Design of peptidase-resistant peptide inhibitors of myosin light chain kinase.

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility including smooth muscle contraction, cell migration, cytokinesis, receptor capping, secretion, etc. Inhibition of MLCK activity in endothelial and epithelial monolayers using cell-permeant peptide Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys (PIK, Peptide Inhibitor of Kinase) allows protecting the barrier capacity, suggesting a potential medical use of PIK. However, low stability of L-PIK in a biological milieu prompts for development of more stable L-PIK analogues for use as experimental tools in basic and drug-oriented biomedical research. Previously, we designed PIK1, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 , that was 2.5-fold more resistant to peptidases in human plasma in vitro than L-PIK and equal to it as MLCK inhibitor. In order to further enhance proteolytic stability of PIK inhibitor, we designed the set of six site-protected peptides based on L-PIK and PIK1 degradation patterns in human plasma as revealed by 1 H-NMR analysis. Implemented modifications increased half-live of the PIK-related peptides in plasma about 10-fold, and these compounds retained 25-100% of L-PIK inhibitory activity toward MLCK in vitro. Based on stability and functional activity ranking, PIK2, H-(Nα Me)Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-D-Arg-Lys-NH2 , was identified as the most stable and effective L-PIK analogue. PIK2 was able to decrease myosin light chain phosphorylation in endothelial cells stimulated with thrombin, and this effect correlated with the inhibition by PIK2 of thrombin-induced endothelial hyperpermeability in vitro. Therefore, PIK2 could be used as novel alternative to other cell-permeant inhibitors of MLCK in cell culture-based and in vivo studies where MLCK catalytic activity inhibition is required. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Myosin light chain kinase and myosin light chain phosphatase from Dictyostelium: effects of reversible phosphorylation on myosin structure and function.

We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which was partially purified, selectively removes phosphate from the 18,000-D light chain but not from the heavy chain of Dictyostelium myosin. Phosphatase-treated Dictyostelium myosin has less than or equal to 0.01 mol Pi/mol 18,000-D light chain. Phosphatase-treated myosin could be rephosphorylated to greater than or equal to 0.96 mol Pi/mol 18,000-D light chain by incubation with MLCK and ATP. We found myosin thick filament assembly to be independent of the extent of 18,000-D light-chain phosphorylation when measured as a function of ionic strength. However, actin-activated Mg2+-ATPase activity of Dictyostelium myosin was found to be directly related to the extent of phosphorylation of the 18,000-D light chain. MLCK-treated myosin moved in an in vitro motility assay (Sheetz, M. P., and J. A. Spudich, 1983, Nature (Lond.), 305:31-35) at approximately 1.4 micron/s whereas phosphatase-treated myosin moved only slowly or not at all. The effects of phosphatase treatment on the movement were fully reversed by subsequent treatment with MLCK.

Structure and expression of a calcium-binding protein gene contained within a calmodulin-regulated protein kinase gene.

We have determined the first genomic structure and characterized the mRNA and protein products of a novel vertebrate gene that encodes a calcium-binding protein with amino acid sequence identity to a protein kinase domain. The elucidation of the complete DNA sequence of this transcription unit and adjacent genomic DNA, Southern blot and polymerase chain reaction analyses of cellular genomic DNA, and examination of mRNA and protein species revealed that the calcium-binding kinase-related protein (KRP)-encoding gene is contained within the gene for a calmodulin-regulated protein kinase, myosin light-chain kinase (MLCK). The KRP gene transcription unit is composed of three exons and a 5'-flanking sequence containing a canonical TATA box motif. The TATA box, the transcription initiation site, and the first 109 nucleotides of the 5' noncoding region of the KRP mRNA correspond to an MLCK gene intron sequence. Both KRP and MLCK are produced in the same adult chicken tissue in relatively high abundance from a single contiguous stretch of genomic DNA and utilize the same reading frame and common exons to produce distinct mRNAs (2.7 and 5.5 kb, respectively) that encode proteins with dissimilar biochemical functions. There appears to be no precedent in vertebrate molecular biology for such a relationship. This may represent a mechanism whereby functional diversity can be achieved within the same vertebrate tissue by use of common exons to produce shuffled domains with identical amino acid sequences in different molecular contexts.

Intrasteric regulation of myosin light chain kinase: the pseudosubstrate prototope binds to the active site.

We previously proposed a molecular mechanism for the activation of smooth muscle myosin light chain kinase (smMLCK) by calmodulin (CaM). According to this model, smMLCK is autoinhibited in the absence of Ca2+/CaM due to the interaction of a pseudosubstrate prototope, contained within the CaM binding/regulatory region, with the active site of the enzyme. Binding of Ca2+/CaM releases the autoinhibition and allows access of the protein substrate to the active site of the enzyme, resulting in phosphorylation of the myosin light chains. We now provide direct experimental evidence that the pseudosubstrate prototope can associate with the active site. We constructed a smMLCK mutant in which the five-amino acid phosphorylation site of the myosin light chain substrate was inserted into the pseudosubstrate sequence of the CaM binding domain without disrupting the ability of the enzyme to bind Ca2+/CaM. We demonstrate that this mutant undergoes intramolecular autophosphorylation at the appropriate inserted serine residue in the absence of CaM and that this autophosphorylation activates the enzyme. Binding of Ca2+/CaM to the mutant enzyme stimulated myosin light chain substrate phosphorylation but strongly inhibited autophosphorylation, presumably by removing the pseudosubstrate from the active site. These results confirm that the pseudosubstrate sequence has access to the catalytic site and that the activation of the enzyme is accompanied by its removal from this position due to Ca2+/CaM binding as predicted by the model.

Inhibitory Ca(2+)-regulation of myosin light chain kinase in the lower eukaryote, Physarum polycephalum: role of a Ca(2+)-dependent inhibitory factor.

ATP-dependent interactions between myosin and actin in the lower eukaryote, Physarum polycephalum, are inhibited by micromolar levels of Ca2+. This inhibition is mediated by the binding of Ca2+ to myosin, the phosphorylation of which is required if Ca2+ is to inhibit the activities of myosin (Kohama, K., Trends Pharmacol. Sci. 11, 433-435 (1990)). As the first step to examine whether Ca2+ also regulates phosphorylation in the actomyosin system, we purified myosin light chain kinase (MLCK) of 55 kDa almost to homogeneity. The MLCK activity was high whether or not Ca2+ was present. However, a Ca(2+)-dependent inhibitory factor (CIF) purified from Physarum (Okagaki et al., Biochem. Biophys. Res. Commun. 176, 564-570 (1991)) was shown to reduce the MLCK activity in a Ca(2+)-dependent manner. Using crude preparations, not only MLCK but also myosin heavy chain kinase and actin kinase were shown to be inhibited by Ca2+ half-maximally at micromolar levels. Since CIF is the only Ca(2+)-binding protein in the preparations, we propose that this inhibitory Ca(2+)-regulation of the kinases for actomyosin is mediated by CIF.

Heterogeneity of smooth muscle myosin light chain kinase. Characterization of isoelectric variants.

Purified chicken gizzard myosin light chain kinase (MLCK) analyzed by anion-exchange high-performance liquid chromatography (HPLC) can be consistently resolved into three well separated peaks, termed alpha, beta, gamma. These peaks are shown to correspond to differently charged forms of MLCK with the charge difference between alpha and beta twice as large as between beta and gamma. The isoelectric point and elution position of the peaks as well as their amplitudes are modified by phosphorylation or by autophosphorylation of MLCK suggesting that the observed charge differences are related to their different phosphate content. The three forms appear to have similar apparent affinity for both the substrates, ATP and the isolated regulatory light chain, but their specific activities are different.

Preparation and properties of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.

Peptides corresponding to the calmodulin-binding domain of MLCK and other target enzymes promise to be useful tools in the study of calmodulin action. Not only will they help to elucidate the molecular interactions of calmodulin with specific target enzymes, but they should also prove to be useful calmodulin antagonists because of their high affinity and high specificity. Given the lack of sequence similarity in the peptides presently known to avidly bind calmodulin, it seems as likely as not that there will be little sequence homology found between calmodulin-binding domains from different calmodulin-dependent enzymes. If this is the case, it may be difficult to identify the calmodulin-binding domain of a target enzyme by simple inspection of the enzyme's sequence. However, the techniques described in this chapter require only nominal amounts of target enzyme and should, therefore, prove useful in a situation where the sequence of a target enzyme is known from DNA cloning work, but only a small quantity of pure enzyme is available for structure-function studies.

The kinase activity of the giant protein projectin of the flight muscle of Locusta migratoria.

Projectin is a member of the functionally and structurally heterogeneous family of myosin light chain kinases associated to myosin of synchronous as well as asynchronous insect muscles. We examined the phosphotransferase activity of projectin from flight muscle of Locusta migratoria. Isolated projectin exhibits an unstimulated autophosphorylation activity in vitro. We observed differences in the formation of synthetic filaments with myosin, and paramyosin depending on if projectin was autophosphorylated in vitro or not. Aggregates of native projectin with myosin and paramyosin (molar ratio 0.08:1:0.5) showed diameters 20-50 nm similar to those of myosin filaments. When in vitro autophosphorylated projectin was used we predominantly obtained, however, subfilament-like structures of only 7-10 nm in diameter. The in vitro autophosphorylation of projectin was suppressed in the presence of either acto-myosin, actin-filaments or myosin, but still seems to exhibit a phosphorylation activity: Projectin added to actomyosin resulted in the phosphorylation of three polypeptides of apparent molecular masses of 200, 165 and 100 kDa, respectively. These data suggest that the autophosphorylation activity of projectin is regulated by its environment. We conclude, therefore, a dual function of its kinase domain: at first, a role of its autophosphorylation in the formation of myosin filaments (association of subfilaments to filaments); secondly, the transphosphorylation activity of projectin modulates the contractile response of the actomyosin system by phosphorylating some of its components. Moreover, we could stimulate in vitro the projectin autophosphorylation 3.4-fold by calmodulin (EC50 = 17.8 nM). However, the transphosphorylations described above were not stimulated by calmodulin.

Characterization of regulatory light chain-a myosin kinase from smooth muscle of scallop, Patinopecten yessoensis.

The protein kinase that phosphorylates the regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was isolated from scallop smooth muscle (Sohma, H. & Morita, F. (1986) J. Biochem. 100, 1155-1163). The enzymatic properties of this kinase (aMK) were investigated using RLC-a as the substrate. The Km value for ATP was 6.5 microM in the presence of 27 microM RLC-a at pH 7.0, and that for RLC-a was 133 microM in the presence of 1 mM ATP. The Vm value at saturation of both RLC-a and ATP was 0.25 s-1 at pH 7.0. The pH activity curve for aMK was bell-shaped with a maximum at around pH 7.8. The aMK activity was inhibited strongly by an increase in the KCl concentration. aMK required Mg2+, but was inhibited by high concentrations of Mg2+. The optimum activity was seen at 3 mM MgCl2. The mode of inhibition of the aMK activity by Ca2+ was studied. Assuming that the binding of Ca2+ to aMK induces the inhibition, the dissociation constant of Ca2+ was estimated to be 64 microM. aMK also phosphorylated LC20 of chicken gizzard myosin at a similar rate to that for RLC-a and the DTNB light chain of rabbit skeletal muscle myosin at a more lower rate. The helix and beta-sheet contents of aMK were estimated to be 19 and 30%, respectively, from the CD spectrum.

Isolation from bovine brain of 155 kDa component exhibiting myosin light chain kinase activity.

1) Taking myosin light chain kinase (MLCK) activity as the index, bovine extract was fractionated by the use of anion-exchange chromatography, cation-exchange chromatography, and calmodulin affinity chromatography. The kinase activity of the fraction thus obtained was elevated up to about 12,400 times over that of the original crude extract. 2) The fraction mentioned above was subjected again to anion exchange chromatography. The kinase activities were divided into two parts, i.e., part I which contained the 155 kDa component and part II which was virtually free of 155 kDa component. The MLCK activity of part I was considerably lower than that of part II. 3) Part I was subjected to gel filtration using AcA 34 gel and the 155 kDa component was isolated. The fraction contained the 155 kDa component in a homogeneous state and showed myosin specific kinase activity, which was about 2 X 10(5) times that of the original crude extract. 4) The high kinase activity of part II seemed to be ascribable to the 130 kDa component, in accord with the report of Hathaway, Adelstein, and Klee (J. Biol. Chem. 256, 8183-8189, 1981).

Preparation of protein components exhibiting myosin light chain kinase activities from bovine aorta: discrepancies between its enzyme activity and actomyosin activating effect.

1) Two protein components, 155 and 130 kDa in their electrophoretic molecular weights, respectively, were isolated in a homogeneous state from bovine aorta; they showed both the superprecipitation-inducing effect on desensitized natural actomyosin and the myosin light chain kinase (MLCK) action on gizzard myosin. 2) The superprecipitating activity of the 155 kDa component was 5 time higher than that of the 130 kDa component on the basis of equivalent MLCK activity. 3) The same procedure was applied to bovine stomach, giving rise to a 155 kDa component in a homogeneous state as in the case of aorta, but the 130 kDa component thus prepared was contaminated by higher molecular weight components. 4) If compared on the basis of equivalent MLCK activity, bovine stomach 155 kDa component showed more than 10 times higher superprecipitating activity than the fraction that contained the 130 kDa component as the main constituent. 5) The discrepancy between the superprecipitating activity and MLCK activity mentioned above was discussed in relation to the Ca2+ regulation mechanism in smooth muscle contraction. The possibility that the 130 kDa component might be a proteolytic product of the 155 kDa component was also discussed.

Purification of a protein kinase phosphorylating myosin regulatory light chain-a (RLC-a) from smooth muscle of scallop, Patinopecten yessoensis.

A protein kinase activity phosphorylating regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was found to be present in scallop smooth muscle homogenate. The kinase was purified to homogeneity and named RLC-a myosin kinase (aMK). aMK was extracted from the muscle homogenate with a low salt solution and was purified by successive DE-32 ion exchange chromatography, gel filtration on Ultrogel AcA 44, and affinity chromatography on Sepharose 4B-6-aminohexyl-1-pyrophosphate. The molecular weight of aMK was estimated to be 40-kDa from the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 35-kDa from the elution volume on Sephadex G-150 gel filtration. The phosphorylation site of RLC-a by aMK was determined to be Ser residue(s). Only RLC-a was phosphorylated; the other regulatory light chain, RLC-b, was not. The phosphorylatable Ser of RLC-a is, therefore, considered to be Ser-11, which is located in the N-terminal region having a different amino acid sequence from that of RLC-b. RLC-a was phosphorylated by aMK 3 times faster in the free state than in the bound state to myosin. aMK does not require calmodulin and is rather inhibited by CaCl2.

Purification of caldesmon and myosin light chain (MLC) kinase from arterial smooth muscle: comparisons with gizzard caldesmon and MLC kinase.

We have developed a simple and conventional purification method for caldesmon and MLC kinase from bovine arterial smooth muscle, and compared the arterial and gizzard proteins. Arterial caldesmon shares the alternative binding to calmodulin or F-actin in a Ca2+-dependent manner and the antigenic determinants with the gizzard protein. Both caldesmons have the same association constant with F-actin (1.3-1.7 X 10(7) M-1) and the same maximum binding (1 caldesmon per 12-14 actins). However, the molecular weight of arterial caldesmon (dimer of a 148 kDa polypeptides) was slightly different from that of gizzard caldesmon (heterodimer of 150/147 kDa polypeptides). The molecular weight of arterial MLC kinase (160 kDa) was much larger than that of the gizzard enzyme (135 kDa). The enzyme activities of both MLC kinases were comparable (Km = 9.5 microM, Vmax = 12.5 mumol/min X mg). The association constant of the arterial enzyme to F-actin (5.1 X 10(6) M-1) was much larger than that of the gizzard enzyme (9.0 X 10(5) M-1) but the maximum binding was the same (1 enzyme per 12-13 actins). Immunocytochemical examinations showed that caldesmon and MLC kinase in cultured arterial cells have a restricted localization along the stress fibers, suggesting functional linkages between both proteins and actin filaments in vivo.

Anti-gizzard MLCK monoclonal antibody MM13 inhibits superprecipitation and phosphorylation of bovine aortic smooth muscle actomyosin.

We prepared monoclonal antibodies directed against chicken gizzard myosin light chain kinase (MLCK) and used them to study the contractile system of aortic smooth muscle. One monoclonal antibody, MM13, dose dependently inhibited actomyosin superprecipitation of bovine aortic smooth muscle, in accord with the suppression of 20 kDa myosin light chain phosphorylation by endogenous kinase. Immunoblotting analysis demonstrated that MM13 cross-reacted with the 150,000 Mr peptide of bovine aortic actomyosin preparation. The bovine aortic MLCK was purified approximately 2,400-fold to apparent homogeneity by three steps of column chromatography. The purified enzyme has a molecular weight of 150,000 and a slower mobility than chicken gizzard MLCK (130,000 Mr), as determined by SDS-polyacrylamide gel electrophoresis. MM13 also cross-reacted with purified bovine aortic MLCK and inhibited the kinase activity, in vitro. We interpret these findings to mean that binding of the anti-gizzard MLCK monoclonal antibody directly to aortic smooth muscle MLCK (150,000 Mr) decreases the phosphorylation of the 20 kDa myosin light chain, thus suppressing the aortic smooth muscle myosin-actin interaction.

Effect of vanadate on force and myosin light chain phosphorylation in skinned aortic smooth muscle.

The effects of vanadate were examined on Ca2+-activated force and phosphorylation of 20-kDa myosin light chain in membrane-permeabilized rabbit aortic smooth muscle strips. Addition of vanadate during maximum contraction reduced the force in a dose-dependent manner, and inhibited it almost completely at 1 mM. Two-dimensional polyacrylamide gel electrophoretic analyses revealed that vanadate also reduced the phosphorylation of 20- kDa myosin light chain in a dose-dependent manner from approximately 50% in the absence of vanadate to approximately 20% in the presence of 1 mM vanadate. The effects of 1 mM vanadate on purified myosin light chain kinase and phosphatase were then examined using purified myosin as substrate, and it was found that vanadate neither inhibited myosin light chain kinase nor activated myosin light chain phosphatase. These results indicate that the reduction in the 20-kDa myosin light chain phosphorylation level by vanadate may be effected through its inhibition of the force generation in skinned smooth muscle strip, as evidenced by the finding that vanadate eliminated the enhancement of myosin light chain kinase activity brought about by the interaction between purified myosin and actin.

Increased myosin light chain kinase content in sensitized canine saphenous vein.

Our previous studies revealed that smooth muscle from sensitized canine saphenous vein (SCSV) demonstrated greater active shortening capacity, maximum shortening velocity, and prolonged relaxation vis-a-vis the control muscle. These changes could be responsible for the in vivo hyperreactivity of venous smooth muscle observed in anaphylactic shock. Because smooth muscle cross-bridge cycling is regulated by myosin light chain kinase (MLCK)-dependent phosphorylation of the 20-kDa myosin light chain (MLC20), we studied MLC20 and MLCK phosphorylation in homogenates of SCSV and veins from littermate control dogs. We found that phosphorylation of MLC20 in SCSV homogenate was higher (42.26 +/- 5.10%) compared with control homogenates (26.69 +/- 3.30%; P < 0.05); MLCK content was significantly higher in SCSV homogenates [0.169 +/- 0.019 (SE) mu g/mg protein] than in control homogenates (0.075 +/- 0.004 mu g/mg protein; P < 0.05). Total MLCK activity increased from 6.16 +/- 0.60 x 10(-5) nmol Pi x mg fresh weight of tissue-1 x min-1 in control homogenates to 12.50 +/- 2.50 x 10(-5) nmol Pi x mg fresh weight of tissue-1 x min-1 in sensitized homogenates (P < 0.05). Specific MLCK activity was, however, similar in sensitized and control homogenates. The results of our study suggest that elevation of MLCK content in the homogenate could account for the increased contractility of the SCSV in anaphylactic shock.

Ca(2+)-dependent phosphorylation of the tail domain of myosin-V, a calmodulin-binding myosin in vertebrate brain.

1. Myosin-V from vertebrate brain is a novel molecular motor with a myosin-like head domain, a calmodulin-binding neck region and a unique tail domain of unknown function. Previous studies showed brain myosin-V to be a phosphoprotein substrate for Ca2+/calmodulin-dependent protein kinase associated with actomyosin. In the present study we describe the preparation of a specific actin-cytoskeletal fraction which is enriched in brain myosin-V. 2. We show that Ca2+/calmodulin-dependent protein kinase activity is also associated with this preparation and phosphorylates brain myosin-V. 3. Calpain, a Ca(2+)-dependent protease, generates a M(r) 80,000 fragment from the COOH terminal region of brain myosin-V containing most or all of the phosphorylation sites. 4. These results suggest that the unique tail domain of this novel myosin is subject to Ca2+ control via phosphorylation by kinase activity associated with the actin cytoskeleton.