Kinetic characterization of the function of myosin loop 4 in the actin-myosin interaction

Myosin interacts with actin during its enzymatic cycle, and actin stimulates myosin's ATPase activity. There are extensive interaction surfaces on both actin and myosin. Several surface loops of myosin play different roles in actomyosin interaction. However, the functional role of loop 4 in actin binding is still ambiguous. We explored the role of loop 4 by either mutating its conserved acidic group, Glu-365, to Gln (E365Q), or by replacing the entire loop with three glycines (DeltaAL) in a Dictyostelium discoideum myosin II motor domain (MD) containing a single tryptophan residue. This native tryptophan (Trp-501) is located in the relay loop and is sensitive to nucleotide binding and lever-arm movement. Fluorescence and fast kinetic measurements showed that the mutations in loop 4 do not alter the enzymatic steps of the ATPase cycle in the absence of actin. By contrast, actin binding was significantly weakened in the absence and presence of ADP and ATP in both mutants. Because the strength of actin-myosin interaction increases in the order of rigor, ADP, and ATP complex, we conclude that loop 4 is a functional actin-binding region that stabilizes actomyosin complex, particularly in weak actin-binding states.     

Addition of lysines to the 50/20 kDa junction of myosin strengthens weak binding to actin without affecting the maximum ATPase activity

Much interest has centered on two surface loops in the motor domain to explain the differences in enzymatic and mechanical properties of myosin isoforms. We showed that two invariant lysines at the C-terminal end of loop 2, which is part of the actin-binding interface, are required to obtain actin activation [Joel et al. (2001) J. Biol. Chem. 276, 2998-3003]. Here we investigate the effects of increasing positive charge in the variable portion of loop 2 of smooth muscle heavy meromyosin (smHMM). Increasing the net positive charge by +4 increased the affinity for actin in the presence and absence of ATP. The K(m) for actin-activated ATPase activity decreased 15-fold, but V(max) was unchanged, showing that "weak binding" of myosin for actin can be significantly strengthened without increasing the rate-limiting step for V(max). The mutant HMM had slower rates of in vitro motility and ADP release compared to WT HMM. ADP release and motility, which were both salt-dependent, correlated linearly with each other. Loop 2 thus plays a major role in setting the affinity for actin but also affects ADP release and motility. Because the actin- and nucleotide-binding regions communicate, mutations to one region can impact multiple facets of myosin's mechanical and enzymatic properties.     

The sequence of the myosin 50-20K loop affects Myosin's affinity for actin throughout the actin-myosin ATPase cycle and its maximum ATPase activity

We are interested in the role that solvent-exposed, proteolytically sensitive surface loops play in myosin function. The 25-50K loop, or loop 1, is near the ATP binding site, while the 50-20K loop (loop 2) is in the actin binding site. Through chimeric studies, we have found that loop 1 affects ADP release [Murphy, C. T., and Spudich, J. A. (1998) Biochemistry 37, 6738-44], while loop 2 affects the actin-activated ATPase activity [Uyeda, T. Q.-P., et al. (1994) Nature 368, 567-9]. In the study described here, we have found that the kcat of the actin-activated ATPase activity is changed by the loop 2 substitutions in a manner that reflects the relative actin-activated ATPase activities of the donor myosins. Additionally, changes in loop 2 affect the affinity of myosin for actin both in the presence and in the absence of nucleotides. Pre-steady-state studies together with the ATPase and affinity data suggest that while loop 2 does not affect interactions between myosin and nucleotide, it plays a role in determining the affinity of myosin for actin in various nucleotide states and in the rate-limiting transition allowing phosphate release.     

The structural basis of myosin V processive movement as revealed by electron cryomicroscopy

The processive motor myosin V has a relatively high affinity for actin in the presence of ATP and, thus, offers the unique opportunity to visualize some of the weaker, hitherto inaccessible, actin bound states of the ATPase cycle. Here, electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional (3D) reconstructions provide the atomic models of myosin V in both weak and strong actin bound states. One structure shows that ATP binding opens the long cleft dividing the actin binding region of the motor domain, thus destroying the strong binding actomyosin interface while rearranging loop 2 as a tether. Nucleotide analogs showed a second new state in which the lever arm points upward, in a prepower-stroke configuration (lever arm up) bound to actin before phosphate release. Our findings reveal how the structural elements of myosin V work together to allow myosin V to step along actin for multiple ATPase cycles without dissociating.     

Kinetic mechanism of blebbistatin inhibition of nonmuscle myosin IIb

We examined the effect of blebbistatin on the kinetic properties of nonmuscle myosin IIB subfragment 1 (NMIIB S1). Blebbistatin is a small molecule that affects cell blebbing during the process of cell division, which has been shown to decrease the myosin ATPase activity of a number of myosins [Straight et al. (2003) Science 299, 1743-1747]. The steady-state actin-activated ATPase activity of NMIIB S1 was decreased approximately 90% at 40 microM actin in the presence of blebbistatin. Stopped-flow techniques were employed to elucidate the effect of blebbistatin on the various steps of the NMIIB S1 cross-bridge cycle. Blebbistatin did not affect ATP binding and hydrolysis. Binding to actin in the presence of ADP (0.57 +/-0.08 microM(-1) s(-1)) was reduced slightly in the presence of blebbistatin (0.38 +/- 0.03 microM(-1) s(-1)), while mantADP dissociation from acto-NMIIB S1 was reduced (approximately 30%). P(i) release was blocked in the presence of blebbistatin. Accordingly, the apparent affinity of NMIIB S1 for actin in the presence of ATP was greatly reduced. Based on the above data, we surmise that blebbistatin inhibits the ATPase activity of NMIIB S1 primarily by blocking entry into the strong binding state; secondarily, it reduces the rate of ADP release. These effects are likely mediated by binding of blebbistatin within the myosin cleft that progressively closes in forming the acto-myosin rigor state.     

Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V(max) of the actin-activated Mg2+-ATPase activity, possibly through P(i) release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b1IQ, was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b1IQ; rate of ADP release from actin-Myo1b1IQ; and the affinity of actin for Myo1b1IQ and Myo1b1IQ.ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, gamma-P(i)-, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.     

Structural dissection of ATP turnover in the prototypical GHL ATPase TopoVI

GHL proteins are functionally diverse enzymes defined by the presence of a conserved ATPase domain that self-associates to trap substrate upon nucleotide binding. The structural states adopted by these enzymes during nucleotide hydrolysis and product release, and their consequences for enzyme catalysis, have remained unclear. Here, we have determined a complete structural map of the ATP turnover cycle for topoVI-B, the ATPase subunit of the archaeal GHL enzyme topoisomerase VI. With this ensemble of structures, we show that significant conformational changes in the subunit occur first upon ATP binding, and subsequently upon release of hydrolyzed P(i). Together, these data provide a structural framework for understanding the role of ATP hydrolysis in the type II topoisomerase reaction. Our results also suggest that the GHL ATPase module is a molecular switch in which ATP hydrolysis serves as a prerequisite but not a driving force for substrate-dependent structural transitions in the enzyme.     

Structural basis for actin assembly, activation of ATP hydrolysis, and delayed phosphate release

Assembled actin filaments support cellular signaling, intracellular trafficking, and cytokinesis. ATP hydrolysis triggered by actin assembly provides the structural cues for filament turnover in vivo. Here, we present the cryo-electron microscopic (cryo-EM) structure of filamentous actin (F-actin) in the presence of phosphate, with the visualization of some α-helical backbones and large side chains. A complete atomic model based on the EM map identified intermolecular interactions mediated by bound magnesium and phosphate ions. Comparison of the F-actin model with G-actin monomer crystal structures reveals a critical role for bending of the conserved proline-rich loop in triggering phosphate release following ATP hydrolysis. Crystal structures of G-actin show that mutations in this loop trap the catalytic site in two intermediate states of the ATPase cycle. The combined structural information allows us to propose a detailed molecular mechanism for the biochemical events, including actin polymerization and ATPase activation, critical for actin filament dynamics.     

A structural pathway for activation of the kinesin motor ATPase

Molecular motors move along actin or microtubules by rapidly hydrolyzing ATP and undergoing changes in filament-binding affinity with steps of the nucleotide hydrolysis cycle. It is generally accepted that motor binding to its filament greatly increases the rate of ATP hydrolysis, but the structural changes in the motor associated with ATPase activation are not known. To identify the conformational changes underlying motor movement on its filament, we solved the crystal structures of three kinesin mutants that decouple nucleotide and microtubule binding by the motor, and block microtubule-activated, but not basal, ATPase activity. Conformational changes in the structures include a disordered loop and helices in the switch I region and a visible switch II loop, which is disordered in wild-type structures. Switch I moved closer to the bound nucleotide in two mutant structures, perturbing water-mediated interactions with the Mg2+. This could weaken Mg2+ binding and accelerate ADP release to activate the motor ATPASE: The structural changes we observe define a signaling pathway within the motor for ATPase activation that is likely to be essential for motor movement on microtubules.     

Nucleotide effects on the structure and dynamics of actin

Adenosine 5'-triphosphate or ATP is the primary energy source within the cell, releasing its energy via hydrolysis into adenosine 5'-diphosphate or ADP. Actin is an important ATPase involved in many aspects of cellular function, and the binding and hydrolysis of ATP regulates its polymerization into actin filaments as well as its interaction with a host of actin-associated proteins. Here we study the dynamics of monomeric actin in ATP, ADP-Pi, and ADP states via molecular dynamics simulations. As observed in some crystal structures we see that the DNase-I loop is an alpha-helix in the ADP state but forms an unstructured coil domain in the ADP-Pi and ATP states. We also find that this secondary structure change is reversible, and by mimicking nucleotide exchange we can observe the transition between the helical and coil states. Apart from the DNase-I loop, we also see several key structural differences in the nucleotide binding cleft as well as in the hydrophobic cleft between subdomains 1 and 3 where WH2-containing proteins have been shown to interact. These differences provide a structural basis for understanding the observed differences between the various nucleotide states of actin and provide some insight into how ATP regulates the interaction of actin with itself and other proteins.     

Cooperative turning on of myosin subfragment 1 adenosinetriphosphatase activity by the troponin-tropomyosin-actin complex

In the field of muscle regulation, there is still controversy as to whether Ca2+, alone, is able to shift muscle from the relaxed to the fully active state or whether cross-bridge binding also contributes to turning on muscle contraction. Our previous studies on the binding of myosin subfragment 1 (S-1) to the troponin-tropomyosin-actin complex (regulated actin) in the absence of ATP suggested that, even in Ca2+, the binding of rigor cross-bridges is necessary to turn on regulated actin fully. In the present study, we demonstrate that this is also the case for the turning on of the acto.S-1 ATPase activity. By itself, Ca2+ does not fully turn on the acto.S-1 ATPase activity; at low actin concentration, there is almost a 10-fold increase in ATPase activity when the regulated actin is fully turned on by the binding of rigor cross-bridges in the presence of Ca2+. This large increase in ATPase activity does not occur because the binding of S-1.ATP to actin is increased; the binding of S-1.ATP is almost the same to maximally turned-off and maximally turned-on regulated actin. The increase in ATPase activity occurs because of a marked increase in the rate of Pi release so that when the regulated actin is fully turned on, Pi release becomes so rapid that the rate-limiting step precedes the Pi release step. These results suggest that, while Ca2+, alone, does not fully turn on the regulated actin filament in solution, the binding of rigor cross-bridges can turn it on fully. If force-producing cross-bridges play the same role in vivo as rigor cross-bridges in vitro, there may be a synergistic effect of Ca2+ and cross-bridge binding in turning on muscle contraction which could greatly sharpen the response of the muscle fiber to Ca2+.     

Mechanism of regulation of Acanthamoeba myosin-IC by heavy-chain phosphorylation

The ATPase activity of myosin-Is from lower eukaryotes is activated by phosphorylation by the p21-activated kinase family at the TEDS site on an actin-binding surface-loop. This actin-binding loop is the site of a cardiac myosin-II mutation responsible for some forms of familial hypertrophic cardiomyopathy. To determine the mechanism of myosin-I regulation by heavy-chain phosphorylation (HCP) and to better understand the importance of this loop in the function of all myosin isoforms, we performed a kinetic investigation of the regulatory mechanism of the Acanthamoeba myosin-IC motor domain (MIC(IQ)). Phosphorylated and dephosphorylated MIC(IQ) show actin-activated ATPase activity; however, HCP increases the ATPase activity >20-fold. HCP does not greatly affect the rate of phosphate release from MIC in the absence of actin, as determined by single turnover experiments. Additionally, HCP does not significantly affect the affinity of myosin for actin in the absence or presence of ATP, the rate of ATP-induced dissociation of actoMIC(IQ), the affinity of ADP, or the rate of ADP release. Sequential-mix single-turnover experiments show that HCP regulates the rate of phosphate release from actin-bound MIC(IQ). We propose that the TEDS-containing actin-binding loop plays a direct role in regulating phosphate release and the force-generating (A-to-R) transition of myosin-IC.     

Phosphate enhances myosin-powered actin filament velocity under acidic conditions in a motility assay

Elevated levels of inorganic phosphate (P(i)) are believed to inhibit muscular force by reversing myosin's force-generating step. These same levels of P(i) can also affect muscle velocity, but the molecular basis underlying these effects remains unclear. We directly examined the effect of P(i) (30 mM) on skeletal muscle myosin's ability to translocate actin (V(actin)) in an in vitro motility assay. Manipulation of the pH enabled us to probe rebinding of P(i) to myosin's ADP-bound state, while changing the ATP concentration probed rebinding to the rigor state. Surprisingly, the addition of P(i) significantly increased V(actin) at both pH 6.8 and 6.5, causing a doubling of V(actin) at pH 6.5. To probe the mechanisms underlying this increase in speed, we repeated these experiments while varying the ATP concentration. At pH 7.4, the effects of P(i) were highly ATP dependent, with P(i) slowing V(actin) at low ATP (<500 μM), but with a minor increase at 2 mM ATP. The P(i)-induced slowing of V(actin), evident at low ATP (pH 7.4), was minimized at pH 6.8 and completely reversed at pH 6.5. These data were accurately fit with a simple detachment-limited kinetic model of motility that incorporated a P(i)-induced prolongation of the rigor state, which accounted for the slowing of V(actin) at low ATP, and a P(i)-induced detachment from a strongly bound post-power-stroke state, which accounted for the increase in V(actin) at high ATP. These findings suggest that P(i) differentially affects myosin function: enhancing velocity, if it rebinds to the ADP-bound state, while slowing velocity, if it binds to the rigor state.     

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.     

The rate-limiting step in the actomyosin adenosinetriphosphatase cycle

We have previously shown that myosin does not have to detach from actin during each cycle of ATP hydrolysis. In the present study, using the A-1 isoenzyme of myosin subfragment 1, we have investigated the nature of the rate-limiting steps in the ATPase cycle. Our results show that, at 15 degrees C, at very low ionic strength, KATPase determined from the double-reciprocal plot of ATPase activity vs. actin concentration is more than 6-fold stronger than KBINDING determined by directly measuring the binding of A-1 myosin subfragment 1 to actin during steady-state ATP hydrolysis. Computer modeling shows that this large difference between KATPase and KBINDING is not compatible with Pi release being the rate-limiting step in the ATPase cycle. If Pi release is not rate limiting, it is possible that the ATP hydrolysis step, itself, is rate limiting. However, this predicts that, at high actin concentration, the value of the initial Pi burst should be close to zero. Therefore, we measured the magnitude of the initial Pi burst in the presence of actin, using both direct measurement and measurement of relative fluorescence magnitude. Our results suggest that the magnitude of the initial Pi burst in the presence of actin is considerably higher than would be expected if the ATP hydrolysis step were the rate-limiting step in the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human myosin III is a motor having an extremely high affinity for actin

Myosin IIIA is expressed in photoreceptor cells and thought to play a critical role in phototransduction processes, yet its function on a molecular basis is largely unknown. Here we clarified the kinetic mechanism of the ATPase cycle of human myosin IIIA. The steady-state ATPase activity was markedly activated approximately 10-fold with very low actin concentration. The rate of ADP off from actomyosin IIIA was 10 times greater than the overall cycling rate, thus not a rate-determining step. The rate constant of the ATP hydrolysis step of the actin-dissociated form was very slow, but the rate was markedly accelerated by actin binding. The dissociation constant of the ATP-bound form of myosin IIIA from actin is submicromolar, which agrees well with the low K(actin). These results indicate that ATP hydrolysis predominantly takes place in the actin-bound form for actomyosin IIIA ATPase reaction. The obtained K(actin) was much lower than the previously reported one, and we found that the autophosphorylation of myosin IIIA dramatically increased the K(actin), whereas the V(max) was unchanged. Our kinetic model indicates that both the actin-attached hydrolysis and the P(i) release steps determine the overall cycle rate of the dephosphorylated form. Although the stable steady-state intermediates of actomyosin IIIA ATPase reaction are not typical strong actin-binding intermediates, the affinity of the stable intermediates for actin is much higher than conventional weak actin binding forms. The present results suggest that myosin IIIA can spend a majority of its ATP hydrolysis cycling time on actin.     

Mechanism of inhibition of skeletal muscle actomyosin by N-benzyl-p-toluenesulfonamide

N-Benzyl-p-toluenesulfonamide (BTS) is a small organic molecule that specifically inhibits the contraction of fast skeletal muscle fibers. To determine the mechanism of inhibition by BTS, we performed a kinetic analysis of its effects on the elementary steps of the actomyosin subfragment-1 ATPase cycle. BTS decreases the steady-state acto-S1 ATPase rate approximately 10-fold and increases the actin concentration for half-maximal activation. BTS primarily affects three of the elementary steps of the reaction pathway. It decreases the rate of P(i) release >20-fold in the absence of actin and >100-fold in the presence of actin. It decreases the rate of S1.ADP dissociation from 3.9 to 0.8 s(-)(1) while decreasing the S1.ADP dissociation constant from 2.3 to 0.8 microM. BTS weakens the apparent affinity of S1.ADP for actin, increasing the K(d) from 7.0 to 29.5 microM. ATP binding to S1, hydrolysis, and the affinity of nucleotide-free S1 for actin are unaffected by BTS. Kinetic modeling indicates that the binding of BTS to myosin depends on actin association/dissociation and on nucleotide state. Our results suggest that the reduction of the acto-S1 ATPase rate is due to the inhibition of P(i) release, and the suppression of tension is due to inhibition of P(i) release in conjunction with the decreased apparent affinity of S1.ADP.P(i) and S1.ADP for actin.     

Temperature dependence of nucleotide association and kinetic characterization of myo1b

Myo1b is a widely expressed myosin-I isoform that concentrates on endosomal and ruffling membranes and is thought to play roles in membrane trafficking and dynamics. It is one of the best characterized myosin-I isoforms and appears to have unique biochemical properties tuned for tension sensing or tension maintenance. We determined the key biochemical rate constants that define the actomyo1b ATPase cycle at 37 degrees C and measured the temperature dependence of ATP binding, ADP release, and the transition from a nucleotide-inaccessible state to a nucleotide-accessible state (k(alpha)). The rate of ATP binding is highly temperature sensitive, with an Arrhenius activation energy 2-3-fold greater than other characterized myosins (e.g., myosin-II and myosin-V). ATP hydrolysis is fast, and phosphate release is slow and rate limiting with an actin dependence that is nearly identical to the steady-state ATPase parameters (Vmax and K(ATPase)). ADP release is not as temperature dependent as ATP binding. The rates and temperature dependence of ADP release are similar to k(alpha) suggesting that a similar structural change is responsible for both transitions. We calculate a duty ratio of 0.08 based on the biochemical kinetics. However, this duty ratio is likely to be highly sensitive to strain.     

The phosphatase activity of the isolated H4-H5 loop of Na+/K+ ATPase resides outside its ATP binding site.

The structural stability of the large cytoplasmic domain (H(4)-H(5) loop) of mouse alpha(1) subunit of Na(+)/K(+) ATPase (L354-I777), the number and the location of its binding sites for 2'-3'-O-(trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) and p-nitrophenylphosphate (pNPP) were investigated. C- and N-terminal shortening revealed that neither part of the phosphorylation (P)-domain are necessary for TNP-ATP binding. There is no indication of a second ATP site on the P-domain of the isolated loop, even though others reported previously of its existence by TNP-N(3)ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)-anisotropy measurements reveal a considerable stability of the nucleotide (N)-domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N-domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in the alpha(1-4) isoforms of Na(+)/K(+) ATPase, whereas N398 is only conserved in the vertebrates' alpha(1) subunit. The phosphatase activity of the isolated H(4)-H(5) loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na(+)/K(+) ATPase.     

Dual regulation of mammalian myosin VI motor function

Myosin VI is expressed in a variety of cell types and is thought to play a role in membrane trafficking and endocytosis, yet its motor function and regulation are not understood. The present study clarified mammalian myosin VI motor function and regulation at a molecular level. Myosin VI ATPase activity was highly activated by actin with K(actin) of 9 microm. A predominant amount of myosin VI bound to actin in the presence of ATP unlike conventional myosins. K(ATP) was much higher than those of other known myosins, suggesting that myosin VI has a weak affinity or slow binding for ATP. On the other hand, ADP markedly inhibited the actin-activated ATPase activity, suggesting a high affinity for ADP. These results suggested that myosin VI is predominantly in a strong actin binding state during the ATPase cycle. p21-activated kinase 3 phosphorylated myosin VI, and the site was identified as Thr(406). The phosphorylation of myosin VI significantly facilitated the actin-translocating activity of myosin VI. On the other hand, Ca(2+) diminished the actin-translocating activity of myosin VI although the actin-activated ATPase activity was not affected by Ca(2+). Calmodulin was not dissociated from the heavy chain at high Ca(2+), suggesting that a conformational change of calmodulin upon Ca(2+) binding, but not its physical dissociation, determines the inhibition of the motility activity. The present results revealed the dual regulation of myosin VI by phosphorylation and Ca(2+) binding to calmodulin light chain.