Actin Filament Cross-linking by MARCKS

CHARACTERIZATION OF TWO ACTIN-BINDING SITES WITHIN THE PHOSPHORYLATION SITE DOMAIN*

Elena G. Yarmola, Arthur S. EdisonDagger , Robert H. Lenox§, and Michael R. Bubb||

From the Departments of Medicine and Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 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

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 beta 4 and actobindin, has little or no secondary structure by circular dichroism in aqueous buffers (but does fold into more extensive alpha -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.

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 beta 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 alpha -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.

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 - 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.

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 (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.

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, phi , 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 phi  greater than 1, or instances in which phi  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 phi  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.

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 - 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.

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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


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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.

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 alpha  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.

                              
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Table I
Synthetic peptides
Fluorescent labels are amide-linked, and peptides modified with 5-(and 6)-carboxytetramethyl rhodamine succinimidyl ester are indicated by "rh," peptides modified with Oregon Green 488 succinimidyl ester (5-isomer) are shown by "og," and peptides modified with pyrenyl acetic acid are shown by "py." Phosphorylated serines are in boldface type and underlined.


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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 ±2sigma 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.

                              
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Table II
Equilibrium dissociation constants (Kd) for MARCKS peptides organized by experimental technique, ionic conditions, and whether bound to G- or F-actin

The Actin-binding Proteins Thymosin beta 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 beta 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 beta 4 (25, 28), although substantially lower affinity for thymosin beta 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 beta 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 beta 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 beta 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 beta 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 beta 4 with an equilibrium dissociation constant, Kd, of 0.07 ± 0.06 µM for binding of thymosin beta 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.

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.


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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 (phi  = 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 phi  is consistent with the data for full-length labeled PSD (phi  = 2.5 × 103, solid line), and in contrast to B, phi  = 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.

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 phi  (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 phi , then it would have been reasonable to expect a similarly large value of phi  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.

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 Delta 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).

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.


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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

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 alpha  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.

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.

    FOOTNOTES

* 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.

    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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