Turkey Gizzard Myosin Light Chain Research Articles

Modulation of Smooth Muscle Myosin Light Chain Kinase Activity by Ca2+/Calmodulin-Dependent, Oligomeric-Type Modifications

Oligomerization of turkey gizzard myosin light chain kinase (MLCKase) was demonstrated by a zero-length cross-linker, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC), a standard reagent used in investigations of specific protein-protein interaction [Mornet et al. (1989) J. Muscle Res. Cell Motil. 10, 10-24]. This approach revealed that in solution the kinase was not monomeric but the monomers were in equilibrium with the kinase dimeric and oligomeric forms. Addition of Ca2+/calmodulin (CM) shifted this equilibrium in the direction of the kinase dimers, accompanied by a 2-fold decrease of the kinase catalytic activity, in addition to a 2-fold decrease of its apparent affinity for CM [Sobieszek et al. (1993) Biochem. J. 295, 405-411]. The dimer (and/or oligomer) formation was shown to result from an interaction of the kinase autoinhibitory domain with its 24 kDa tryptic fragment containing titin-like domain II-3. The possible significance of the oligomerization in regulation of MLCKase activity is discussed.

Analytical sedimentation studies of turkey gizzard myosin light chain kinase and telokin

Sedimentation equilibrium and velocity studies were performed with turkey gizzard myosin light chain kinase (MLCK) and telokin, a small protein apparently corresponding to the sequence of the COOH-terminal end of MLCK. The measurements carried out with MLCK give values for the monomer molecular weight (M(r)), sedimentation coefficient (S20 degrees,w), and virial coefficient (A2) of 108,000, 3.74 S, and -1.95 x 10(-4) mol.ml.g-2, respectively. In the case of telokin, M(r) = 18,500; S20 degrees, w = 1.63 S; and A2 = 5.81 x 10(-4)mol.ml.g-2. Combination of the results of the two kinds of experiment shows that MLCK is a rod-shaped molecule (a/b = 18.9) with a Stoke's radius of 69 A. Telokin is also elongated (a/b = 8.3) with a Stoke's radius of 29 A. MLCK apparently exhibits self-association, with 15% of the protein sedimenting as a dimer in the experiments.

The secondary structure of turkey gizzard myosin light chain kinase and the nature of its interaction with calmodulin

The enzymatic activities of native myosin light chain kinases are subject to modification by interaction with Ca2(+)-calmodulin (CaM). The interaction between myosin light chain kinase isolated from turkey gizzard (tgMLCK) and calmodulin isolated from bovine testes (CaMbt) and wheat germ (CaMwg) has been examined by means of the intrinsic tryptophan fluorescence of tgMLCK and the fluorescence of extrinsic fluorescent labels located at Cys-27 and Tyr-139 of CaMwg and Tyr-99 of CaMbt. Static and dynamic fluorescence measurements provide evidence for the involvement of the former two sites in the zone of contact with lesser involvement of the site marked by the probe at Tyr-99. Complex formation protected the primary cleavage site in CaMbt (Lys-77) from proteolysis by trypsin. These results are consistent with involvement of the N- and C-terminal lobes of CaM in stabilization of the complex with tgMLCK, but cannot rule out participation of the connecting strand in the interaction. CD measurements extending to 175 nm, obtained using synchroton radiation, indicate the following secondary structure content for tgMLCK: 17 +/- 2% alpha-helix, 22 +/- 3% antiparallel beta-sheet, 3 +/- 1% parallel beta-sheet, 24 +/- 2% beta-turns, and 34 +/- 2% random coil. Similar measurements of the CD spectra of CaMbt and of the 1:1::CaMbt:tgMLCK complex presently indicate that neither protein undergoes major secondary structure rearrangement during their interaction, although subtle changes in the CD spectrum of tgMLCK appear to be correlated with the interaction with CaM.

In situ phosphorylation of human platelet myosin heavy and light chains by protein kinase C

Treatment of human platelets with 162 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in phosphorylation of a number of peptides, including myosin heavy chain and the 20-kDa myosin light chain. The site phosphorylated on the myosin heavy chain was localized by two-dimensional peptide mapping to a serine residue(s) in a single major tryptic phosphopeptide. This phosphopeptide co-migrated with a tryptic peptide that was produced following in vitro phosphorylation of platelet myosin heavy chain using protein kinase C. The sites phosphorylated in the 20-kDa myosin light chain in intact cells were analyzed by two-dimensional mapping of tryptic peptides and found to correspond to Ser1 and Ser2 in the turkey gizzard myosin light chain. In vitro phosphorylation of purified human platelet myosin by protein kinase C showed that in addition to Ser1 and Ser2, a third site corresponding to Thr9 in turkey gizzard myosin light chain is also phosphorylated. The phosphorylatable myosin light chains from human platelets were found to consist of two major isoforms present in approximately equal amounts, but differing in their molecular weights and isoelectric points. A third, minor isoform was also visualized by two-dimensional gel electrophoresis. Following treatment with TPA, both the mono- and diphosphorylated forms of each isoform could be visualized, and the sites of phosphorylation were identified. The phosphate content rose from negligible amounts found prior to treatment with TPA to 1.2 mol of phosphate/mol of myosin light chain and 0.7 mol of phosphate/mol of myosin heavy chain following treatment. These results suggest that TPA mediates phosphorylation of both myosin light and heavy chains in intact platelets by activation of protein kinase C.

Microcalorimetric investigation of the interaction of calmodulin with seminalplasmin and myosin light chain kinase.

Flow microcalorimetric titrations of calmodulin with seminalplasmin at 25 degrees C revealed that the high affinity one-to-one complex in the presence of Ca2+ (Comte, M., Malnoe, A., and Cox, J. A. (1986) Biochem. J. 240, 567-573) is entirely enthalpy-driven (delta H0 = -50 kJ.mol-1; delta S0 = O J.K-1.mol-1; delta Cp0 = O J.K-1.mol-1) and is not influenced by the proton or Mg2+ concentration. The Sr2+- and Cd2+-promoted high affinity complexes are also exothermic for -49 and -45 kJ.mol-1, respectively. The observed low affinity interaction in the absence of divalent ions displays no enthalpy change. No enthalpy changes are observed when calmodulin and seminalplasmin are mixed in the presence of millimolar concentrations of Mg2+, Zn2+, or Mn2+. Enthalpy titrations of the 1:1 calmodulin-seminalplasmin complex with Ca2+ and of partly Ca2+-saturated calmodulin with seminalplasmin revealed that only the species calmodulin.Can greater than or equal to 2 is fully competent for high affinity interaction with seminalplasmin. Binding of the second Ca2+ is strongly enhanced (K2 greater than or equal to 5 X 10(7) M-1) as compared to that in free calmodulin (K2 = 2.6 X 10(5) M-1). This is essentially due to the concomitant strongly exothermic step of isomerization of the calmodulin-seminalplasmin complex from its low to its high affinity form. Binding of the remaining two Ca2+ to the high affinity seminalplasmin-calmodulin complex displays the same affinity constants and endothermic enthalpy change as in free calmodulin. A microcalorimetric study on the complex formation between Ca2+-saturated calmodulin and turkey gizzard myosin light chain kinase revealed that the interaction is strongly exothermic with an important overall gain of order (delta H0 = -85 kJ.mol-1; delta S0 = -122 J.K-1.mol-1) and occurs with significant proton uptake (0.44 H+ per mol at pH 7.5). The observed low affinity interaction (K = 2.2 X 10(5) M-1) in the absence of Ca2+ (Mamar-Bachi, A., and Cox, J. A. (1987) Cell Calcium 8, 473-482) displays neither a change in enthalpy nor in protonation.

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

Interactions between Ca 2+, 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 Ca 2+-binding. The determination of the Ca 2+-binding constants is inherently approximative due to the apparent homotropic cooperativity; therefore a statistical χ 2 analysis was carried out to delimit the formation-, and subsequently the stoichiometric Ca 2+-binding constants. Whereas the first two stoichiometric Ca 2+-binding constants of enzyme-bound CaM do not differ or are at the upmost 10-fold higher than those in free calmodulin, the third Ca 2+ ion binds with an at least 70-fold and more likely 3000-fold higher affinity constant. The binding constant for the fourth Ca 2+ is only 5-fold higher than the corresponding one in free calmodulin, thus creating a plateau at 3 bound Ca 2+ in the isotherm. Direct binding of Ca 2+-free calmodulin to myosin light chain kinase at 10 −7 M free Ca 2+ yielded a 1 1 stoichiometry and an affinity constant of 2.2 × 10 5 M −1. It is thus anticipated that in resting smooth muscle ([Ca 2+] ≤ 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 Ca 2+ revealed that this Ca 2+-free complex is inactive and that activation is concomitant with the formation of the enzyme.calmodulin.Ca 3 complex.

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.

Stimulation of canine cardiac sarcoplasmic reticulum Ca 2+ uptake by dihydropyridine Ca 2+ antagonists

We examined the effects of four Ca 2+ antagonists that possess the ability to bind to calmodulin—felodipine, nitrendipine, prenylamine, and verapamil—as well as the effect of the calmodulin antagonist trifluoperazine on Ca 2+ uptake and Ca 2+ + Mg 2+/ATPase activity in canine cardiac sarcoplasmic reticulum. In the presence of 20–30 μM felodipine and 100–200 μM nitrendipine, Ca 2+ uptake increased from 69 nmoles · mg −1 · min −1 to 107 and 108 nmoles · mg −1 · min −1, respectively, with half-maximal stimulation occurring at 7.5 and 28 μM respectively. Ca 2+ + Mg 2+/ATPase activity was unchanged over the same concentration ranges. In contrast, both Ca 2+ uptake and Ca 2+ + Mg 2+/ ATPase activities were inhibited in the presence of 10–100 μM trifluoperazine ( ic 50 = 25 μ M) , 10–100 μM prenylamine ( ic 50 = 35 μ M) and 100–200 μM verapamil (inhibition insufficient for ic 50 determination). None of the drugs affected membrane permeability to Ca 2+ as determined by passive 45Ca 2+ efflux in the presence of ethyleneglycol bis (β-amenoethyl ether) N, N, N 1-tetraacetic acid (EGTA). Drug inhibition of calmodulin-dependent turkey gizzard myosin light chain kinase activation in a purified protein system was used as a direct measure of calmodulin antagonism, and felodipine, nitrendipine, trifluoperazine, prenylamine, and verapamil blocked this activation at ic 50 values of 9.8, 55, 6.4,31, and 93 μM respectively. None of the drugs studied, however, had any effect upon endogenous phospholamban phosphorylation in our cardiac sarcoplasmic reticulum preparations. These observations indicate that di-hydropyridine Ca 2+ antagonists stimulate cardiac sarcoplasmic reticulum Ca 2+ uptake in vitro either by increasing the efficiency of the transport process or by inhibiting Ca 2+-dependent Ca 2+ release, and suggest that these effects do not result from interference with calmodulin-mediated processes.

Inhibition of Turkey gizzard myosin light chain: Kinase activity by dihydropyridine calcium antagonists

Drugs which block the influx of calcium (Ca 2−) across plasma membranes may additionally have direct effects upon smooth muscle contractile proteins. In a system of purified proteins comprised of calmodulin, turkey gizzard myosin light chains, and turkey gizzard myosin light chain kinase, the inhibition of myosin light chain phosphorylation by the dihydropyridine Ca 2+ antagonists felodipine [3-ethyl-5-methyl-1-1,4-dihydro-2,6-dimethyl-4-(2,3-dichlorophenyl)-3,5-pyridine dicarboxylate] and nitrendipine [3-ethyl-5-methyl-1-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine dicarboxylate] was studied. In the presence of excess myosin light chain kinase, 50% inhibition of myosin light chain phosphorylation occurred at felodipine and nitrendipine concentrationsof 9.8 ± 1.1 × 10 −6 M and 5.6 ± 0.6 × 10 −5 M respectively. Inhibition of light chain kinase activity could not be overcome by increasing the free Ca 2+ concentration from 0.05 to 5.0 mM. Felodipine was unable to inhibit the activity of myosin light chain kinase rendered Ca 2+/calmodulin-independent by limited tryptic digestion. Using molecular sieve chromatography, nitrendipine was found to bind to calmodulin with an apparent dissociation constant ( K app) of 5.2 ± 0.3 × 10 −5 M, and this binding was Ca 2+ dependent. These data suggest that dihydropyridines inhibit the phosphorylation of smooth muscle myosin light chains in vitro by binding to Ca +/calmodulin and inhibiting the activation of myosin light chain kinase.

Protein kinase activity of purified rat liver glucocorticoid receptor

Molybdate-stabilized nonactivated rat liver glucocorticoid receptor (GR) was purified to near homogeneity using a biospecific affinity adsorbent, Bio Gel A 0.5m and DEAE-Sephacel. The purified GR sedimented in the 9–10S region in 5–20% sucrose gradients containing 0.10M KCl and 20mM Na 2MoO 4. SDS-polyacrylamide gel electrophoresis revealed a major single band with an apparent molecular weight of 90,000 ± 2,000. Affinity labeling of GR with [ 3H]-dexamethasone mesylate showed association of the radioactivity with a peptide of 90,000 molecular weight. Purified receptor preparation was dialyzed to remove molybdate and was incubated with different protein substrates in the presence of 50μM [λ- 32P]-ATP and divalent cations. Radioactive phosphate from [λ- 32P]-ATP was seen to be incorporated into calf thymus histones, turkey gizzard myosin light chain kinase and rabbit skeletal muscle kinase in the presence of Mg 2+ and Ca 2+ ions. Addition of steroid ligand exogenously to the reaction mixture appeared to increase the extent of protein phosphorylation. No autophosphorylation of GR was evident under the above conditions. The data suggest that purified rat liver GR displays protein kinase activity.

Inhibition of turkey gizzard myosin light chain kinase activity by BAY K 8644

BAY K 8644, a dihydropyridine with positive inotropic effects in smooth muscle, was found to inhibit calmodulin-dependent turkey gizzard myosin light chain kinase activity with an IC50 of 80 μM. In contrast, when myosin light chain kinase was rendered calmodulin-independent by limited proteolysis, drug inhibition was markedly diminished. BAY K 8644 inhibition was additive with that of the negative-inotropic dihydropyridine felodipine. These results indicate that the inotropic effects of dihydropyridines in smooth muscle are unrelated to their calmodulin-antagonistic properties.

Functional interactions between smooth muscle myosin light chain kinase and calmodulin.

Calmodulin (CaM) binding by turkey gizzard myosin light chain kinase (MLCK) causes subtle changes in the fluorescence emission and polarization excitation spectra of the enzyme. Fluorescence experiments using 9-anthroyl-choline (9AC), which competes with ATP in binding, demonstrate mutually stabilizing interactions between the CaM and ATP binding sites corresponding to delta G = -0.6 to -0.7 kcal/mol. Fluorescence titrations in the presence of 9AC or 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] confirm the stoichiometry of 1 mol of CaM/MLCK. Phosphorylation of MLCK has no effect on either the protein fluorescence or the binding of ATP and 9AC. The dissociation constant for the MLCL-CaM complex is increased approximately 500-fold on phosphorylation. Values of Kd for the phosphorylated enzyme range from 0.5 to 1.1 microM in 0.2 N KCl, pH 7.3, 25 degrees C. We showed competition between MLCK and other CaM binding proteins and peptides by using both fluorescence and catalytic activity measurements. Competition for CaM occurs with ACTH, beta-endorphin, substance P, glucagon, poly(L-arginine), myelin basic protein, troponin I, and histone H2A. Phosphorylation of the last three proteins by the adenosine cyclic 3',5'-phosphate dependent protein kinase diminishes their ability to compete. Phosphorylation of MLCK by the protein kinase gives 0.95 +/- 0.04 and 2.2 +/- 0.4 mol of incorporated 32P in the presence and absence of CaM, respectively. These stoichiometries agree with those recently reported [Conti, M. A. & Adelstein, R. S. (1981) J. Biol. Chem. 256, 3178].

Subcellular localization of myosin light chain kinase in skeletal, cardiac, and smooth muscles.

Antibodies were elicited against turkey gizzard myosin light chain kinase (MLCK), purified by affinity chromatography on the enzyme bound to Sepharose, and used to localize myosin kinase--in rabbit fast skeletal, slow skeletal, cardiac, and smooth muscles--by indirect immunofluorescence. When studied on nitrocellulose replicas of NaDodSO4/polyacrylamide gel electrophoretograms, antibodies were specific for the Mr 140,000 MLCK of gizzard smooth muscle. By using the same technique, they were shown to recognize the Mr 140,000 MLCK and a Mr 75,000 polypeptide--presumably derived from the former by proteolysis--in rat arterial and stomach smooth muscle as well as in rat thyroid cells. The same antibodies reacted only with a Mr approximately equal to 75,000 protein from rat cardiac and skeletal muscle. Antibodies inhibited the activity of smooth and skeletal myosin kinases in an in vitro assay with approximately equal to 11 mole of antibody needed for 50% inhibition of 1 mole of gizzard enzyme. The antibodies stain vascular and gizzard smooth muscle cells with no apparent segregation of the enzyme in a specific part of the cell. In contrast, sarcomeric muscles exhibit a striated staining pattern, superimposable to the staining by antiactin antibodies. This shows that (i) antibodies are not species- or tissue-specific, (ii) they recognize kinases that differ in their molecular weight and ability to be phosphorylated, probably at the level of their common catalytic and calmodulin-binding domains, and (iii) sarcomeric muscle kinases are at least in part bound to the contractile apparatus and their distribution is restricted to a specific part of the sarcomere. This raises the possibility that myosin phosphorylation may be controlled not only by the Ca2+ concentration but also by actin-myosin interaction.

Phosphorylation-dependent regulation of Limulus myosin.

Myosin from Limulus, the horseshoe crab, is shown to be regulated by a calcium-calmodulin-dependent phosphorylation of its regulatory light chains. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of a Limulus myosin preparation reveals three light chain bands. Two of these light chains have been termed regulatory light chains based on their ability to bind to light chain-denuded scallop myofibrils (Sellers, J. R., Chantler, P. D., and Szent-Györgyi, A. G. (1980) J. Mol. Biol. 144, 223-245). Ths other light chain does not bind to these myofibrils and is thus termed the essential light chain. Both Limulus regulatory light chains can be phosphorylated with a highly purified turkey gizzard myosin light chain kinase or with a partially purified myosin light chain kinase which can be isolated from Limulus muscle by affinity chromatography on a calmodulin-Sepharose column. Phosphorylation with both of these enzymes requires calcium and calmodulin. Limulus myosin is isolated in an unphosphorylated form. The MgATPase of this unphosphorylated myosin is only slightly activated by rabbit skeletal muscle actin plus tropomyosin. The calcium-dependent phosphorylation of the myosin results in an increase in the actin-activated MgATPase rate. Once phosphorylated, the actin-activated MgATPase rate is only slightly modified by calcium. This suggests that calcium operates mainly at the level of the myosin kinase-calmodulin system.

Phosphorylation of smooth muscle myosin light chains by cAMP-dependent protein kinase.

The 20,000-dalton light chain of chicken gizzard myosin was phosphorylated in vitro by the cAMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from bovine heart. The enzyme catalyzed incorporation of 1 mol of Pi/mol of light chain in a reaction that was completely dependent upon cAMP and independent of Ca2+. Two-dimensional peptide mapping of alpha-chymotryptic digests, as well as phosphoamino acid analysis of acid hydrolysates, were used to compare the site phosphorylated by cAMP-dependent protein kinase to that phosphorylated by turkey gizzard myosin light chain kinase. The results indicate that both enzymes phosphorylate the same serine residue. However, the light chains were a better in vitro substrate for myosin light chain kinase than for cAMP-dependent protein kinase. The amino acid sequence around the phosphorylated serine is characteristic of substrates of cAMP-dependent protein kinases.