| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 282, Issue 8, 5871-5879, February 23, 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1
1234
From the
Department of Cell Biology and Physiology and the Department of Biomedical Engineering and Center for Computational Biology, Washington University, St. Louis, Missouri 63110
Received for publication, October 19, 2006 , and in revised form, December 1, 2006.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
-subunit. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The 
/ heterodimeric capping protein, referred to here as CP,5 is a capper found in essentially every eukaryotic organism and in every vertebrate cell and tissue type (reviewed in Ref. 1). In vertebrate striated muscle, a sarcomere-specific isoform of CP at the Z-line caps the barbed ends of the actin-based thin filaments, leading to the name CapZ. Cells lacking CP show a range of morphologic and developmental defects, based on improper actin assembly and cell motility. In Drosophila, loss of CP is lethal during embryogenesis.
Typical vertebrate cultured cells contain lamellipodial and filopodial processes on their surface, which are based on actin filaments organized into branched networks and straight bundles, respectively. In each case, barbed ends are oriented predominantly toward the plasma membrane, and the local capping of barbed ends is crucial. The branched networks of filaments in lamellipodia are nucleated from Arp2/3 complex, as described in the dendritic nucleation model (2). Capping of the barbed ends over time is a necessary feature of this model in order to keep the filaments short, which makes the branched network stiff, and to restrict or "funnel" polymerization to the newly created barbed ends near the membrane. Inhibition of CP essentially abolishes the formation of lamellipodia and branched networks (3). In such cells, filopodia formation is greatly enhanced, which contributes to the notion that localized inhibition of CP at one place on the membrane may be essential for a filopodium to form.
In vitro, actin-based motility can be reconstituted from pure proteins with a mixture of Arp2/3 complex, ADF/cofilin, and CP (4). Here, the dependence of motility on CP concentration was a bell-shaped curve, suggesting that optimal levels of capping may be crucial for actin-based motility in cells. Indeed, Arp2/3-mediated actin assembly as part of endocytosis in yeast does depend on optimal CP activity (5).
CP is active in the absence of calcium and under a wide range of buffer conditions. However, cells contain a number of proteins that are able to bind to and inhibit CP, such as V-1/myotrophin, CKIP-1, CD2AP, and CARMIL (6-9). In addition, polyphosphoinositides such as phosphatidylinositol 4,5-bisphosphate (PIP2) can bind and inhibit CP (10). Of note, PIP2 appears to be able to remove CP from capped barbed ends (11), which may help to stimulate actin assembly in certain situations in cells. During platelet activation, an initial step appears to be release of CP from the actin cytoskeleton by PIP2 (12); later in the process, CP appears to return to the actin cytoskeleton, presumably binding newly formed barbed ends. Association of CP with the actin cytoskeleton is also seen with actin assembly in Dictyostelium cells responding to chemoattractant (13).
The crystal structure of CP shows the - and -subunits arranged with a pseudo-2-fold rotational axis of symmetry (14). In molecular dynamics simulations, the C-terminal region of the -subunit is highly mobile, and the C-terminal region of the -subunit remains closely apposed to the surface of the protein (6), as suggested by the crystal structure. A recent cryo-electron microscopy analysis of CP bound to the barbed end of the actin filament shows the top surface of CP in contact with actin, with the C-terminal regions of each subunit as likely sites of close contact (15). In biochemical studies, the C-terminal region of each subunit appears to be able to bind to the barbed end, and those interactions are independent of each other (16, 17). When both actin-binding sites are intact, CP binds to the barbed end with subnanomolar affinity because of a low off-rate constant (17).
Our current view of the interaction of CP with the filament barbed end includes the hypothesis that when CP is attached solely by its -tentacle, it is able to wobble. This exposes the top surface of the protein, which includes the actin-binding site near the -subunit C terminus. Recent findings with the protein V-1/myotrophin support this view (6). V-1 appears to bind the -subunit C terminus, and V-1 inhibits capping but does not uncap, as predicted by the hypothesis. Another prediction of the hypothesis is that a molecule that is able to uncap should bind to the top surface of the protein, and this interaction should be independent of the -tentacle.
In this study, to gain insight into how PIP2 binds CP, how PIP2 binding inhibits CP, and how CP binds actin, we wanted to perform a structure/function analysis with site-directed mutagenesis to identify regions of CP necessary for interaction with PIP2. We also performed computational modeling of CP/PIP2 interactions to help guide the mutagenesis and to provide independent and complementary evidence regarding the nature of the interaction. In addition, we sought new evidence for the uncapping effect of PIP2, with direct visualization of actin filament growth in real time by TIRF microscopy. Finally, we wanted to use these results to test the wobble model for the interaction of CP with the filament barbed end.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Plasmid Construction and Mutagenesis—Site-directed mutations were created in a chicken CP 11 pET bacterial expression plasmid (pBJ994) (18) by PCR using the QuikChange method (Stratagene, La Jolla, CA). Primers and plasmids are listed in supplemental Tables I and II, respectively. pET-3d/CP(K256A), encoding a K256A mutation in the -subunit, was constructed using forward primer KKT 255 and reverse primer KKT 256. pET-3d/CP(R260A), encoding an R260A mutation in the -subunit, was constructed using KKT 216 and 217. pET-3d/CP(K95A), encoding a K95A mutation in the -subunit, was constructed using KKT 205 and KKT 206. pET-3d/CP(KR256, 260AA), encoding a R256A, K260A mutation in the -chain, was constructed using pET-3d/CP(K256A) as template and primers KKT 216 and KKT 217. pET3d/CP(RK266, 268AA), encoding an RK266, 268AA mutation in the -subunit, was constructed using KKT 214 and KKT 215. pET-3d/CP(R256A, K260A)(R225A), encoding a R256A, K260A mutation in the -subunit and an R225A mutation in the -subunit, was constructed with primers KKT 257 and KKT 258 using pET-3d/CP(R256A, K260A) as a template plasmid.
Proteins—CP was expressed in bacteria and purified as described (16) with minor modifications for mutants. Purified proteins were stored at -20 °C in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, and 50% glycerol. Muscle actin was purified and labeled with pyrene as described (17). Spectrin-F-actin seeds were prepared from human erythrocytes as described (19). Actin capping assays were performed as described (16).
Rabbit skeletal muscle myosin (Cytoskeleton, Inc., Denver, CO) (10 µM) was dialyzed against 10 mM imidazole, pH 7, 0.5 M KCl, 10 mM EDTA for 2 h. Following a 1-h incubation with 1 mM N-ethylmaleimide on ice, 1 mM DTT was added for 1 h on ice. N-ethylmaleimide-myosin was dialyzed overnight against 50% glycerol in 10 mM imidazole, pH 7, 0.5 M KCl, 10 mM EDTA, 1mM DTT and stored at -20 °C.
Fitting of Actin Polymerization Reactions—Binding constants for CP with actin and lipids were determined by least-squares fitting of full time course data using Berkeley Madonna 8.3 as described (17) with the following kinetic mechanism. In these reactions, A is actin monomer, Nb is free barbed end, CP is capping protein, and L is lipid. In this simple scheme, the capped barbed end, CPNb, can neither add nor lose actin subunits; the complex of CP with lipid, CPL, cannot interact with a barbed end.
For Reaction 1, k+ was 11.6 µM -1 s-1 and k- was 1.4 s-1 (20). The rate constants for capping in Reaction 2 were determined by fitting the experimental data for seeded actin assembly in the presence of CP. A range of CP concentrations was used, generating a family of curves, and they were fit together. The rate constants for CP binding to lipid in Reaction 3 were determined by fitting a set of curves produced by addition of PIP2 at various concentrations.
Tryptophan Quenching Assay for CP/Lipid Interaction—The equilibrium dissociation constant, Kd, for the binding of CP to phospholipid was determined by measuring the quenching of intrinsic tryptophan fluorescence as described (21) with minor modifications. Briefly, fluorescence emission spectra (300-400 nm) were collected using a PTI Quantmaster spectrofluorometer (Photon Technology International, Birmingham, NJ) with excitation at 292 nm. The mixture of CP and phospholipid was incubated for 5 min before was fluorescence measured. The maximum fluorescence intensity (
F) was plotted against the total concentration of PIP2, and Kd was determined by leastsquares fitting as described (21).
TIRF Microscopy—To image the polymerization of individual actin filaments, we used TIRF microscopy essentially as described (22). The system included an inverted microscope (IX-81, Olympus America, Center Valley, PA), an electronmultiplication back-thinned frame transfer charge-coupled device video camera (Model C9100-12, Hamamatsu Photonics, Bridgewater, NJ) with a 60x 1.45 numerical aperture PlanApo oil objective. SlideBook software (Intelligent Imaging Innovations, Denver, CO) operated the system and collected the images. Frames of 1500-ms duration were collected every 2-3 min. Measurements of filament length versus time were obtained.
To construct the microscope flow chamber, No. 0 sapphire coverslips were sonicated in a water bath sonicator for 45 min in 2% (v/v) VersaClean detergent in hot tap water, rinsed in hot tap water, and sonicated for 30 min in hot tap water. Coverslips were rinsed in distilled water, incubated for 3 h in 1 M KOH at 42 °C, rinsed in deionized distilled water, and incubated overnight in 1 M HCl at 42 °C. They were cooled to room temperature, rinsed in deionized distilled water, sonicated for 30 min in deionized distilled water, rinsed twice in 5 mM EDTA, and sonicated for 30 min in EDTA. They were rinsed in 70% ethanol, sonicated for 30 min in 70% ethanol, rinsed in absolute ethanol, sonicated for 30 min in absolute ethanol, and rinsed and stored in absolute ethanol.
Flow cell chambers were prepared as described (23). A clean sapphire coverslip was removed from ethanol and dried. Parafilm strips were stretched to approximately three times their length and placed across the long axis of the coverslip. A conventional glass slide was placed on and perpendicular to the parafilm strips. Pressure was applied and the chamber was flamed briefly to seal it. Solutions were flowed into the chamber via capillary action.
For actin polymerization experiments, unlabeled and Alexa Fluor 488-labeled rabbit muscle actin (Invitrogen) were dialyzed overnight against G buffer and then centrifuged at 100,000 x g for 2 h. The upper two-thirds of the supernatant was taken, and the actin concentration was measured by absorbance at 290 nm. Ca-ATP-actin was converted to Mg-ATP-actin by incubation with a 1/10 volume of 10 mM EGTA/0.2 mM MgCl2 at 23 °C for 2 min. N-ethylmaleimide-inactivated myosin at 0.1 µM in high salt Tris-buffered saline (HS-TBS; 50 mM Tris-Cl, pH 7.6, 600 mM NaCl) was flowed into the chamber and incubated for 1 min at room temperature. The chamber was washed with 1% bovine serum albumin in HS-TBS followed by 1% bovine serum albumin in low salt Tris-buffered saline (50 mM Tris-HCl, pH 7.6, 50 mM NaCl).
To polymerize actin filaments, Mg-ATP-actin with 10% Alexa-labeled actin was mixed 1:1 with 2x polymerization buffer (100 mM KCl, 0.2 mM MgCl2, 2 mM EGTA, 20 mM imidazole, pH 7.0, 100 mM DTT, 0.4 mM ATP, 30 mM glucose, 2% methylcellulose, 40 µg/ml catalase, 200 µg/ml glucose oxidase) giving a final actin concentration of 2 µM. 0.25 nM spectrin-F actin seeds were added immediately. After 10 min at room temperature, 10 nM CP 11 was added. After another 10 min, 10 µl of this mixture was loaded into the flow chamber. The actin filaments were monitored for several min to document the absence of growth, and then various concentrations of PIP2 were added along with 2 µM Mg-ATP actin (30% labeled) in polymerization buffer. Actin filament growth was monitored. The rate of filament growth was measured, as was the percentage of filaments growing over time. The rate of incorporation was converted to subunits/s assuming 370 subunits/µm (24).
Molecular Simulations—The starting point for our computational work was a molecular dynamics simulation of CP (Protein Data Bank code 1IZN
[PDB]
) performed using NAMD (25). We used the CHARMM27 force field, TIP3P waters, particle mesh Ewald, Berendsen temperature, and pressure coupling (NPT ensemble) and 2-fs time steps. The heating was performed in 50,000 steps with -carbon constraints, equilibrated at 300,000 without constraints, followed by a 5-ns production trajectory. Five representative structures of CP were extracted from the simulation at 800-ps intervals. Because the macromolecule is held rigid in molecular docking simulations, using multiple protein snapshots allowed us to capture some degree of side chain and backbone flexibility, resulting in significantly improved docking results (26). The molecular docking was carried out using AutoDock 3.0 (27). As PIP2 is somewhat cumbersome to deal with as a full molecule, we used a modified form where the alkyl tails were truncated, leaving just the inositol and diacylglycerol groups (supplemental Fig. 1). This truncated version of PIP2 was minimized using the Tripos force field, and Gasteiger-Marsili charges were assigned, all using Sybyl 6.8 (Tripos Inc., St. Louis, MO). Docking was performed using the Lamarckian genetic algorithm in AutoDock. Initial docking runs using the entire CP structure showed a preference for the region around the base of the -tentacle. Based on these results, we constructed 0.2-Å spaced grids centered on the -tentacle. 20 docking runs were completed for each of the five CP structures resulting in a total of 100 predictions. These results were clustered with a 3-Å cutoff, resulting in single, top-predicted structure that was used in subsequent analysis.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
|
|
0.3 µM (Fig. 1D), comparable with the value for PIP2, which is less than the expected CMC value. To test physical binding, we used quenching of intrinsic tryptophan fluorescence (10). PIP2 quenched the intrinsic tryptophan fluorescence of CP, with no change in the maximum emission wavelength. The effect was saturable at high PIP2 concentration (Fig. 2A). A simple 1:1 binding model fit the data well (Fig. 2A, gray lines), with a Kd of 5.2 ± 0.2 µM, similar to a previous value for mouse CP in this type of assay (10). diC4-PIP2 and diC8-PIP2 had little effect, whereas diC16-PIP2 produced saturable quenching with a Kd of 7 µM (Fig. 2B), close to the value for purified PIP2. These result support the notion that the ability of PIP2 to assemble in micelles is important for binding and inhibition of CP, presumably because of multimerization of the anionic head groups.
Next, we tested the head group alone, by using IP3 (inositol-1,4,5 triphosphate). In an actin polymerization seeded growth assay, IP3 at 100 µM had no effect on the capping activity of 4 nM CP (data not shown). In a tryptophan fluorescence titration, addition of IP3 to concentrations >100 µM had no effect on the fluorescence of CP (Fig. 2B). As part of this experiment, we tested diacylglycerol, to represent the lipid portion of PIP2. Again, no effect was seen (Fig. 2B). Thus, neither the head group nor the lipid backbone of the PIP2 molecule is sufficient to bind CP, even at high concentrations.
Computational Docking Analysis of PIP2/CP Interaction—To predict potential sites of interaction between PIP2 and CP, we used a computational molecular docking approach. We began with a collection of CP structures produced by molecular dynamics simulation. The use of multiple structures for CP allowed us to sample different side chain and backbone conformations, and because we performed flexible docking where the rotatable bonds in PIP2 were allowed to move freely, this approach captured some degree of flexibility for both molecules. The molecular docking studies resulted in a clear single prediction for the structure of the complex of PIP2 and CP. As one may have anticipated based on the anionic character of PIP2, we saw interaction primarily with a group of basic residues on the surface of CP. This group included Lys-256 and Arg-260 of the -subunit and Arg-225 of the -subunit (Fig. 3, A and B). Given the residues and charged groups involved, the interactions are predominantly electrostatic in nature, with the addition of several hydrogen bonds (Fig. 3B). The inositol ring sits in a pocket formed by these residues, between the two subunits, and the location and orientation of these basic side chains suggests some degree of specificity for PI(4,5)P2 versus other phosphoinositides, as observed previously (28).
|
-subunit (Fig. 3C). Among these conserved basic residues, the CP crystal structure shows that Lys-256, Arg-260, Arg-266, and Lys-268 are on the surface and exposed to solvent (Fig. 3A). In contrast, Arg-259 is conserved but buried, where it forms an ionic bond with the conserved acidic residue Glu-221 of the -subunit. Residue Arg-225 of CP is also highly conserved in sequence alignments and exposed to solvent in the crystal structure. The C-terminal region of the -subunit has been implicated in binding to actin (17), so the presence of a PIP2 micelle at this site would be expected to provide an effective steric block to actin binding, as seen for villin, for example (31).
Mutation of the Conserved Basic Residues—To test for involvement of this basic patch in binding PIP2, we introduced point and truncation mutations into the region. First, we tested binding with tryptophan quenching as shown in Fig. 2 and Table 1. For the residues implicated by the computational docking analysis, the single mutants CP 1(K256A)1, CP 1(R260A)1 and CP 11(R225A) bound PIP2 normally. The double mutant CP 1(R256A, K260A) 1 bound more weakly, with a Kd of 12 µM, compared with 5 µM for wild-type CP. The triple mutant CP 1(R256A, K260A)1(R225A) had a higher Kd of 19 µM.
|
-subunit double mutant KR266, 268AA bound to PIP2 with normal affinity (Table 1). Truncation of 28 residues from the C terminus of the -subunit removes the final helix, including Arg-260, Lys-266, and Arg-268, and leaves Leu-258 as the C-terminal residue. This mutant is known to bind actin poorly (17). The affinity of the CP 28 truncated protein for PIP2 was decreased, with a Kd of 17 µM (Fig. 2, Table 1).
The C-terminal region of the -subunit of CP also appears to function as a binding site for actin, independently of the C terminus (17). Truncation of the -subunit by 34 residues had no effect on the ability of CP to bind PIP2 in the tryptophan quenching assay (Fig. 2, Table 1).
We next tested all of these CP mutants for interaction with PIP2 based on inhibition of capping activity in actin polymerization assays. As part of this analysis, we first determined the affinity of the CP mutants for the F-actin barbed end, i.e."Kcap." These values are required in order to calculate the binding constant for the CP/PIP2 interaction. Wild-type CP (11) inhibited actin polymerization at barbed ends of actin filaments created by spectrin-actin seeds in a dose-dependent manner (Fig. 4A, black lines). Kinetic modeling gave good fits (Fig. 4A, gray lines), with a Kcap of 0.1 ± 0.05 nM (Table 2), similar to previous results (17). The -subunit single and double mutations K256A, R260A, K266A, R268A, and R256A, K260A produced small to moderate increases in Kcap, with values of 0.18, 0.36, 0.4, and 1.3 nM, respectively (Fig. 4, Table 2). The -subunit single mutant R225A also showed a moderate increase in Kcap to 1.2 nM. The triple mutant CP(R256A, K260A)(R225A) capped actin weakly (Fig. 4F), with a Kcap of 45 nM, which was mainly because of an increase in the off-rate constant (Table 2). The -subunit truncation mutant CP(28) capped actin filaments weakly, with a Kcap of 1570 nM, and the -subunit truncation mutant CP(34) capped actin with a Kcap of 10.5 nM (Table 2), consistent with previous studies (17).
|
|
-subunit single and double mutations K256A, R260A, K266A, R268A, and R256A, K260A produced small to moderate increases in the Kd, with values of 0.31, 0.36, 0.41 and 3.9 µM, respectively (Table 3). The -subunit single mutant R225A showed no increase in Kd. The triple mutant CP (R256A, K260A)(R225A) gave a 60-fold increase in Kd to 21 µM (Fig. 5F and Table 3). For the -subunit truncation mutant CP(C28), the Kd was 42 µM, 130-fold greater than the value for wild-type CP (Fig. 5A, Table 3). The -subunit truncation mutant CP(34) had a Kd of 1.6 µM (Fig. 5B, gray lines, and Table 3).
|
-subunit. One interacts with Lys-95, and the other interacts with the helix dipole of helix 5. We mutated Lys-95 to alanine to make CP (K95A). We found that this mutant capped actin with a normal affinity (Table 2), and its affinity for PIP2 was the same as that of wildtype CP in both the actin capping and tryptophan fluorescence assays (Tables 1 and 3).
Direct Observation of Uncapping by PIP2—One prediction of the wobble model is that an inhibitor of capping that does not bind to the CP -subunit tentacle, about which the wobbling is proposed to occur, should be able to uncap. Indeed, previous studies showed that addition of PIP2 to capped actin filaments in a polymerization reaction leads to an increase in the polymerization rate consistent with complete and rapid uncapping (11). Here, we tested uncapping with a more direct assay in which individual actin filaments are visualized by TIRF microscopy. In these experiments, movies reveal actin filaments growing from their ends. When CP was added, the growth stopped. Subsequent addition of PIP2 caused the ends to return to the growing state (Fig. 6A and supplemental Movie 1). The mean rate of growth was 3.4 ± 0.3 subunits/s before capping and 2.8 ± 0.4 subunits/s after uncapping, in the presence of 2.0 µM monomeric actin. The effect of PIP2 was nearly complete and relatively rapid, which is comparable with previous results with assays in solution (11). The fraction of ends converted from the capped to the growing state increased with PIP2 concentration (Fig. 6B). The concentration of PIP2 producing a half-maximal effect in this assay was 100 µM, substantially higher than those seen with the tryptophan fluorescence and actin capping assays above. This may reflect sequestration of PIP2 by the relatively large surface of bovine serum albumin-coated glass in this assay or perhaps a decreased accessibility of the actin filaments because of tethering at the surface.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Nature of the Interaction between CP and PIP2—We sought evidence of what regions of CP were needed for its interaction with PIP2 by site-directed mutagenesis, changing single amino acids and truncating the C termini of the two subunits of the heterodimer. In previous studies, clusters of basic residues on the surface of proteins were implicated in binding PIP2. For example, crystal structures of an AP180 protein complexed with phosphatidylinositols reveal a surface-exposed binding site composed of three lysine residues and one histidine, which contact the phosphate groups (32). Sequence analysis of related proteins, combined with mutagenesis, identified a consensus binding sequence of KX9KX(K/R)(H/Y). PIP2 binds and inhibits a number of actin-binding proteins, including profilin and the gelsolin family, and a number of studies identify (K/R)X3-5(K/R)X(K/R)(K/R) as a consensus binding site (31, 33, 34).
In addition, we know that CP from every organism tested, including yeast, plants and mammals, is inhibited by PIP2 (10). Therefore, we focused on conserved clusters of basic residues on the surface of the protein. The largest and most distinct cluster is in and around the C-terminal region of the -subunit, including its connection to the body of the protein. A recent study of phosphoinositides interacting with Arabidopsis CP also noted the existence of this conserved basic region and suggested that it might be important (10). Indeed, we found that a cluster of three conserved basic residues on the surface, near the C terminus, were important for binding PIP2. These include Lys-256 and Arg-260 of the -subunit and Arg-225 of the -subunit. In contrast, two nearby basic residues, also conserved and on the surface, were not important.
Computational docking studies provide complementary evidence for the primary role of these three residues. Of note, the docking studies were done largely in advance of the mutagenesis studies, and they helped us to narrow and define our list of potential mutants. A recent computational study of CP interacting with lipids, including ones other than polyphosphoinositides, raises the possibility that portions of CP may become partially buried in the lipid bilayer (35). One of several regions identified as potentially important in that study was the 215-232 region of the -subunit, which includes Arg-225, one of the three residues identified as important in our results here. The notion of lipid binding to the interior of CP may be supported by an early study in which, by gel filtration analysis, micelles of PIP2 appeared to partially dissociate the - and -subunits (28).
Implications for How CP Binds Actin—As part of our analysis, we tested the ability of a number of new point mutants of CP to cap actin filaments. Previous studies, largely with truncations, had implicated the C-terminal region of the -subunit as important, and these new point mutations help to define the residues important for binding actin more closely. Of note, a cluster of three conserved basic residues on the surface, near the C terminus, were found to be important, including Lys-256 and Arg-260 of the -subunit and Arg-225 of the -subunit. Two other basic conserved surface residues located nearby, Arg-266 and Lys-268 of the -subunit, were found to be much less important. These results do not show that these residues bind actin directly. This will require further studies, including structural ones. A recent cryo-electron microscopy study indicates that Arg-260, Arg-266, and Lys-268 are among a cluster of basic of residues making close contact with nearby Lys-256 (15).
Implications for the Wobble Hypothesis—As originally suggested by the analysis of the crystal structure of CP (14), we currently hypothesize that CP has two independent sites for binding to the barbed end of the actin filament. One includes the C-terminal "tentacle" of the -subunit, so-named because it is surrounded by solvent in the crystal structure and is highly mobile in molecular dynamics simulations (6, 14). The other involves the C-terminal region of the -subunit. However, in this case, the region does not appear to be mobile, based on S100 inhibitor and molecular dynamics studies (6, 14). CP bound to the barbed end of the actin filament appears to utilize both sites (17). We hypothesize that they can dissociate independently, which predicts that CP bound only by its -tentacle should be relatively mobile and "wobble" on the barbed end of the filament. One prediction of the model is that a molecule that binds to CP at the second actin-binding site, near the C terminus, should be able to bind to the wobble state and thereby promote dissociation and thus "uncapping."
Our results here with PIP2 confirm this prediction. First, the point mutations near the C terminus show that this region is involved in binding actin and PIP2. Second, PIP2 can induce uncapping, as visualized here in real time with TIRF microscopy of polymerizing actin filaments. A complementary prediction and confirmation of the model was provided by our recent results with V-1/myotrophin, which binds to CP and inhibits actin binding but does so by interacting with the -tentacle (6). In this case, the model predicts that uncapping will not occur, and that was the result.
Implications for Actin Assembly in Cells—CP is an essential element of the dendritic nucleation model, and consistent with that view, loss of CP leads to loss of lamellipodia in cultured cells (3). In this model, barbed ends are created near the plasma membrane and grow for a time before being capped by CP. One simple hypothesis based on our results is that PIP2 in the plasma membrane, acting as a second messenger, will inhibit capping and thus promote actin polymerization at the membrane. Indeed, overexpression of phosphatidylinositol 5-kinase can lead to rocketing of membrane vesicles in the cell (36, 37).
A more interesting and speculative hypothesis is that PIP2 or other molecules that can cause uncapping, such as the protein CARMIL (9), might be responsible for promoting disassembly of actin filaments in zones of depolymerization or for providing a source of free barbed ends. The latter has been suggested by studies of platelet activation, where explosive actin polymerization occurs (12).
|
FOOTNOTES |
|---|
The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables I and II, Fig. 1, and Movie 1.
1 These authors made equal contributions to this work.
2 Supported in part by a Washington University/Howard Hughes Medical Inst. summer undergraduate research fellowship funded by an Undergraduate Biological Sciences Education Program Grant from the Howard Hughes Medical Institute to Washington University.
3 Supported by National Institutes of Health Institutional Training Grant T32 HL007873.
4 To whom correspondence should be addressed: Campus Box 8228, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-3964; Fax: 314-362-7463; E-mail: jcooper{at}wustl.edu.
5 The abbreviations used are: CP, capping protein; PIP2, phosphatidylinositol 4,5-bisphosphate; TIRF, total internal reflection fluorescence; DTT, dithiothreitol.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Pasic, T. Kotova, and D. A. Schafer Ena/VASP Proteins Capture Actin Filament Barbed Ends J. Biol. Chem., April 11, 2008; 283(15): 9814 - 9819. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. R. Kuhn and T. D. Pollard Single Molecule Kinetic Analysis of Actin Filament Capping: POLYPHOSPHOINOSITIDES DO NOT DISSOCIATE CAPPING PROTEINS J. Biol. Chem., September 21, 2007; 282(38): 28014 - 28024. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||
