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.     

A kinetic model for the molecular basis of the contractile activity of Acanthamoeba myosins IA and IB

Acanthamoeba myosins IA and IB are single-headed, monomeric molecules consisting of one heavy chain and one light chain. Both have high actin-activated Mg2+-ATPase activity, when the heavy chain is phosphorylated, but neither seems to be able to form the bipolar filaments that are generally thought to be required for actomyosin-dependent contractility. In this paper, we show that, at fixed F-actin concentration, the actin-activated Mg2+-ATPase activities of myosins IA and IB increase about 5-fold in specific activity in a cooperative manner as the myosin concentration is increased. The myosin concentration range over which this cooperative change occurs depends on the actin concentration. More myosin I is required for the cooperative increase in activity at high concentrations of F-actin. The cooperative increase in specific activity at limiting actin concentrations is caused by a decrease in the KATPase for F-actin. The high and low KATPase states of the myosin have about the same Vmax at infinite actin concentration. Both myosins are completely bound to the F-actin long before the Vmax values are reached. Therefore, much of the actin activation must be the result of interactions between F-actin and actomyosin. These kinetic data can be explained by a model in which the cooperative shift of myosin I from the high KATPase to the low KATPase state results from the cross-linking of actin filaments by myosin I. Cross-linking might occur either through two actin-binding sites on a single molecule or by dimers or oligomers of myosin I induced to form by the interaction of myosin I monomers with the actin filaments. The ability of Acanthamoeba myosins IA and IB to cross-link actin filaments is demonstrated in the accompanying paper (Fujisaki, H., Albanesi, J.P., and Korn, E.D. (1985) J. Biol. Chem. 260, 11183-11189).     

ATPase activities and actin-binding properties of subfragments of Acanthamoeba myosin IA

Previous studies had led to the conclusion that the globular, single-headed myosins IA and IB from Acanthamoeba castellanii contain two actin-binding sites: one associated with the catalytic site and whose binding to F-actin activates the Mg2+-ATPase activity and a second site whose binding results in the cross-linking of actin filaments and makes the actin-activated ATPase activity positively cooperative with respect to myosin I concentration. We have now prepared a 100,000-Da NH2-terminal peptide and a 30,000-Da COOH-terminal peptide by alpha-chymotryptic digestion of the myosin IA heavy chain. The intact 17,000-Da light chain remained associated with the 100,000-Da fragment, which also contained the serine residue that must be phosphorylated for expression of actin-activated ATPase activity by native myosin IA. The 30,000-Da peptide, which contained 34% glycine and 21% proline, bound to F-actin with a KD less than 0.5 microM in the presence or absence of ATP but had no ATPase activity. The 100,000-Da peptide bound to F-actin with KD = 0.4-0.8 microM in the presence of 2 mM MgATP and KD less than 0.01 microM in the absence of MgATP. In contrast to native myosin IA, neither peptide cross-linked actin filaments. The phosphorylated 100,000-Da peptide had actin-activated ATPase activity with the same Vmax as that of native phosphorylated myosin IA but this activity displayed simple, noncooperative hyperbolic dependence on the actin concentration in contrast to the complex cooperative kinetics observed with native myosin IA. These results provide direct experimental evidence for the presence of two actin-binding sites on myosin IA, as was suggested by enzyme kinetic and filament cross-linking data, and also for the previously proposed mechanism by which monomeric myosins I could support contractile activities.     

Experimental evidence for the contractile activities of Acanthamoeba myosins IA and IB

The low-shear viscosity of 5-30 microM F-actin was greatly increased by the addition of 0.1-0.5 microM unphosphorylated Acanthamoeba myosins IA and IB. The increase in viscosity was about the same in 2 mM ADP as in the absence of free nucleotide but was much less in 2 mM ATP. The single-headed monomolecular Acanthamoeba myosins were as effective as an equal molar concentration of two-headed muscle heavy meromyosin and much more effective than single-headed muscle myosin subfragment-1. These results suggest that Acanthamoeba myosins IA and IB can cross-link actin filaments as proposed in the accompanying paper (Albanesi, J. P., Fujisaki, H., and Korn, E. D. (1985) J. Biol. Chem. 260, 11174-11179) to explain the actin-dependent cooperative increase in actin-activated Mg2+-ATPase activity as a function of the concentration of myosin I. Superprecipitation occurred when phosphorylated myosin IA or IB was mixed with F-actin. In addition to myosin I heavy chain phosphorylation, superprecipitation required Mg2+ and ATP. ATP hydrolysis was linear during the time course of the superprecipitation, and inhibitors of ATP hydrolysis inhibited superprecipitation. A small, dense contracted gel was formed when the reaction was carried out in a cuvette, and a birefringent actomyosin thread resulted from superprecipitation in a microcapillary. The rate and extent of superprecipitation depended on the actin and myosin I concentrations with maximum superprecipitation occurring at an actin:myosin ratio of 7:1. These results provide strong evidence for the ability of Acanthamoeba myosins IA and IB to perform contractile and motile functions.     

Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II

It has been previously demonstrated that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II is inhibited by phosphorylation of its two heavy chains (Collins, J. H., and Korn, E. D. (1980) J. Biol. Chem. 255, 8011-8014). In this paper, it is shown that a partially purified kinase preparation from Acanthamoeba catalyzes the incorporation of 3 mol of phosphate into each mole of myosin II heavy chain. Tryptic digestion of the 32P-myosin, followed by two-dimensional peptide mapping, indicates that two of the three sites phosphorylated by the kinase in vitro correspond to the two major phosphorylation sites on the myosin heavy chain in vivo. Phosphorylation of myosin II in vitro by the kinase fraction completely inhibits the actin-activated Mg2+-ATPase activity of myosin II. Myosin II can be isolated in a highly phosphorylated, enzymatically inactive form, then dephosphorylated to an active form, and finally rephosphorylated to an inactive form. The Acanthamoeba kinase fraction catalyzes the phosphorylation of all three sites on the heavy chain of myosin II at virtually the same rate. From a comparison of the decrease in actin-activated Mg2+-ATPase activity with the amount of phosphate incorporated into myosin II, and from the results obtained previously by dephosphorylating myosin II (Collins, J. H., and Korn, E. D., (1980) J. Biol. Chem. 255, 8011-8014), it can be inferred that two of the sites phosphorylated in vitro act in a synergistic manner to inhibit the actin-activated myosin II Mg2+-ATPase.     

Purification and characterization of actin-activatable, Ca2+-sensitive myosin II from Acanthamoeba

Approximately 8-10 mg of highly actin-activatable, CA2+-sensitive Acanthamoeba myosin II can be isolated in greater than 98% purity from 100 g of amoeba by the new procedure described in detail in this paper. The enzyme isolated by this procedure can be activated by actin because its heavy chains are not fully phosphorylated (Collins, J. H., and Korn, E. D. (1980) J. Biol Chem. 255, 8011-8014). We now show that Acanthamoeba myosin II Mg2+-ATPase activity is more highly activated by Acanthamoeba actin than by muscle actin. Also, actomyosin II ATPase is inactive at concentrations of free Mg2+ lower than about 3 mM and fully active at Mg2+ concentrations greater than 4 mM. Actomyosin II Mg2+-ATPase activity is stimulated by micromolar Ca2+ when assayed over the narrow range of about 3-4 mM Mg2+ but is not affected by Ca2+ at either lower or higher concentrations of Mg2+. The specific activity of te actomyosin II Mg2+-ATPase also increases with increasing concentrations of myosin II when the free Mg2+ concentration is in the range of 3-4 mM but is independent of the myosin II concentration at lower or higher concentrations of Mg2+ . This marked effect of the Mg2+ concentration on the Ca2+-sensitivity and myosin concentration-dependence of th specific activity of actomyosin II ATPase activity does not seem to be related to the formation of myosin filaments, and to be related to the formation of myosin filaments, and myosin II is insoluble only at high concentrations of free Mg2+ (6-7 mM) were neither of these effects is observed. Also, the Mg2+ requirements for actomyosin II ATPase activity and myosin II insolubility can be differentially modified by EDTA and sucrose.     

Purification and characterization of a third isoform of myosin I from Acanthamoeba castellanii

A third isoform of myosin I has been isolated from Acanthamoeba and designated myosin IC. Peptide maps and immunoassays indicate that myosin IC is not a modified form of myosin IA, IB, or II. However, myosin IC has most of the distinctive properties of a myosin I. It is a globular protein of native Mr approximately 162,000, apparently composed of a single 130-kDa heavy chain and a pair of 14-kDa light chains. It is soluble in MgATP at low ionic strength, conditions favoring filament assembly by myosin II. Myosin IC has high Ca2+- and (K+,EDTA)-ATPase activities. Its low Mg2+-ATPase activity is stimulated to a maximum rate of 20 s-1 by the addition of F-actin if its heavy chain has been phosphorylated by myosin I heavy chain kinase. The dependence of the Mg2+-ATPase activity of myosin IC on F-actin concentration is triphasic; and, at fixed concentrations of F-action, this activity increases cooperatively as the concentration of myosin IC is increased. These unusual kinetics were first demonstrated for myosins IA and IB and shown to be due to the presence of two actin-binding sites on each heavy chain which enable those myosins I to cross-link actin filaments. Myosin IC is also capable of cross-linking F-actin, which, together with the kinetics of its actin-activated Mg2+-ATPase activity, suggests that it, like myosins IA and IB, possesses two independent actin-binding domains.     

Localization of the actin-binding sites of Acanthamoeba myosin IB and effect of limited proteolysis on its actin-activated Mg2+-ATPase activity

Acanthamoeba myosin IB contains a 125-kDa heavy chain that has high actin-activated Mg2+-ATPase activity when 1 serine residue is phosphorylated. The heavy chain contains two F-actin-binding sites, one associated with the catalytic site and a second which allows myosin IB to cross-link actin filaments but has no direct effect on catalytic activity. Tryptic digestion of the heavy chain initially produces an NH2-terminal 62-kDa peptide that contains the ATP-binding site and the regulatory phosphorylation site, and a COOH-terminal 68-kDa peptide. F-actin, in the absence of ATP, protects this site and tryptic cleavage then produces an NH2-terminal 80-kDa peptide. Both the 62- and the 80-kDa peptides retain the (NH+4,EDTA)-ATPase activity of native myosin IB and both bind to F-actin in an ATP-sensitive manner. However, only the 80-kDa peptide retains a major portion of the actin-activated Mg2+-ATPase activity. This activity requires phosphorylation of the 80-kDa peptide by myosin I heavy chain kinase but, in contrast to the activity of intact myosin IB, it has a simple, hyperbolic dependence on the concentration of F-actin. Also unlike myosin IB, the 80-kDa peptide cannot cross-link F-actin filaments indicating the presence of only a single actin-binding site. These results allow the assignment of the actin-binding site involved in catalytic activity to the region near, and possibly on both sides of, the tryptic cleavage site 62 kDa from the NH2 terminus, and the second actin-binding site to the COOH-terminal 45-kDa domain. Thus, the NH2-terminal 80 kDa of the myosin IB heavy chain is functionally similar to the 93-kDa subfragment 1 of muscle myosin and most likely has a similar organization of functional domains.     

Cooperative dependence of the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II on the extent of filament phosphorylation

The actin-activated Mg2+-ATPase of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues at the tip of the tail of each of its two heavy chains; only dephosphorylated myosin II is active, whereas the phosphorylated and dephosphorylated forms have identical Ca2+-ATPase activities and Mg2+-ATPase activities in the absence of F-actin. We have now chemically modified phosphorylated and dephosphorylated myosin II with N-ethylmaleimide (NEM). The modification occurred principally at a single site within the NH2-terminal 73,000 Da of the globular head of the heavy chain. NEM-myosin II bound to F-actin and formed filaments normally, but the Ca2+- and Mg2+-ATPase activities of phosphorylated and dephosphorylated myosin II and the actin-activated Mg2+-ATPase activity of NEM-dephosphorylated myosin II were inhibited. Only filamentous myosin II has actin-activated Mg2+-ATPase activity. Native phosphorylated myosin II acquired actin-activated Mg2+-ATPase activity when it was co-polymerized with NEM-inactivated dephosphorylated myosin II, and the increase in its activity was cooperatively dependent on the fraction of NEM-dephosphorylated myosin II in the filaments. From this result, we conclude that the specific activity of each molecule within a filament is independent of its own state of phosphorylation, but is highly cooperatively dependent upon the state of phosphorylation of the filament as a whole. This enables the actin-activated Mg2+-ATPase activity of myosin II filaments to respond rapidly and extensively to small changes in the level of their phosphorylation.     

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.     

Purification and characterization of an Acanthamoeba nuclear actin-binding protein

Immunolocalization of monoclonal antibodies to Acanthamoeba myosin I showed a cross-reactive protein in nuclei (Hagen, S. J., D. P. Kiehart, D. A. Kaiser, and T. D. Pollard. 1986. J. Cell Biol. 103:2121-2128). This protein is antigenically related to myosin I in that nine monoclonal antibodies and three polyclonal antibodies are cross-reactive. However, studies with affinity-purified antibodies and two-dimensional peptide maps show that the protein is not a proteolytic product of myosin I. We have used cell fractionation and column chromatography to purify this protein. It is a dimer of 34-kD polypeptides with a Stokes' radius of 4 nm. A polyclonal antisera generated against the purified protein confirms the nuclear localization seen with the cross-reactive monoclonal antibodies. The 34-kD protein binds actin filaments in an ATP-insensitive manner with a Kd of approximately 0.25 microM without cross-linking, severing, or capping. No ATPase activity was detected in the presence or absence of actin. It also binds to DNA. These unique properties suggest we have discovered a new class of actin-binding protein. We have given this protein the name NAB for "nuclear actin-binding" protein.     

Purification from Dictyostelium discoideum of a low-molecular-weight myosin that resembles myosin I from Acanthamoeba castellanii

A low-molecular-weight myosin has been purified 1500-fold from extracts of Dictyostelium discoideum, based on the increase in K+,EDTA-ATPase specific activity. The purified enzyme resembles the single-headed, low-molecular-weight myosins IA and IB from Acanthamoeba castellanii, and differs from the conventional two-headed, high-molecular-weight myosin previously isolated from Dictyostelium, in several ways. It has higher K+,EDTA-ATPase activity than Ca2+-ATPase activity; it has a native molecular mass of about 150,000 and a single heavy chain of about 117,000; the 117,000-dalton heavy chain is phosphorylated by Acanthamoeba myosin I heavy chain kinase; phosphorylation of its heavy chain enhances its actin-activated Mg2+-ATPase activity; and the 117,000-dalton heavy chain reacts with antibodies raised against the heavy chain of Acanthamoeba myosin IA. None of these properties is shared by the low-molecular-weight active fragment that can be produced by chymotryptic digestion of conventional Dictyostelium myosin. We conclude that Dictyostelium contains an enzyme of the myosin I type previously isolated only from Acanthamoeba.     

The effects of smooth muscle caldesmon on actin filament motility.

The movement of reconstituted thin filaments over an immobilized surface of thiophosphorylated smooth muscle myosin was examined using an in vitro motility assay. Reconstituted thin filaments contained actin, tropomyosin, and either purified chicken gizzard caldesmon or the purified COOH-terminal actin-binding fragment of caldesmon. Control actin-tropomyosin filaments moved at a velocity of 2.3 +/- 0.5 microns/s. Neither intact caldesmon nor the COOH-terminal fragment, when maintained in the monomeric form by treatment with 10 mM dithiothreitol, had any effect on filament velocity; and yet both were potent inhibitors of actin-activated myosin ATPase activity, indicating that caldesmon primarily inhibits myosin binding as reported by Chalovich et al. (Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599, 85-99). Inhibition of filament motion was, however, observed under conditions where cross-linking of caldesmon via disulfide bridges was present. To determine if monomeric caldesmon could "tether" actin filaments to the myosin surface by forming an actin-caldesmon-myosin complex as suggested by Chalovich et al., we looked for caldesmon-dependent filament binding and motility under conditions (80 mM KCl) where filament binding to myosin is weak and motility is not normally seen. At caldesmon concentrations > or = 0.26 microM, actin filament binding was increased and filament motion (2.6 +/- 0.6 microns/s) was observed. The enhanced motility seen with intact caldesmon was not observed with the addition of up to 26 microM COOH-terminal fragment. Moreover, a molar excess of the COOH-terminal fragment competitively reversed the enhanced binding seen with intact caldesmon. These results show that tethering of actin filaments to myosin by the formation of an actin-caldesmon-myosin complex enhanced productive acto-myosin interaction without placing a significant mechanical load on the moving filaments.     

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.     

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility

The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.     

Functional characterization of the secondary actin binding site of myosin II

The role of the interaction between actin and the secondary actin binding site of myosin (segment 565-579 of rabbit skeletal muscle myosin, referred to as loop 3 in this work) has been studied with proteolytically generated smooth and skeletal muscle myosin subfragment 1 and recombinant Dictyostelium discoideum myosin II motor domain constructs. Carbodiimide-induced cross-linking between filamentous actin and myosin loop 3 took place only with the motor domain of skeletal muscle myosin and not with those of smooth muscle or D. discoideum myosin II. Chimeric constructs of the D. discoideum myosin motor domain containing loop 3 of either human skeletal muscle or nonmuscle myosin were generated. Significant actin cross-linking to the loop 3 region was obtained only with the skeletal muscle chimera both in the rigor and in the weak binding states, i.e., in the absence and in the presence of ATP analogues. Thrombin degradation of the cross-linked products was used to confirm the cross-linking site of myosin loop 3 within the actin segment 1-28. The skeletal muscle and nonmuscle myosin chimera showed a 4-6-fold increase in their actin dissociation constant, due to a significant increase in the rate for actin dissociation (k(-)(A)) with no significant change in the rate for actin binding (k(+A)). The actin-activated ATPase activity was not affected by the substitutions in the chimeric constructs. These results suggest that actin interaction with the secondary actin binding site of myosin is specific for the loop 3 sequence of striated muscle myosin isoforms but is apparently not essential either for the formation of a high affinity actin-myosin interface or for the modulation of actomyosin ATPase activity.     

Myosin surface loop 4 modulates inhibition of actomyosin 1b ATPase activity by tropomyosin

Structural studies of the class I myosin, MyoE, led to the predictions that loop 4, a surface loop near the actin-binding region that is longer in class I myosins than in other myosin subclasses, might limit binding of myosins I to actin when actin-binding proteins, like tropomyosin, are present, and might account for the exclusion of myosin I from stress fibers. To test these hypotheses, mutant molecules of the related mammalian class I myosin, Myo1b, in which loop 4 was truncated (from an amino acid sequence of RMNGLDES to NGLD) or replaced with the shorter and distinct loop 4 found in Dictyostelium myosin II (GAGEGA), were expressed in vitro and their interaction with actin and with actin-tropomyosin was tested. Saturating amounts of expressed fibroblast tropomyosin-2 resulted in a decrease in the maximum actin-activated Mg2+-ATPase activity of wild-type Myo1b but had little or no effect on the actin-activated Mg2+-ATPase activity of the two mutants. In motility assays, few actin filaments bound tightly to Myo1b-WT-coated cover slips when tropomyosin-2 was present, whereas actin filaments both bound and were translocated by Myo1b-NGLD or Myo1b-GAGEGA in both the presence and absence of tropomyosin-2. When expressed in mammalian cells, like the wild type, the mutant myosins were largely excluded from tropomyosin-containing actin filaments, indicating that in the cell additional factors besides loop 4 determine targeting of myosins I to specific subpopulations of actin filaments.     

The myosin relay helix to converter interface remains intact throughout the actomyosin ATPase cycle

Crystal structures of the myosin motor domain in the presence of different nucleotides show the lever arm domain in two basic angular states, postulated to represent prestroke and poststroke states, respectively (Rayment, I. (1996) J. Biol. Chem. 271, 15850-15853; Dominguez, R., Freyzon, Y., Trybus, K. M., and Cohen, C. (1998) Cell 94, 559-571). Contact is maintained between two domains, the relay and the converter, in both of these angular states. Therefore it has been proposed by Dominguez et al. (cited above) that this contact is critical for mechanically driving the angular change of the lever arm domain. However, structural information is lacking on whether this contact is maintained throughout the actin-activated myosin ATPase cycle. To test the functional importance of this interdomain contact, we introduced cysteines into the sequence of a "cysteine-light" myosin motor at position 499 on the lower cleft and position 738 on the converter domain (Shih, W. M., Gryczynski, Z., Lakowicz, J. L., and Spudich, J. A. (2000) Cell 102, 683-694). Disulfide cross-linking could be induced. The cross-link had minimal effects on actin binding, ATP-induced actin release, and actin-activated ATPase. These results demonstrate that the relay/converter interface remains intact in the actin strongly bound state of myosin and throughout the entire actin-activated myosin ATPase cycle.     

Functional consequences of the proteolytic removal of regulatory serines from the nonhelical tailpiece of Acanthamoeba myosin II

The actin-activated Mg2(+)-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serines in its 29-residue, nonhelical, COOH-terminal tailpiece, i.e., serines-1489, -1494, and -1499 or, in reverse order, residues 11, 16, and 21 from the COOH terminus. To investigate the essential requirements for regulation, myosin II filaments in the presence of F-actin were digested by arginine-specific submaxillary gland protease. Two-dimensional peptide mapping of purified, cleaved myosin II showed that the two most terminal phosphorylation sites, serines-1494 and -1499, had been removed. Cleaved dephosphorylated myosin II retained full actin-activated Mg2(+)-ATPase activity (with no change in Vmax or Kapp) and the ability to form filaments similar to those of the native enzyme. However, higher Mg2+ concentrations were required for both filament formation and maximal ATPase activity. The one remaining regulatory serine in the cleaved myosin II was phosphorylatable by myosin II heavy-chain kinase, and phosphorylation inactivated the actin-activated Mg2(+)-ATPase activity, as in the case of the native myosin II. Also as in the case of the native myosin II, phosphorylated cleaved myosin II inhibited the actin-activated Mg2(+)-ATPase activity of dephosphorylated cleaved myosin II when the two were copolymerized. These results suggest that at least 18 of the 29 residues in the nonhelical tailpiece of the heavy chain are not required for either actin-activated Mg2(+)-ATPase activity or filament formation and that phosphorylation of Ser-1489 is sufficient to regulate the actin-activated Mg2(+)-ATPase activity of myosin II.     

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.