Composition of the myosin light chain kinase from chicken gizzard.

The Ca²⁺-dependent protein kinase (ATP:myosin light chain phosphotransferase) from chicken gizzard smooth muscle requires two proteins for enzymatic activity. These have approximate molecular weights of 105,000 and 17,000 daltons. The isolation procedure for each component is described. Neither component alone markedly alters either the actin-moderated ATPase activity or the phosphorylation of myosin. Activation of ATPase activity by a combination of the two components occurred only in the presence of Ca²⁺ and was always accompanied by the phosphorylation of myosin. The simultaneous activation of ATPase activity and myosin phosphorylation establishes a direct correlation between the two events.     

Functional domains of chicken gizzard myosin light chain kinase

The proteolytic susceptibility of chicken gizzard myosin light chain kinase, a calmodulin-dependent enzyme, has been utilized to define the relative location of the catalytic and regulatory domains of the enzyme. Myosin light chain kinase isolated from this source exhibits a Mr of 130,000 and is extremely sensitive to trypsin at 24 degrees C; however, the molecule is divided into susceptible and resistant domains such that proteolysis proceeds rapidly and at multiple sites in the sensitive regions even at 4 degrees C while the rest of the molecule remains relatively resistant to digestion. One of these sensitive areas is the calmodulin-binding domain. On the other hand, Staphylococcus aureus V8 protease digestion generates a calmodulin-binding fragment (Mr = 70,000) that retains Ca2+/calmodulin-dependent enzymatic activity and both of the phosphorylation sites recognized by cAMP-dependent protein kinase. In contrast, treatment with chymotrypsin produces a 95,000 Mr calmodulin-binding fragment that contains only the calmodulin-modulated phosphorylation site. Sequential proteolytic digestion studies demonstrated that the chymotryptic cleavage site responsible for the generation of this 95,000 Mr peptide is within 3,000 Mr of the V8 protease site which produces the 70,000 Mr fragment. Moreover, the non-calmodulin-modulated phosphorylation site must exist in this 3,000 Mr region. A calmodulin-Sepharose affinity adsorption protocol was developed for the digestion and used to isolate both the 70,000 and 95,000 Mr fragments for further study. Taken together, our results are compatible with a model for chicken gizzard myosin light chain kinase in which there is no overlap between the active site, the calmodulin-binding region, and the two sites phosphorylated by cAMP-dependent protein kinase with regard to their relative position in the primary sequence of the molecule.     

Modulator protein as a component of the myosin light chain kinase from chicken gizzard.

The Ca2+-dependent regulation of smooth muscle actomyosin involves a myosin light chain kinase (ATP: myosin light chain phosphotransferase). It has been shown (Dabrowska, R., Aromatorio, D., Sherry, J.M.F., and Hartshorne, D.J. 1977, Biochem. Biophys. Res. Commun. 78, 1263) that the kinase is composed of two proteins of approximate molecular weights 105 000 and 17 000. In this communication it is demonstrated that the 17 000 component is the modulator protein. This conclusion is based on: (1) the identical behavior of the 17 000 kinase component and modulator protein in assays of actomyosin Mg2+-ATPase activity, phosphorylation of myosin, and phosphodiesterase activity, and, (2) the similarity of the 17 000 kinase component and the modulator protein with respect to amino acid composition, absorption spectrum, and electrophoresis in urea-polyacrylamide gels. It is shown also that the modulator protein from smooth muscle and troponin C are distinct proteins.     

The phosphorylation site for casein kinase II on 20,000-Da light chain of gizzard myosin

The 20-kDa light chain isolated from gizzard myosin has recently been reported to be phosphorylated by casein kinase II at a site distinct from that phosphorylated by Ca²⁺- and calmodulin-dependent myosin light-chain kinase. In the present study, the site phosphorylated by casein kinase II has been analyzed through procedures including tryptic digestion of the radioactively phosphorylated light chain and CNBr cleavage of the purified tryptic phosphopeptide, followed by amino acid analysis of these phosphopeptides. Comparison of the amino acid compositions of these peptides with the previously reported sequence has indicated that the phosphorylation site is threonine-134 of the light chain. The significance of the phosphorylation of the light chain by casein kinase II, as well as the substrate specificity of the protein kinase, is discussed on the basis of the result.     

Domain organization of chicken gizzard myosin light chain kinase deduced from a cloned cDNA

Myosin light chain kinases (MLCK) are the most studied of the calmodulin-activated enzymes; however, minimal sequence information is available for the smooth muscle form of the enzyme. The production of an antibody against the enzyme and the use of expression vectors for constructing cDNA libraries have facilitated the isolation of a cDNA for this kinase. The derived amino sequence was found to contain a region of high homology (54%) to the rabbit skeletal muscle enzyme and also very significant homology (35%) to the catalytic subunit of phosphorylase b kinase and cGMP-dependent protein kinase. All of these homologies were found in the known catalytic domains of these enzyme, thus enabling us to predict the location of the catalytic domain for the chicken gizzard myosin light chain kinase. Within the catalytic domain a consensus sequence for an ATP-binding site was located. Subcloning and expression of different regions of the cDNA defined a 192 base pair fragment coding for the calmodulin-binding domain of MLCK. Both of the cAMP-dependent protein kinase phosphorylation sites were identified by sequence homology. A linear model for MLCK is presented placing the various domains in relative position. Northern blot analysis and S1 protection and mapping experiments have revealed that the mRNA for MLCK is 5.5 kilobases in length, but there also exists a second mRNA of 2.7 kilobases that shares a high degree of homology with about 520 base pairs at the 3' end of the cDNA for MLCK.     

Phosphorylation and dephosphorylation of a light chain of the chicken gizzard myosin molecule.

Chicken gizzard myosin was incubated with ATP and/or "native" tropomyosin (NTM) of gizzard muscle in the presence or absence of calcium ions. One of the two light chains of the myosin molecule was phosphorylated in the presence of Ca, but not in its absence. The phosphorylated gizzard myosin was dephosphorylated by a crude preparation of myosin light-chain phosphatase obtained from gizzard muscle. In a superprecipitation test in the presence of EGTA, actomyosin reconstituted from dephosphorylated gizzard myosin did not superprecipitate, whereas actomyosin reconstituted from phosphorylated gizzard myosin showed superprecipitation activity which was inhibited by skeletal NTM and reactivated by Ca.     

Embryonic chicken gizzard: expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase

The patterns of expression of the smooth muscle regulatory proteins caldesmon and myosin light chain kinase were investigated in the developing chicken gizzard. Immunofluorescent studies revealed that both proteins were expressed as early as E5 throughout the mesodermal gizzard anlage, together with actin, -actinin and a small amount of nonmuscle myosin. These proteins appear to form the scaffold for smooth muscle development, defined by the onset of smooth muscle myosin expression. During E6, a period of extensive cell division, smooth muscle myosin begins to appear in the musculi laterales close to the serosal border and, later, also in the musculi intermedii. Until about E10, myosin reactivity expands into the pre-existing thin filament scaffold. Later in development, the contractile and regulatory proteins co-localize and show a regular uniform staining pattern comparable to that seen in adult tissue. By using immunoblotting techniques, the low-molecular mass form of caldesmon and myosin light chain kinase were detected as early as E5. During further development, the expression of caldesmon switched from the low-molecular mass to the high-molecular mass form; in neonatal and adult tissue, high-molecular mass caldesmon was the only isoform expressed. The level of expression of myosin light chain kinase increased continously during embryonic development, but no embryospecific isoform with a different molecular mass was detected.     

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.     

Purification and characterization of bovine cardiac calmodulin-dependent myosin light chain kinase.

Myosin light chain kinase, which is located primarily in the soluble fraction of bovine myocardium, has been isolated and purified approximately 1200-fold with 16% yield by a three-step procedure. The approximate content of soluble myosin light chain kinase in heart is calculated to be 0.63 microM. The isolated kinase is active only as a ternary complex consisting of the kinase, calmodulin, and Ca2+; the apparent Kd for calmodulin is 1.3 nM. The enzyme also exhibits a requirement for Mg2+ ions. Myosin light chain kinase is a monomeric enzyme with Mr = 85,000. The enzyme exhibits a Km for ATP of 175 microM, and a K0.5 for the regulatory light chain of cardiac myosin of 21 microM. The optimum pH is 8.1. Kinase activity is specific for the regulatory light chain of myosin. The specific activity of the isolated enzyme (30 nmol 32P/min/mg of protein) is considerably less than and corresponding values reported for the skeletal and smooth muscle light chain kinases. This is probably due to proteolysis during extraction of the myocardium, a phenomenon which has, as yet, proven impossible to eliminate. In contrast to the smooth muscle enzyme (Adelstein, R.S., Conti, M.A., Hathaway, D.R., and Klee, C.B. (1978) J. Biol. Chem. 253, 8347-8350), the cardiac kinase is not phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

Activation mechanism of rabbit skeletal muscle myosin light chain kinase. 5'-p-fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme

5'-p-fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3 = -0.040 min-1 and -0.038 min-1, and Ki = 0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation.     

Two-site phosphorylation of the phosphorylatable light chain (20-kDa light chain) of chicken gizzard myosin

When prepared under specified conditions chicken gizzard myosin was obtained which when incubated with ATP gave rise to a diphosphorylated as well as the monophosphorylated form of P light chain. Formation of the diphosphorylated light chain occurred more readily with these myosin preparations, but could also be obtained by prolonged incubation of the isolated whole light chain fraction with kinase preparations from rabbit skeletal and chicken gizzard muscles. Using isolated light chains as substrate the more readily formed monophosphorylated light chain contained serine phosphate while the diphosphorylated form contained serine and threonine phosphates.     

Structure of the pseudosubstrate recognition site of chicken smooth muscle myosin light chain kinase

The structure of the chicken smooth muscle myosin light chain kinase pseudosubstrate sequence MLCK(774–807)amide was studied using two-dimensional proton NMR spectroscopy. Resonance assignments were made with the aid of totally correlated and nuclear Overhauser effect spectroscopy. A distance geometry algorithm was used to process the body of NMR distance and angle data and the resulting family of structures was further refined using dynamic simulated annealing. The major structural features determined include two helical segments extending from Asp-777 to Lys-785 and from Arg-790/Met-791 to Trp-800 connected by a turn region from Leu-786 to Asp-789 enabling the helices to interact in solution. The C-terminal helix incorporates the bulk of the pseudosubstrate recognition site which is partially overlapped by the calmodulin binding site while the N-terminal helix forms the bulk of the connecting peptide. The demonstrated turn between the helices may assist in enabling the autoregulatory or pseudosubstrate recognition sequence to be rotated out of the active site of the catalytic core following calmodulin binding.     

Identification of the native form of chicken gizzard myosin light chain kinase with the aid of monoclonal antibodies

Examination, by immunoblotting, of myosin light chain kinase-containing fractions obtained during purification of the enzyme from chicken gizzard has shown that a single species (Mr = 136,000) exists in the muscle and that this enzyme is degraded, primarily to a 130,000-dalton fragment, during purification. These conclusions were confirmed by phosphorylation of the different species of myosin light chain kinase by the isolated catalytic subunit of cyclic AMP-dependent protein kinase.     

Localization and activity of myosin light chain kinase isoforms during the cell cycle

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.     

Dinitrophenylation of chicken gizzard myosin: reactivity of the 17 000-dalton light chain

Chicken gizzard myosin rapidly incorporated 3 mol of 1-fluoro-2,4-dinitrobenzene per 4.7 x 10(5) g of protein with little change in the ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity. During an interval when 2 additional mol of the reagent were bound the K+-ATPase activity in the presence of EDTA was inhibited and the Ca2+-ATPase activity was altered to a lesser extent. Cysteine residues were modified in the dinitrophenylated gizzard myosin. The dinitrophenyl group was located mainly in the active proteolytic fragment, subfragment 1. Dinitrophenylation of the heavy and light chains was observed but major changes in the ATPase activity occurred when the 17 000-dalton light chain and some heavy chains were modified as judged by dissociation experiments in sodium dodecyl sulfate. Thiolysis of the dinitrophenylated gizzard myosin with 2-mercaptoethanol restored the ATPase activity and approx. 2 mol of the dinitrophenyl group were removed. The restoration of the enzymic activity, however, occurred when 1 mol of the label was thiolytically cleaved from cysteine residues of the 17 000-dalton light chain. Substrate Mg-ATP(2-) or MgADP did not protect the ATPase activity of modified gizzard myosin. In the presence of nucleotide there was an increase in the incorporation of the reagent, and a change in its distribution into the light and heavy chains. Calcium had no effect on the dinitrophenylation of this myosin. these results indicate that the reagent, 1-fluoro-2,4-dinitrobenzene, could detect chemical differences in smooth muscle myosin when compared to the reactivity of other myosins. Thiol groups of the 17 000-light chain (and some heavy chains) are probably located peripheral to the active site region of gizzard myosin and they are involved in maintaining the enzymic activity of this protein.     

A simple and rapid method to remove light chain phosphatase from chicken gizzard myosin

A simple and rapid method to remove myosin light chain phosphatase from gizzard myosin using hydroxyapatite chromatography was developed. Stable phosphorylated myosin without light chain phosphatase and kinase was also prepared by the same procedure. A convenient method using the urea gel system to detect minor contamination by phosphatase of myosin is also described.     

Insulin receptor capping and its correlation with calmodulin-dependent myosin light chain kinase

Both fluorescence microscopy and fluorometric analysis techniques have been used to characterize insulin receptor capping in IM-9 human lymphoblastoid cells. Morphologically, insulin caps appear similar to lectin or antiimmunoglobulin-induced caps displaying a preferential accumulation of actin, myosin, and actin-binding protein directly underneath the cap structure. Using the fluorescent calcium indicator quin2 we have detected no change in the calcium activity following insulin stimulation. However, in the presence of a number of calmodulin inhibitors, such as W-5, W-7, W-12, and trifluoperazine (TFP), insulin capping is significantly inhibited, which implies that a calmodulin-regulated process is involved. Using double immunofluorescence microscopy, we have found that the calmodulin-dependent myosin light chain kinase (MLCK) is concentrated directly beneath insulin caps. Upon treatment with trifluoperazine (TFP), the redistribution of both MLCK and insulin receptors are inhibited concomitantly. Our data indicate that the calmodulin-dependent myosin light chain kinase may be directly responsible for the activation of actomyosin-mediated contractility during insulin receptor capping.     

Changes in myosin and myosin light chain kinase during myogenesis

Myosins and myosin light chain kinases have been isolated from a cloned line of myoblasts (L5/A10) as this cell line undergoes differentiation toward adult muscle. At least three myosin isozymes were obtained during this developmental process. Initially a nonmuscle type of myosin was found in the myoblasts. The molecular weights of the myoblast light chains were 20 000 and 15 000. Myosin isolated from early myotubes had light chains with molecular weights of 20 000 and 19 500. Myosin isolated from myotubes which contained sarcomeres had light chains with molecular weights of 23 000, 18 500, and 16 000. This last myosin was similar in light chain complement to adult rat thigh muscle. Two forms of the myosin light chain kinase activity were detected: a calcium-independent kinase in the myoblasts and a calcium-dependent kinase in the myotubes with sarcomeres. No myosin light chain kinase activity was detected in the early myotubes.     

Ca2+-independent gizzard myosin light chain kinase produced by cross-linking of the enzyme with calmodulin using glutaraldehyde

Cross-linked complex of gizzard myosin light chain kinase (MLCK) and calmodulin (CM) was produced by glutaraldehyde treatment of a mixture of these proteins in a high Ca2+ (0.1 mM) solution. Although the specific activity was reduced, this complex showed MLCK activity in a Ca2+-independent manner, different from the original MLCK whose activity was Ca2+-dependent. Chlorpromazine, one of the CM antagonists, was no longer able to inhibit the MLCK activity of this complex. These observations support the previously proposed hypothesis on the regulatory mechanism of MLCK activity via Ca2+. This complex could be regarded as another kind of Ca2+-independent MLCK different from that obtained by chymotryptic digestion of MLCK (Walsh, M.P., Dabrowska, R., Hinkins, S., & Hartshorne, D.J. (1982) Biochemistry 21, 1919-1925). This complex caused superprecipitation of gizzard actomyosin and enhanced actin-activated ATPase of myosin Ca2+-independently.