca2+.
This was done to ensure that the decrease in F-actin binding at
submaximal annexin II concentrations is not due to a shift in the
Ca2+ dependence of the F-actin-annexin II interaction. As
shown in Fig. 2B, the mutant annexin II proteins showed
approximately a 5-fold reduction in F-actin binding in the presence of
1 mM Ca2+ compared with WT annexin II. Thus,
the decrease in F-actin binding by mutant annexin II was not due to a
decrease in the Ca2+ dependence of F-actin binding.
Phospholipid Aggregation and Binding by Mutant Annexin II--
The
Ca2+-dependent binding of annexin II to
phospholipid liposomes is the most studied phenomenon of the protein
(reviewed in Ref. 24). Fig. 3A
is a comparison of the relative abilities of WT and mutant annexin II
to aggregate phospholipid vesicles. In the presence of EGTA, none of
the proteins were able to aggregate phospholipid vesicles. However, all
proteins aggregated phospholipid liposomes equally in the presence of
either 20 or 200 µM Ca2+.

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Fig. 3.
Annexin II-mediated phospholipid vesicle
interaction. A, Ca2+ dependence of
phospholipid vesicle aggregation by WT, CT 9, CT 13, and CT 16
annexin II. Phospholipid vesicles consisting of phosphatidylserine,
phosphatidylethanolamine, and cholesterol were incubated at 20 °C
with 30 mM HEPES, pH 7.5, 50 mM KCl, and either
200 µM CaCl2 (dark gray bars), 20 µM CaCl2 (light gray bars), or 3 mM EGTA (black bars). Immediately after taking
the first absorbance reading, WT, CT 9, CT 13, or CT 16 annexin
II (1.4 µM) was added to the reaction mixture, and the
A450 was continuously recorded for 15 min.
Results are shown as a percentage of starting
A450 (no annexin II added), and are expressed as
mean ± S.D. (n = 3). Inset,
Ca2+ dependence of phospholipid vesicle binding by WT,
CT 9, CT 13, and CT 16 annexin II. After completion of the
aggregation reactions, the reaction mixtures were centrifuged at
14,000 × g and annexin II bound to the phospholipid
vesicles was determined by SDS-PAGE, followed by Coomassie Blue
staining of the gel. Results are representative of three independent
experiments. B, concentration dependence of phospholipid
vesicle binding by WT, CT 9, CT 13, and CT 16 annexin II.
Phospholipid vesicles were prepared and incubated as described above in
the presence of 200 µM CaCl2. WT
(filled circles), CT 9 (open circles), CT 13
(filled triangles), or CT 16 (open triangles)
annexin II was then added to the reaction mixture at the indicated
concentrations. After a 15-min incubation period, the reaction mixtures
were centrifuged at 14,000 × g and phospholipid
vesicle associated annexin II was resolved by SDS-PAGE. The amount of
annexin bound was then quantitated by densitometric analysis of the
Coomassie Blue-stained gel. The results are displayed as percentage of
maximal binding displayed by WT annexin II and are representative of
three independent experiments.
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We next examined the ability of the mutant annexin II to bind to
phospholipid vesicles in a Ca2+-dependent
manner (Fig. 3A, inset). The CT
9, CT
13, and
CT
16 annexin II, in a similar affinity and capacity to WT annexin
II, pelleted with the phospholipid vesicles in the presence of
Ca2+ (either 20 or 200 µM Ca2+).
In the absence of Ca2+, neither WT annexin II nor mutant
annexin II bound to the phospholipid vesicles.
The dose dependence of phospholipid vesicle binding by WT, CT
9,
CT
13, and CT
16 annexin II is shown in Fig. 3B. In the
presence of Ca2+, the WT, CT
9, CT
13, and CT
16
annexin II all bound phospholipid vesicles with similar affinity and
capacity. Thus, truncation of the C terminus of annexin II did not
affect the phospholipid binding activity of the protein.
F-actin Bundling and Binding by Mutant AIIt--
Although annexin
II contains the phospholipid- and F-actin-binding sites, it is the
tetrameric form of the protein that has been localized to the cortical
actin cytoskeleton (20, 21, 23, 43, 44). It is known that the
interaction of the annexin II subunits with the p11 subunits causes a
change in the conformation of both proteins (45). Therefore, it is
unclear whether the C terminus of the annexin II subunit within AIIt is
equivalent in structure or function to that of annexin II monomer.
Upon Ca2+-dependent binding to F-actin, AIIt,
but not annexin II, rapidly and reversibly forms anisotropic F-actin
bundles (25). We therefore analyzed the ability of C-terminally
truncated AIIt to bundle actin filaments using light scattering as
described previously (25, 27). Shown in Fig.
4A, the C-terminally truncated AIIt mutants are unable to cause any significant F-actin
filament-filament interaction as measured by light scattering.

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Fig. 4.
Annexin II tetramer-mediated F-actin
interaction. A, concentration dependence of F-actin
bundling by WT, CT 9, CT 13, and CT 16 AIIt. F-actin (0.6 µM) was incubated at 20 °C in a buffer containing 25 mM MOPS, pH 7.0, 50 mM KCl, 0.33 mM
ATP, 0.5 mM DTT, and 500 µM CaCl2
(600 µl of total volume). WT (filled circles), CT 9
(open circles), CT 13 (filled triangles), or
CT 16 (open triangles) AIIt was added at the
indicated concentrations to initiate the reaction. After a 10-min
incubation period, the reactions were analyzed for F-actin bundling
activity by light scattering (mean ± S.D., n = 5). B, concentration dependence of F-actin binding by WT,
CT 9, CT 13, and CT 16 AIIt. F-actin (0.6 µM) was
incubated at 20 °C in a buffer containing 25 mM MOPS, pH
7.0, 50 mM KCl, 0.33 mM ATP, 0.5 mM
DTT, and 500 µM CaCl2 (200 µl of total
volume). WT (filled circles), CT 9 (open
circles), CT 13 (filled triangles), or CT 16
(open triangles) AIIt were added at the indicated
concentrations and the reaction mixture was incubated at 20 °C for
10 min. After the incubation period, the samples were centrifuged at
400,000 × g for 30 min and then resolved by SDS-PAGE.
The AIIt bound to F-actin was quantitated by densitometric analysis of
the Coomassie Blue-stained gel. Results are expressed as the percentage
of maximal binding displayed by WT AIIt and are representative of three
independent experiments.
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We next examined whether or not the lack of F-actin bundling activity
displayed by the AIIt mutants was due to a decreased binding affinity
for F-actin. As shown in Fig. 4B, the mutant forms of AIIt
display only about 15-20% of the F-actin binding activity of WT AIIt.
Thus, the truncation of the C terminus of annexin II affects the
binding affinity of AIIt for F-actin, which in turn directly affects
the F-actin bundling activity of the protein.
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DISCUSSION |
Annexin II tetramer (AIIt) is a multifunctional protein that
interacts in a Ca2+-dependent manner with
anionic phospholipids, F-actin, and heparin (18). Due to its ability to
bind simultaneously to actin and acidic phospholipids, it is thought to
act within cells as a membrane-cytoskeleton linking protein (24). It is
well established that the annexin II subunit of the protein complex
houses the binding sites for the aforementioned ligands, as well as the
sites for Ca2+ coordination (18). However, the exact
location of the F-actin-binding sites within the protein has remained
elusive. The similarity in function between the ERM proteins and AIIt,
and the presence of the F-actin binding domain within the C terminus of
the ERM proteins, led us to suspect that the C-terminal region of the annexin II subunit of AIIt might be involved in F-actin binding. It is
known that the N terminus of annexin II is responsible for binding of
p11 (23, 46, 47), however, a role has not been attributed to the
C-terminal region of the protein. Therefore, we have used site-directed
mutagenesis to truncate the C terminus of annexin II to assess its
possible function in F-actin binding.
We initially observed that truncation of the last 16 amino acid
residues of the C terminus of the annexin II subunit of AIIt inhibited
the F-actin bundling activity of the protein by about 90%. This led us
to suspect that the C terminus was involved in the interaction with
F-actin. We then examined the F-actin binding activity of the isolated
annexin II subunit. Analysis of the F-actin binding properties of
several C-terminal truncations of annexin II revealed that removal of
16 or even as few as 9 amino acid residues also inhibited the F-actin
binding activity of the protein. Furthermore, the truncations in the C
terminus of annexin II had no effect on the interaction of annexin II
with phospholipid vesicles or heparin. The C-terminally truncated
annexin II mutant proteins bound to and aggregated acidic phospholipid
vesicles in a Ca2+-dependent manner with a
similar affinity and capacity as WT annexin II. Furthermore, mutant
annexin II bound to a heparin-Sepharose affinity column in a similar
manner as WT annexin II (data not shown). Finally, to determine whether
the annexin II monomer behaved similarly to annexin II that was
complexed with p11, we assessed the ability of the AIIt, composed of
wild-type p11 subunits and C-terminally truncated annexin II subunits
to bundle F-actin filaments. Clearly, AIIt composed of wild-type p11
subunits and C-terminally truncated annexin II subunits was unable to
bundle F-actin. Further investigation revealed that the loss in F-actin
bundling was due to a decrease in the binding of the mutant AIIt to
F-actin.
Our results therefore identify for the first time that the C-terminal
sequence of the annexin II subunit of AIIt, LLYLCGGDD, contains an
F-actin binding domain of AIIt. The localization of the F-actin binding
domain to the C-terminal 9 amino acid residues of annexin II represents
one of the smallest F-actin binding domains discovered to date.
Thymosin
4 also contains a relatively small actin-binding site. In
this protein, a region comprising the first 12 amino acid residues of
the protein forms the actin-binding site (48, 49). However, thymosin
4 is a well-characterized G-actin-sequestering protein, with only a
weak affinity for F-actin (50-52). Known F-actin binding domains,
however, are quite variable in length, with no consensus sequence
length among them. For example, the F-actin-binding sites of the ERM
proteins vary from 22 to 34 amino acids (14, 38). As well, an F-actin
binding domain recently localized in myosin light-chain kinase spans
amino acid residues 2-42 of the protein (53). Even in light of the
above examples, most F-actin-binding sites encompass substantially
larger portions of the protein. For example, the F-actin-binding site of vinculin is 123 amino acid residues in length (35). In addition, the
three identified F-actin-binding sites within the D. discoideum 34-kDa F-actin-bundling protein vary in length from 16 to 123 amino acid residues in length, although the 16-amino acid
residue domain has only weak binding to F-actin (34).
Although the overall length of the F-actin-binding site of annexin II
is relatively small compared with known sites, other requirements for
F-actin binding within this site appear to be met. For example, the
structural requirements of prototypical F-actin-binding sites are
fulfilled when discussing the C terminus of annexin II. It is known
that the majority of F-actin-binding sites consist mostly of an
-helical structure (8). Interestingly, the region of annexin II that
we propose contains the F-actin-binding site known to be comprised of
mainly
-helical secondary structure (54).
We expected that a peptide to the C-terminal 16 amino acid residues of
annexin II would inhibit the binding of annexin II or AIIt to F-actin.
However we found that the peptide to the C-terminal 16 amino acid
residues of annexin II was unable to inhibit F-actin binding by either
annexin II or AIIt (data not shown). We therefore examined the
possibility that this peptide was conformationally distinct to the
C-terminal region of annexin II. It was very likely that this peptide
was not in the same conformation as the C-terminal region of annexin
II, because circular dichroism spectra of this peptide revealed a large
portion of random coil structure, with very little
-helical
structure (data not shown).
The inability of a peptide to the C-terminal 16 amino acids of annexin
II to directly bind to F-actin presents two possibilities. First, it is
possible that the C-terminal 9 amino acid residues of annexin II may
only be part of the F-actin-binding site and other amino acids
residues, contributed by distinct regions of annexin II, may
participate in assembly of the intact site. Second, although the
C-terminal region of annexin II binds F-actin, it is possible that the
C terminus requires interaction with other amino acids residues to
stabilize a conformation that allows these C-terminal amino acid
residues to bind F-actin. Although speculative, it is reasonable to
suspect that the peptide was unable to assume the expected
-helical
structure, because it required the presence of amino acid residues from
other regions of annexin II to stabilize its
-helical structure.
It was also interesting that the C-terminal truncations of annexin II
most dramatically affected the F-actin binding activity of AIIt
compared with the monomeric annexin II. This was an important observation, because it is AIIt and not annexin II that has been localized to the subcortical F-actin cytoskeleton. Thus, the binding of
the p11 subunit to annexin II may place the C-terminal region of
annexin II in a conformation that allows the 9 amino acid residues to
more autonomously form the F-actin-binding site. In other words, it is
possible that the proposed requirement for distal amino acids to
stabilize the interaction of the 9 C-terminal amino acid residues with
F-actin is minimized by the binding of p11 to annexin II.
The data obtained from x-ray crystallography is consistent with our
localization of the F-actin-binding site of annexin II to the C
terminus. It is known that annexin II has an overall curved, planar
shape with opposing concave and convex surfaces. The convex side of the
molecule is thought to face the plasma membrane and is known to contain
the Ca2+-binding sites and presumably the phospholipid
binding domains (54). The concave face of the protein is postulated to
face the cytosol and contains both the N- and C-terminal domains of the
protein. It is well established that the N terminus interacts with p11
to form AIIt, and the fact that the C terminus also faces the cytosol
places it in an ideal proximity to interact with the actin cytoskeleton
within the cell. Thus, if AIIt is fulfilling its proposed role as a
membrane-cytoskeletal linker, the C-terminal domain of annexin II
appears to be in the correct orientation to bind to and potentially
modulate the actin cytoskeleton.