Comparison between the gelsolin and adseverin domain structure.

Adseverin (74-kDa protein, scinderin) is a calcium- and phospholipid-modulated actin-binding protein that promotes actin polymerization, severs actin filaments, and caps the barbed end of the actin filament, with its NH2-terminal half retaining these properties (Sakurai, T., Kurokawa, H., and Nonomura, Y. (1991) J. Biol. Chem. 266, 4581-4585). Further proteolysis of this NH2-terminal half generated five fragments, and two of them (Mr 15,000 and 31,000) showed Ca(2+)-dependent binding to monomeric actin. The Mr 31,000 fragment especially caused actin filament fragmentation, although its severing activity was also inhibited by several acidic phospholipids as was found in adseverin and its NH2-terminal half. Amino acid sequencing demonstrated that the two fragments' NH2 terminus were blocked in the same manner as the NH2 terminus of adseverin, and thus these two fragments are possibly located at the NH2-terminal of the adseverin molecule. This would then indicate that NH2-terminal fragments had a Ca(2+)-sensitive actin-binding function that relates to actin severing. The other two fragments' NH2-terminal sequencing showed a similar homology to the amino acid sequences of gelsolin and villin. Based on these observations, we propose that adseverin has a functional domain structure similar to that of the gelsolin and villin core.     

Structure of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Masses of chains and sites of ultraviolet-induced vanadate-dependent cleavage

We report here on the UV-induced vanadate-dependent cleavage of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Both polypeptides are cleaved at a single site (termed the V1 site) by UV irradiation in the presence of Mg2+, ATP, and vanadate. The alpha chain yields fragments of Mr 290,000 and 190,000. Fragments of Mr 255,000 and 185,000 are obtained from the beta chain. Ultraviolet irradiation of the alpha and beta chains in the presence of vanadate and Mn2+ (but no nucleotide) induces cleavage of both molecules at sites (termed the V2 sites) distinct from the V1 sites. The single V2 site within the beta chain is located 75,000 daltons from the site of V1 cleavage within the Mr 255,000 V1 fragment. The alpha chain contains three distinct sites of V2 cleavage; all are located within the Mr 290,000 V1 fragment, 60,000, 90,000, and 100,000 daltons from the site of V1 cleavage. From these studies, we estimate the masses of the alpha and beta heavy chains to be 480,000 and 440,000 daltons, respectively.     

Mapping the glycoprotein Ib-binding site in the von willebrand factor A1 domain

The von Willebrand factor (vWF) mediates platelet adhesion to exposed subendothelium at sites of vascular injury. It does this by forming a bridge between subendothelial collagen and the platelet glycoprotein Ib-IX-V complex (GPIb). The GPIb-binding site within vWF has been localized to the vWF-A1 domain. Based on the crystal structure of the vWF-A1 domain (Emsley, J., Cruz, M., Handin, R., and Liddington, R. (1998) J. Biol. Chem. 273, 10396-10401), we introduced point mutations into 16 candidate residues that might form all or part of the GPIb interaction site. We also introduced two mutations previously reported to impair vWF function yielding a total of 18 mutations. The recombinant vWF-A1 mutant proteins were then expressed in Escherichia coli, and the activity of the purified proteins was assessed by their ability to support flow-dependent platelet adhesion and their ability to inhibit ristocetin-induced platelet agglutination. Six mutations located on the front and upper anterior face of the folded vWF-A1 domain, R524S, G561S, H563T, T594S/E596A, Q604R, and S607R, showed reduced activity in all the assays, and we suggest that these residues form part of the GPIb interaction site. One mutation, G561S, with impaired activity occurs in the naturally occurring variant form of von Willebrand's disease-type 2M underscoring the physiologic relevance of the mutations described here.     

Effect of actin-binding protein on the sedimentation properties of actin

Actin and actin-binding protein (ABP) have recently been purified from human platelet cytoskeletons (S. Rosenberg, A. Stracher, and R.C. Lucas, 1981, J. Cell Biol. 91:201-211). Here, the effect of ABP on the sedimentation of actin was studied. When ABP was added to preformed F- actin filaments, it bound until a maximum ratio of 1:9 (ABP:actin, mol:mol) was reached. however, when actin was polymerized in the presence of ABP, two and a half times more ABP was able to bind to the actin- that is, every 3.4 actin monomers were now bound by an ABP dimer. ABP was not able to induce the sedimentation of actin under nonpolymerizing conditions but was able to reduce the time and concentration of actin required for sedimentation under slow polymerizing conditions. ABP, therefore, exerts its effect of G-actin by either nucleating polymerization or by cross-linking newly formed oligomers into a more sedimentable form.     

Isolation and characterization of actin and actin-binding protein from human platelets

Human blood platelets, which are highly motile cells essential for the maintenance of hemostasis, contain large quantities of actin and other contractile proteins. We have previously introduced a method (Lucas, R. C., T. C. Detwiler, and A. Stracher, J. Cell Biol., 1976, 70(2, Pt. 2):259 a) for the quantitative recovery of the platelets' cytoskeleton using a solution containing 1% Triton X-100 and 10 mM EGTA. This cytoskeleton contains most of the platelets' actin, actin-binding protein (ABP, subunit molecular weight = 260,000), and a 105,000-dalton protein. Negative staining of this Triton-insoluble residue on an EM grid shows it to consist of branched cables of actin filaments aligned in parallel. When this cytoskeletal structure is dissolved in high-salt solutions, the actin and ABP dissociate and can subsequently be separated. Here we will present simple and rapid methods for the individual purifications of platelet actin and platelet ABP. When purified actin and ABP are recombined in vitro, they are shown to be both necessary and sufficient for the reformation of the cytoskeletal complex. The reformed structure is visualized as a complex array of fibers, which at the EM level are seen to be bundles of actin filaments. The reformation of the cytoskeleton requires only that the actin be in the filamentous form--no accessory proteins, chelating agents, divalent cations, or energy sources are necessary. In vivo, however, the state of assembly of the platelets' cytoskeleton appears to be under the control of the intracellular concentration of free calcium. Under conditions where proteolysis is inhibited and EGTA is omitted from the Triton-solubilization step, no cytoskeleton can be isolated. The ability of Ca+2 to control the assembly and disassembly of the platelets' cytoskeleton provides a mechanism for cytoskeletal involvement in shape change and pseudopod formation during platelet activation.     

Isolation and sequence of a tropomyosin-binding fragment of turkey gizzard calponin

Limited chymotryptic cleavage of turkey gizzard calponin yields a 13 kDa fragment which could be purified by its ability to bind to Sepharose-immobilized tropomyosin. This 13 kD polypeptide is shown to be derived from a 22 kDa fragment. Complete amino acid sequence analysis of the 13 kD and 22 kD fragments reveals high homology with the formerly characterized smooth muscle-specific protein SM22 alpha (Pearlstone, J.R., Weber, M., Lees-Miller, J.P., Carpenter, M.R. and Smillie L.B., 1987, J. Biol. Chem. 262, 5985-5991) and the product of gene mp20 of Drosophila (Ayme-Southqate, A., Lasko, P., French, C, and Pardue, M.L. [(1989) J. Cell Biol. 108, 521-531]. Futhermore we recognize sequence elements of a putative actin-binding domain of alpha-actinin, the calpactin I or p 36 sequence, and a consensus motif present in the repeats of the gene product of the candidate unc-87 gene of C. elegans (S.D. Goetinck and R.H. Waterston, personal communication).     

Alignment of biologically active domains in the fibronectin molecule

Gelatin-binding material was isolated from a human plasma cryoprecipitate by affinity chromatography on gelatin-Sepharose. Individual fragments of fibronectin with Mr = 170,000, 100,000, and 80,000 and a mixture of fragments with Mr = 205,000 and 190,000 (200K fraction) were isolated from this material. These fragments reacted with antifibronectin and with antibodies to a gelatin-binding Mr = 70,000 tryptic fragment of fibronectin. They all shared the same NH2-terminal amino acid sequence. The 205K and 190K fragments bound also to heparin-Sepharose, whereas the smaller fragments did not. The 200K fraction and the 170K fragment mediated cell attachment when used to coat plastic, whereas the 100K and 80K fragments were inactive in this assay. Further digestion of the 205K and 190K fragments with chymotrypsin yielded separate sets of smaller fragments that bound to either gelatin-Sepharose or heparin-Sepharose, as well as fragments that did not show either of these binding activities but mediated cell attachment. Since the NH2-terminal ends of the 205K, 190K, 100K, and 80K fragments are the same, the results define the order of the active sites in the fibronectin molecule as gelatin-binding site, cell attachment site, and heparin-binding site.     

Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane.

Platelet glycoprotein (GP) V is a Mr 82,000 plasma membrane protein of unknown function that is cleaved by the potent platelet agonist, thrombin, to yield a Mr 69,500 fragment (GPVf1). Platelet GPIb, a disulfide-linked alpha beta heterodimer (Mr 160,000) that forms a noncovalent complex with GPIX (Mr 22,000), functions as the platelet adhesion receptor for surface-bound von Willebrand factor. Association between GPV and GPIb-IX has been suggested by the finding that both proteins are deficient in the Bernard-Soulier syndrome, a bleeding disorder characterized by giant platelets and defective interaction with von Willebrand factor. Here we report that GPV and GPIb-IX are coprecipitated by monoclonal antibodies (mAbs) against GPV, GPIb, or GPIX when platelets are solubilized in the mild detergent, digitonin. Treatment of digitonin immunopreciptates with the nonionic detergent, Nonidet P-40, released GPV from anti-GPIb and anti-GPIX mAb precipitates and GPIb-IX from the anti-GPV mAb precipitate. Removal of the Mr 45,000 amino-terminal part of GPIb alpha by treatment with elastase did not abrogate association of GPV with GPIb-IX, showing that the leucine-rich repeat sequences in GPIb alpha are not required for complex formation. Binding studies with 125I-labeled mAbs showed the presence of 24,370 GPIb-IX complexes and 11,170 molecules of GPV/platelet (n = 5). These data show that the leucine-rich glycoproteins GPV and GPIb-IX form a noncovalent complex in the platelet membrane. GPV may play a role in the interaction of platelets with von Willebrand factor.     

Ezrin has a COOH-terminal actin-binding site that is conserved in the ezrin protein family

Ezrin, previously also known as cytovillin, p81, and 80K, is a cytoplasmic protein enriched in microvilli and other cell surface structures. Ezrin is postulated to have a membrane-cytoskeleton linker role. Recent findings have also revealed that the NH2-terminal domain of ezrin is associated with the plasma membrane and the COOH-terminal domain with the cytoskeleton (Algrain, M., O. Turunen, A. Vaheri, D. Louvard, and M. Arpin. 1993. J. Cell Biol. 120: 129-139). Using bacterially expressed fragments of ezrin we now demonstrate that ezrin has an actin-binding capability. We used glutathione-S-transferase fusion proteins of truncated ezrin in affinity chromatography to bind actin from the cell extract or purified rabbit muscle actin. We detected a binding site for filamentous actin that was localized to the COOH-terminal 34 amino acids of ezrin. No binding of monomeric actin was detected in the assay. The region corresponding to the COOH- terminal actin-binding site in ezrin is highly conserved in moesin, actin-capping protein radixin and EM10 protein of E. multilocularis, but not in merlin/schwannomin. Consequently, this site is a potential actin-binding site also in the other members of the protein family. Furthermore, the actin-binding site in ezrin shows sequence homology to the actin-binding site in the COOH terminus of the beta subunit of the actin-capping protein CapZ and one of the potential actin-binding sites in myosin heavy chain. The actin-binding capability of ezrin supports its proposed role as a membrane-cytoskeleton linker.     

A calmodulin-binding peptide of caldesmon

Caldesmon is a major actin-binding protein identified in smooth muscle and many non-muscle cells. It also interacts with calmodulin and a number of other acidic proteins. We have shown previously that the polypeptide stretch from Val629 to Ser666 near the C terminus contains a calmodulin binding site (Wang, C.-L. A., Wang, L.-W. C., Xu, S., Lu, R. C., Saavedra-Alanis, V., and Bryan, J. (1991) J. Biol. Chem. 266, 9166-9172). On the other hand, Bartegi et al. (Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238) reported a cyanogen bromide fragment beginning at Trp659 which is also capable of binding both calmodulin and actin. A comparison of the overlapping sequence between these two peptides suggests that this calmodulin binding site is localized in a 7-residue segment, 659Trp-Glu-Lys-Gly-Asn-Val-Phe665. We have chemically synthesized an 18-residue peptide (GS17C, from Gly651 to Ser667 with an added cysteine at the C terminus) that contains this segment. This peptide was purified by high performance liquid chromatography and labeled with fluorescent probes at the terminal cysteine residue. We found that GS17C indeed binds calmodulin in a Ca(2+)-dependent manner (Kd = 8 x 10(-7) M) and appears to compete with caldesmon. Interestingly, this synthetic peptide also co-sediments with F-actin, binding to actin being displaceable by calmodulin, as in the case of the native caldesmon. But GS17C does not have any effect on the actomyosin ATPase activity, indicating that this peptide segment does not contain the inhibitory region.     

Actin-binding and dimerization domains of HeLa cell filamin

HeLa cell filamin is a linear, bivalent, homodimer of high molecular weight subunits (Mr 250,000 that may cross-link actin filaments in vivo into supramolecular structures such as networks and bundles. We used millimolar Ca protease from chicken breast muscle to cleave the subunit into smaller fragments that we mapped with respect to the overall structure of the dimer. The enzyme cleaves HeLa filamin into a larger (Mr 192,000) and a smaller (Mr 104,000) fragment; the smaller fragment is the precursor of a still smaller (Mr 92,000) fragment. Only the larger fragment bound to actin in a cosedimentation test, suggesting that it contains the actin-binding region of the subunit. Digestion of HeLa filamin that had been cross-linked with dimethyl adipimidate produced a good yield of the Mr 192,000 fragment but a poor yield of the Mr 104,000/92,000 fragments. Since native filamins are head-to-head dimers, it was expected that cross-linking would proceed most readily at the dimerization site and, therefore, it appears that the Mr 192,000 fragment is cleaved from cross-linked filamin because it is distal to the dimerization region, while the Mr 104,000/92,000 fragments are absent because they lie at the dimerization region and were cross-linked to a form that was not identifiable by sodium dodecyl sulfate electrophoresis.     

Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro

In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.     

Further characterization of a conserved actin-binding 27-kDa fragment of actinogelin and alpha-actinins and mapping of their binding sites on the actin molecule by chemical cross-linking

A conserved actin-binding domain (Mr = 27,000) of rat hepatic actinogelin, rat skeletal muscle, and chicken gizzard alpha-actinins (Mimura, N., and Asano, A. (1986) J. Biol. Chem. 261, 10680-10687) was separated into two components having different isoelectric points (peptides A and B) by chromatofocusing. Thermolysin digestion of peptide A generated peptide B with concomitant loss of peptide A. Amino acid compositions and tryptic maps of peptides A and B also demonstrated that peptide A is a precursor of peptide B upon thermolysin digestion. All of peptides A and B retained the activity to bind with F-actin competitively to each other. By the gel-filtration method, it was also shown that the native actin-binding 27-kDa fragments are monomeric and globular. The non-actin-binding 50- or 53.5-kDa fragment of actinogelin/alpha-actinins was, however, found to be asymmetric and dimeric in the native state. Chemical cross-linking of the 27-kDa fragment with F-actin with a water-soluble carbodiimide produced at least four different complexes (I-IV). Chemical cleaving analysis of the cross-linked products (complexes I and II) indicated that the 27-kDa fragment possesses two possible binding sites on actin at the NH2-terminal residues 1-12 (for complex I) and at residues spanning 86-119 or 123 (for complex II).     

Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. I. Inhibition by N-ethylmaleimide and trypsin

We have shown previously (Brooker, R.J., and Slayman, C.W. (1982) J. Biol. Chem. 257, 12051-12055; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 222-226) that the plasma membrane [H+]-ATPase of Neurospora crassa is inhibited by N-ethylmaleimide (NEM), which reacts at an essential nucleotide-protectable site on the Mr = 104,000 polypeptide. The present study demonstrates that Mg2+ has a biphasic effect on NEM inhibition. At low concentrations (0.01-0.1 mM, Mg2+ decreases the sensitivity of the enzyme to NEM, while at high concentrations (greater than 1 mM), it enhances sensitivity. These effects are seen in the presence or absence of nucleotides (ATP, ADP). Mg2+ also acts in a concentration-dependent way to influence the degradation of the ATPase by trypsin. Low concentrations of Mg2+ have little or no effect on tryptic inactivation of ATPase activity or on the disappearance of the Mr = 104,000 polypeptide and the stepwise appearance of Mr = 100,000 and 91,000 tryptic fragments. High concentrations of Mg2+ decrease the rate of inactivation, and a new fragment of Mr = 98,000 is seen. Taken together, the NEM and trypsin results indicate that the Neurospora [H+]-ATPase possesses high and low affinity Mg2+ binding sites which affect the conformation of the enzyme. The divalent cation specificity of the sites has also been investigated. Co2+, Mn2+, and (to a lesser extent) Ni2+ mimic the behavior of Mg2+, but Ca2+ has a different effect, at least at the high affinity site. It appears to bind to that site, based on its ability to inhibit ATP hydrolysis (in the presence of Mg2+), but does not offer protection against NEM inhibition. The results suggest a way in which Ca2+ may serve as a physiological regulator of the ATPase.     

On the association of glycoprotein Ib and actin-binding protein in human platelets

Glycoprotein (GP) Ib was purified from lysates of human platelets prepared in the presence or absence of inhibitors of the endogenous calcium-activated neutral protease (CANP) by immunoaffinity chromatography, employing the GPIb-specific murine monoclonal antibody, AP1, coupled to Sepharose CL4B. When derived from lysates prepared in the presence of EDTA or leupeptin, the eluate from the AP1-affinity column contained a 240,000-260,000-mol-wt protein in addition to GPIb. In SDS PAGE, this protein was stained by Coomassie Blue R, but not by the periodic acid-Schiff reagent, and it was not labeled with 125I in intact platelets by the lactoperoxidase-catalyzed method. When derived from lysates prepared in the absence of CANP inhibitors, the eluate contained only GPIb and its proteolytic derivative, glycocalicin. A change in the electrophoretic mobility of GPIb consistent with its association with the 240,000-260,000-mol-wt protein was confirmed by crossed immunoelectrophoresis. By an immunoblot technique involving transfer of proteins eluted from the AP1-affinity column and separated by SDS PAGE onto a nitrocellulose membrane, the 240,000-260,000-mol-wt protein bound polyclonal goat antibody raised against rabbit macrophage actin-binding protein (ABP). On the basis of these results, we conclude the GPIb is tightly associated with ABP under conditions in which the endogenous CANP is inhibited, and that this apparent transmembrane complex of GPIb-ABP can be isolated in lysates of nonactivated human platelets.     

Interaction of the Mr = 90,000 heat shock protein with the steroid-binding domain of the glucocorticoid receptor

We have investigated the physiochemical characteristics of trypsin-treated, molybdate-stabilized glucocorticoid-receptor complexes from rat liver in the presence of 10 mM sodium molybdate by high performance ion-exchange chromatography, high performance size-exclusion chromatography, and sedimentation analysis. Trypsin treatment was performed under conditions previously reported to degrade the monomeric Mr approximately 94,000 steroid-binding protein to an Mr approximately 27,000 ligand-binding entity (Wrange, O., and Gustafsson, J.-A. (1978) J. Biol. Chem. 253, 856-865). Also in the presence of molybdate, an Mr approximately 27,000 steroid-binding fragment was obtained by limited trypsinization. However, no major differences in the tested physicochemical parameters were seen when trypsin-treated glucocorticoid-receptor complexes were compared with crude cytosolic complexes. Furthermore, the Mr approximately 27,000 steroid-binding fragment generated in the presence of molybdate could be immunoprecipitated by antibodies specific for the glucocorticoid receptor-associated Mr approximately 90,000 heat shock protein. These results provide direct evidence for an interaction of the Mr approximately 90,000 heat shock protein with the steroid-binding domain of the glucocorticoid receptor, known to correspond to the C-terminal third of the receptor protein.     

The glycoprotein Ib complex of human blood platelets

Human glycoprotein Ib (GPIb) is a major integral membrane protein on human blood platelets responsible for the initial attachment of platelets to the exposed vascular subendothelium. In this report we describe an isolation method for a 'GPIb complex' as well as for its individual components. A three-step procedure involving Triton X-114 phase-partition, affinity chromatography on wheat germ agglutinin and ion-exchange chromatography on DEAE-Sephacel yielded milligram quantities of purified GPIb complex. The single components of the complex were further purified by gel filtration on AcA34 in 0.1% sodium dodecyl sulfate. As well as GPIb, the complex contains GP17, actin-binding protein, actin and a series of unidentified proteins with different molecular masses. In contrast to GPIb alpha, which is very rich in O-linked oligosaccharides, sugar analysis revealed that GPIb beta and GP17 seem to have only N-linked chains of the lactosamine type. The C-terminal alpha-chain remnant, which probably spans the plasma membrane, was identified and isolated for the first time. Western blotting with polyclonal rabbit anti-GPIb antibodies and silver-staining of one- or two-dimensional dodecyl sulfate/polyacrylamide gels revealed that it has an apparent molecular mass of 20 kDa and is linked to GPIb beta by a disulfide bridge close to the membrane. The thrombin-binding site on GPIb is located near the N-terminus on a 40-kDa fragment of GPIb alpha. A disulfide bridge in the N-terminal region is not essential for thrombin binding to GPIb.     

Location of a dicyclohexylcarbodiimide-reactive glutamate residue in the Neurospora crassa plasma membrane H+-ATPase

The proton pump (H+-ATPase) found in the plasma membrane of the fungus Neurospora crassa is inactivated by dicyclohexylcarbodiimide (DCCD). Kinetic and labeling experiments have suggested that inactivation at 0 degrees C results from the covalent attachment of DCCD to a single site in the Mr = 100,000 catalytic subunit (Sussman, M. R., and Slayman, C. W. (1983) J. Biol. Chem. 258, 1839-1843). In the present study, when [14C]DCCD-labeled enzyme was treated with the cleavage reagent, N-bromosuccinimide, a single major radioactive peptide fragment migrating at about Mr = 5,300 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was produced. The fragment was coupled to glass beads and partially sequenced by automated solid-phase Edman degradation at the amino terminus and at an internal tryptic cleavage site. By comparison to the DNA-derived amino acid sequence for the entire Mr = 100,000 polypeptide (Hager, K., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697), the fragment has been identified as arising by cleavage at tyrosine 100 and tryptophan 141. Covalently incorporated [14C]DCCD was released at a position corresponding to glutamate 129. The DCCD-reactive glutamate is located in the middle of the first of eight predicted transmembrane sequences. When the sequence surrounding the DCCD site is compared to that surrounding the DCCD-reactive residue of two other proton pumps, the F0F1-ATPase and cytochrome c oxidase, no homology is apparent apart from an abundance of hydrophobic amino acids.     

Actin filament cross-linking by MARCKS: characterization of two actin-binding sites within the phosphorylation site domain

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.     

Kinetic analysis of F-actin depolymerization in the presence of platelet gelsolin and gelsolin-actin complexes

Platelet gelsolin (G), a 90,000-mol-wt protein, binds tightly to actin (A) and calcium at low ionic strength to form a 1:2:2 complex, GA2Ca2 (Bryan, J., and M. Kurth, 1984, J. Biol. Chem. 259:7480-7487). Chromatography of actin and gelsolin mixtures in EGTA-containing solutions isolates a stable binary complex, GA1Ca1 (Kurth, M., and J. Bryan, 1984, J. Biol. Chem. 259:7473-7479). The effects of platelet gelsolin and the binary gelsolin-actin complex on the depolymerization kinetics of rabbit skeletal muscle actin were studied by diluting pyrenyl F-actin into gelsolin or complex-containing buffers; a decrease in fluorescence represents disassembly of filaments. Dilution of F- actin to below the critical concentration required for filament assembly gave a biphasic depolymerization curve with both fast and slow components. Dilution into buffers containing gelsolin, as GCa2, increased the rate of depolymerization and gave a first order decay. The rate of decrease in fluorescence was found to be gelsolin concentration dependent. Electron microscopy of samples taken shortly after dilution into GCa2 showed a marked reduction in filament length consistent with filament severing and an increase in the number of ends. Conversely, occupancy of the EGTA-stable actin-binding site by an actin monomer eliminated the severing activity. Dilution of F-actin into the gelsolin-actin complex, either as GA1Ca1 or GA1Ca2, resulted in a decrease in the rate of depolymerization that was consistent with filament end capping. This result indicates that the EGTA-stable binding site is required and must be unoccupied for filament severing to occur. The effectiveness of gelsolin, GCa2, in causing filament depolymerization was dependent upon the ionic conditions: in KCI, actin filaments appeared to be more stable and less susceptible to gelsolin, whereas in Mg2+, actin filaments were more easily fragmented. Finally, a comparison of the number of kinetically active ends generated when filaments were diluted into gelsolin versus the number formed when gelsolin can function as a nucleation site suggests that gelsolin may sever more than once. The data are consistent with a mechanism where gelsolin, with both actin-binding sites unoccupied, can sever but not cap F-actin. Occupancy of the EGTA-stable binding site yields a gelsolin-actin complex that can no longer sever filaments, but can cap filament ends.