From the Departments of Medicine and Biochemistry and
Molecular Biology, University of Florida, Gainesville, Florida 32610, the § Departments of Psychiatry, Pharmacology, and
Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania
19104, and the ¶ Research Service, Malcom Randall Department of
Veterans Affairs Medical Center, Gainesville, Florida 32608
Received for publication, February 15, 2001, and in revised form, March 19, 2001
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ABSTRACT |
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We recently identified conformational changes
that occur upon phosphorylation of myristoylated alanine-rich protein
kinase C substrate (MARCKS) that preclude efficient cross-linking of actin filaments (Bubb, M. R., Lenox, R. H., and Edison,
A. S. (1999) J. Biol. Chem. 274, 36472-36478).
These results implied that the phosphorylation site domain of MARCKS
has two actin-binding sites. We now present evidence for the existence
of two actin-binding sites that not only mutually compete but also
specifically compete with the actin-binding proteins thymosin
Dynamic remodeling of the actin cytoskeleton is a requisite for
the morphological changes that occur at synaptic junctions as a result
of learning and development (1-4). While several actin-binding
proteins have been identified that may contribute to the alteration of
cytoskeletal dynamics that occurs during synapse maturation,
myristoylated alanine-rich protein kinase C substrate
(MARCKS)1 has demonstrable
in vitro and in vivo properties that correlate with known signaling events and observed alterations in structural plasticity (5-8). In the nonphosphorylated state, MARCKS
cross-links actin filaments and localizes to the plasma membrane of
neuronal dendrites in culture, features that would be expected to
contribute to membrane stability in the quiescent neuron (7, 9).
Repetitive stimulation activates protein kinase C and phosphorylates
MARCKS, coincident with membrane reorganization into dynamic pre-
and postsynaptic filopodial structures (10, 11). Corresponding to this
change in cytoskeletal function, phosphorylated MARCKS neither
efficiently cross-links actin nor localizes to plasma membrane (6, 7,
9).
In an effort to better understand the actin filament cross-linking
activity of MARCKS, we have identified and biophysically characterized
two actin-binding sites within the 25-amino acid basic phosphorylation
site domain (PSD). This domain not only interacts with actin but also
can bind to plasma membrane or to calmodulin. Our previous studies (12)
showed that nonphosphorylated MARCKS PSD is a monomeric, extended rod,
with no significant helical structure. Phosphorylated MARCKS PSD, in
contrast, has a more compact conformation. These differences result
from the partial neutralization of polycationic charges at each end of
the PSD by phosphate. Since phosphorylated MARCKS PSD did not
cross-link actin filaments as well as nonphosphorylated MARCKS PSD, we
speculated that either steric and ionic effects of phosphorylation
resulted in a loss of site-specific affinity at either of two
postulated actin-binding sites or that the conformation changes in the
PSD resulted in a dynamic structure that had an inaccessible
actin-binding site. In the current work, we recognize that this
dichotomy could be incomplete and, indeed, present evidence that this
is the case. Moreover, the constraints imposed by conformation could be
less restrictive than previously speculated. For example, perhaps a change in orientation of the two binding sites due to phosphorylation could disrupt cooperative or localizing interactions between MARCKS and
F-actin, without an actual change in the stoichiometry of interaction.
Conformational regulation of the cytoskeletal dynamics by nonglobular
proteins such as MARCKS has ample precedent. Various microtubule-associated proteins bind to and cross-link actin, and like
MARCKS PSD, this activity is inhibited by phosphorylation (13). These
microtubule-associated proteins, like MARCKS, appear to be loosely
folded proteins in solution that may change conformation on binding to
tubulin or actin (14). Synapsins are a family of synaptic vesicle
phosphoproteins that cross-link and bundle F-actin. Phosphorylation of
synapsin I induces major conformational changes in the molecule and
simultaneously abolishes actin bundling activity (15). Synapsin I, like
the actin-binding proteins thymosin Several polycationic ligands have been previously shown to bind to
actin (20). The polycationic segments of the MARCKS PSD have been
assumed to contribute to the affinity of this ligand for actin, and
like other polycationic actin-binding domains such as that of myosin
(21), this region may interact with the highly charged N terminus of
actin (22). Specificity of binding is probably the result of additional
interactions between spatially distinct regions, and in the case of
MARCKS, we have identified a hexapeptide that is repeated in both the
N- and C-terminal halves of the PSD. In the current work, we show that
nonionic characteristics of this hexapeptide contribute to actin
binding activity. The specificity of MARCKS-actin interactions is
further supported by the finding that certain, but not all,
actin-binding proteins compete with MARCKS peptides for actin.
Materials--
Rabbit skeletal muscle actin was prepared from
frozen muscle (Pel-Freez, Rogers AR) in buffer G (5.0 mM
Tris-HCl, 0.2 mM ATP, 0.2 mM dithiothreitol,
0.1 mM CaCl2, and 0.01% sodium azide, pH 7.8)
(24), and pyrenyl-labeled
actin2 was prepared with
0.7-0.95 mol of label/mol of protein using the method of Kouyama and
Mihashi (23). Recombinant human profilin and thymosin Analytical Ultracentrifugation--
Sedimentation equilibrium
experiments were performed using absorption optics with data collected
at 535 nm (the absorption maximum for rhodamine-labeled MARCKS
peptides) in a Beckman XLA centrifuge. All samples contained 3.5 µM labeled MARCKS peptide and variable amounts of
unlabeled Ca2+-actin (4.0 or 20 µM). Samples
of 110 µl in buffer G (except 40 mM KCl and 10 µM ATP) reached equilibrium in 48 h at 14,400 r.p.m. (after initially overspeeding to 15,000) at 4 °C. Buffer density was
determined by pycnometry, and partial specific volumes were as
previously reported for actin or calculated from the amino acid
sequence for MARCKS (27). With no actin present, the data (expressed as
concentration as a function of radius) were compared with that
predicted by the Svedberg equation assuming a mass calculated from the
sequence of the MARCKS peptide. For experiments employing full-length
PSD peptide, the actin concentration was 20 µM, and the
data were fit so as to test the assumption that peptide was saturated
by actin. In this case, the apparent molecular weight of the peptide is
that of peptide plus any bound actin, and specific hypotheses tested
included saturation of peptide with either one or two actin subunits.
With saturating actin at 20 µM, the samples also
contained 24 µM latrunculin A to ensure that the actin
did not polymerize. Anisotropy experiments mentioned below provided evidence that latrunculin A does not affect actin-MARCKS interactions. When the actin concentration was not saturating (4.0 µM),
then the gradient was analyzed assuming that equilibrium binding
conditions were satisfied at all radii (as is appropriate for a
sector-shaped cell) according to a previously described method of
implicit constraints (28). These experiments were the source of the
equilibrium dissociation constants listed in Table II. In brief, at 535 nm only labeled MARCKS has a measurable extinction coefficient. The
other sample components are invisible. Therefore, at this wavelength,
the optical density at any radius is directly proportional to the sum
of the concentration of all allowable MARCKS species (assuming, for
example, 1:1 stoichiometry, these would include MARCKS and MARCKS plus one actin subunit). Curve fitting is constrained by the initial concentration of all components, and the fitting parameters include only Kd, the dissociation constant for MARCKS
peptide and actin, and the concentration of each component at an
arbitrary radius, rb (28). Error estimates were
based on an analysis of the sum of squares of deviations as a function
of Kd (29).
The use of a buffer with low ATP concentration is necessitated by the
tendency of the full-length PSD peptide to aggregate at higher
concentrations of ATP (e.g. 0.1 mM). Aggregation
is slow, requiring more than 2 h at room temperature to produce
significant deviation from monomers. Both unlabeled and labeled
peptides are soluble to more than 5 mM in the absence of
ATP, and although the unanticipated aggregation was discovered only
because of the formation of a colored precipitate for labeled PSD, both
labeled and unlabeled peptides show similar extents of aggregation by sedimentation equilibrium (data not shown). Aggregation was not observed when actin is added to MARCKS simultaneously with the addition
of ATP.
Fluorescence Anisotropy--
Data were collected on a Photon
Technology International (South Brunswick, NJ) spectrofluorimeter.
Tetramethylrhodamine-labeled peptides were excited with vertically
polarized light at 552 nm. The horizontal, (Ih) and
vertical (Iv) components of the emitted light were
measured at 577 nm for ~20 s for each component. The fluorescence
anisotropy, f, is calculated as follows: f = (Iv
Fitting parameters for assays of direct binding of labeled MARCKS
peptide to actin included the equilibrium dissociation constant for
labeled peptide and actin and the terms rf, the
anisotropy of free peptide and rb, the
anisotropy of the complex of labeled peptide with G- or F-actin. Data
from competitive binding assays were evaluated with fixed
rf, rb, and Kd
as determined from a direct binding assay and fit only for the
equilibrium dissociation constant for competing substance and actin.
The data were analyzed as previously described (25), assuming only that
the concentration of free labeled peptide, [L], and
unlabeled peptide [P] satisfy the relation,
[L]/KdL
For experiments involving full-length MARCKS PSD, the data were fit
assuming the presence of two binding sites with site-specific affinity
determined as above. Bound fluorescently labeled peptide was assumed to
have the same anisotropy, rb, independent of which
and how many sites were occupied. In this case, these binding constants
were fixed, and the data were fit to an additional parameter,
F-actin Pelleting Assay--
A 20 µM stock of
actin was converted to Mg2+-actin as before and was
polymerized by the addition of MgCl2 to a final
concentration of 2.0 mM. In some cases, jasplakinolide was
added at a ratio of 1:3 with actin, a sufficient amount to lower the
critical concentration to very close to zero. Peptide affinity for
actin was measured using centrifugation conditions that pellet F-actin
plus bound peptide but not free peptide. Labeled peptides (6 µM) were pelleted with various concentrations of F-actin
in Mg2+-F-buffer with 20 mM KCl and 0.5%
bovine serum albumin for 30 min at 150,000 × g. The
supernatants were compared for relative fluorescence intensity (345-nm
excitation, 388-nm emission for pyrene-labeled peptide; 553-nm
excitation, 574-nm emission for rhodamine-labeled peptide). The
fraction of specifically bound peptide was calculated by comparison
with an unspun control. Data were fit assuming a single binding site.
For curve fitting purposes, it is convenient to switch the dependent
and independent variable and fit the function, At = Pt × Fb + Kd × Fb/(1 NMR Spectroscopy--
Two-dimensional nuclear Overhauser effect
spectroscopy (31) and total correlation spectroscopy (32) experiments
were carried out on full-length Ala-substituted PSD (Table I). Data
were collected on a 600-MHz Bruker Avance spectrometer. The mixing
times were 400 and 60 ms for the nuclear Overhauser effect spectroscopy
and total correlation spectroscopy experiments, respectively. Spectral widths in all dimensions were 8333.3 Hz, which were digitized with 2048 and 256 complex points in the acquisition and indirect dimensions,
respectively. Data were processed using NMRPipe (33) and analyzed using
NMRView (34).
Sedimentation Equilibrium Experiments Show That the MARCKS PSD Can
Be Saturated by Two Actin Subunits--
In our previous work (12), we
showed that when MARCKS and actin are mixed at a ratio of 1:2, the
gradient obtained by sedimentation equilibrium deviates from that of
monomeric actin, implying that MARCKS PSD induces an aggregated state
of actin. The data were consistent with the formation of a small amount
of dimer, but other states of oligomerization could not be excluded.
Now we have used rhodamine-labeled MARCKS PSD to make a highly
sensitive technique for detection of specific actin oligomers. When
fluorescently labeled MARCKS PSD is mixed with unlabeled actin, the
optical absorbance of the label as measured by sedimentation
equilibrium reflects only the apparent molecular weight of the PSD.
Thus, by adding saturating amounts of actin (invisible at 540 nm), the stoichiometry of interaction is shown to be two actin per PSD (Fig.
1). The labeled PSD, by itself, is shown
to be monomeric.
Peptides That Correspond to the N- and C-terminal Halves of the PSD
Show That Both Halves Have an Actin Binding Site and That the Two
Binding Sites Compete with Each Other--
Given the presence of two
binding sites on the PSD, two observations enabled us to select likely
portions of the PSD that would bind actin. First, since polycations are
known ligands of actin (20), both polycationic termini of the PSD were
probably contributing to the interaction. Second, we observed that the mouse PSD sequence contained an identically repeated hexapeptide, FSFKKS, at residues 151-156 and 162-167, and this hexapeptide resembles the VTVKKV segment that contributes to the actin-binding activity of Acanthamoeba actobindin (35). Peptides
synthesized to facilitate identification of the actin-binding sites on
MARCKS are shown and named in Table I.
The N-terminal and C-terminal PSD peptides each contain both the
hexapeptide motif and a polycationic region. Representative
equilibrium binding data from sedimentation equilibrium (Fig. 1),
fluorescence anisotropy (Fig. 2), and
F-actin pelleting (Fig. 3) assays are
shown. The results of these quantitative assays are summarized in Table
II. Competitive binding anisotropy assays
employing unlabeled peptides yield systematically lower affinity than
direct binding assays of labeled peptides (Fig. 2), suggesting that the
label is not completely inert but may contribute to binding. Perhaps
these bulky hydrophobic labels serve to replace the loss of
phenylalanine in the intact PSD. The competitive binding assays allow
for direct comparison of unlabeled C- and N-terminal peptides and imply
that the N-terminal peptide probably binds with slightly higher
affinity. There are also systematic differences between the results
obtained using different assay methods to evaluate the same peptide,
although when assays employ the same method, the relative affinity of
different peptides is independent of method. The addition of four
residues to the N-terminal PSD peptide, "longer rhodamine-labeled
N-terminal PSD," had no significant effect on affinity as determined
by pelleting and sedimentation equilibrium assays, suggesting that
these residues may be important only to prevent steric inhibition by
putting a finite distance between the two actin-binding sites (19 Å between the The Actin-binding Proteins Thymosin A Comparison of MARCKS PSD Binding to F- and G-actin Reveals
Localizing Interactions between MARCKS and F-actin--
Binding of the
N-terminal PSD peptide to F-actin is increased relative to G-actin by a
factor of less than 2 (Fig.
5A, inset). Theoretical arguments indicate that the loss of entropy for MARCKS upon
binding to G-actin is less than that upon binding to F-actin. This
difference can be quantitatively estimated as ~0.7 kcal/mol when
full-length MARCKS binds to actin (38), but negligible when the MARCKS
PSD peptide binds to actin. Thus, full-length MARCKS might bind to
G-actin with similar or even higher affinity than to F-actin.
Binding of the PSD peptide to G-actin can be entirely explained by
assuming that the full-length PSD peptide has two actin binding sites
each with site-specific Kd in the ratio reported for
binding to G-actin by unlabeled N- and C-terminal peptides in Table II
(Fig. 5B). In contrast, binding of the full-length PSD
peptide to F-actin, assuming the same ratio, but with site-specific Kd as reported in Table II for binding of
rhodamine-labeled N-terminal PSD to F-actin, can only be explained by a
highly cooperative interaction between the PSD and F-actin or an effect
of localization of binding sites (Fig. 5C). The fitting
algorithm allowed the value of anisotropy for bound peptides,
rb, to float because the labels are at different
sites on the full-length and the N-terminal peptide and therefore
probably have different constraints on rotational diffusion when bound
to actin. Furthermore, rb varies depending on
whether the peptide is bound to G- or F-actin (Fig. 5A), and
this may reflect differences in the local environment of the label
rather than global differences in peptide motion.
The best fit to the data in Fig. 5C requires that the
interaction parameter Phosphorylation or Alanine Substitution Results in a Modest
Decrease in Site-specific Equilibrium Dissociation Constants for PSD
Peptides--
A comparison of binding by rhodamine-labeled N-terminal
nonphosphorylated (Kd = 2.0 µM) and
phosphorylated peptide (Kd = 6.8 µM)
in Fig. 6A and binding by
C-terminal nonphosphorylated (Kd = 28 µM) and phosphorylated peptide (Kd = 62 µM) in Fig. 6B and additional data shown in
Table II imply that phosphorylation results in a decrease in affinity
corresponding to Differences in Cross-linking of Actin Filaments between
Phosphorylated and Nonphosphorylated MARCKS Peptides Cannot Be
Explained by the Differences in Site-specific Affinity but Are
Consistent with Conformational Regulation of Function--
We find, as
previously reported (9, 12), that the phosphorylated PSD cross-links
actin less efficiently than nonphosphorylated PSD (Fig.
7), and because of uncertainty regarding
the relationship between the extents of cross-linking and
light-scattering, the difference between the activity of two peptides
is potentially very great (as reported by others (9)). Our current data
also show that the Ala-substituted PSD cross-links F-actin much better than phosphorylated PSD (Fig. 7). Since the results in Table II show
that the effects of alanine substitution are at least as great as that
of phosphorylation on the affinity of each site-specific actin-binding
site, these cross-linking results imply that something other than the
effect of phosphorylation on the site-specific Kd is
responsible for the loss of cross-linking activity. Thus, rather than
steric or ionic effects on the site-specific Kd,
these results are consistent with the hypothesis that conformational
changes induced by phosphorylation result in a more
compact structure that cannot effectively cross-link actin filaments.
The alanine-substituted peptide, although demonstrating a similar
decrease in site-specific affinity, has little or no structure by NMR
as evidenced by nearly complete overlap resonances and only two weak
sequential amide to amide NOEs between Phe13 and
Lys14 and between Gly17 and Ala18
(data not shown). No other nuclear Overhauser effects are observable in
the amide region. Therefore, unlike phosphorylated PSD (12), the
alanine-substituted peptide does not fold into a compact structure and
therefore can still cross-link actin filaments.
A single domain of MARCKS, the PSD, is able to reproduce the known
actin binding functions of the intact protein (9). The unfolded state
of the intact molecule (42) and its lack of self-association (43) imply
that the function of the PSD is unlikely to be influenced by
neighboring domains, other than by small entropic factors, making the
study of a peptide corresponding to the PSD particularly appropriate
for detailed investigation. Our previous work (12) suggested that the
PSD undergoes conformation changes related to phosphorylation, changing
from an extended, rigid structure to a more compact state that can be
logically related to partial neutralization of the polycationic regions
by the phosphate groups. Although more compact, the phosphorylated PSD
is a structurally dynamic molecule that, like nonphosphorylated PSD,
lacks canonical structure. This contrasts with a strong propensity for
the formation of Relatively large differences were observed in the values for
equilibrium dissociation constants reported in Table II, and most of
these differences can be attributed to the steep dependence of these
constants on ionic conditions. Other differences were suggestive of
systematic variation related to methodology. The sedimentation
equilibrium data reflect 2-3 times higher affinity than that obtained
by anisotropy, possibly related to the differences in pressure effects
on the noncovalent complex. (45). Differences between Ca2+-
and Mg2+-actin could also be responsible for differences
between centrifugation and anisotropy assays, but the prolonged
duration for centrifugation requires the use of more stable (and less
physiologic) Ca2+-actin. Some differences are probably
related to true differences between binding to F- and G-actin, since
these differences were observed even when using the same method at
nearly identical ionic conditions (Fig. 5A). The observed
dissociation constants are of the same magnitude as the 12 µM concentration reported for MARCKS in calf brain (42),
and while our own interpretation of the original data (43) suggests
that this reported value may be an overestimate by a factor of 10, localized concentrations in neurites, as seen by indirect
immunofluorescence (7), almost certainly equal or exceed this value.
Excluded volume effects in the molecularly crowded environment of the
synapse could substantially increase the effective concentration of
MARCKS (46).
Recently published work by Wohnsland et al. (41) evaluated
MARCKS PSD peptides, some of which were similar to those in the current
study. Although the assays differed significantly in that Wohnsland
et al. included measurements of function and not binding affinity, it is interesting to evaluate our conclusions in light of
these published data. (a) Consistent with our results, an
electron microscopy assay of actin filament cross-linking showed that
substitution at the phenylalanine residues had minimal effect on this
function. (b) Despite the absence of effect on cross-linking
activity, substitution at the phenylalanine residues had a significant
effect on the ability of the peptide to stimulate actin polymerization,
consistent with our results that showed an unambiguous effect on the
site-specific Kd. (c) substitutions or
truncations at either of the terminal regions that we have defined as
actin-binding sites resulted in diminished cross-linking function,
consistent with our hypothesis that these two actin-binding sites are
necessary for activity.
Because of its ligands, calmodulin and phosphatidylinositol
4,5-bisphosphate (7), and its post-translational modification by
phosphorylation, MARCKS (and in particular, the MARCKS PSD) integrates
several important signaling cascades to provide an output signal that
regulates actin-filament cross-linking activity. Given its localization
to the plasma membrane (6) and to the synaptic junction (7),
cytoskeletal remodeling by MARCKS may function to alter morphologic
plasticity of the synapse, with focal changes in plasma membrane
rigidity conceivably affecting ion channel activity, receptor protein
coupling to second messenger systems, vesicle-mediated release at
presynaptic sites, and structural maturation of dendritic spine (47).
Characterization of actin-binding sites on MARCKS, in conjunction with
studies to delineate the specific functional implications of
MARCKS-actin interactions, will facilitate drug development that
targets these key features of actin-membrane plasticity within the brain.
4 and actobindin to bind to actin. The effects of
substitution of alanine for phenylalanine within a repeated hexapeptide
segment suggest that the noncharged region of the domain contributes to
binding affinity, but the binding affinity of peptides corresponding to
each binding site has a steep dependence on salt concentration,
consistent with presumed electrostatic interactions between these
polycationic peptides and the polyanionic N terminus of actin.
Phosphorylation decreases the site-specific affinity by no more than
0.7 kcal/mol, which is less than the effect of alanine substitution.
However, phosphorylation has a much greater effect than alanine
substitution on the loss of actin filament cross-linking activity.
These results are consistent with the hypothesis that the compact
structure resulting from conformational changes due to phosphorylation, in addition to modest decreases in site-specific affinity, explains the loss of cross-linking activity in phosphorylated MARCKS.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 and actobindin,
has little or no secondary structure by circular dichroism in aqueous
buffers (but does fold into more extensive
-helix in
trifluoroethanol) (16-18). Thus, it appears that the conformational
changes we have observed in MARCKS PSD are probably representative of
an important mechanism to regulate actin dynamics and neuroplasticity.
This idea is consistent with the paradigm proposed by Wright and Dyson
(19) that unfolded, nonglobular proteins control key regulatory checkpoints.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
were purified as previously described (24, 25), and actobindin was
isolated from Acanthamoeba castellanii (26). All peptides
were synthesized at the University of Florida; the MARCKS peptides are
identified by name in Table I. Peptides were covalently modified with
fluorophores while still bound to resin, and in the case of N-terminal
fluorophores, these are exclusively
-amino modifications. The
peptides were purified by HPLC and then shown to be greater than 96%
labeled by analytical HPLC and mass spectroscopy. Concentrations of
proteins and peptides were confirmed by amino acid analysis.
Latrunculin A (BIOMOL Research Laboratories, Plymouth Meeting, PA) and
jasplakinolide (Molecular Probes, Inc., Eugene, OR) were stored as
concentrated stock solutions in Me2SO at
80 °C.
Fluorescent chemicals were obtained from Molecular Probes.
GIh)/ (Iv + 2GIh). The G factor was determined for
the peptide in solution excited with horizontally polarized light and
averaged over ~100 measurements. The total intensity of the labeled
peptide fluorescence, Iv + 2GIh
did not change significantly upon actin binding, and the observed
anisotropy was therefore assumed to be a linear function of the
fraction of peptide bound to actin. The experiments were performed in
0.3-ml samples in glass cuvettes with 0 or 40 mM KCl in
Mg2+-G (buffer G in which Ca2+-actin has been
converted to Mg2+-actin by the addition of 125 µM EGTA and 50 µM MgCl2 10 min
prior to the experiment) to measure binding to G-actin or in
Mg2+-F-buffer (buffer G in which the actin was first
converted to Mg2+-actin and then 10 min later polymerized
by the addition of MgCl2 to a final concentration of 2.0 mM) to measure binding to F-actin. For direct binding
assays at a peptide concentration of 0.35 or 2.0 µM,
anisotropy was measured as a function of actin subunit concentration.
For competition assays, the direct binding assay is used to determine a
nonsaturating amount of actin that will bind about two-thirds of the
labeled peptide, and the anisotropy is measured as a function of
concentration of competing substance. Because polycations have been
previously reported to stimulate actin polymerization, assays involving
full-length PSD and G-actin were repeated in the presence of saturating
amounts of latrunculin A, after first determining that latrunculin A
had no effect on the assay, i.e. did not compete with or
augment actin-MARCKS interactions as determined by anisotropy.
(1 + [P]/KdP), where KdL
and KdP are the respective equilibrium dissociation
constants for labeled and unlabeled peptide-actin complex. In some
cases, additional iterations were added to the fitting algorithm to
ensure accuracy. To make the fit more robust, in some competition
experiments data were collected at more than one concentration of
actin, and all data were fit simultaneously. Representative experiments
were performed in triplicate, and these results were used to generate error estimates.
, the ratio of equilibrium dissociation constants for binding
at one site relative to that for the same site when the other site is
occupied. This additional parameter describes either cooperative
interactions between the two sites, in which case positive
cooperativity is indicated by
greater than 1, or instances in which
is greater than 1 because of localization of binding sites
(i.e. where binding results in a higher local concentration
of ligand than that found in a dispersed solution) (30). For example, a
bivalent PSD might bind to two contiguous subunits on an actin filament
with no alteration in site-specific Kd, in which
case localization of the second binding site by occupancy of the first
would result in
greater than 1. Similarly, cross-linking of actin
filaments by one MARCKS peptide could increase the local concentration
of available binding sites for the second binding site of
other MARCKS peptides bound to the cross-linked filaments.
Fb), where
Pt is the total peptide, At is
the total actin, and Fb is the fraction of peptide
bound. Error estimates are based on three replicates.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Sedimentation equilibrium analysis of the
MARCKS PSD and actin. When rhodamine-labeled PSD is centrifuged
alone, it behaves as though it is monomeric with a molecular
mass consistent with sequence (3.9 kDa) (squares).
The top panel shows the residuals with respect to
the expected curve for monomer. When centrifuged in the presence of
nearly saturating amounts of actin (20 µM), the apparent
molecular mass of the PSD (3.5 µM) shifts to that of a
much larger protein complex (circles). The accompanying
solid line show the expected results if all of the MARCKS
was saturated with two actin, so that its apparent molecular mass was
(42 × 2) + 3.9, or 87.9 kDa. The result if PSD should bind to
only one actin at saturation is shown by the line to which
there is no associated data; this line is easily distinguished from
that of the actin-saturated PSD. The third set of equilibrium data is
for 3.5 µM rhodamine N-terminal PSD and 4 µM actin (triangles). These data have been fit
for both centrifugal and chemical equilibrium, yielding
Kd = 0.90 µM.
carbons of Ser156 and Phe162 at
the respective end and beginning of the repeated hexapeptide motif as
per the model in Ref. 12). Competitive binding studies between the N-
and C-terminal peptides (Fig. 2) imply that the peptides either bind to
the same site on actin or allosterically affect each other's binding
site.
Synthetic peptides
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Fig. 2.
Fluorescence anisotropy assay showing
competition between N and C termini. Titration of 0.35 µM rhodamine labeled N-terminal PSD peptide solutions is
shown with the same, but unlabeled, peptide (circles,
solid line) or with C-terminal PSD peptide
(triangles, dashed line) in the presence of 0.8 µM Mg2+ G-actin. Inset, results of
a direct binding assay in which rhodamine-labeled N-terminal PSD
peptide was titrated with actin. All samples contain
Mg2+-actin in no KCl. Lines represent the best
simultaneous fit to all data with Kd = 0.03 for
labeled N-terminal peptide (inset), Kd = 0.12 for unlabeled N-terminal peptide, and Kd = 0.13 for unlabeled C-terminal peptide.
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Fig. 3.
Identification of actin binding segments of
MARCKS using an F-actin pelleting assay. Results of a pelleting
assay showing that both N- and C-terminal PSD peptides bind to F-actin.
Bound peptide is shown as a function of total F-actin. Error
bars are ±2 for samples done in triplicate and are shown
only for the longer rhodamine-labeled N-terminal PSD
(circles) for clarity. Based on these data using labeled
peptides in 20 mM KCl, the longer rhodamine-labeled
N-terminal PSD binds with Kd of 0.4 µM, and pyrene-labeled C-terminal PSD
(triangles) binds with Kd of 0.9 µM.
Equilibrium dissociation constants (Kd) for MARCKS peptides
organized by experimental technique, ionic conditions, and whether
bound to G- or F-actin
4 and
Actobindin, but Not Profilin, Compete with MARCKS PSD to Bind
Actin--
The specificity of the interaction between MARCKS PSD and
actin was revealed by competitive binding studies that showed that either thymosin
4 or actobindin inhibits binding by
N-terminal MARCKS PSD peptide (Fig. 4).
The quantitative results were consistent with our previously reported
binding constants for actobindin and thymosin
4 (25,
28), although substantially lower affinity for thymosin
4 and actin has been reported based on other steady state methods (36). The site-specific nature of the interaction was
further confirmed by a global fit of the thymosin
4 data for two different concentrations of actin. The failure to completely displace MARCKS peptides at near saturation is perhaps due to the
inability of thymosin
4 or actobindin to completely
shield the postulated binding site at the charged N terminus of actin, resulting in weak residual interactions with MARCKS peptide. Profilin, with an actin-binding site near to, but not identical to, that of
thymosin
4 (37), did not compete with N-terminal MARCKS PSD. The slight increase in anisotropy observed in the presence of
profilin may either reflect that an increase in the affinity of actin
for MARCKS or perhaps the steady-state anisotropy of the ternary
complex is simply a little higher than that for the MARCKS-actin
complex alone. Similar experiments with the actin-binding marine
macrolide, latrunculin A, show that concentrations of up to 100 µM had no effect on the anisotropy of the N-terminal
MARCKS PSD-actin complex, indicating that latrunculin A was also unable to compete with the N-terminal PSD (data not shown). Both the data for
alanine-substituted peptides and for competition with actobindin
support the hypothesis that a repeated hexapeptide within the PSD
contributes to the free energy of binding.
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Fig. 4.
Competition between MARCKS and other
actin-binding proteins. Titration is shown of 0.35 µM rhodamine-labeled N-terminal PSD peptide and
Mg2+-G-actin in 40 mM KCl with thymosin
4 in the presence of 1.5 (circles) and 3.0 µM (squares) of actin, with profilin
(triangles) in the presence of 2.0 µM actin,
and with actobindin (inset, circles) in the
presence of 2.0 µM actin. The lines represent
the best global fit to all of the data for thymosin
4
with an equilibrium dissociation constant, Kd, of
0.07 ± 0.06 µM for binding of thymosin
4 to actin. The best fit to the actobindin data was
obtained with Kd of 2.2 ± 0.3 µM. The profilin data are depicted with an arbitrary
line.
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Fig. 5.
Comparison of F- and G-actin binding
properties of MARCKS. Titration of 0.35 µM
full-length labeled PSD (closed symbols) or
rhodamine-labeled N-terminal PSD (open symbols) with F- or
G-Mg2+-actin in 40 mM KCl. As a control, the
assays with G-actin were repeated in the presence of saturating amounts
of latrunculin A to exclude the possibility that the PSD may have
stimulated actin polymerization (41). A, a comparison of
binding of labeled PSD to F-actin (triangles), G-actin
(squares), and G-actin in the presence of 40 µM latrunculin A (circles) shows a large
increase in apparent affinity for F-actin relative to G-actin. In
contrast, the rhodamine-labeled N-terminal PSD (inset) binds
to F-actin (triangles) with Kd = 1.5 µM (solid line) and to G-actin
(squares) with Kd = 2.0 µM
(dashed line). The use of 40 µM latrunculin A
(circles) did not significantly change the results for G-actin. B, the data in
A are replotted to illustrate the difference between binding
of labeled PSD (closed symbols) or rhodamine-labeled
N-terminal PSD (open symbols) to G-actin. Using the best
estimate for site-specific affinity of the C-terminal actin binding
site (Kd = 1.5 times that of the N-terminal actin
binding site) and the best fit for the N-terminal binding site
(dashed line), the data for full-length labeled PSD can be
fit assuming independent binding sites (solid line) but
cannot be well fit assuming any significant extent of cooperativity
( = 50, dotted line). Inset, magnified
portion of the plot at low concentrations of actin. C, the
data in A are replotted to illustrate the difference between
binding of labeled PSD (closed symbols) or rhodamine-labeled
N-terminal PSD (open symbols) to F-actin. Curve fitting as
in B reveals that only a large, positive value for
is
consistent with the data for full-length labeled PSD (
= 2.5 × 103, solid line), and in contrast to
B,
= 1 provides a very poor fit (dotted
line). The dashed line is the best fit to the
rhodamine-labeled N-terminal PSD data, and the inset shows a
magnified portion of the plot at low concentrations of actin.
(see "Experimental Procedures") be
2.5 × 103, indicating that occupancy of one site of
the PSD has localized the second binding site near its ligand, causing
a relative increase in the amount bound. Alternatively, if positive
cooperativity between the two binding sites on the PSD were responsible
for the large value of
, then it would have been reasonable to
expect a similarly large value of
in the interaction of PSD with
G-actin. Thus, the discrepancy between results with G- and F-actin is
consistent with localization of PSD near its ligand, resulting in a
higher effective concentration of ligand, but not with cooperativity between the two postulated PSD binding sites. Assuming that
interactions with F-actin are occurring at a single site, then, because
only 19 Å separates the repeated hexapeptide motifs, models of F-actin (39, 40) limit this localization phenomenon to interfilament cross-linking. Localization of MARCKS binding sites on F-actin is
therefore more likely due to cross-linking of filaments than to
occupancy of adjacent sites on a single filament.
G = 0.7 kcal/mol (range: 0.3-1.4
kcal/mol) in 40 mM KCl. Assuming the hypothesis that MARCKS
has two actin-binding sites that localize to the N and C terminus, then
this decrease represents site-specific changes in affinity related to
either steric or ionic effects. Decreases similar or greater in
magnitude were observed after substituting alanine for phenylalanine
within the repeated hexapeptide segments of the PSD with
Kd = 7.1 µM for rhodamine-labeled N-terminal Ala-substituted PSD and Kd = 125 µM for C-terminal Ala-substituted PSD (Fig. 6). These
results are consistent with other reports that the interaction between
PSD and actin is not entirely mediated by polycationic effects (41)
and, moreover, imply that the interaction between the repeated
hexapeptide and actin may contribute specificity to the interaction
between MARCKS and actin beyond that of polycationic interactions.
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Fig. 6.
Fluorescence anisotropy assay showing the
effect of phosphorylation or alanine substitution on site-specific
binding of N-terminal and C-terminal PSD peptides. A,
titration of 0.35 µM rhodamine-labeled N-terminal PSD
peptides with Mg2+-G-actin in the presence of 40 mM KCl. The lines represent the simultaneous fit
to all three sets of data for rhodamine-labeled N-terminal PSD
(squares, solid line), rhodamine-labeled
N-terminal phosphorylated PSD (circles, dashed
line), and rhodamine-labeled N-terminal Ala-substituted PSD
(triangles, dotted line). B, titration
of 0.35 µM rhodamine-labeled N-terminal PSD peptide with
C-terminal unlabeled peptides in the presence of 2 µM
Mg2+-G-actin and 40 mM KCl. The
lines represent the simultaneous fit to all three sets of
data for competitive binding to rhodamine-labeled N-terminal PSD by
C-terminal PSD (squares, solid line), C-terminal
phosphorylated PSD (circles, dashed line), and
C-terminal Ala-substituted PSD (triangles, dotted
line).
View larger version (15K):
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Fig. 7.
Light scattering results indicative of actin
filament cross-linking by MARCKS PSD. Actin (7.8 µM)
polymerized in 2 mM MgCl2 and 20 mM
KCl is cross-linked by nonphosphorylated PSD (circles),
Ala-substituted PSD (triangles), and phosphorylated PSD
(squares). The data represent mean values for samples done
in triplicate with S.D. indicated by error bars.
The lines are arbitrary.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helix, as per structure prediction algorithms
(44). MARCKS is therefore an example of the class of proteins described
in the paradigm of Wright and Dyson (19) for nonglobular proteins that
perform key regulatory functions, in which intrinsically unstructured
proteins may adopt secondary structure only upon binding of appropriate ligands.
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FOOTNOTES |
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* This work was supported by the Medical Research Service of the Department of Veterans Affairs and the National High Magnetic Field Laboratory.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Box 100277, Dept.
of Medicine, University of Florida, Gainesville, FL 32610. Tel.: 352-392-4681; Fax: 352-374-6170; E-mail: bubb@medicine.ufl.edu.
Published, JBC Papers in Press, April 6, 2001, DOI 10.1074/jbc.M101457200
2 Actin was labeled on Cys374 with N-(1-pyrene)iodoacetamide.
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ABBREVIATIONS |
---|
The abbreviations used are: MARCKS, myristoylated alanine-rich protein kinase C substrate, PSD, phosphorylation site domain; HPLC, high pressure liquid chromatography..
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REFERENCES |
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