Adenosine 3':5'-monophosphate-mediated inhibition of myosin light chain phosphorylation in bovine aortic actomyosin.

Ca2+-dependent phosphorylation of the myosin light chains in bovine aortic native actomyosin is markedly depressed in the presence of cyclic AMP and its dependent protein kinase. This inhibition occurs with either cardiac, skeletal, or aortic protein kinase plus cyclic AMP, while little or no inhibition occurs with either cyclic AMP or protein kinase alone. The extent of inhibition is related to the concentration of protein kinase and approaches a maximum of approximately 50%. Concomitant with the inhibition of myosin light chain phosphorylation is (a) an increased phosphorylation of a 100,000-dalton moiety which possibly corresponds to the myosin light chain kinase present in the native actomyosin preparation and (b) a decrease in the actomyosin Mg2+-ATPase activity. These findings suggest that modulation of actin-myosin interactions by the cAMP system directly at the level of the contractile proteins may represent a mechanism by which beta adrenergic relaxation occurs in mammalian vascular smooth muscle.     

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.     

Calcium-sensitivity of pig-carotid-actomyosin ATPase in relation to phosphorylation of the regulatory light chain

Ca2+-dependent phosphorylation of the 20000-Mr regulatory light chain was found to be a necessary condition for the Ca2+-sensitivity of the Mg2+-dependent ATPase activity and superprecipitation of pig carotid actomyosin. Actin-myosin interaction independent of phosphorylation and Ca2+ (ATPase activity and superprecipitation) were demonstrated in aged actomyosin preparations and in preparations from which the regulatory light chains were removed by papain digestion.     

Ca2+ regulation not associated with phosphorylation of myosin light chain in aortic intima smooth muscle. II. Effects of regulatory proteins on the actin-myosin interaction

Contractile and regulatory proteins were prepared from bovine aortic intima, and actin from bovine stomach smooth and rabbit skeletal muscles. In the desensitized and reconstituted actomyosin system, the superprecipitation activity was measured by the turbidity method. Superprecipitation of each system was not exhibited even in the presence of Ca ions, but was observable only in the presence of tropomyosin and Ca ions, while 20,000-dalton light chain of myosin remained dephosphorylated during the reaction. Addition of tropomyosin to the reconstituted acto-myosin digest system (trypsin-digested myosin was devoid of 20,000-dalton light chain) also restored the Ca2+-sensitivity. These results indicate that the phosphorylation of myosin light chain is not a crucial step in the contraction of aortic intima smooth muscle. For full activation of the actin-myosin-ATP interaction, additional factors other than the myosin light chain kinase are required, although some contribution of the kinase to the full activation cannot be ruled out.     

Phosphatase-mediated modulation of actin-myosin interaction in bovine aortic actomyosin and skinned porcine carotid artery

Since the Ca2+-regulatory mechanism for actin-myosin interaction in smooth muscle involves phosphorylation of the 20,000-Da myosin light chains, it was hypothesized that such interaction should be influenced by myosin phosphatase. Accordingly, we studied the effects of an aortic myosin light-chain phosphatase on Ca1+-dependent actin-myosin interaction in detergent-skinned porcine carotid artery and bovine aortic native actomyosin. In skinned preparations, the aortic phosphatase (16 U/ml) markedly inhibited the rate of isometric contraction in low Ca2+ (6.8 X 10(-7) M) and responsiveness to Ca2+ (force attained with 6.8 X 10(-7) Ca2+/force attained with 1.6 X 10(-6) M Ca2+), whereas relaxation was accelerated. Ca2+-dependent actomyosin ATPase activity and phosphorylation of the light chains were significantly and progressively depressed in the presence of increasing concentrations of phosphatase (0.1-0.9 U/ml). The concentration of Ca2+ (1.1 X 10(-6) M) required for half-maximal activation of either ATPase activity or light-chain phosphorylation increased by 70% in the presence of 0.1 U phosphatase/ml. Neither the maximal rate of Ca2+-sensitive ATP hydrolysis (39 +/- 0.8 nmole/min/mg actomyosin) nor the extent of phosphorylation (0.68 +/- 0.05 mole PO4/mole light chain) was altered at greater than 5 X 10(-6) M Ca2+. ATPase activity was correlated to light-chain phosphorylation under diverse conditions including the presence or absence of 1 microM calmodulin, different concentrations of phosphatase (0-0.9 U/ml), and different concentrations of Ca2+ (10(-8) to 1.25 X 10(-5) M). However, significant phosphorylation was present (20-25% of maximum) in the absence of Ca2+-dependent ATPase activity and only 15% of the maximal rate of ATP hydrolysis was expressed until phosphorylation attained 50% of its maximal value. These findings are consistent with the ordered model of myosin phosphorylation suggested by A. Persechini and D. J. Hartshorne [Science (Washington, DC), 213:1383-285, 1961] (36). They also suggest that myosin phosphatase may participate in modulating actin-myosin interactions in vascular smooth muscle.     

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.     

Suppression of superprecipitation of contractile proteins from chicken gizzard muscle by preincubation in the presence of adenosine triphosphate without Ca2+

Superprecipitation of reconstituted actomyosin composed of smooth muscle myosin, skeletal muscle actin and smooth muscle native tropomyosin was studied. When the actomyosin solution was preincubated in the presence of ATP and the absence of Ca2+, or in the relaxed state, superprecipitation was markedly suppressed. The extent of suppression was correlated with the inhibition of the phosphorylation of the 20,000-dalton light chain of smooth muscle myosin. This is consistent with the theory that the interaction of smooth muscle actomyosin is regulated by the phosphorylation of myosin light chain through a system of myosin light chain kinase and phosphatase. However, further studies showed that the myosin light chain kinase and phosphatase system could not explain the present suppression of superprecipitation, even if a cyclic AMP-dependent protein kinase system was also involved. A new regulatory factor should be taken into account in the regulation of smooth muscle actomyosin interaction.     

平滑肌肌球蛋白B的超沉淀与肌球蛋白轻链磷酸化

本文报导了牛胃肌球蛋白B(天然肌动球蛋白)的超沉淀性质。当钙离子、钙调蛋白和ATP存在时,肌球蛋白B出现超沉淀,在pH6.8和7.5处,有两个峰值。Ca~(2+)(PCa值8-4)对超沉淀影响的浓度-反应曲线呈典型的S形,表明当Ca~(2+)浓度处于微摩尔水平时产生超沉淀。伴随超沉淀发生了肌球蛋白调节轻链磷酸化。这说明肌球蛋白轻链的Ca~(2+)-CaM依赖性磷酸化可能包含在脊椎动物平滑肌收缩活动的调节机制中。     

Effect of myosin DTNB light chain on the actin-myosin interaction in the presence of ATP.

The influence of the DTNB light chain of myosin on its enzymatic activities was examined by studying the superprecipitation of actomyosin and the actin-activated ATPase of heavy meromyosin (HMM) [EC 3.6.1.3]. Although the Ca2+-, Mg2+-, and EDTA-ATPase activities of control and DTNB myosin were practically the same, the superprecipitation of actomyosin prepared from actin and DTNB myosin occurred more slowly than that of control myosin. The apparent binding constant obtained from double-reciprocal plots of actin-activated ATPase of DTNB HMM was lower than that of control HMM. Recombination of DTNB myosin and HMM with DTNB light chains restored the original properties of myosin and HMM. The removal of DTNB light chain from myosin had no effect on the formation of the rigor complex between actin and myosin. These results suggest that the DTNB light chain participates in the interaction of myosin with actin in the presence of ATP.     

Calcium regulation of porcine aortic myosin

Calcium regulation of actin-activated porcine aortic myosin MgATPase was studied. The MgATPase of the purified actomyosin was stimulated about 10-fold by 0.1 mM Ca2+. The 20,000 molecular weight light chain subunit (LC20) of myosin was phosphorylated by an endogenous kinase that required Ca2+. Half-maximal activation of both kinase and ATPase occurred at about 0.9 microM Ca2+. Phosphorylated and unphosphorylated myosins, free of actin, kinase, and phosphatase, were purified by gel filtration. The MgATPase of phosphorylated myosin was activated by rabbit skeletal muscle actin; unphosphorylated myosin was actin activated to a much lesser extent. Actin activation was maximal in the presence of Ca2+. Regulation of the aortic myosin MgATPase seems to involve both direct interaction of calcium with phosphorylated myosin and calcium activation of the myosin kinase. The MgATPase of trypsin-treated actomyosin did not require Ca2+ for full activity. The trypsin-treated actomyosin was devoid of LC20. When purified unphosphorylated aortic myosin was treated with trypsin, the LC20, was cleaved and the MgATPase, which was not appreciably actin activated before exposure to protease, was increased and was activated by skeletal muscle actin. After incubation of this light chain-depleted myosin with light chain from rabbit skeletal muscle myosin, the actin activation but not the increased activity, was abolished. Unphosphorylated LC20 seems to inhibit actin activation in this smooth muscle.     

Regulation of embryonic smooth muscle myosin by protein kinase C

Phosphorylation of the 20-kDa light chain regulates adult smooth muscle myosin; phosphorylation by the Ca2+/calmodulin-dependent enzyme myosin light chain kinase stimulates the actomyosin ATPase activity of adult smooth muscle myosin; the simultaneous phosphorylation of a separate site on the 20-kDa light chain by the Ca2+/phospholipid-dependent enzyme protein kinase C attenuates the myosin light chain kinase-induced increase in the actomyosin ATPase activity of adult myosin. Fetal smooth muscle myosin, purified from 12-day-old fertilized chicken eggs, is structurally different from adult smooth muscle myosin. Nevertheless, phosphorylation of a single site on the 20-kDa light chain of fetal myosin by myosin light chain kinase results in stimulation of the actomyosin ATPase activity of this myosin. Protein kinase C, in contrast, phosphorylates three sites on the fetal myosin 20-kDa light chain including a serine or threonine residue on the same peptide phosphorylated by myosin light chain kinase. Interestingly, phosphorylation by protein kinase C stimulates the actomyosin ATPase activity of fetal myosin. Moreover, unlike adult myosin, there is no attenuation of the actomyosin ATPase activity when fetal myosin is simultaneously phosphorylated by myosin light chain kinase and protein kinase C. These data demonstrate, for the first time, the in vitro activation of a smooth muscle myosin by another enzyme besides myosin light chain kinase and raise the possibility of alternate pathways for regulating smooth muscle myosin in vivo.     

Ca-linked phosphorylation of a light chain of vertebrate smooth-muscle myosin.

In vertebrate smooth muscle actomyosin and myofibrils a myosin light chain of molecular weight about 20,000 becomes phosphorylated at the same Ca2+ concentration as required to stimulate the actin-activated ATPase activity of myosin. Further, the degree of phosphorylation in the preparations as well as in various reconstituted actomyosins is proportional to their measured Ca2+ sensitivity. The phosphorylation process is very rapid and is essentially completed before the rise in ATPase activity. The enzyme responsible for the observed myosin phosphoylation is a specific myosin light chain kinase which is routinely co-purified with myosin. This kinase is normally present in actomyosin and its removal together with tropomyosin leads to a complete loss of the actin-activated ATPase activity. It is suggested that the Ca-dependent phosphorylation of the light chain via the light chain kinase represents the initial step in the activation of myosin that leads to contraction. Relaxation is probably effected by an as yet uncharacterised light chain phosphatase.     

Purification and characterization of a sea urchin egg Ca2+-calmodulin-dependent kinase with myosin light chain phosphorylating activity

The crude actomyosin precipitate from sea urchin (Arbacia punctulata) egg extracts contains Ca2+-sensitive myosin light chain kinase activity. Activity can be further increased by exogenous calmodulin (CaM). Egg myosin light chain kinase activity is purified from total egg extract by fractionating on three different chromatographic columns: DEAE ion exchange, gel filtration on Sephacryl-300, and Affi-Gel-CaM affinity. The purified egg kinase depends totally on Ca2+ and CaM for activity. Unphosphorylated egg myosin has very little actin-activated ATPase. After phosphorylation of the phosphorylable light chain by either egg kinase or gizzard myosin light chain kinase, the actin-activated ATPase of egg myosin is enhanced several fold. However, the egg kinase bears some unique characteristics which are very different from conventional myosin light chain kinases of differentiated tissues. The purified egg kinase has a native molecular mass of 405 kDa, while on sodium dodecyl sulfate-polyacrylamide electrophoresis it shows a single subunit of 56 kDa. The affinity of egg kinase for CaM (Ka = 0.4 microM) is relatively weaker than that of the gizzard myosin light chain kinase. The egg kinase autophosphorylates in the presence of Ca2+ and CaM and has a rather broad substrate specificity. The possible relationship between this egg Ca2+-CaM-dependent kinase and the Ca2+-CaM-dependent kinases from brain and liver is discussed.     

Amino acid sequence of the phosphorylation site of bovine cardiac myosin light chain

Amino acid sequences of peptides containing the phosphorylation site of bovine cardiac myosin light chain (L2) were determined. The site was localized to a serine residue in the tentative amino terminus of the light chain and is homologous to phosphorylation sites in other myosin light chains. Phosphorylation of bovine cardiac light chain by chicken gizzard myosin light chain kinase was Ca2+-calmodulin dependent. Kinetic data gave a Km of 107; microM and a Vmax of 23.6 mumol min-1 mg-1. In contrast to what has been observed with smooth muscle light chains, neither the phosphorylation site fragment of the cardiac light chain nor a synthetic tetradecapeptide containing the phosphorylation site were effectively phosphorylated by the chicken gizzard kinase. Phosphorylation of cardiac myosin light chains by chicken gizzard myosin light chain kinase, therefore, requires other regions of the light chain in addition to a phosphate acceptor site.     

K-252a, a novel microbial product, inhibits smooth muscle myosin light chain kinase

Effects of K-252a, (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta [cde]trinden-1-one, purified from the culture broth of Nocardiopsis sp., on the activity of myosin light chain kinase were investigated. 1) K-252a (1 x 10(-5) M) affected three characteristic properties of chicken gizzard myosin-B, natural actomyosin, to a similar degree: the Ca2+-dependent activity of ATPase, superprecipitation, and the phosphorylation of the myosin light chain. 2) K-252a inhibited the activities of the purified myosin light chain kinase and a Ca2+-independent form of the enzyme which was constructed by cross-linking of myosin light chain kinase and calmodulin using glutaraldehyde. The degrees of inhibition by 3 x 10(-6) M K-252a were 69 and 48% of the control activities with the purified enzyme and the cross-linked complex, respectively. Chlorpromazine (3 x 10(-4) M), a calmodulin antagonist, inhibited the native enzyme, but not the cross-linked one. These results suggested that K-252a inhibited myosin light chain kinase by direct interaction with the enzyme, whereas chlorpromazine suppressed the enzyme activation by interacting with calmodulin. 3) The inhibition by K-252a of the cross-linked kinase was affected by the concentration of ATP, a phosphate donor. The concentration causing 50% inhibition was two orders magnitude lower in the presence of 100 microM ATP than in the presence of 2 mM ATP. 4) Kinetic analyses using [gama-32P]ATP indicated that the inhibitory mode of K-252a was competitive with respect to ATP (Ki = 20 nM). These results suggest that K-252a interacts at the ATP-binding domain of myosin light chain kinase. The direct action of the compound on the enzyme would explain the multivarious inhibition of myosin ATPase, of superprecipitation, and of the contractile response of smooth muscle.     

The effects of phosphorylation and dephosphorylation of brain myosin on its actin-activated Mg2+-ATPase and contractile activities

Purified bovine brain myosin contained approximately 1 and 3 mol of protein-bound phosphate/mol myosin in the light chains and heavy chains, respectively. Large portions of this light chain- and heavy chain-bound phosphate (about 0.8 and 2.4 mol, respectively) were removed by incubation with a brain phosphoprotein phosphatase and potato acid phosphatase, respectively. Upon phosphorylation of the dephosphorylated brain myosin with myosin light chain kinase and casein kinase II, about 1.6 and 3.0 mol of phosphate was incorporated into the light chains and heavy chains, respectively, while much lower levels of phosphate were incorporated into the non-dephosphorylated brain myosin under the same conditions. The actin-activated Mg2+-ATPase activity of brain myosin rephosphorylated with myosin light chain kinase was about twice as high as that of dephosphorylated brain myosin (about 30 and 15 nmol phosphate/mg/min, respectively). On the other hand, whereas the rephosphorylated brain myosin superprecipitated rapidly with F-actin, the rate of superprecipitation of the dephosphorylated brain myosin was extremely low. Under appropriate conditions, a loose network of tiny superprecipitates, which formed initially throughout the solution, contracted to form eventually a large and dense particle. These results indicate that phosphorylation of the light chains of brain myosin is a prerequisite for the contraction of brain actomyosin. The role of phosphorylation of the heavy chains by casein kinase II remains to be elucidated.     

An aortic spontaneously active phosphatase dephosphorylates myosin and inhibits actin-myosin interaction

Actin-myosin interaction in aortic actomyosin reportedly requires phosphorylation of the 20,000 dalton myosin light chains. A spontaneously active phosphatase which dephosphorylates phosphorylase $\text{a}$ and isolated phosphorylated cardiac myosin light chains was extracted from bovine aortic smooth muscle. This enzyme, when added to aortic native actomyosin (a) significantly suppressed phosphorylation of the light chains of the native hexameric smooth muscle myosin, (b) accelerated the rate and increased the magnitude of myosin light chain dephosphorylation in actomyosin that had been prephosphorylated, and (c) markedly attenuated the rate of actin-myosin interaction. These results support the hypothesis that myosin phosphorylation and subsequent actin-myosin interactions (contractility) in vascular smooth muscle may be modulated by spontaneously active aortic phosphatase.     

Phosphorylation in skeletal myosin light chain modulates the actin--myosin interaction in the presence of regulatory proteins in vitro

The effect of phosphorylation in skeletal myosin light chain (LC2) on the actomyosin and acto-heavymeromyosin (HMM) ATPase activities was investigated in the presence or absence of regulatory proteins (tropomyosin-troponin complex). Phosphorylation in LC2 did not modulate the actin-myosin and actin-HMM interactions over a wide range of KCl concentrations from 30 to 150 mM without regulatory proteins. In the presence of regulatory proteins, phosphorylation in myosin LC2 enhanced the ATPase activity of actomyosin with calcium ions, but the removal of calcium ions made little difference in the ATPase activity between phosphorylated and dephosphorylated myosins. Ca2+-sensitivity of the regulated actomyosin was slightly changed by phosphorylation in myosin LC2. However, both the ATPase activity and Ca2+-sensitivity of the regulated acto-HMM were unaffected by phosphorylation in HMM LC2.     

Ca2+ stimulates the Mg2(+)-ATPase activity of brush border myosin I with three or four calmodulin light chains but inhibits with less than two bound.

Brush border myosin I from chicken intestinal microvilli is a membrane-associated, single-headed myosin composed of a 119-kDa heavy chain and several calmodulin light chains. We first describe in detail a new procedure for the rapid purification of brush border myosin I in greater than 99% purity with a yield of 40%, significantly higher than for previous methods. The subunit stoichiometry was determined to be 4 calmodulin light chains/myosin I heavy chain by amino acid compositional analysis of the separated subunits. We have studied the effects of Ca2+ and temperature on dissociation of calmodulin from myosin I and on myosin I Mg2(+)-ATPase and contractile activities. At 30 degrees C the actin-activable ATPase activity is stimulated 2-fold at 10-700 microM Ca2+. Dissociation of 1 calmodulin occurs at 25-50 microM Ca2+, but this has no effect on actin activation. The contractile activity of myosin I, expressed as superprecipitation, is greatly enhanced by Ca2+ under conditions in which 1 calmodulin is dissociated. This calmodulin is thus not essential for actin activation or superprecipitation. Myosin I was found to be highly temperature-sensitive, with an increase to 37 degrees C resulting in dissociation of 1 calmodulin at below 10(-7) M Ca2+ and an additional 1.5 calmodulins at 1-10 microM Ca2+. A complete loss of actin activation accompanies the Ca2(+)-induced calmodulin dissociation at 37 degrees C. Our conclusion is that physiological levels of Ca2+ can either stimulate or inhibit the mechanoenzyme activities of brush border myosin I in vitro, with the mode of regulation determined by the number of associated calmodulin light chains.     

Properties of phosphorylated myofibrils from gizzard smooth muscle

Phosphorylation of chicken gizzard myosin light chain in myofibril and its effect on myofibrillar ATPase activity were investigated in the contracted state of myofibrils. When myofibrils were incubated for two hours at 30 degreeds C with ATP, magnesium and calcium, the myosin light chain was phosphorylated by endogenous light-chain kinase. Standing overnight, the phosphorylated light chain was dephosphorylated by endogenous light-chain phosphatase. Control myofibril had much higher ATPase activity than phosphorylated and phosphorylated-dephosphorylated myofibrils. It was very interesting that the phosphorylated and phosphorylated-dephosphorylated myofibrils were quite similar in ATPase activity. However, phosphorylated myofibril differed from phosphorylated-dephosphorylated myofibril in Ca2+ dependency of Mg2+-ATPase activity. The phosphorylated-dephosphorylated myofibril was not affected by the presence or absence of Ca2+. In contrast, phosphorylated myofibril apparently showed a negative Ca2+-sensitivity. On the other hand, the results indicating that the superprecipitation gel formed by phosphorylated-dephosphorylated myosin could not be dissolved in 0.6 M NaCl, suggest that the phosphorylation-dephosphorylation process of the actomyosin system in gizzard myofibril results in stronger actin-myosin interaction.