Activation of Arp2/3 complex by Wiskott-Aldrich Syndrome protein is linked to enhanced binding of ATP to Arp2.

In response to signaling, the Arp2/3 complex (actin-related proteins 2 and 3 complex) is activated by binding the C-terminal (WA) domain of proteins of the Wiskott-Aldrich Syndrome family to promote the formation of a branched actin filament array, responsible for cell protrusion. The Arp2/3 complex exists in different structural/functional states: the inactive Arp2/3, the activated WA.Arp2/3 complex, the ternary G-actin.WA.Arp2/3 complex, which branches the filaments. This work addresses the role of ATP binding in Arp2/3 function. Using photo-cross-linking, hydrodynamic, and fluorescence techniques, we show that in the inactive Arp2/3 complex only one rapidly exchangeable ATP is tightly bound to Arp3 with an affinity of 10(8) m(-1). Upon activation of the Arp2/3 complex by WA, ATP binds to Arp2 with high affinity (10(7) m(-1)), implying that a large structural change of Arp2 is linked to Arp2/3 activation. ATP is rapidly exchangeable on Arp2 and Arp3 in WA.Arp2/3 and G-actin.WA.Arp2/3 complexes. ATP is not hydrolyzed in inactive Arp2/3, in WA.Arp2/3, nor in G-actin.WA.Arp2/3. Arp2 has a greater specificity than Arp3 for ATP versus ATP analogs. Using functional assays of actin polymerization in branched filaments, we show that binding of ATP to Arp2 is required for filament branching.

Listeria protein ActA mimics WASp family proteins: it activates filament barbed end branching by Arp2/3 complex.

Actin-based propulsion of the bacteria Listeria and Shigella mimics the forward movement of the leading edge of motile cells. While Shigella harnesses the eukaryotic protein N-WASp to stimulate actin polymerization and filament branching through Arp2/3 complex, the Listeria surface protein ActA directly activates Arp2/3 complex by an unknown mechanism. Here we show that the N-terminal domain of ActA binds one actin monomer, in a profilin-like fashion, and Arp2/3 complex and mimics the C-terminal domain of WASp family proteins in catalyzing filament barbed end branching by Arp2/3 complex. No evidence is found for side branching of filaments by ActA-activated Arp2/3 complex. Mutations in the conserved acidic (41)DEWEEE(46) and basic (146)KKRRK(150) regions of ActA affect Arp2/3 binding but not G-actin binding. The motility properties of wild-type and mutated Listeria strains in living cells and in the medium reconstituted from pure proteins confirm the conclusions of biochemical experiments. Filament branching is followed by rapid debranching. Debranching is 3-4-fold faster when Arp2/3 is activated by ActA than by the C-terminal domain of N-WASp. VASP is required for efficient propulsion of ActA-coated beads in the reconstituted motility medium, but it does not affect the rates of barbed end branching/debranching by ActA-activated Arp2/3 nor the capping of filaments. VASP therefore affects another still unidentified biochemical reaction that plays an important role in actin-based movement.

The Arp2/3 complex branches filament barbed ends: functional antagonism with capping proteins.

The Arp2/3 complex is an essential regulator of actin polymerization in response to signalling and generates a dendritic array of filaments in lamellipodia. Here we show that the activated Arp2/3 complex interacts with the barbed ends of filaments to initiate barbed-end branching. Barbed-end branching by Arp2/3 quantitatively accounts for polymerization kinetics and for the length correlation of the branches of filaments observed by electron microscopy. Filament branching is visualized at the surface of Listeria in a reconstituted motility assay. The functional antagonism between the Arp2/3 complex and capping proteins is essential in the maintenance of the steady state of actin assembly and actin-based motility.

Control of actin filament length and turnover by actin depolymerizing factor (ADF/cofilin) in the presence of capping proteins and ARP2/3 complex.

The effect of Arabidopsis thaliana ADF1 and human ADF on the number of filaments in F-actin solutions has been examined using a seeded polymerization assay. ADF did not sever filaments in a catalytic fashion, but decreased the steady-state length distribution of actin filaments in correlation with its effect on actin dynamics. The increase in filament number was modest as compared with the large increase in filament turnover. ADF did not decrease the length of filaments shorter than 1 micrometer. ADF promoted the rapid turnover of gelsolin-capped filaments in a manner dependent on the number of pointed ends. To explain these results, we propose that, as a consequence of the cooperative binding of ADF to F-actin, two populations of energetically different filaments coexist in solution pending a flux of subunits from one to the other. The ADF-decorated filaments depolymerize rapidly from their pointed ends, while undecorated filaments polymerize. ADF also promotes rapid turnover of gelsolin-capped filaments in the presence of the pointed end capper Arp2/3 complex. It is shown that the Arp2/3 complex steadily generates new barbed ends in solutions of gelsolin-capped filaments, which represents an important aspect of its function in actin-based motility.

Synergy between actin depolymerizing factor/cofilin and profilin in increasing actin filament turnover.

The mechanism of control of the steady state of actin assembly by actin depolymerizing factor (ADF)/cofilin and profilin has been investigated. Using Tbeta4 as an indicator of the concentration of ATP-G-actin, we show that ADF increases the concentration of ATP-G-actin at steady state. The measured higher concentration of ATP-G-actin is quantitatively consistent with the increase in treadmilling, caused by the large increase in the rate of depolymerization from the pointed ends induced by ADF (Carlier, M.-F. , Laurent, V., Santolini, J., Didry, D., Melki, R., Xia, G.-X., Hong, Y., Chua, N.-H., and Pantaloni, D. (1997) J. Cell Biol. 136, 1307-1322). Experiments demonstrate that profilin synergizes with ADF to further enhance the turnover of actin filaments up to a value 125-fold higher than in pure F-actin solutions. Profilin and ADF act at the two ends of filaments in a complementary fashion to increase the processivity of treadmilling. Using the capping protein CapZ, we show that ADF increases the number of filaments at steady state by 1. 3-fold, which cannot account for the 25-fold increase in turnover rate. Computer modeling of the combined actions of ADF and profilin on the dynamics of actin filaments using experimentally determined rate constants generates a distribution of the different actin species at steady state, which is in quantitative agreement with the data.

Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins. Comparison of plant and human ADFs and effect of phosphorylation.

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF)1, human ADF, and S6D mutant ADF1 protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP.G-actin in rapid equilibrium (k+ = 155 microM-1.s-1 and k- = 16 s-1 at 4 degreesC under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J. Mol. Biol. 245, 598-607). In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP.actin self-assembly indicates that ADF stabilizes lateral interactions in the filament. Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics. Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.

Hydrolysis of GTP associated with the formation of tubulin oligomers is involved in microtubule nucleation.

Hydrolysis of GTP is known to accompany microtubule assembly. Here we show that hydrolysis of GTP is also associated with the formation of linear oligomers of tubulin, which are precursors (prenuclei) in microtubule assembly. The hydrolysis of GTP on these linear oligomers inhibits the lateral association of GTP-tubulin that leads to the formation of a bidimensional lattice. Therefore GTP hydrolysis interferes with the nucleation of microtubules. Linear oligomers are also formed in mixtures of GTP-tubulin and GDP-tubulin. The hydrolysis of GTP associated with heterologous interactions between GTP-tubulin and GDP-tubulin in the cooligomer takes place at a threefold faster rate than upon homologous interactions between GTP-tubulins. The implication of these results in a model of vectorial GTP hydrolysis in microtubule assembly is discussed.

Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility.

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical-chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADP-bound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi-bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

Role of nucleotide exchange and hydrolysis in the function of profilin in action assembly.

Profilin, an essential G-actin-binding protein, has two opposite regulatory functions in actin filament assembly. It facilitates assembly at the barbed ends by lowering the critical concentration (Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014); in contrast it contributes to the pool of unassembled actin when barbed ends are capped. We proposed that the first of these functions required an input of energy. How profilin uses the ATP hydrolysis that accompanies actin polymerization and whether the acceleration of nucleotide exchange on G-actin by profilin participates in its function in filament assembly are the issues addressed here. We show that 1) profilin increases the treadmilling rate of actin filaments in the presence of Mg2+ ions; 2) when filaments are assembled from CaATP-actin, which polymerizes in a quasireversible fashion, profilin does not promote assembly at the barbed ends and has only a G-actin-sequestering function; 3) plant profilins do not accelerate nucleotide exchange on G-actin, yet they promote assembly at the barbed end. The enhancement of nucleotide exchange by profilin is therefore not involved in its promotion of actin assembly, and the productive growth of filaments from profilin-actin complex requires the coupling of ATP hydrolysis to profilin-actin assembly, a condition fulfilled by Mg-actin, and not by Ca-actin.

Kinetics of association of myosin subfragment-1 to unlabeled and pyrenyl-labeled actin.

The kinetics of reaction of myosin subfragment-1 (S1) with F-actin have been monitored by the changes in light scattering and in pyrenyl-actin fluorescence at 20 degrees C, pH 7.5, and physiological ionic strength. The association rate constant of S1 to F-actin decreases about 10-fold as the molar ratio of bound S1 increases from 0 to 1. This decrease in k+ is most likely due to the steric hindrance of available binding sites by initially bound S1. The apparent rate constant for association of S1 to bare filaments is 9 microM-1 s-1, a value 1 order of magnitude higher than the one previously estimated from experiments in which S1 was in excess over F-actin. The anticooperative binding kinetics of S1 to F-actin are consistent with the negative cooperativity displayed in the equilibrium binding curves of S1 to pyrenyl-F-actin. Fluorescence titration curves of partially labeled pyrenyl-F-actin by S1 are sigmoidal, consistent with a 4-fold higher affinity of S1 for unlabeled than for labeled action. This conclusion is strengthened by kinetic data of S1 binding to partially labeled F-actin, which exhibit a biphasic behavior due to the slower dissociation of S1 from unlabeled than from labeled actin.

Tbeta 4 is not a simple G-actin sequestering protein and interacts with F-actin at high concentration.

Thymosin beta 4 is acknowledged as a major G-actin binding protein maintaining a pool of unassembled actin in motile vertebrate cells. We have examined the function of Tbeta 4 in actin assembly in the high range of concentrations (up to 300 micron) at which Tbeta 4 is found in highly motile blood cells. Tbeta 4 behaves as a simple G-actin sequestering protein only in a range of low concentrations (<20 micron). As the concentration of Tbeta 4 increases, its ability to depolymerize F-actin decreases, due to its interaction with F-actin. The Tbeta 4-actin can be incorporated, in low molar ratios, into F-actin, and can be cross-linked in F-actin using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. As a result of the copolymerization of actin and Tbeta 4-actin complex, the critical concentration is the sum of free G-actin and Tbeta 4-G-actin concentrations at steady state, and the partial critical concentration of G-actin is decreased by Tbeta 4-G-actin complex. The incorporation of Tbeta 4-actin in F-actin is associated to a structural change of the filaments and eventually leads to their twisting around each other. In conclusion, Tbeta 4 is not a simple passive actin-sequestering agent, and at high concentrations the ability of Tbeta 4-actin to copolymerize with actin reduces the sequestering activity of G-actin-binding proteins. These results question the evaluation of the unassembled actin in motile cells. They account for observations made on living fibroblasts overexpressing beta-thymosins.

Interaction of profilin with G-actin and poly(L-proline).

The interaction of bovine spleen profilin with ATP- and ADP-G-actin and poly(L-proline) has been studied by spectrofluorimetry, analytical ultracentrifugation, and rapid kinetics in low ionic strength buffer. Profilin binding to G-actin is accompanied by a large quenching of tryptophan fluorescence, allowing the measurement of an equilibrium dissociation constant of 0.1-0.2 microM for the 1:1 profilin-actin complex, in which metal ion and nucleotide are bound. Fluorescence quenching monitored the bimolecular reaction between G-actin and profilin, from which association and dissociation rate constants of 45 microM-1 s-1 and 10 s-1 at 20 degrees C could be derived. The tryptophan(s) which are quenched in the profilin-actin complex are no longer accessible to solvent, which points to W356 in actin as a likely candidate, consistent with the 3D structure of the crystalline profilin-actin complex [Schutt, C. E., Myslik, J. C., Rozycki, M. D., Goonesekere, N. C. W., & Lindberg, U. (1993) Nature 365, 810-816]. Upon binding poly(L-proline), the fluorescence of both tyrosines and tryptophans of profilin is enhanced 2.2-fold. A minimum of 10 prolines [three turns of poly(L-proline) helix II] is necessary to obtain binding (KD = 50 microM), the optimum size being larger than 10. Binding of poly(L-proline) is extremely fast, with k+ > 200 microM-1 s-1 at 10 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin subfragment-1-induced polymerization of G-actin. Formation of partially decorated filaments at high actin-S1 ratios.

Myosin subfragment-1-induced polymerization of G-actin into arrowhead-decorated F-actin-myosin subfragment-1 (S1) filaments has been studied at low ionic strength and in the absence of ATP, using a combination of light scattering, fluorescence of 4-nitrobenz-2-oxa-1,3-diazol-7-yl- or pyrenyl-labeled actin, sedimentation, and electron microscopy techniques. When G-actin is in excess over myosin subfragment-1, the initial formation of fully decorated F-actin-S1 filaments, in which the actin:S1 molar ratio is 1:1, is followed by further incorporation of G-actin subunits in the polymer concomitant with the redistribution of the myosin heads along the polymer, leading to partially decorated filaments containing less than one S1/actin, in equilibrium with G-actin. This process leads to an overshoot in the light-scattering polymerization curves at high actin:S1 ratios. The concentration of G-actin at equilibrium with partially decorated filaments is a nonlinear function of the molar fraction of S1 in the polymer, indicating that actin-actin-S1 interactions are energetically more favorable than actin-actin or actin-S1-actin-S1 interactions.

Interaction between G-actin and myosin subfragment-1 probed by covalent cross-linking.

The topography of rapid equilibrium complexes formed between G-actin and myosin subfragment-1, which are the first kinetic intermediates in the polymerization process into F-acto-S1 filaments, has been probed by chemical cross-linking. In the absence of ATP, cross-linking of G-actin-S1 complexes by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) yielded a major 165-170-kDa and a fainter 200-205-kDa doublet polypeptide. The actin:S1 molar ratio was 1 in the EDC-cross-linked complexes, using either double labeling techniques or the method combining EDC + N-hydroxysuccinimide. Chemical cleavages of the covalently cross-linked complexes by formic acid and N-hydroxylamine (Sutoh, K. (1983) Biochemistry 22, 1579-1585) showed that in the main cross-linked 165-kDa polypeptide, the 1-12 acidic N-terminal region of actin was covalently linked to the lysine-rich region connecting the central 50-kDa domain to the C-terminal 20-kDa domain of S1, as in F-acto-S1 complexes. G-actin, but not F-actin, was covalently cross-linked to S1 by N,N'-paraphenylenedimaleimide (p-PDM). A major 195-kDa and a minor 165-kDa polypeptide were obtained, could be separated from actin and S1 by DEAE-cellulose chromatography, and did not exhibit actin-activated Mg-ATPase activity. Both EDC-cross-linked and p-PDM-cross-linked complexes between G-actin and S1 could be incorporated into F-acto-S1 decorated filaments. The C-terminal cysteine 374 of actin is involved in the p-PDM cross-linked 195-kDa complex. Accordingly, a covalent photocross-linked 200-kDa conjugate was formed between S1 heavy chain and benzophenone-G-actin, obtained by covalent modification of Cys374 by benzophenonemaleimide (Tao, T., Lamkin, M., and Scheiner, C. J. (1985) Arch. Biochem. Biophys. 240, 627-634). These results demonstrate that (i) G-actin-S1 and F-actin-S1 complexes display a large similarity in the EDC-cross-linked electrostatic close contacts and (ii) a change in the environment of Cys374 is linked to the polymerization into F-actin-S1 decorated filaments.

Interaction of myosin subfragment-1 with F- and G-actin in equilibrium.

The structural changes of the F-actin-myosin head (S1) complex during the cross-bridge cycle are essential in muscle contraction. Although a large body of evidence has accumulated showing that the actin: S1 stoichiometry in the decorated F-actin-S1 filament is 1:1 at saturation by S1, a recent report by Andreev and Borejdo (1991, Biochem. Biophys. Res. Comm. 177, 350-356) indicated that under some conditions, the actin: S1 stoichiometry could be 2:1 at saturation by S1. Because of the important implications of this result in the mechanism of acto-myosin motility, we have re-investigated this issue. It is shown here that evidence for the 2:1 stoichiometry was circumstantial and was only observed under conditions where 50% of the actin was F-actin, i.e. at a total actin concentration twice as large as the critical concentration. The interaction of S1 with both F- and G-actin in dynamic equilibrium is studied in detail. The present data fully support the 1:1 actin: S1 stoichiometry in the decorated filament at saturation by S1.

Interaction between chromium GTP and tubulin. Stereochemistry of GTP binding, GTP hydrolysis, and microtubule stabilization.

Chromium GTP (CrGTP) has been used to probe the stereochemistry of metal-GTP binding to exchangeable site of tubulin and to examine the fate and role of nucleotide-bound metal ion in GTP hydrolysis associated with microtubule assembly. The absolute stereoconfiguration of the two pairs of diastereomers of beta,gamma-bidentate CrGTP has been determined by comparison of their visible circular dichroism spectra with those of the beta,gamma-CrATP isomers whose configurations have been established (Lin, I., and Dunaway-Mariano, D. (1988) J. Am. Chem. Soc. 110, 950-956). Tubulin binds metal-GTP preferentially in the delta pseudoaxial configuration. CrGTP-tubulin shows a high propensity to undergo tubulin-tubulin interactions with associated hydrolysis of CrGTP. Hydrolysis of CrGTP in microtubule assembly develops in two consecutive steps: cleavage of the gamma-phosphate followed by release of Pi and chromium. In contrast to other NTPases (actin, hexokinase) tubulin appears able to catalyze the dissociation of the stable chromium-phosphate bonds, which implies a highly nucleophilic environment of the binding site of the metal-triphosphate moiety of GTP. Microtubules assembled from CrGTP-tubulin are made of 90% GDP subunits, and their stability is linked to a 10% proportion of CrGDP-Pi subunits, scattered along the microtubule, from which Pi does not dissociate. The possibility is evoked that some tubulin variants do not catalyze release of Pi and metal ion efficiently, and their presence could affect microtubule dynamics.

Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GDP-Pi using phosphate analogues.

Beryllium fluoride (BeF3-) has previously been shown to bind tightly to microtubules as a structural analogue of Pi and to mimic the GDP-Pi transient state in tubulin polymerization [Carlier, M.-F., Didry, D., Melki, R., Chabre, M., & Pantaloni, D. (1988) Biochemistry 27, 3555-3559]. The interaction of BeF3- with tubulin is analyzed here in greater detail. BeF3- binds to and dissociates from microtubule GDP subunits at very slow rates (k+ congruent to 100 M-1 s-1; k- congruent to 6 x 10(-4) s-1), suggesting that a slow conformation change of tubulin, linked to the stabilization of the microtubule structure, follows BeF3- binding. The possibility is evoked that BeF3- acts as a transition-state analogue in the GTPase reaction of tubulin. BeF3- does not bind to dimeric nor to oligomeric GDP-tubulin with high affinity. Substoichiometric binding of BeF3- to microtubules provides extensive stabilization of the structure. An original mechanistic model that accounts for the data is proposed. The kinetic parameters for microtubule elongation in the presence of GTP- and GDP-tubulin with and without BeF3- have been determined. Data support the following views: (i) Microtubules at steady state and in a regime of slow growth in the presence of GTP are stabilized by a cap of GDP-Pi subunits functionally similar to GDP-BeF3 subunits. (ii) In the presence of BeF3-, microtubules elongate from GDP-tubulin within the following sequence of reactions: initial nonproductive binding of GDP-tubulin to microtubule ends is followed by the binding of BeF3- and the associated conformation change allowing sustained elongation.

Analysis of the membrane glycoproteins of platelets in the Wiskott-Aldrich syndrome.

We have examined the plasma membrane glycoproteins of platelets from three unrelated patients with the Wiskott-Aldrich syndrome. Single- or two-dimensional SDS-polyacrylamide gel electrophoresis was performed. Glycoproteins were located by staining for carbohydrate, or by autoradiography when the platelets had been surface-labelled with 125I prior to solubilization. In one patient a slight decrease in the 125I-labelling intensity of GP Ib, GP Ia and a 125I-labelled polypeptide of Mr 168,000 were noted. For the two other patients the glycoprotein profiles were indistinguishable from those of normal subjects. These results clearly indicate that abnormalities in platelet membrane glycoproteins are not a common trait among Wiskott-Aldrich patients, and thus cannot be regarded as primary defects in this disease.

Cross-linking of alpha and gamma-thrombin to distinct binding sites on human platelets.

The interaction of thrombin with proteins at the platelet surface was assessed by chemical cross-linking with the membrane-impermeable reagents bis(sulphosuccinimidyl)suberate and dithiobis(sulphosuccinimidyl propionate) under conditions which induced no modification of intracellular proteins and minimal cross-linking of membrane glycoproteins. The proteins covalently linked to 125I-labelled alpha and gamma-thrombin were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. 125I-alpha-thrombin was detected in high-molecular-mass complexes (a) at the top of a 3% acrylamide stacking gel and (b) with a Mr approximately equal to 400,000. In addition, two complexes of 240 kDa and 78 kDa were characterized. Hirudin prevented the formation of each of these complexes. The 78-kDa complex occurred spontaneously in the absence of bifunctional reagents, was only observed with active alpha-thrombin and was not dissociated by hirudin. Such characteristics are similar to those of a serpin serine-protease complex. The 240-kDa complex was formed with 0.8-100 nM alpha-thrombin, was observed after a short incubation time (30 s) and occurred with TosLysCH2Cl-inactivated alpha-thrombin. After analysis of Triton-X-100-soluble extracts of cross-linked platelets by crossed immunoelectrophoresis against a rabbit antiserum to platelets, two principal precipitates contained 125I-alpha-thrombin. These were a precipitate containing GPIIb-IIIa complexes and a precipitate in the position of GPIb. Indirect immunoprecipitation of GPIb, using a murine monoclonal antibody, confirmed it to be the major platelet component in the 240-kDa complex. Significantly, 125I-gamma-thrombin, which activates platelets with a prolonged lag phase, failed to bind to GPIb and complexes in the 240-kDa and 78-kDa molecular mass range were not observed. We conclude that several binding sites for alpha-thrombin are present at the platelet surface, and that GPIb is one of them. The studies with gamma-thrombin suggest that binding to GPIb is not obligatory for platelet activation although it could be involved in an initial step of the platelet response.

Stabilization of microtubules by inorganic phosphate and its structural analogues, the fluoride complexes of aluminum and beryllium.

In order to elucidate how the elementary reactions of GTP cleavage and subsequent inorganic phosphate (Pi) release, which accompany microtubule assembly, regulate microtubule dynamics, the effect of Pi and of its structural analogues AlF4- and BeF3- on the stability of GDP-microtubules has been investigated. Inorganic phosphate binds to microtubules with a low affinity (KD = 25 mM) and slows down the rate of GDP-subunit dissociation by about 2 orders of magnitude. AlF4- and BeF3- exhibit phosphate-like effects with 1000-fold higher affinity. Evidence has been obtained for direct binding of BeF3- to microtubules with a stoichiometry of 1 mol of BeF3- per mole of GDP-subunit and an equilibrium dissociation constant of 12-15 microM. AlF4- and Pi compete for this site. Phosphate analogues abolish oscillatory polymerization kinetics and slow down microtubule turnover at steady state. In view of these results, we propose that Pi and its structural analogues bind to the site of the gamma-phosphate of GTP in the E site and reconstitute a GDP-Pi-microtubule, from which tubulin subunits dissociate very slowly. We therefore understand that, following GTP cleavage on microtubules, Pi release in the medium is accompanied by a structural change resulting in a large destabilization of the polymer. A cap of slowly dissociating GDP-Pi-subunits prevents depolymerization of the microtubule GDP-core at steady state. The similarity with the actin system [Carlier, M.-F., & Pantaloni, D. (1988) J. Biol. Chem. 263, 817-825] is underlined.