The effect of phosphorylation of myosin light chains on the structural state of tropomyosin in thin filaments, decorated with heavy meromyosin

The structural state of tropomyosin (TM) modified by 5-(iodoacetamidoethyl)-aminonaphthalene-1-sulfonate (1.5-IAEDANS) upon F-actin decoration with myosin subfragment 1 (S1) and heavy meromyosin (HMM) in glycerinated myosin- and troponin-free muscle fibers was studied. HMM preparations contained native phosphorylated myosin light chains, while S1 preparations did not. The changes in the polarized fluorescence of 1.5-IAEDANS-TM during the F-actin interaction with S1 were independent of light chains phosphorylation and Ca2+ concentration, but were dependent on these factors during the F-actin interaction with HMM. The binding of myosin heads to F-actin is supposed to initiate conformational changes in TM which are accompanied by changes in the flexibility and molecular arrangement of TM. In the presence of light chains, the structural changes in TM depend on light chains phosphorylation and Ca2+ concentration. The conformational changes in TM seem to be responsible for the mechanisms of coupling of the myosin and tropomyosin modulation system during the actin-myosin interaction in skeletal muscles.     

Phosphorylation of light chains of myosin from rabbit skeletal muscles affects the type of conformation changes of F-actin induced by heavy meromyosin

The changes in F-actin conformation in myosin-free single ghost fiber induced by the binding of heavy meromyosin (HMM) with dephosphorylated or phosphorylated light chains-2 (LC2) have been studied by measuring intrinsic tryptophan polarized fluorescence of F-actin. It has been found that at low concentrations of Ca2+ (pCa greater than or equal to 8), the binding of HMM with dephosphorylated LC2 to F-actin in ghost fibres increases, whereas the binding of HMM with phosphorylated LC2 decreases the anisotropy of polarized tryptophan fluorescence. The effect is reversed at high concentrations of Ca2+ (pCa = 5). It has been assumed that this effect of myosin light chains phosphorylation may be due to its influence on the type of myosin head binding to F-actin.     

Regulation of Limulus skeletal muscle contraction

Skeletal muscle contraction of Limulus polyphemus, the horseshoe crab, seemed to be regulated in a dual manner, namely Ca2+ binding to the troponin complex as well phosphorylation of the myosin light chains (MLC) by a Ca2+/calmodulin-dependent myosin light chain kinase. We investigated muscle contraction in Limulus skinned fibers in the presence of Ca2+ and of Ca2+/calmodulin to find out which of the two mechanisms prevails in Limulus skeletal muscle contraction. Although skinned fibers revealed high basal MLC mono- and biphosphorylation levels (0.48 mol phosphate/mol 31 kDa MLC; 0.52 mol phosphate/mol 21 kDa MLC), the muscle fibers were fully relaxed at pCa 8. Upon C2+ or Ca2+/calmodulin activation, the fibers developed force (357+/-78.7 mN/mm2; 338+/-69.7 mN/mm2, respectively) while the MLC phosphorylation remained essentially unchanged. We conclude that Ca2+ activation is the dominant regulatory mechanism in Limulus skeletal muscle contraction.     

Phosphorylation of the regulatory light chains of myosin affects Ca2+ sensitivity of skeletal muscle contraction.

The role of phosphorylation of the myosin regulatory light chains (RLC) is well established in smooth muscle contraction, but in striated (skeletal and cardiac) muscle its role is still controversial. We have studied the effects of RLC phosphorylation in reconstituted myosin and in skinned skeletal muscle fibers where Ca2+ sensitivity and the kinetics of steady-state force development were measured. Skeletal muscle myosin reconstituted with phosphorylated RLC produced a much higher Ca2+ sensitivity of thin filament-regulated ATPase activity than nonphosphorylated RLC (change in -log of the Ca2+ concentration producing half-maximal activation = approximately 0.25). The same was true for the Ca2+ sensitivity of force in skinned skeletal muscle fibers, which increased on reconstitution of the fibers with the phosphorylated RLC. In addition, we have shown that the level of endogenous RLC phosphorylation is a crucial determinant of the Ca2+ sensitivity of force development. Studies of the effects of RLC phosphorylation on the kinetics of force activation with the caged Ca2+, DM-nitrophen, showed a slight increase in the rates of force development with low statistical significance. However, an increase from 69 to 84% of the initial steady-state force was observed when nonphosphorylated RLC-reconstituted fibers were subsequently phosphorylated with exogenous myosin light chain kinase. In conclusion, our results suggest that, although Ca2+ binding to the troponin-tropomyosin complex is the primary regulator of skeletal muscle contraction, RLC play an important modulatory role in this process.     

Fluorescence polarization study on Ca2+-sensitivity of conformational changes in F-actin induced by the formation of F-actin-subfragment-1 complex

Ca2+-dependent conformational changes in F-actin during myosin subfragment-1 binding with thin filament (in the absence of troponin and tropomyosin) were found in myosin-free ghost fibres by polarized UV microscopy. The pattern of the conformational changes in F-actin changed cooperatively within the range of free Ca2+ concentrations from 10(-7) mol/l to 10(-6) mol/l. It should be suggested that in skeletal muscle of vertebrates there exists a myosin-linked modulation of contraction by Ca2+.     

The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibers

We have studied the effect of myosin P-light chain phosphorylation on the isometric tension generated by skinned fibers from rabbit psoas muscle at 0.6 and 10 microM Ca2+. At the lower Ca2+ concentration, which produced 10-20% of the maximal isometric tension obtained at 10 microM Ca2+, addition of purified myosin light chain resulted in a 50% increase in isometric tension which correlated with an increase in P-light chain phosphorylation from 0.10 to 0.80 mol of phosphate/mol of P-light chain. Addition of a phosphoprotein phosphatase reversed the isometric tension response and dephosphorylated P-light chain. At the higher Ca2+ concentration, P-light chain phosphorylation was found to have little effect on isometric tension. Fibers prepared and stored at -20 degrees C in a buffer containing MgATP, KF, and potassium phosphate incorporated 0.80 mol of phosphate/mol of P-light chain. Addition of phosphoprotein phosphatase to these fibers incubated at 0.6 microM Ca2+ caused a reduction in isometric tension and dephosphorylation of the P-light chain. There was no difference before and after phosphorylation of P-light chain in the normalized force-velocity relationship for fibers at the lower Ca2+ concentration, and the extrapolated maximum shortening velocity was 2.2 fiber lengths/s. Our results suggest that in vertebrate skeletal muscle, P-light chain phosphorylation increases the force level at submaximal Ca2+ concentrations, probably by affecting the interaction between the myosin cross-bridge and the thin filament.     

Conformational changes of contractile proteins accompanying modulation of skeletal muscle contraction. Polarized microfluorometry investigations

Results of studies on the modulation of skeletal muscle contraction by phosphorylation of myosin regulatory light chains and by exchange of magnesium for calcium in myosin heads were reviewed. The polarized fluorescence method was used in these studies, and conformational changes of contractile proteins accompanying modulation of skeletal muscle contraction were investigated. It was found that both the exchange of bound magnesium for calcium on myosin heads and the phosphorylation of myosin regulatory light chains control the ability of myosin heads to induce, upon binding to actin, conformational changes of thin filament leading to decrease or increase of its flexibility. The changes in actin filament flexibility may be caused by alteration of both the inter- and the intramonomer structural organization.     

Calcium/calmodulin regulation of ATPase activity and endogenous phosphorylation of mammalian brain actomyosin

Rabbit brain actomyosin showed several fold stimulation of the MgATPase activity by Ca2+ alone and by Ca2+/calmodulin. The calmodulin-binding drug, fluphenazine, abolished the stimulated activity. In the presence of Ca2+, exogenous calmodulin had a biphasic effect on ATPase activity at low concentrations (less than 0.15 microM) and activated the ATPase activity by 60-70% at about 1 microM. Tropomyosin-troponin complex from skeletal muscle did not stimulate the ATPase activity of brain actomyosin, but conferred Ca2+ sensitivity to a skeletal muscle myosin/brain actomyosin mixture. These results indicate the presence of myosin-linked, calmodulin-dependent, Ca2+-regulatory system for brain actomyosin. Heavy and light chains of brain myosin were found to be rapidly phosphorylated by endogenous Ca2+-dependent protein kinase(s). Ca2+-independent phosphorylation of one of the light chains was also observed.     

Comparison of Ca2+-dependent effects of caldesmon-tropomyosin-calmodulin and troponin-tropomyosin complexes on the structure of F-actin in ghost fibers and its interaction with myosin heads

Comparison of two types of Ca2+-regulated thin filament, reconstructed in ghost fibers by incorporating either caldesmon-gizzard tropomyosin-calmodulin or skeletal muscle troponin-tropomyosin complex, was performed by polarized microphotometry. The changes in actin structure under the influence of these regulatory complexes, as well as those upon the binding of the myosin heads, were followed by measurements of F-actin intrinsic tryptophan fluorescence and the fluorescence of phalloidin-rhodamine complex attached to F-actin. The results show that in the presence of smooth muscle tropomyosin and calmodulin, caldesmon causes Ca2+-dependent alterations of actin conformation and flexibility similar to those induced by skeletal muscle troponin-tropomyosin complex. In both cases, transferring of the fiber from '-Ca2+' to '+Ca2+' solution increases the number of turned-on actin monomers. However, whereas troponin in the absence of Ca2+ potentiates the effect of skeletal muscle tropomyosin, caldesmon-calmodulin complex inhibits the effect of smooth muscle tropomyosin. This difference seems to be due to the qualitatively different alterations in the structure and flexibility of F-actin in ghost fibers evoked by smooth and skeletal muscle tropomyosins. Troponin can bind to F-actin-smooth muscle tropomyosin-caldesmon complex and, in the presence of Ca2+, release the restraint by caldesmon for S-1-induced alterations of conformation, and reduce that for flexibility of actin in ghost fibers. This effect seems to be related to the abolishment by troponin of the potentiating effect of tropomyosin on caldesmon-induced inhibition of actomyosin ATPase activity.     

The disappearance of the dependence of actin-myosin interaction on the phosphorylation of myosin light chains in the "freezing" of the structure of heavy meromyosin by a bifunctional reagent

Using glycerinated muscle fibers, free of myosin, tropomyosin and troponin, a study was made of the structural state of F-actin modified by N-(iodoacetyl)-N'-(1-naphthyl-5-sulfo)-ethylendiamine (1.5-IAEDANS) and by rhodaminyl--phalloin at decoration of thin filaments with a proteolytic fragment of myosin--heavy meromyosin containing phosphorylated and dephosphorylated myosin light chains. The heavy meromyosin used has three SH-groups of heavy chain SH1, SH2 and SH chi modified by bifunctional reagent N,N'-n-phenylmaleimide (SH1-SH2, SH2-SH chi). At decoration of thin filaments with heavy meromyosin, some changes in polarized fluorescence of rhodaminyl--phalloin and 1.5-IAEDANS independent of phosphorylation of myosin light chains were found. Fluorescence anisotropy of the fiber was found to depend primarily on the character of heavy chain of SH-group modification. The ability of heavy chains to change their conformations is supposed to play an important role in the mechanism of myosin system modulation of muscle contraction.     

Phosphorylation of chicken gizzard myosin: Myosin filament hypothesis of calcium regulation

Myosin from chicken gizzard smooth muscle was found to be characteristically different from rabbit skeletal striated myosin: i) ATP induced a profound change in the conformation of gizzard myosin molecules. ii) ATP also induced disassembling of gizzard myosin filaments. iii) Enzymic phosphorylation of gizzard myosin light chains rendered both the myosin conformation and the myosin filaments resistant to the actions of ATP. iv) Very high concentrations of magnesium were required for formation of the ATP-resistant filaments as well as for superprecipitation (a model contraction) of actomyosin suspensions. v) ITP was a very poor substrate for MLCK, and was accordingly incapable of inducing “Ca-tension” in glycerinated fibers of gizzard muscle, but it did induce “Mg-tension.” Primarily from these findings, it was proposed that tje mechanism of gizzard muscle contraction involves ATP-induced changes in the morphology of myosin filaments which are reversibly altered by enzymic phosphorylation and dephosphorylation of myosin light chains in the presence of relatively high concentrations of magnesium.     

The effect of myosin light chain phosphorylation and Mg2+ on the conformation of myosin in thick filaments of glycerinated fibers of rabbit skeletal muscle

It has been shown by polarization microfluorimetry that phosphorylation of myosin light chain 2, in stretched single glycerinated fibers of rabbit skeletal muscle, results in changes in polarized fluorescence anisotropy of both the tryptophan residues of myosin molecules and the fluorescent label, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine, associated with the fast-reacting thiol group in myosin heads. These changes are also dependent on the presence or absence of Mg2+ in the medium: they are most pronounced in the presence of 5 mM MgCl2. It is assumed that both Mg2+ binding to myosin and phosphorylation of light chain 2 associated with myosin heads induce structural changes in myosin filaments of muscle fibres which are expressed as changes in the orientation of myosin heads and in the conformation of myosin rods.     

Studies on Ca2+-Mg2+ binding sites of frog skeletal muscle myosin.

From skeletal muscle myosin light chains readily dissociate from the myosin oligomer in the absence of divalent cations, and unlike rabbit skeletal muscle myosin light chains, the released light chains of frog skeletal muscle myosin have a high Ca2+ binding affinity. Whereas each Ca2+ binding light chain of frog skeletal muscle myosin, when in association with the heavy chains bound 1 mol of Ca2+, when in the dissociated state bound 0.5 mol of Ca2+; the latter were readily displaced with low Mg2+ concentrations. Whereas 10(-5) M Mg2+ displaced all of the Ca2+ binding sites on the released light chains at Ca2+ concentration ranges of 10(-7) to 10(-4) M, there was negligible displacement of the Ca2+ binding sites with native frog skeletal muscle myosin under these same conditions.     

Myosin light chain kinase-regulated endothelial cell contraction: the relationship between isometric tension, actin polymerization, and myosin phosphorylation

The phosphorylation of regulatory myosin light chains by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) has been shown to be essential and sufficient for initiation of endothelial cell retraction in saponin permeabilized monolayers (Wysolmerski, R. B. and D. Lagunoff. 1990. Proc. Natl. Acad. Sci. USA. 87:16-20). We now report the effects of thrombin stimulation on human umbilical vein endothelial cell (HUVE) actin, myosin II and the functional correlate of the activated actomyosin based contractile system, isometric tension development. Using a newly designed isometric tension apparatus, we recorded quantitative changes in isometric tension from paired monolayers. Thrombin stimulation results in a rapid sustained isometric contraction that increases 2- to 2.5-fold within 5 min and remains elevated for at least 60 min. The phosphorylatable myosin light chains from HUVE were found to exist as two isoforms, differing in their molecular weights and isoelectric points. Resting isometric tension is associated with a basal phosphorylation of 0.54 mol PO4/mol myosin light chain. After thrombin treatment, phosphorylation rapidly increases to 1.61 mol PO4/mol myosin light chain within 60 s and remains elevated for the duration of the experiment. Myosin light chain phosphorylation precedes the development of isometric tension and maximal phosphorylation is maintained during the sustained phase of isometric contraction. Tryptic phosphopeptide maps from both control and thrombin-stimulated cultures resolve both monophosphorylated Ser-19 and diphosphorylated Ser-19/Thr-18 peptides indicative of MLCK activation. Changes in the polymerization of actin and association of myosin II correlate temporally with the phosphorylation of myosin II and development of isometric tension. Activation results in a 57% increase in F-actin content within 90 s and 90% of the soluble myosin II associates with the reorganizing F-actin. Furthermore, the disposition of actin and myosin II undergoes striking reorganization. F- actin initially forms a fine network of filaments that fills the cytoplasm and then reorganizes into prominent stress fibers. Myosin II rapidly forms discrete aggregates associated with the actin network and by 2.5 min assumes a distinct periodic distribution along the stress fibers.     

Characteristics of Ca2+- and Mg2+-induced tension development in chemically skinned smooth muscle fibers

Chemically skinned fibers from guinea pig taenia caecum were prepared by saponin treatment to study the smooth muscle contractile system in a state as close to the living state as posible. The skinned fibers showed tension development with an increase of Ca2+ in the solution, the threshold tension occurring as 5 X 10(-7) M Ca2+. The maximal tension induced with 10(-4) M Ca2+ was as large and rapid as the potassium-induced contracture in the intact fibers. The slope of the pCa tension curve was less steep than that of skeletal muscle fibers and shifted in the direction of lower pCa with an increase of MgATP. The presence of greater than 1 mM Mg2+ was required for Ca2+-induced contraction in the skinned fibers as well as for the activation of ATPase and superprecipitation in smooth muscle myosin B. Mg2+ above 2 mM caused a slow tension development by itself in the absence of Ca2+. Such a Mg2+-induced tension showed a linear relation to concentrations up to 8 mM in the presence of MgATP. Increase of MgATP concentration revealed a monophasic response without inhibition of Ca2+-induced tension development, unlike the biphasic response in striated muscle. When MgATP was removed from the relaxing solution, the tension developed slowly and slightly, even though the Mg2+ concentrations was fixed at 2 mM. These results suggest a substantial difference in the mode of actin-myosin interaction between smooth and skeletal muscle.     

Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin

F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1:1 molar ratio. Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca2+. Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor. The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension. On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution. It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development.     

Properties of filament-bound myosin light chain kinase

Myosin light chain kinase binds to actin-containing filaments from cells with a greater affinity than to F-actin. However, it is not known if this binding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells. Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca2+-dependent phosphorylation of myosin regulatory light chain and contraction of stress fibers. Thus, dissociation of myosin light chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.     

Myosin light chain phosphatase. Effect on the activation and relaxation of gizzard smooth muscle skinned fibers

Skinned cells of chicken gizzard were used to study the effect of a smooth muscle phosphatase (SMP-IV) on activation and relaxation of tension. SMP-IV has previously been shown to dephosphorylate light chains on myosin. When this phosphatase was added to submaximally Ca2+-activated skinned cells, tension increased while phosphorylation of myosin light chains decreased. In contrast, when the myosin phosphatase was added to cell bundles activated in the absence of Ca2+ by a Ca2+-insensitive myosin light chain kinase, tension and phosphorylation of the myosin light chains both decreased. These data suggest that Ca2+ inhibits the deactivation of tension even when myosin light chains are dephosphorylated to a low level. Furthermore, comparison of Ca2+-activated cells caused to relax in CTP, in the presence or absence of Ca2+, shows that cells in the presence of Ca2+ do not relax completely, whereas in the absence of Ca2+ cells completely relax. Solutions containing Ca2+ and CTP, however, are incapable of generating tension from the resting state. Endogenous myosin light chain kinase is not active in solutions containing CTP and dephosphorylation of myosin light chains occurs in CTP solutions both in the presence and absence of Ca2+. These data imply that Ca2+ inhibits relaxation even though myosin light chains are dephosphorylated. These data are consistent with a model wherein an obligatory Ca2+-activated myosin light chain phosphorylation is followed by a second Ca2+ activation process for further tension development or maintenance.     

Interaction of isoforms of subfragment-1 of myosin, containing fluorescently labelled alkaline light chains, with muscle fiber actin

Using polarization microfluorimetry, the interaction of myosin subfragment 1 (S1) isoforms containing alkali light chains A1 and A2 respectively (S1(A1) and S1(A2] with F-actin of single glycerinated rabbit skeletal muscle fibers was studied. The alkali light chains of S1 were substituted by reassociation for A1 or A2 chains modified by a fluorescent label (1.5-IAEDANS) at the single SH-group located in the C-terminus. It was found that in S1(A1) bound to muscle fiber F-actin the mobility of the fluorescent label is lower than in S1(A2). At the same time the S1(A1) and S1(A2) interaction with F-actin induces similar changes in polarized fluorescence of rhodamine linked to falloidine which, in turn, is specifically bound to F-actin. It is concluded that the both S1 isoforms bind to F-actin and produce similar effects on the conformational state of actin filaments in muscle fibers. Local differences between S1(A1) and S1(A2) seem to be due to the interaction of the N-terminus of A1 within S1(A1) with the C-terminal region of actin.     

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.