Calmodulin and myosin light-chain kinase of rabbit fast skeletal muscle.

1. It is confirmed that myosin light-chain kinase is a protein of mol.wt. about 80,000 that is inactive in the absence of calmodulin. 2. In the presence of 1 mol of calmodulin/mol of kinase 80-90% of the maximal activity is obtained. 3. Crude preparations of the whole light-chain fraction of rabbit fast-skeletal-muscle myosin contain enough calmodulin to activate the enzyme. A method for the preparation of calmodulin-free P light chain is described. 4. A procedure is described for the isolation of calmodulin from rabbit fast skeletal muscle. 5. Rabbit fast-skeletal-muscle calmodulin is indistinguishable from bovine brain calmodulin in its ability to activate myosin light-chain kinase. The other properties of these two proteins are also very similar. 6. Rabbit fast-skeletal-muscle troponin C was about 10% as effective as calmodulin as activator for myosin light-chain kinase. 7. By chromatography on a Sepharose-calmodulin affinity column evidence was obtained for the formation of a Ca2+-dependent complex between calmodulin and myosin light-chain kinase. 8. Troponin I from rabbit fast skeletal muscle and histone IIAS were phosphorylated by fully activated myosin light-chain kinase at about 1% of the rate of the P light chain.     

Purification and characterization of myosin light-chain kinase from porcine myometrium and its phosphorylation and modulation by cyclic AMP-dependent protein kinase

Myosin light-chain kinase was purified from porcine myometrium to apparent homogeneity at about 262-fold with an Mr of 130 000 as determined by SDS-polyacrylamide gel electrophoresis and a sedimentation coefficient of 4.5 S. The approximate content of the soluble myosin light-chain kinase was estimated to be about 0.85 microM. The purified enzyme exhibited strict substrate specificity only for 20-kDa myosin light chain and Ka values of 0.6 nM and 0.3 microM for calmodulin and Ca2+, respectively. The enzyme was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase, which resulted in a decrease in the affinity for calmodulin of 4-7-fold without effect on the Vmax. The maximal amount of phosphate incorporated into the enzyme was 0.5-0.8 and 1.0-1.4 mol per mol of the enzyme in the presence and absence of Ca2+ and calmodulin, respectively. In the presence of a subsaturating concentration of calmodulin, the enzyme showed a lower sensitivity for Ca2+ by phosphorylation.     

Structure and dynamics of calmodulin in solution.

To characterize the dynamic behavior of calmodulin in solution, we have carried out molecular dynamics (MD) simulations of the Ca2+-loaded structure. The crystal structure of calmodulin was placed in a solvent sphere of radius 44 A, and 6 Cl- and 22 Na+ ions were included to neutralize the system and to model a 150 mM salt concentration. The total number of atoms was 32,867. During the 3-ns simulation, the structure exhibits large conformational changes on the nanosecond time scale. The central alpha-helix, which has been shown to unwind locally upon binding of calmodulin to target proteins, bends and unwinds near residue Arg74. We interpret this result as a preparative step in the more extensive structural transition observed in the "flexible linker" region 74-82 of the central helix upon complex formation. The major structural change is a reorientation of the two Ca2+-binding domains with respect to each other and a rearrangement of alpha-helices in the N-terminus domain that makes the hydrophobic target peptide binding site more accessible. This structural rearrangement brings the domains to a more favorable position for target binding, poised to achieve the orientation observed in the complex of calmodulin with myosin light-chain kinase. Analysis of solvent structure reveals an inhomogeneity in the mobility of water in the vicinity of the protein, which is attributable to the hydrophobic effect exerted by calmodulin's binding sites for target peptides.     

The gamma-subunit of skeletal muscle phosphorylase kinase contains two noncontiguous domains that act in concert to bind calmodulin

Phosphorylase kinase is a Ca2+-regulated, multisubunit enzyme that contains calmodulin as an integral subunit (termed the delta-subunit). Ca2+-dependent activity of the enzyme is thought to be regulated by direct interaction of the delta-subunit with the catalytic subunit (the gamma-subunit) in the holoenzyme complex. In order to systematically search for putative calmodulin (delta-subunit)-binding domain(s) in the gamma-subunit of phosphorylase kinase, a series of 18 overlapping peptides corresponding to the C terminus of the gamma-subunit was chemically synthesized using a tea bag method. The calmodulin-binding activity of each peptide was tested for its ability to inhibit Ca2+/calmodulin-dependent activation of myosin light chain kinase. Data were obtained indicating that two distinct regions in the gamma-subunit, one spanning residues 287-331 (termed domain-N) and the other residues 332-371 (domain-C), are capable of binding calmodulin with nanomolar affinity. Peptides from both of these two domains also inhibited calmodulin-dependent reactivation of denatured gamma-subunit. The interactions of peptides from both domain-N and domain-C with calmodulin were found to be Ca2+-dependent. Dixon plots obtained using mixtures of peptides from domain-N and domain-C indicate that these two domains can bind simultaneously to a single molecule of calmodulin. Multiple contacts between the gamma-subunit and calmodulin (delta-subunit), as indicated by our data, may help to explain why strongly denaturing conditions are required to dissociate these two subunits, whereas complexes of calmodulin with most other target enzymes can be readily dissociated by merely lowering Ca2+ to submicromolar concentrations. Comparison of the sequences of the two calmodulin-binding domains in the gamma-subunit of phosphorylase kinase with corresponding regions in troponin I indicates similarities that may have functional and evolutionary significance.     

Isolation of the native form of chicken gizzard myosin light-chain kinase.

A simple and rapid procedure for the purification of the native form of chicken gizzard myosin light-chain kinase (Mr 136000) is described which eliminates problems of proteolysis previously encountered. During this procedure, a calmodulin-binding protein of Mr 141000, which previously co-purified with the myosin light-chain kinase, is removed and shown to be a distinct protein on the basis of lack of kinase activity, different chymotryptic peptide maps, lack of cross-reactivity with a monoclonal antibody to turkey gizzard myosin light-chain kinase, and lack of phosphorylation by the purified catalytic subunit of cyclic AMP-dependent protein kinase. This Mr-141000 calmodulin-binding protein is identified as caldesmon on the basis of Ca2+-dependent interaction with calmodulin, subunit Mr, Ca2+-independent interaction with skeletal-muscle F-actin, Ca2+-dependent competition between calmodulin and F-actin for caldesmon, and tissue content.     

An enzymatically active cross-linked complex of calmodulin and rabbit skeletal muscle myosin light chain kinase

The interaction between bovine testes calmodulin and rabbit fast skeletal muscle myosin light chain kinase was investigated with the zero-length cross-linking reagent N,N'-dicyclohexylcarbodiimide. A cross-linked product of 110 kDa was produced only in the presence of Ca2+. The reaction mixture was separated on diethylaminoethyl cellulose, and a fraction containing the cross-linked complex of calmodulin and myosin light chain kinase was found to have an elevated kinase activity in the absence of Ca2+, which constituted approximately 50% of the maximally stimulated kinase activity of control, and additional kinase activity in the presence of Ca2+, which constituted the remaining 50% of control activity. Calmodulin added exogenously to the cross-linked complex had no effect on the measured Ca2+ dependence or the maximal extent of kinase activity, which is consistent with the cross-linking of calmodulin in close proximity to a regulatory region of myosin light chain kinase. Moreover, the results are consistent with a mechanism whereby the association of calmodulin is sufficient to stimulate kinase activity and the binding of Ca2+ to bound calmodulin increases catalytic efficiency.     

Insulin-induced myosin light-chain phosphorylation during receptor capping in IM-9 human B-lymphoblasts.

We have examined further the interaction between insulin surface receptors and the cytoskeleton of IM-9 human lymphoblasts. Using immunocytochemical techniques, we determined that actin, myosin, calmodulin and myosin light-chain kinase (MLCK) are all accumulated directly underneath insulin-receptor caps. In addition, we have now established that the concentration of intracellular Ca2+ (as measured by fura-2 fluorescence) increases just before insulin-induced receptor capping. Most importantly, we found that the binding of insulin to its receptor induces phosphorylation of myosin light chain in vivo. Furthermore, a number of drugs known to abolish the activation properties of calmodulin, such as trifluoperazine (TFP) or W-7, strongly inhibit insulin-receptor capping and myosin light-chain phosphorylation. These data imply that an actomyosin cytoskeletal contraction, regulated by Ca2+/calmodulin and MLCK, is involved in insulin-receptor capping. Biochemical analysis in vitro has revealed that IM-9 insulin receptors are physically associated with actin and myosin; and most interestingly, the binding of insulin-receptor/cytoskeletal complex significantly enhances the phosphorylation of the 20 kDa myosin light chain. This insulin-induced phosphorylation is inhibited by calmodulin antagonists (e.g. TFP and W-7), suggesting that the phosphorylation is catalysed by MLCK. Together, these results strongly suggest that MLCK-mediated myosin light-chain phosphorylation plays an important role in regulating the membrane-associated actomyosin contraction required for the collection of insulin receptors into caps.     

Purification and characterization of myosin light-chain kinase from the rat pancreas.

We have partially purified a protein kinase from rat pancreas that phosphorylates two light-chain subunits of pancreatic myosin, a doublet with components of 18 and 20 kDa. This protein kinase was purified approx. 1000-fold by sequential (NH4)2SO4 fractionation, gel filtration, ion-exchange and affinity chromatography on calmodulin-Sepharose 4B. The resultant enzyme preparation is free of cyclic AMP-dependent protein kinase, protein kinase C and calmodulin-dependent type I or II kinase activities. The purified protein kinase is completely dependent on Ca2+ and calmodulin, and phosphorylates a 20 kDa light-chain subunit of intact gizzard myosin, suggesting that it belongs to a class of enzymes known as myosin light-chain kinase (MLCK). The apparent Km values of the putative pancreatic MLCK for ATP (73 microM), gizzard myosin light chains (18 microM) and calmodulin (2 nM) are similar to those reported for MLCKs isolated from smooth muscle, platelet and other sources. The enzyme is half-maximally activated at a free Ca2+ concentration of 2.5 microM. A single component of the affinity-purified kinase reacts with antibodies to turkey gizzard MLCK. The apparent molecular mass of this component is 138 kDa. Immunoprecipitation of a pancreatic homogenate with these antibodies decreases calmodulin-dependent kinase activity for pancreatic myosin by over 85%. The immunoprecipitate contains a single electrophoretic band of 138 kDa. Tryptic phosphopeptide analyses of pancreatic myosin, phosphorylated by either gizzard or pancreatic MLCK, are identical. Thus the enzyme that we have purified from rat pancreas is a MLCK, as judged by (1) absolute dependence on Ca2+ and calmodulin, (2) high affinity for calmodulin, (3) narrow substrate specificity for the light-chain subunit of myosin, and (4) reactivity with antibodies to turkey gizzard MLCK. These studies establish the existence of a pancreatic MLCK which may be responsible for regulating myosin phosphorylation and enzyme secretion in situ.     

Calmodulin activates bovine-cardiac myosin light-chain kinase by increasing the affinity for myosin light-chain 2

The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was less than 4 parts/million and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 microM. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and in the presence of calcium with a V of 11.1 and 11.0 mumol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of calcium and was suppressed by chelating free calcium either before or during a phosphorylation reaction. The apparent KA for calcium was 1.2 microM and 0.4 microM in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the 'specific' calmodulin antagonists W-7, trifluoperazine and R24571 with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibiotics raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. These results suggest that calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinity for its substrate myosin light-chain 2.     

Biological activities of the peptides obtained by digestion of troponin C and calmodulin with thrombin.

1. Troponin C and calmodulin were not digested by thrombin at a significant rate in the presence of Ca2+. 2. In the presence of EGTA, troponin C was digested by thrombin to yield three peptides, TH1 (residues 1--120), TH3 (residues 1--100) and TH2 (residues 121--159). 3. In the presence of EGTA calmodulin was digested by thrombin giving two peptides, TM1 (residues 1--106) and TM2 (residues 107--148). 4. The electrophoretic mobilities of peptides TH1 and TM1 were increased at pH 8.6 by Ca2+ both in the presence and absence of urea. The mobilities of peptides TH2 and TM2 were unaltered under these conditions. 5. Peptides TH1, TH2 and tM1 formed complexes with troponin I on polyacrylamide gels at pH 8.6 in the presence of Ca2+. 6. The phosphorylation of troponin I by cyclic AMP-dependent protein kinase was significantly inhibited by peptides TH1 and TH3 and to a lesser extent by peptide TM1. 7. The calmodulin peptide TM1 activated myosin light-chain kinase when present in large molar excess. Peptide TM2 did not activate the enzyme.     

Calcium binding to complexes of calmodulin and calmodulin binding proteins

The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.     

Calmodulin conformational changes in the activation of protein kinases

The conformation of Ca2+/calmodulin changes from extended when free in solution to compact when bound in peptide complexes. The extent and kinetics of calmodulin compaction in association with Ca2+/calmodulin-dependent protein kinases (CaMKs), as well as target peptides, were investigated by fluorescence, resonance energy transfer and stopped-flow kinetics. Compaction of Ca2+/calmodulin labelled with resonance energy-transfer probes in association with target peptides was rapid (>350 s(-1)). With the target enzymes smooth-muscle myosin light-chain kinase, CaMKIV and CaMKII, the rates of calmodulin compaction were one-two orders of magnitude lower compared with those of the peptides and in the case of alphaCaMKII, ATP binding and Thr(286) auto-phosphorylation were required for calmodulin compaction. In the absence of nucleotides, Ca2+/calmodulin bound to alphaCaMKII in extended conformations, initially probably attached by one lobe only. Kinetic data suggest that in the activation process of Ca2+/calmodulin-dependent protein kinases, productive as well as unproductive complexes are formed. The formation of productive complexes with Ca2+/calmodulin thus may determine the rate of activation.     

Fluorescent adducts of wheat calmodulin implicate the amino-terminal region in the activation of skeletal muscle myosin light chain kinase

Considerable attention is being directed toward defining a binding site in the central region of calmodulin that forms a high affinity interaction with certain enzymes and amphiphilic peptides. However, other regions of calmodulin are also known to be involved in the activation of enzymes such as myosin light chain kinase, regions which may not be directly involved in the binding of small peptides, e.g. mastoparan X. We investigated the properties of wheat calmodulin fluorescent derivatives, which were modified chemically in the first calcium binding site at Cys-27, in the activation of rabbit fast skeletal muscle myosin light chain kinase. Unmodified wheat calmodulin stimulated myosin light chain kinase to a greater maximal velocity than wheat calmodulin that was modified at Cys-27 by any of four fluorescent compounds, IAANS (2-[4'-iodoacetamidoanilino]naphthalene-6-sulfonic acid), 5-[2'-[[iodoacetyl]amino]ethyl]aminonaphthalene]-1-sulfonic acid, 5-iodoacetamidofluorescein, and 7-diethylamino-3-[4'-maleimidylphenyl]-4-methylcoumarin; the midpoints for activation of myosin light chain kinase were not significantly different for unmodified wheat calmodulin and three of the four wheat calmodulin derivatives. Myosin light chain kinase, but not mastoparan X, enhanced the fluorescence emission intensity of wheat calmodulin-IAANS. Mastoparan X reversed, in a dose-dependent manner, the changes in fluorescence intensity of a preformed complex of myosin light chain kinase and wheat calmodulin-IANNS. Thus, we propose that the region vicinal to Cys-27 participates in the activation but not the high affinity association of myosin light chain kinase. Lastly, a comparison of mammalian and plant calmodulin showed that the Vmax for the stimulation of myosin light chain kinase was 1.6-fold greater for bovine than wheat calmodulin. The difference between the two calmodulins was more pronounced at lower Ca2+ because less Ca2+ was needed to saturate the kinase rate when stimulated by bovine calmodulin.     

Novel aspects of calmodulin target recognition and activation.

Several crystal and NMR structures of calmodulin (CaM) in complex with fragments derived from CaM-regulated proteins have been reported recently and reveal novel ways for CaM to interact with its targets. This review will discuss and compare features of the interaction between CaM and its target domains derived from the plasma membrane Ca2+-pump, the Ca2+-activated K+-channel, the Ca2+/CaM-dependent kinase kinase and the anthrax exotoxin. Unexpected aspects of CaM/target interaction observed in these complexes include: (a) binding of the Ca2+-pump domain to only the C-terminal part of CaM (b) dimer formation with fragments of the K+-channel (c) insertion of CaM between two domains of the anthrax exotoxin (d) binding of Ca2+ ions to only one EF-hand pair and (e) binding of CaM in an extended conformation to some of its targets. The mode of interaction between CaM and these targets differs from binding conformations previously observed between CaM and peptides derived from myosin light chain kinase (MLCK) and CaM-dependent kinase IIalpha (CaMKIIalpha). In the latter complexes, CaM engulfs the CaM-binding domain peptide with its two Ca2+-binding lobes and forms a compact, ellipsoid-like complex. In the early 1990s, a model for the activation of CaM-regulated proteins was developed based on this observation and postulated activation through the displacement of an autoinhibitory or regulatory domain from the target protein upon binding of CaM. The novel structures of CaM-target complexes discussed here demonstrate that this mechanism of activation may be less general than previously believed and seems to be not valid for the anthrax exotoxin, the CaM-regulated K+-channel and possibly also not for the Ca2+-pump.     

Properties of caldesmon isolated from chicken gizzard.

Chicken gizzard smooth muscle contains two major calmodulin-binding proteins: caldesmon (11.1 microM; Mr 141 000) and myosin light-chain kinase (4.6 microM; Mr 136 000), both of which are associated with the contractile apparatus. The amino acid composition of caldesmon is distinct from that of myosin light-chain kinase and is characterized by a very high glutamic acid content (25.5%), high contents of lysine (13.6%) and arginine (10.3%), and a low aromatic amino acid content (2.4%). Caldesmon lacked myosin light-chain kinase and phosphatase activities and did not compete with either myosin light-chain kinase or cyclic nucleotide phosphodiesterase (both calmodulin-dependent enzymes) for available calmodulin, suggesting that calmodulin may have distinct binding sites for caldesmon on the one hand and myosin light-chain kinase and cyclic nucleotide phosphodiesterase on the other. Consistent with the lack of effect of caldesmon on myosin phosphorylation, caldesmon did not affect the assembly or disassembly of myosin filaments in vitro. As previously shown [Ngai & Walsh (1984) J. Biol. Chem. 259, 13656-13659], caldesmon can be reversibly phosphorylated. The phosphorylation and dephosphorylation of caldesmon were further characterized and the Ca2+/calmodulin-dependent caldesmon kinase was purified; kinase activity correlated with a protein of subunit Mr 93 000. Caldesmon was not a substrate of myosin light-chain kinase or phosphorylase kinase, both calmodulin-activated protein kinases.     

Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation.

Ca(2+)-activated calmodulin (CaM) regulates many target enzymes by docking to an amphiphilic target helix of variable sequence. This study compares the equilibrium Ca2+ binding and Ca2+ dissociation kinetics of CaM complexed to target peptides derived from five different CaM-regulated proteins: phosphorylase kinase. CaM-dependent protein kinase II, skeletal and smooth myosin light chain kinases, and the plasma membrane Ca(2+)-ATPase. The results reveal that different target peptides can tune the Ca2+ binding affinities and kinetics of the two CaM domains over a wide range of Ca2+ concentrations and time scales. The five peptides increase the Ca2+ affinity of the N-terminal regulatory domain from 14- to 350-fold and slow its Ca2+ dissociation kinetics from 60- to 140-fold. Smaller effects are observed for the C-terminal domain, where peptides increase the apparent Ca2+ affinity 8- to 100-fold and slow dissociation kinetics 13- to 132-fold. In full-length skeletal myosin light chain kinase the inter-molecular tuning provided by the isolated target peptide is further modulated by other tuning interactions, resulting in a CaM-protein complex that has a 10-fold lower Ca2+ affinity than the analogous CaM-peptide complex. Unlike the CaM-peptide complexes, Ca2+ dissociation from the protein complex follows monoexponential kinetics in which all four Ca2+ ions dissociate at a rate comparable to the slow rate observed in the peptide complex. The two Ca2+ ions bound to the CaM N-terminal domain are substantially occluded in the CaM-protein complex. Overall, the results indicate that the cellular activation of myosin light chain kinase is likely to be triggered by the binding of free Ca2(2+)-CaM or Ca4(2+)-CaM after a Ca2+ signal has begun and that inactivation of the complex is initiated by a single rate-limiting event, which is proposed to be either the direct dissociation of Ca2+ ions from the bound C-terminal domain or the dissociation of Ca2+ loaded C-terminal domain from skMLCK. The observed target-induced variations in Ca2+ affinities and dissociation rates could serve to tune CaM activation and inactivation for different cellular pathways, and also must counterbalance the variable energetic costs of driving the activating conformational change in different target enzymes.     

Quantitative analysis of the free energy coupling in the system calmodulin, calcium, smooth muscle myosin light chain kinase

Interactions between Ca2+, calmodulin and turkey gizzard myosin light chain kinase have been studied by equilibrium gel filtration and analyzed in terms of the theory of free energy coupling as formulated by Huang and King for calmodulin-regulated systems (Current Topics in Cellular Regulation 27, 1966-1971, 1985). Direct binding studies revealed that upon interaction with the enzyme, calmodulin acquires strong positive cooperativity in Ca2+-binding. The determination of the Ca2+-binding constants is inherently approximative due to the apparent homotropic cooperativity; therefore a statistical chi 2 analysis was carried out to delimit the formation-, and subsequently the stoichiometric Ca2+-binding constants. Whereas the first two stoichiometric Ca2+-binding constants of enzyme-bound CaM do not differ or are at the upmost 10-fold higher than those in free calmodulin, the third Ca2+ ion binds with an at least 70-fold and more likely 3000-fold higher affinity constant. The binding constant for the fourth Ca2+ is only 5-fold higher than the corresponding one in free calmodulin, thus creating a plateau at 3 bound Ca2+ in the isotherm. Direct binding of Ca2+-free calmodulin to myosin light chain kinase at 10(-7) M free Ca2+ yielded a l/l stoichiometry and an affinity constant of 2.2 x 10(5) M-1. It is thus anticipated that in resting smooth muscle ([Ca2+] less than or equal to 10(-7) M) more than half of the enzyme is bound to metal-free calmodulin. Analysis of the enzymatic activation of myosin light chain kinase at different concentrations of calmodulin and Ca2+ revealed that this Ca2+-free complex is inactive and that activation is concomitant with the formation of the enzyme.calmodulin.Ca3 complex.     

Inhibition by melittin of phospholipid-sensitive and calmodulin-sensitive Ca2+-dependent protein kinases.

Effects of melittin, an amphipathic polypeptide, on various species of protein kinases were investigated. It was found that melittin inhibited the newly identified phospholipid-sensitive Ca2+-dependent protein kinase (from heart, brain, spleen and neutrophils) and the cardiac myosin light-chain kinase, a calmodulin-sensitive Ca2+-dependent enzyme. In contrast, melittin had little or no effect on either the holoenzymes of the cardiac cyclic AMP-dependent and cyclic GMP-dependent protein kinases or the catalytic subunit of the former. Kinetic analysis indicated that melittin inhibited phospholipid-sensitive Ca2+-dependent protein kinase non-competitively with respect to ATP (Ki = 1.3 microM); although exhibiting complex kinetics, its inhibition of the enzyme was overcome by phosphatidylserine (a phospholipid cofactor), but not by protein substrate (histone H1) or Ca2+. On the other hand, melittin inhibited myosin light-chain kinase non-competitively with respect to ATP (Ki = 1.4 microM) or Ca2+ (Ki = 1.9 microM), and competitively with respect to calmodulin (Ki = 0.08 microM); although exhibiting complex kinetics, its inhibition of the enzyme was reversed by myosin light chains (substrate protein). The present findings indicate the presence of functionally important hydrophobic or hydrophilic loci on the Ca2+-dependent protein kinases, but not on the cyclic nucleotide-dependent class of protein kinase, with which melittin can interact. Moreover, the kinetic data suggest that melittin inhibited myosin light-chain kinase by interacting with a site on the enzyme the same as, or proximal to, the calmodulin-binding site, thus interfering with the formation of active enzyme-calmodulin-Ca2+ complex.     

Site-specific dephosphorylation of smooth muscle myosin light chain kinase by protein phosphatases 1 and 2A.

Smooth muscle myosin light chain kinase is phosphorylated at two sites (A and B) by different protein kinases. Phosphorylation at site A increases the concentration of Ca2+/calmodulin required for kinase activation. Diphosphorylated myosin light chain kinase was used to determine the site-specificity of several forms of protein serine/threonine phosphatase. These phosphatases readily dephosphorylated myosin light chain kinase in vitro and displayed differing specificities for the two phosphorylation sites. Type 2A protein phosphatase specifically dephosphorylated site A, and binding of Ca2+/calmodulin to the kinase had no effect on dephosphorylation. The purified catalytic subunit of type 1 protein phosphatase dephosphorylated both sites in the absence of Ca2+/calmodulin but only dephosphorylated site A in the presence of Ca2+/calmodulin. A protein phosphatase fraction was prepared from smooth muscle actomyosin by extraction with 80 mM MgCl2. On the basis of sensitivity to okadaic acid and inhibitor 2, this activity was composed of multiple protein phosphatases including type 1 activity. This phosphatase fraction dephosphorylated both sites in the absence of Ca2+/calmodulin. However, dephosphorylation of both sites A and B was completely blocked in the presence of Ca2+/calmodulin. These results indicate that two phosphorylation sites of myosin light chain kinase are dephosphorylated by multiple protein serine/threonine phosphatases with unique catalytic specificities.     

Purification, characterization and substrate specificity of calmodulin-dependent myosin light-chain kinase from bovine brain.

A substrate-specific calmodulin-dependent myosin light-chain kinase (MLCK) was purified 45,000-fold to near homogeneity from bovine brain in 12% yield. Bovine brain MLCK phosphorylates a serine residue in the isolated turkey gizzard myosin light chain (MLC), with a specific activity of 1.8 mumol/min per mg of enzyme. The regulatory MLC present in intact gizzard myosin is also phosphorylated by the enzyme. The Mr-19,000 rabbit skeletal-muscle MLC is a substrate; however, the rate of its phosphorylation is at best 30% of that obtained with turkey gizzard MLC. Phosphorylation of all other protein substrates tested is less than 1% of that observed with gizzard MLC as substrate. SDS/polyacrylamide-gel electrophoresis of purified MLCK reveals the presence of a major protein band with an apparent Mr of 152000, which is capable of binding 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of MLCK by the catalytic subunit of cyclic-AMP-dependent protein kinase results in the incorporation of phosphate into the Mr-152,000 protein band and a marked decrease in the affinity of MLCK for calmodulin. The presence of Ca2+ and calmodulin inhibits the phosphorylation of the enzyme. Bovine brain MLCK appears similar to MLCKs isolated from platelets and various forms of muscle.