Phosphorylation of a single head of smooth muscle myosin activates the whole molecule

Regulatory light chain (RLC) phosphorylation activates smooth and non-muscle myosin II, but it has not been established if phosphorylation of one head turns on the whole molecule. Baculovirus expression and affinity chromatography were used to isolate heavy meromyosin (HMM) containing one phosphorylated and one dephosphorylated RLC (1-P HMM). Motility and steady-state ATPase assays indicated that 1-P HMM is nearly as active as HMM with two phosphorylated heads (2-P HMM). Single-turnover experiments further showed that both the dephosphorylated and phosphorylated heads of 1-P HMM can be activated by actin. Singly phosphorylated full-length myosin was also an active species with two cycling heads. Our results suggest that phosphorylation of one RLC abolishes the asymmetric inhibited state formed by dephosphorylated myosin [Liu, J., et al. (2003) J. Mol. Biol. 329, 963-972], allowing activation of both the phosphorylated and dephosphorylated heads. These findings help explain how smooth muscles are able to generate high levels of stress with low phosphorylation levels.     

Role of actin in the myosin-linked Ca(2+)-regulation of ATP-dependent interaction between actin and myosin of a lower eukaryote, Physarum polycephalum.

Actin-activated ATPase activity of myosin from Physarum polycephalum decreases when it binds Ca2+ and increases when it loses Ca2+. This Ca-inhibition is observed with phosphorylated myosin [Kohama, K. (1990) Trend, Pharmacol. Sci. 11, 433-435]. The activity of dephosphorylated myosin remained at a low level both in the presence and absence of Ca2+, although Ca(2+)-binding ability was much the same as that of the phosphorylated myosin. The effect of phosphorylation has been studied at a conventional actin concentration, which is comparable with that of myosin by weight. When the concentration of actin was increased by 10 times, the dephosphorylated myosin became actin-activatable in the absence of Ca2+, and Ca-inhibition was recovered. As actin exists quite abundantly in non-muscle cells of Physarum, myosin phosphorylation plays virtually no role in regulating actin-myosin-ATP interaction in vivo. Physiologically the interaction may be regulated by Ca2+ by binding to and subsequent release from myosin. Latex beads coated by either phosphorylated or dephosphorylated myosin moved ATP-dependently on the actin cables of Characeae cells to the same extent in the absence of Ca2+, but the movement was abolished by increasing Ca2+. When the interaction was examined by monitoring the movement of actin filaments on myosin fixed on a coverslip, the movement and Ca-inhibition of the movement were detected with phosphorylated, not dephosphorylated, myosin [Okagaki, T., Higashi-Fujime, S., & Kohama, K. (1989) J. Biochem. 106, 955-957].(ABSTRACT TRUNCATED AT 250 WORDS)     

Filamentous smooth muscle myosin is regulated by phosphorylation

The enzymatic activity of filamentous dephosphorylated smooth muscle myosin has been difficult to determine because the polymer disassembles to the folded conformation in the presence of MgATP. Monoclonal antirod antibodies were used here to "fix" dephosphorylated myosin in the filamentous state. The steady-state actin-activated ATPase of phosphorylated filaments was 30-100-fold higher than that of antibody- stabilized dephosphorylated filaments, suggesting that phosphorylation can activate ATPase activity independent of changes in assembly. The degree of regulation may exceed 100-fold, because steady-state measurements slightly overestimate the rate of product release from dephosphorylated filaments. Single-turnover experiments in the absence of actin showed that although dephosphorylated folded myosin released products at the low rate of 0.0005 s-1 (Cross, R. A., K. E. Cross, A. Sobieszek. 1986. EMBO [Eur. Mol. Biol. Organ.] J. 5:2637-2641) the rate of product release from dephosphorylated filaments was only 3-12-fold higher, depending on the ionic strength. The addition of actin did not increase this rate to any appreciable extent. Dephosphorylated filaments and dephosphorylated heavy meromyosin (Sellers, J. R. 1985. J. Biol. Chem. 260:15815-15819) thus have similar low rates of phosphate release both in the presence and absence of actin. These results show that light chain phosphorylation alone, without invoking other mechanisms, is an effective switch for regulating the activity of smooth muscle myosin filaments.     

Myosin light chain phosphorylation inhibits muscle fiber shortening velocity in the presence of vanadate

We have shown that myosin light chain phosphorylation inhibits fiber shortening velocity at high temperatures, 30 degrees C, in the presence of the phosphate analog vanadate. Vanadate inhibits tension by reversing the transition to force-generating states, thus mimicking a prepower stroke state. We have previously shown that at low temperatures vanadate also inhibits velocity, but at high temperatures it does not, with an abrupt transition in inhibition occurring near 25 degrees C (E. Pate, G. Wilson, M. Bhimani, and R. Cooke. Biophys J 66: 1554-1562, 1994). Here we show that for fibers activated in the presence of 0.5 mM vanadate, at 30 degrees C, shortening velocity is not inhibited in dephosphorylated fibers but is inhibited by 37 +/- 10% in fibers with phosphorylated myosin light chains. There is no effect of phosphorylation on fiber velocity in the presence of vanadate at 10 degrees C. The K(m) for ATP, defined by the maximum velocity of fibers partially inhibited by vanadate at 30 degrees C, is 20 +/- 4 microM for phosphorylated fibers and 192 +/- 40 microM for dephosphorylated fibers, showing that phosphorylation also affects the binding of ATP. Fiber stiffness is not affected by phosphorylation. Inhibition of velocity by phosphorylation at 30 degrees C depends on the phosphate analog, with approximately 12% inhibition in fibers activated in the presence of 5 mM BeF(3) and no inhibition in the presence of 0.25 mM AlF(4). Our results show that myosin phosphorylation can inhibit shortening velocity in fibers with large populations of myosin heads trapped in prepower stroke states, such as occurs during muscle fatigue.     

Phosphorylation of smooth muscle myosin heads regulates the head-induced movement of tropomyosin

It has been shown that skeletal and smooth muscle myosin heads binding to actin results in the movement of smooth muscle tropomyosin, as revealed by a change in fluorescence resonance energy transfer between a fluorescence donor on tropomyosin and an acceptor on actin (Graceffa, P. (1999) Biochemistry 38, 11984-11992). In this work, tropomyosin movement was similarly monitored as a function of unphosphorylated and phosphorylated smooth muscle myosin double-headed fragment smHMM. In the absence of nucleotide and at low myosin head/actin ratios, only phosphorylated heads induced a change in energy transfer. In the presence of ADP, the effect of head phosphorylation was even more dramatic, in that at all levels of myosin head/actin, phosphorylation was necessary to affect energy transfer. It is proposed that the regulation of tropomyosin position on actin by phosphorylation of myosin heads plays a key role in the regulation of smooth muscle contraction. In contrast, actin-bound caldesmon was not moved by myosin heads at low head/actin ratios, as uncovered by fluorescence resonance energy transfer and disulfide cross-linking between caldesmon and actin. At higher head concentration caldesmon was dissociated from actin, consistent with the multiple binding model for the binding of caldesmon and myosin heads to actin (Chen, Y., and Chalovich, J. M. (1992) Biophys. J. 63, 1063-1070).     

Comparison of Orientation and Rotational Motion of Skeletal Muscle Cross-bridges Containing Phosphorylated and Dephosphorylated Myosin Regulatory Light Chain

Calcium binding to thin filaments is a major element controlling active force generation in striated muscles. Recent evidence suggests that processes other than Ca²⁺ binding, such as phosphorylation of myosin regulatory light chain (RLC) also controls contraction of vertebrate striated muscle (Cooke, R. (2011) Biophys. Rev. 3, 33–45). Electron paramagnetic resonance (EPR) studies using nucleotide analog spin label probes showed that dephosphorylated myosin heads are highly ordered in the relaxed fibers and have very low ATPase activity. This ordered structure of myosin cross-bridges disappears with the phosphorylation of RLC (Stewart, M. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 430–435). The slower ATPase activity in the dephosporylated moiety has been defined as a new super-relaxed state (SRX). It can be observed in both skeletal and cardiac muscle fibers (Hooijman, P., Stewart, M. A., and Cooke, R. (2011) Biophys. J. 100, 1969–1976). Given the importance of the finding that suggests a novel pathway of regulation of skeletal muscle, we aim to examine the effects of phosphorylation on cross-bridge orientation and rotational motion. We find that: (i) relaxed cross-bridges, but not active ones, are statistically better ordered in muscle where the RLC is dephosporylated compared with phosphorylated RLC; (ii) relaxed phosphorylated and dephosphorylated cross-bridges rotate equally slowly; and (iii) active phosphorylated cross-bridges rotate considerably faster than dephosphorylated ones during isometric contraction but the duty cycle remained the same, suggesting that both phosphorylated and dephosphorylated muscles develop the same isometric tension at full Ca²⁺ saturation. A simple theory was developed to account for this fact.     

Calcium ions modulate regulation of smooth muscle contraction mediated by phosphorylation of myosin regulatory light chains

The effect of calcium ions on conformational changes of F-actin initiated by decoration of thin filaments with phosphorylated and dephosphorylated heavy meromyosin from smooth muscles was studied by fluorescence polarization spectroscopy. It is shown that heavy meromyosin with phosphorylated regulatory light chains (pHMM) promotes structural changes of F-actin which are typical for the "strong" binding of actin to the myosin heads. Heavy meromyosin with dephosphorylated regulatory light chains (dpHMM) causes conformational changes of F-actin which are typical for the "weak" binding of actin to the myosin heads. The presence of calcium enhances the pHMM effect and attenuates the dpHMM effect. We propose that a Ca2+-dependent mechanism exists in smooth muscles which modulates the regulation of actin--myosin interaction occurring via phosphorylation of myosin regulatory light chains.     

Effects of Ca2+ and Mg2+ on the actomyosin adenosine-5'-triphosphatase of stably phosphorylated gizzard myosin

There are conflicting reports on the effect of Ca2+ on actin activation of myosin adenosine-triphosphatase (ATPase) once the light chain is fully phosphorylated by a calcium calmodulin dependent kinase. Using thiophosphorylated gizzard myosin, Sherry et al. [Sherry, J. M. F., Gorecka, A., Aksoy, M. O., Dabrowska, R., & Hartshorne, D. J. (1978) Biochemistry 17, 4417-4418] observed that the actin activation of ATPase was not inhibited by the removal of Ca2+. Hence, it was suggested that the regulation of actomyosin ATPase activity of gizzard myosin by calcium occurs only via phosphorylation. In the present study, phosphorylated and thiophosphorylated myosins were prepared free of kinase and phosphatase activity; hence, the ATPase activity could be measured at various concentrations of Ca2+ and Mg2+ without affecting the level of phosphorylation. The ATPase activity of myosin was activated either by skeletal muscle or by gizzard actin at various concentrations of Mg2+ and either at pCa 5 or at pCa 8. The activation was sensitive to Ca2+ at low Mg2+ concentrations with both actins. Tropomyosin potentiated the actin-activated ATPase activity at all Mg2+ and Ca2+ concentrations. The calcium sensitivity of phosphorylated and thiophosphorylated myosin reconstituted with actin and tropomyosin was most pronounced at a free Mg2+ concentration of about 3 mM. The binding of 125I-tropomyosin to actin showed that the calcium sensitivity of ATPase observed at low Mg2+ concentration is not due to a calcium-mediated binding of tropomyosin to F-actin. The actin activation of both myosins was insensitive to Ca2+ when the Mg2+ concentration was increased above 5 mM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Asymmetric photocross-linking of singly phosphorylated smooth muscle heavy meromyosin

Although activities of smooth muscle myosin are regulated by phosphorylation, the molecular mechanisms of regulation have not been fully established. Phosphorylation of both heads of myosin is known to activate ATPase and motor activities, but the effects of phosphorylation of only one of the heads have not been established. Such information on singly phosphorylated myosin can serve to elucidate the molecular mechanism of the phosphorylation-dependent regulation. To understand the structural properties of the singly phosphorylated state, we prepared singly phosphorylated heavy meromyosin (HMM) containing a photoreactive benzophenone-labeled RLC and examined its photocross-linking reactivity. The two heads in the singly phosphorylated HMM showed different reactivities. The dephosphorylated RLC in the singly phosphorylated HMM was cross-linked to a heavy chain, like that in the dephosphorylated HMM, whereas the phosphorylated RLC did not react, like that in the fully phosphorylated HMM. These results indicate that the two heads of the singly phosphorylated HMM have an asymmetric structure, suggesting that phosphorylation of one head can to some extent activate smooth muscle HMM.     

Proteolysis and actin-binding properties of 10S and 6S smooth muscle myosin: identification of a site protected from proteolysis in the 10S conformation and by the binding of actin

It was shown previously [Ikebe, M., & Hartshorne, D. J. (1985) Biochemistry 24, 2380-2387] that the conformation of gizzard myosin, either 10S or 6S, influences proteolysis of myosin at two regions designated sites A and B. The studies reported here are focused on site A, which is located approximately 68,000 daltons from the N-terminus of the myosin heavy chain. With papain, Staphylococcus aureus protease, and actinidin, it is shown that the formation of 10S myosin reduces proteolysis at site A. Binding of actin to 6S myosin also hinders cleavage at site A for each of these proteases. To investigate binding of actin to 6S and 10S myosins, adenosine 5'-(beta,gamma-imidotriphosphate) (AMPPNP) is used as a substitute for ATP. In the presence of AMPPNP, it is shown that the 6S to 10S transition occurs and that 10S myosin binds actin with lower affinity than 6S myosin. For 6S myosin at high salt (0.35 M KCl) the dissociation constant of actin from the actin-myosin-nucleotide complex (K3) is approximately the same for phosphorylated (1.9 mol of P/mol of myosin) and dephosphorylated myosin, i.e., 1.3-2.4 microM, respectively. At lower ionic strength (0.17 M KCl) K3 for dephosphorylated myosin (10S myosin) is 42 microM and K3 for phosphorylated myosin (6S myosin) is 0.3 microM. These data show that the conformation of myosin influences the actin-myosin interaction. The constant (K4) for the dissociation of nucleotide from the actin-myosin-nucleotide complex varies slightly (in the range of 0.2-1.3 mM), but there is no marked change as a result of either a conformational change or phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The apparently negatively cooperative phosphorylation of smooth muscle myosin at low ionic strength is related to its filamentous state

The correlation curve between phosphorylation and MgATPase activity suggests that the 20,000-dalton light chain of both heads of a smooth muscle myosin or heavy meromyosin (HMM) molecule must be phosphorylated before the MgATPase activity of either head can be activated by actin. The two heads of HMM appear to be phosphorylated randomly at equal rates, while those of myosin are phosphorylated in a negatively cooperative manner (Persechini, A., and Hartshorne, D.J. (1981) Science, 213, 1383-1385; Ikebe, M., Ogihara, S., and Tonomura, Y. (1982) J. Biochem. 91, 1809-1812). We have investigated the cause of this difference between HMM and myosin. We find that if myosin is first phosphorylated at high ionic strength (0.6 M KCl), where it is monomeric, and then assayed for MgATPase activity (in 0.05 M KCl), the data support a model where the two heads are phosphorylated randomly with equal rates (i.e. similarly to HMM). The correlation curves between MgATPase activity and dephosphorylation of fully phosphorylated myosin, both in a filamentous and monomeric state, are also best explained by a model where dephosphorylation of one head is sufficient to deactivate the entire molecule. With monomeric myosin, the dephosphorylation appears to occur randomly with equal rates, whereas with filamentous myosin the dephosphorylation appears to be negatively cooperative. The correlation between dephosphorylation of HMM and its MgATPase activity is more complex and is consistent with a positively cooperative dephosphorylation. Direct analyses of the time courses of phosphorylation of HMM and monomeric myosin show that a single exponential is sufficient to fit the data through greater than 90% of the reaction. However, when phosphorylation is carried out at low ionic strength (0.02 M KCl), where myosin is present as filaments, the time course consists of two exponential functions where the rate constant for the phosphorylation of one myosin head is 6-10 times greater than that for the other head which is located on the same molecule. This suggests that when myosin is polymerized into filaments the two previously indistinguishable heads either become nonequivalent or are subject to head-head interactions leading to a negatively cooperative phosphorylation reaction.     

Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation

Smooth muscle heavy meromyosin (HMM) is phosphorylated by the Ca2+-activated phospholipid-dependent protein kinase, i.e. protein kinase C, at three sites on each 20,000-dalton light chain. Phosphorylation of three sites also is observed with isolated 20,000-dalton light chain and HMM subfragment 1. The phosphorylation sites are serine 1, serine 2, and threonine 9. Threonine is phosphorylated most rapidly followed by either serine 1 or 2. Phosphorylation of the third site occurs only on prolonged incubation. Phosphorylation is a random process. HMM phosphorylated at two sites per light chain by protein kinase C can be dephosphorylated, as shown using two phosphatase preparations. Increasing levels of phosphorylation of HMM by protein kinase C causes a progressive inhibition of the subsequent rate of phosphorylation of serine 19 by myosin light chain kinase and causes a progressive inhibition of actin-activated ATPase activity of HMM, prephosphorylated by myosin light chain kinase. Inhibition of ATPase activity is due to a decreased affinity of HMM for actin rather than a change in Vmax. Previous results with HMM and protein kinase C (Nishikawa, M., Sellers, J. R., Adelstein, R. S., and Hidaka, H. (1984) J. Biol. Chem. 259, 8808-8814) examined effects induced by phosphorylation of the threonine residues. Our results confirm these and consider also the influence of higher levels of phosphorylation by protein kinase C.     

Assembly of smooth muscle myosin minifilaments: effects of phosphorylation and nucleotide binding

Small bipolar filaments, or "minifilaments," are formed when smooth muscle myosin is dialyzed against low ionic strength pyrophosphate or citrate/Tris buffers. Unlike synthetic filaments formed at approximately physiological ionic conditions, minifilaments are homogeneous as indicated by their hypersharp boundary during sedimentation velocity. Electron microscopy and hydrodynamic techniques were used to show that 20-22S smooth muscle myosin minifilaments are 380 nm long and composed of 12-14 molecules. By varying solvents, a continuum of different size polymers in the range of 15-30S could be obtained. Skeletal muscle myosin, in contrast, preferentially forms a stable 32S minifilament (Reisler, E., P. Cheung, and N. Borochov. 1986. Biophys. J. 49:335-342), suggesting underlying differences in the assembly properties of the two myosins. Addition of salt to the smooth muscle myosin minifilaments caused unidirectional growth into a longer "side-polar" type of filament, whereas bipolar filaments were consistently formed by skeletal muscle myosin. As with synthetic filaments, addition of 1 mM MgATP caused dephosphorylated minifilaments to dissociate to a mixture of folded monomers and dimers. Phosphorylation of the regulatory light chain prevented disassembly by nucleotide, even though it had no detectable effect on the structure of the minifilament. These results suggest that differences in filament stability as a result of phosphorylation are due largely to conformational changes occurring in the myosin head, and are not due to differences in filament packing.     

Random phosphorylation of the two heads of thymus myosin and the independent stimulation of their actin-activated ATPases

Like other vertebrate nonmuscle myosins, thymus myosin contains two phosphorylatable light chains. Phosphorylation of these light chains regulates the actin-activated ATPase of this myosin. The time courses for the phosphorylation of both monomeric and filamentous thymus myosin by gizzard myosin light chain kinase fitted single exponentials to greater than 85% phosphorylation. This indicates that the two heads of thymus myosin are phosphorylated at the same rate and suggests that these phosphorylations are random processes. The actin-activated ATPases of thymus myosins with different levels of light chain phosphorylation were also determined. A linear relationship was obtained between the extent of light chain phosphorylation and stimulation of the actin-activated ATPase. Since thymus myosin appears to be phosphorylated randomly, this linear relationship indicates that phosphorylation of one head of thymus myosin stimulates the actin-activated ATPase of that head independently of the phosphorylation of the second head. The apparent random phosphorylation of thymus myosin light chains contrasts with the reported ordered phosphorylation of the light chains of filamentous smooth (gizzard) muscle myosin. Also, while the actin-activated ATPases of the two heads of thymus myosin are regulated independently, both heads of gizzard myosin must be phosphorylated before the ATPase of either head is activated by actin.     

Light chain phosphorylation regulates the movement of smooth muscle myosin on actin filaments

In smooth muscles there is no organized sarcomere structure wherein the relative movement of myosin filaments and actin filaments has been documented during contraction. Using the recently developed in vitro assay for myosin-coated bead movement (Sheetz, M.P., and J.A. Spudich, 1983, Nature (Lond.)., 303:31-35), we were able to quantitate the rate of movement of both phosphorylated and unphosphorylated smooth muscle myosin on ordered actin filaments derived from the giant alga, Nitella. We found that movement of turkey gizzard smooth muscle myosin on actin filaments depended upon the phosphorylation of the 20-kD myosin light chains. About 95% of the beads coated with phosphorylated myosin moved at velocities between 0.15 and 0.4 micron/s, depending upon the preparation. With unphosphorylated myosin, only 3% of the beads moved and then at a velocity of only approximately 0.01-0.04 micron/s. The effects of phosphorylation were fully reversible after dephosphorylation with a phosphatase prepared from smooth muscle. Analysis of the velocity of movement as a function of phosphorylation level indicated that phosphorylation of both heads of a myosin molecule was required for movement and that unphosphorylated myosin appears to decrease the rate of movement of phosphorylated myosin. Mixing of phosphorylated smooth muscle myosin with skeletal muscle myosin which moves at 2 microns/s resulted in a decreased rate of bead movement, suggesting that the more slowly cycling smooth muscle myosin is primarily determining the velocity of movement in such mixtures.     

Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments

In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAMP. To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KCl, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that approximately 80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KCl (less than 10 mM) the Dictyostelium myosin was also soluble at 100,000 g. But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCl, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 mol Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of approximately 0.43 micron. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of approximately 5 micrograms/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCl, magnesium, ATP, or H+ strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli.     

Effect of phosphorylation of myosin light chains on the interaction of myosin minifilaments with F-actin

The effect of phosphorylation of light chains-2 (LC2) of rabbit skeletal muscle myosin on the interaction of myosin minifilaments with F-actin as well as on the actin-stimulated Mg2+-ATPase of minifilaments was studied. It was shown that in the absence of KCl the degree of F-actin-induced stimulation of myosin minifilament Mg2+-ATPase with phosphorylated LC2 exceeds 2-4-fold that with unphosphorylated LC2. Phosphorylation of LC2 considerably increases the rate of actin-stimulated Mg2+-ATPase reaction of myosin minifilaments but exerts only a very weak influence on the affinity of minifilaments for F-actin. After addition of KCl the differences in the actin-stimulated Mg2+-ATPase activity disappear in a great degree; in the presence of 50 mM KCl they do not exceed 50%. It was assumed that the observed specific influence of LC2 phosphorylation on the kinetic parameters of actin-stimulated Mg2+-ATPase reaction of myosin minifilaments is due to unique properties of the minifilaments (e.g., their ability to ordered self-assembly as a result of interaction between the heads of myosin molecules) which reflect their structural peculiarities.     

Phosphorylated smooth muscle heavy meromyosin shows an open conformation linked to activation

Smooth muscle myosin and smooth muscle heavy meromyosin (smHMM) are activated by regulatory light chain phosphorylation, but the mechanism remains unclear. Dephosphorylated, inactive smHMM assumes a closed conformation with asymmetric intramolecular head-head interactions between motor domains. The "free head" can bind to actin, but the actin binding interface of the "blocked head" is involved in interactions with the free head. We report here a three-dimensional structure for phosphorylated, active smHMM obtained using electron crystallography of two-dimensional arrays. Head-head interactions of phosphorylated smHMM resemble those found in the dephosphorylated state but occur between different molecules, not within the same molecule. The light chain binding domain structure of phosphorylated smHMM differs markedly from that of the "blocked" head of dephosphorylated smHMM. We hypothesize that regulatory light chain phosphorylation opens the inhibited conformation primarily by its effect on the blocked head. Singly phosphorylated smHMM is not compatible with the closed conformation if the blocked head is phosphorylated. This concept has implications for the extent of myosin activation at low levels of phosphorylation in smooth muscle.     

Regulatory light chain influences alterations of myosin head induced by actin.

The effect of magnesium-for-calcium exchange and phosphorylation of regulatory light chain (LC2) on structural organization of rabbit skeletal myosin head was studied by limited tryptic digestion. In the presence of actin, exchange of magnesium bound to LC2 by calcium in dephosphorylated myosin accelerates the digestion of myosin and heavy meromyosin heavy chain and increases the accumulation of a 50 kDa fragment. This effect is significantly diminished in the case of phosphorylated myosin. Thus, both phosphorylation and cation exchange influences the effect of actin binding on the structural organization of myosin head.     

Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity

Purified rabbit skeletal muscle myosin is phosphorylated on one type of light-chain subunit (P-light chain) by calmodulin-dependent myosin light chain kinase and dephosphorylated by phosphoprotein phosphatase C. Analyses of the time courses of both phosphorylation and dephosphorylation of skeletal muscle myosin indicated that both reactions, involving at least 90% of the P-light chain, were kinetically homogeneous. These results suggest that phosphorylation and dephosphorylation of rabbit skeletal muscle myosin heads are simple random processes in contrast to the sequential phosphorylation mechanism proposed for myosin from gizzard smooth muscle. We also examined the effect of phosphorylation of rabbit skeletal muscle myosin on the actin-activated ATPase activity. We observed an apparent 2-fold decrease in the Km for actin, from about 6 microM to about 2.5 microM, with no significant effect on the Vmax (1.8s-1) in response to P-light-chain phosphorylation. There was no significant effect of phosphorylation on the ATPase activity of myosin alone (0.045 s-1). ATPase activation could be fully reversed by addition of phosphatase catalytic subunit. The relationship between the extents of P-light-chain phosphorylation and ATPase activation (at 3.5 microM actin and 0.6 microM myosin) was essentially linear. Thus, in contrast to results obtained with myosin from gizzard smooth muscle, these results suggest that cooperative interactions between the myosin heads do not play an important role in the activation process in skeletal muscle. Since the effect of P-light-chain phosphorylation is upon the Km for actin, it would appear to be associated with a significant activation of ATPase activity only at appropriate concentrations of actin and salt.(ABSTRACT TRUNCATED AT 250 WORDS)