Effect of actin filament length and filament number concentration on the actin-activated ATPase activity of Acanthamoeba myosin I

The actin-activated Mg2+-ATPase activities of phosphorylated Acanthamoeba myosins IA and IB were previously found to have a highly cooperative dependence on myosin concentration (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179). This behavior is reflected in the requirement for a higher concentration of F-actin for half-maximal activation of the myosin Mg2+-ATPase at low ratios of myosin:actin (noncooperative phase) than at high ratios of myosin:actin (cooperative phase). These phenomena could be explained by a model in which each molecule of the nonfilamentous myosins IA and IB contains two F-actin-binding sites of different affinities with binding of the lower affinity site being required for expression of actin-activated ATPase activity. Thus, enzymatic activity would coincide with cross-linking of actin filaments by myosin. This theoretical model predicts that shortening the actin filaments and increasing their number concentration at constant total F-actin should increase the myosin concentration required to obtain the cooperative increase in activity and should decrease the F-actin concentration required to reach half-maximal activity at low myosin:actin ratios. These predictions have been experimentally confirmed by shortening actin filaments by addition of plasma gelsolin, an F-actin capping/severing protein. In addition, we have found that actin "filaments" as short as the 1:2 gelsolin-actin complex can significantly activate Acanthamoeba myosin I.     

Supramolecular regulation of the actin-activated ATPase activity of filaments of Acanthamoeba Myosin II

Acanthamoeba myosin II has three phosphorylation sites clustered near the end of the tail of each of its two heavy chains (six phosphorylation sites/molecule). Myosin II has little or no actin-activated ATPase activity when four to six of these sites are phosphorylated. Maximal actin-activated ATPase activity is obtained when all six sites are dephosphorylated. Under assay conditions, both phosphorylated and dephosphorylated myosin II form bipolar filaments. Filaments of dephosphorylated myosin II have larger sedimentation coefficients than filaments of phosphorylated myosin II but this difference does not explain the difference in their actin-activated ATPase activities. Heteropolymers, formed by mixing soluble dephosphorylated and phosphorylated myosins and then diluting the mixture into low ionic strength buffer containing MgCl2, have sedimentation coefficients close to those of the homopolymer of phosphorylated myosin. The actin-activated ATPase activities of heteropolymers are, under most conditions, lower than the equivalent mixtures of homopolymers of dephosphorylated and phosphorylated myosins. It is concluded, therefore, that the phosphorylation of myosin tails regulates the actin-activated ATPase activity of Acanthamoeba myosin II by affecting the myosin filament as a whole rather than specifically affecting the heads of the phosphorylated myosin molecules only.     

Limited tryptic digestion of Acanthamoeba myosin IA abolishes regulation of actin-activated ATPase activity by heavy chain phosphorylation

Acanthamoeba myosin IA is a globular protein composed of a 140-kDa heavy chain and a 17-kDa light chain. It expresses high actin-activated Mg2+-ATPase activity when one serine on the heavy chain is phosphorylated. We previously showed that chymotrypsin cleaves the heavy chain into a COOH-terminal 27-kDa peptide that can bind to F-actin but has no ATPase activity and a complex containing the NH2-terminal 112-kDa peptide and the light chain. The complex also binds F-actin and has full actin-activated Mg2+-ATPase activity when the regulatory site is phosphorylated. We have now localized the ATP binding site to within 27 kDa of the NH2 terminus and the regulatory phosphorylatable serine to a 20-kDa region between 38 and 58 kDa of the NH2 terminus. Under controlled conditions, trypsin cleaves the heavy chain at two sites, 38 and 112 kDa from the NH2 terminus, producing a COOH-terminal 27-kDa peptide similar to that produced by chymotrypsin and a complex consisting of an NH2-terminal kDa peptide, a central 74-kDa peptide, and the light chain. This complex is similar to the chymotryptic complex but for the cleavage which separates the 38- and 74-kDa peptides. The tryptic complex has full (K+, EDTA)-ATPase activity (the catalytic site is functional) and normal ATP-sensitive actin-binding properties. However, the actin-activated Mg2+-ATPase activity and the F-actin-binding characteristics of the tryptic complex are no longer sensitive to phosphorylation of the regulatory serine. Therefore, cleavage between the phosphorylation site and the ATP-binding site inhibits the effects of phosphorylation on actin binding and actin-activated Mg2+-ATPase activity without abolishing the interactions between the ATP- and actin-binding sites.     

Regulation of the actin-activated ATPase of aorta smooth muscle myosin

Phosphorylation of the 20,000-Da light chains, LC20, of vertebrate smooth muscle myosins is thought to be the primary mechanism for regulating the actin-activated ATPase activities of these myosins and consequently smooth muscle contraction. While actin stimulates the MgATPase activities of phosphorylated smooth muscle myosins, it is generally believed that the MgATPase activities of the unphosphorylated myosins are not stimulated by actin. However, under conditions where both unphosphorylated (5% phosphorylated LC20) and phosphorylated calf aorta myosins are mostly filamentous, the maximum rate, Vmax, of the actin-activated ATPase of the unphosphorylated myosin is one-half that of the phosphorylated myosin. While LC20 phosphorylation causes only a modest increase in Vmax, in the presence of tropomyosin, this phosphorylation does cause up to a 10-fold decrease in Kapp, the actin concentration required to achieve 1/2 Vmax. In the presence of low concentrations of tropomyosin/actin, a linear relationship is obtained between the fraction of LC20 phosphorylated and stimulation of the actin-activated ATPase. The relatively high actin-activated ATPase activity of unphosphorylated aorta myosin suggests that other proteins may be involved in the regulation of smooth muscle contraction. In contrast to the results presented here for aorta myosin, it has been reported that actin does not activate the MgATPase activity of unphosphorylated gizzard myosin and that the actin-activated ATPase of gizzard myosin increases more slowly than LC20 phosphorylation.     

Effect of calponin on actin-activated myosin ATPase activity

Calponin inhibited the actin-activated myosin MgATPase activity in a dose-dependent manner without affecting the phosphorylation level of myosin light chain. This inhibition was Ca2(+)-independent. The decrease in enzymatic activity of myosin was correlated with binding of calponin to actin-tropomyosin filaments. Caldesmon showed a further inhibition of the calponin-induced inhibition of MgATPase activity of the thiophosphorylated myosin. Calponin-induced inhibition of the myosin MgATPase activity was reversed by the addition of calmodulin only in the presence of Ca2+. These results suggest that calponin acts as an inhibitory component of smooth muscle thin filaments.     

Regulation of the actin-activated ATPase and in vitro motility activities of monomeric and filamentous Acanthamoeba myosin II.

The actin-activated Mg(2+)-ATPase activity of filamentous Acanthamoeba myosin II is inhibited by phosphorylation of 3 serine residues at the tip of the tail of each heavy chain. From previous studies, it had been concluded that the activity of each molecule in the filament was regulated by the global state of phosphorylation of the filament and was independent of its own phosphorylation state. The actin-activated Mg(2+)-ATPase activity of monomeric phosphorylated myosin II was not known because it polymerizes under the ionic conditions necessary for the expression of this activity. We have now found conditions to maintain myosin II monomeric and active during the enzyme assay. The actin-activated Mg(2+)-ATPase activities of monomeric dephosphorylated and phosphorylated myosin II were found to be the same as the activity of filamentous dephosphorylated myosin II. These results support the conclusion that phosphorylation regulates filamentous myosin II by affecting filament conformation. Consistent with their equivalent enzymatic activities, monomeric and filamentous dephosphorylated myosin II were equally active in an in vitro motility assay in which myosin adsorbed to a surface drives the movement of F-actin. In contrast to their very different enzymatic activities, however, filamentous and monomeric phosphorylated myosin II had similar activities in the in vitro motility assay; both were much less active than monomeric and filamentous dephosphorylated myosin II. One interpretation of these results is that the rate-limiting steps in the two assays are different and that, while the rate-limiting step for actin-activated Mg(2+)-ATPase activity is regulated only at the level of the filament, the rate-limiting step for motility can also be regulated at the level of the monomer.     

Regulation of the actin-activated ATPase activity of Acanthamoeba myosin II by copolymerization with phosphorylated and dephosphorylated peptides derived from the carboxyl-terminal end of the heavy chain

Myosin II from Acanthamoeba castellanii is a conventional myosin composed of two heavy chains and two pairs of light chains. The amino-terminal approximately 90 kDa of each heavy chain form a globular head that contains the ATPase site and an ATP-sensitive actin-binding site. The carboxyl-terminal approximately 80 kDa of both heavy chains interact to form a coiled coil, helical rod (through which the molecules self-associate into bipolar filaments) ending in a short nonhelical tailpiece. Phosphorylation of 3 serine residues at the tip of the tail (at positions 11, 16, and 21 from the carboxyl terminus) inactivates the actin-activated Mg2(+)-ATPase activity of myosin II filaments. Previous work had indicated that the activity of each myosin II molecule in a filament reflects the global state of phosphorylation of the filament rather than the phosphorylation state of the molecule itself. We have now purified the approximately 28-kDa carboxyl-terminal region of the heavy chain lacking the last two phosphorylation sites, and we have shown that this peptide copolymerizes with and regulates the actin-activated Mg2(+)-ATPase activities of native dephosphorylated and phosphorylated myosin II. It can be concluded from these studies that the biologically relevant enzymatic activity of myosin II is regulated by a phosphorylation-dependent conformational change in the myosin filaments.     

Thick filaments of fish myosin and its actin-activated Mg-ATPase activity

1. Tilapia (Tilapia nilotica) myosin forms short, mini-filaments, and are easily disassembled upon addition of ATP showing no saturated activation in its actin-activated Mg-ATPase activity. 2. The presence of 5-10 mM MgCl2 allows tilapia myosin to form native thick-filaments and are resistant to ATP. 3. The rod portion of Tilapia myosin molecule is responsible for its characteristic filament forming ability. 4. The similar filament forming ability as Tilapia myosin was suggested for other fish myosins.     

Macrophage myosin. Regulation of actin-activated ATPase, activity by phosphorylation of the 20,000-dalton light chain.

Myosin was purified from rabbit alveolar macrophages in a form that could not be activated by actin. This myosin could be phosphorylated by an endogenous myosin light chain kinase, up to 2 mol of phosphate being incorporated/mol of myosin. The site phosphorylated was located on the 20,000-dalton myosin light chain. Phosphorylation of macrophage myosin was found to be necessary for actin activation of myosin ATPase activity. Moreover, the actin-activated ATPase activity was found to vary directly with the extent of myosin phosphorylation, maximal phosphorylation (2 mol of Pi/mol of myosin) resulting in an actin-activated MgATPase activity of approximately 200 nmol of Pi/mg of myosin/min at 37 degrees C. These results establish that phosphyoyration of the 20,000-dalton light chain of myosin is sufficient to regulate the actin-activated ATPase activity of macrophage myosin.     

Uncoupling of actin-activated myosin ATPase activity from actin binding by a monoclonal antibody directed against the N-terminus of myosin light chain 1.

The role of the N-terminal region of myosin light chain 1 (LC1) in actomyosin interaction was investigated using an IgG monoclonal antibody (2H2) directed against the N-terminal region of LC1. We defined the binding site of 2H2 by examining its cross-reactivity with myosin light chains from a variety of species and with synthetic oligopeptides. Our findings suggest that 2H2 is directed against the N-terminal region of LC1 which includes the trimethylated alanine residue at the N-terminus. In the presence of 2H2, the rate of actomyosin superprecipitation was reduced, although the extent was not. 2H2 caused a reduction in the Vmax of both myosin and chymotryptic S1(A1) actin-activated ATPase activity, while the Km appeared to be unaltered. The Mg(2+)-ATPase activity of myosin alone was also unaffected. Binding studies revealed that 2H2 did not prevent the formation of acto-S1 complex, either in the presence or in the absence of ATP, nor did it affect the ability of ATP to dissociate S1 from F-actin. Our findings suggest that the N-terminal region of LC1 is not essential for actin binding but is involved in modulating actin-activated ATPase activity of myosin.     

Enhancement of the actin-activated ATPase activity of myosin from canine cardiac ventricle by purealin

The effects of purealin isolated from the sea sponge, Psammaplysilla purea, on the enzymatic properties of myosin and natural actomyosin (a complex of myosin, actin, tropomyosin and troponin) from canine cardiac ventricle were studied. Purealin increased the ATPase activity of natural actomyosin and the actin-activated ATPase activity of myosin, and accelerated the superprecipitation of natural actomyosin. The Ca2+- and Mg2+-ATPase activities of myosin were inhibited by purealin, whereas the K+-EDTA-ATPase activity was increased. These results suggest that purealin binds to the myosin portion involved in actin-myosin interaction and increases the actin-activated ATPase activity of myosin.     

Correlation between multiple phosphorylation of gizzard myosin light chains and actin-activated myosin ATPase activity

With large amounts of gizzard Mr 135,000 calmodulin-binding protein (myosin light chain kinase), the phosphate incorporation into myosin light chains was determined to be 2 mol/mol of myosin light chain. The actin-activated ATPase activity was dramatically enhanced when myosin light chains were phosphorylated by more than 1 mol of phosphate incorporated/mol of myosin light chain.     

The regulation of actin polymerization and the inhibition of monomeric actin ATPase activity by Acanthamoeba profilin

Profilin inhibits the rate of nucleation of actin polymerization and the rate of filament elongation and also reduces the concentration of F-actin at steady state. Addition of profilin to solutions of F-actin causes depolymerization. The same steady state concentrations of polymerized and nonpolymerized actin are reached whether profilin is added before initiation of polymerization or after polymerization is complete. The KD for formation of the 1:1 complex between Acanthamoeba profilin and Acanthamoeba actin is in the range of 4 to 11 microM; the KD for the reaction between Acanthamoeba profilin and rabbit skeletal muscle actin is about 60 to 80 microM, irrespective of the concentrations of KCl or MgCl2. The critical concentration of actin for polymerization and the KD for the actin-profilin interaction are independent of each other; therefore, a change in the critical concentration of actin alters the amount of actin bound to profilin at steady state. As a consequence, the presence of profilin greatly amplifies the effects of small changes in the actin critical concentration on the concentration of F-actin. Profilin also inhibits the ATPase activity of monomeric actin, the profilin-actin complex being entirely inactive.     

Regulation of sodium channel activity by capping of actin filaments

Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells. The channels were activated by cytochalasin-induced disruption of actin filaments and inactivated by G-actin under ionic conditions promoting rapid actin polymerization. CapZ had no direct effect on channel activity. However, being added together with G-actin, CapZ prevented actin-induced channel inactivation, and this effect occurred at CapZ/actin molar ratios from 1:5 to 1:100. When actin was allowed to polymerize at the plasma membrane to induce partial channel inactivation, subsequent addition of CapZ restored the channel activity. These results can be explained by CapZ-induced inhibition of further assembly of actin filaments at the plasma membrane due to the modification of actin dynamics by CapZ. No effect on the channel activity was observed in response to F-actin, confirming that the mechanism of channel inactivation does not involve interaction of the channel with preformed filaments. Our data show that actin-capping protein can participate in the cytoskeleton-associated regulation of sodium transport in nonexcitable cells.     

Inhibition of Mg ++ ATPase activity of actin-activated Acanthamoeba myosin by muscle troponin-tropomyosin: implications for the mechanism of control of amoeba motility and muscle contraction

Troponin-tropomyosin is known to inhibit the Mg⁺⁺ATPase activity of muscle actomyosin in the absence, but not in the presence, of Ca⁺⁺. In contrast, we have now found that muscle troponin-tropomyosin inhibits the Mg⁺⁺ATPase activity of muscle actin-activated $\text{Acanthamoeba}$ myosin both in the presence and the absence of Ca⁺⁺. Addition of purified tropomyosin and troponins-I, C and T demonstrated that it is troponin-T that acts differently in the two systems which differ only in the source of the myosin. These data suggest that myosin, as well as actin, plays a role in the troponin-tropomyosin control of muscle contraction and make it unlikely that control proteins identical to troponin-tropomyosin function in this amoeba.     

Linear relationship between diphosphorylation of 20 kDa light chain of gizzard myosin and the actin-activated myosin ATPase activity

The addition of large amounts of myosin light chain kinase to the reconstituted gizzard actomyosin shows diphosphorylation of 20 kDa myosin light chain. Accompanying diphosphorylation, the actin-activated myosin ATPase activity was also enhanced. The extent of diphosphorylation and the myosin ATPase activity were clearly demonstrated to be in a linear relationship. From the time course experiment, the conversion of monophosphorylated light chain into one which was diphosphorylated seemed to be a sequential process. Moreover, analyzing phospho-amino acid by using a two-dimensional electrophoresis technique revealed that monophosphorylated light chain contained phosphoserine and diphosphorylated one contained phosphothreonine in addition to phosphoserine.     

EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.     

Filament formation and actin-activated ATPase activity are abolished by proteolytic removal of a small peptide from the tip of the tail of the heavy chain of Acanthamoeba myosin II

Actin-activated Mg2+-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of three serine residues located at the carboxyl-terminal end of each of the two 185,000-Da heavy chains; the phosphorylated molecule has full Ca2+-ATPase activity but no actin-activated Mg2+-ATPase activity. Under controlled conditions, chymotrypsin removes a small peptide containing all three phosphorylation sites from the ends of the myosin II heavy chains producing a molecule with heavy chains of 175,000 Da and undigested light chains. The length of the myosin II tail decreased from 89 to 76 nm. Chymotrypsin-cleaved myosin II has complete Ca2+-ATPase activity but no actin-activated Mg2+-ATPase activity under standard assay conditions and binds to F-actin as well as undigested myosin II in the absence, but not in the presence, of MgATP. In the presence of MgCl2, undigested myosin II forms biopolar filaments but chymotrypsin-cleaved myosin II forms only parallel (monopolar) dimers, as assessed by analytical ultra-centrifugation and rotary shadow electron microscopy. We conclude that the short segment very near the end of the myosin II tail that contains the three phosphorylatable serines is necessary for the formation of biopolar filaments and, probably as a consequence of filament formation, for the high-affinity binding of myosin II to F-actin in the presence of ATP and the actin-activated Mg2+-ATPase activity of native myosin II. This supports our previous conclusion that actin-activated Mg2+-ATPase of native myosin II is expressed only when the enzyme is in bipolar filaments with the proper conformation as determined by the state of phosphorylation of the heavy chains.     

Regulation of Drosophila myosin ATPase activity by phosphorylation of myosin light chains--I. Wild-type fly

1. Two types of myosins with phosphorylated and dephosphorylated myosin light chains were prepared from Drosophila flies. The former had ATPase (Ca2(+)- and Mg2(+)-activited) activities twice those of the latter. 2. The myosin phosphorylated with crude myosin light chain kinase from flies showed ATPase (Ca2(+)- and Mg2(+)-activated) activaties twice those of the dephosphorylated myosin. 3. It is suggested that phosphorylation of myosin light chains several hours after emergence stimulates myosin ATPase activity so as to facilitate the flight function of the fruitfly.     

Acanthamoeba cofactor protein is a heavy chain kinase required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I.

We have purified a cofactor protein previously shown (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697) to be required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I. The purified cofactor protein is a novel myosin kinase that phosphorylates the single heavy chain, but neither of the two light chains, of Acanthamoeba myosin I. Phosphorylation of Acanthamoeba myosin I by the purified cofactor protein requires ATP and Mg2+ but is Ca2+-independent. The Mg2+-ATPase activity of phosphorylated Acanthamoeba myosin I is highly activated by F-actin in the absence of cofactor protein. Actin-activated Mg2+-ATPase activity is lost when phosphorylated Acanthamoeba myosin I is dephosphorylated by platelet phosphatase. Phosphorylation and dephosphorylation have no effect on the (K+,EDTA)-ATPase and Ca2+-ATPase activities of Acanthamoeba myosin I. These results show that cofactor protein is an Acanthamoeba myosin I heavy chain kinase and that phosphorylation of the heavy chain of this myosin is required for actin activation of its Mg2+-ATPase activity.