Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse

Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) initiates smooth muscle contraction and regulates actomyosin-based cytoskeletal functions in nonmuscle cells. The net extent of RLC phosphorylation is controlled by MLCK activity relative to myosin light chain phosphatase activity. We have constructed a CaM-sensor MLCK where Ca(2+)-dependent CaM binding increases the catalytic activity of the kinase domain, whereas coincident binding to the biosensor domain decreases fluorescence resonance energy transfer between two fluorescent proteins. We have created transgenic mice expressing this construct specifically in smooth muscle cells to perform real-time evaluations of the relationship between smooth muscle contractility and MLCK activation in intact tissues and organs. Measurements in intact bladder smooth muscle demonstrate that MLCK activation increases rapidly during KCl-induced contractions but is not maximal, consistent with a limiting amount of cellular CaM. Carbachol treatment produces the same amount of force development and RLC phosphorylation, with much smaller increases in [Ca(2+)](i) and MLCK activation. A Rho kinase inhibitor suppresses RLC phosphorylation and force but not MLCK activation in carbachol-treated tissues. These observations are consistent with a model in which the magnitude of an agonist-mediated smooth muscle contraction depends on a rapid but limited Ca(2+)/CaM-dependent activation of MLCK and Rho kinase-mediated inhibition of myosin light chain phosphatase activity. These studies demonstrate the feasibility of producing transgenic biosensor mice for investigations of signaling processes in intact systems.     

Regulation of scallop myosin by the regulatory light chain depends on a single glycine residue.

Specific Ca2+ binding and Ca2+ activation of ATPase activity in scallop myosin require a regulatory light chain (RLC) from regulated (molluscan or vertebrate smooth) myosin; hybrids containing vertebrate skeletal RLCs do not bind Ca2+ and their ATPase activity is inhibited. Chimeras between scallop and chicken skeletal RLCs restore Ca2+ sensitivity to RLC-free myosin provided that residues 81-117 are derived from scallop. Six mutants (R90M, A94K, D98P, N105K, M116Q, and G117C) were generated by replacing amino acids of the scallop RLC with the corresponding skeletal RLC residues in positions conserved in either regulated or nonregulated myosins. Ca2+ binding was abolished by a G117C and a G117A mutation; however, these mutants have a decreased affinity for the heavy chain. None of the other mutations affected RLC function. Replacement of the respective cysteine with glycine in the skeletal RLC has markedly changed the regulatory properties of the molecule. The single cysteine to glycine mutation conferred to this light chain the ability to restore Ca2+ binding and regulated ATPase activity, although Ca2+ activation of the actin-activated ATPase was lower than with scallop RLC. The presence of amino acids other than glycine at this position in vertebrate skeletal myosin RLCs may explain why these are not fully functional in the scallop system. The results are in agreement with x-ray crystallography data showing the central role of G117 in stabilizing the Ca(2+)-binding site of scallop myosin.     

Site-directed spin labeling reveals a conformational switch in the phosphorylation domain of smooth muscle myosin

We have used site-directed spin labeling and EPR spectroscopy to detect structural changes within the regulatory light chain (RLC) of smooth muscle myosin upon phosphorylation. Smooth muscle contraction is activated by phosphorylation of S19 on RLC, but the structural basis of this process is unknown. There is no crystal structure containing a phosphorylated RLC, and there is no crystal structure for the N-terminal region of any RLC. Therefore, we have prepared single-Cys mutations throughout RLC, exchanged each mutant onto smooth muscle heavy meromyosin, verified normal regulatory function, and used EPR to determine dynamics and solvent accessibility at each site. A survey of spin-label sites throughout the RLC revealed that only the N-terminal region (first 24 aa) shows a significant change in dynamics upon phosphorylation, with most of the first 17 residues showing an increase in rotational amplitude. Therefore, we focused on this N-terminal region. Additional structural information was obtained from the pattern of oxygen accessibility along the sequence. In the absence of phosphorylation, little or no periodicity was observed, suggesting a lack of secondary structural order in this region. However, phosphorylation induced a strong helical pattern (3.6-residue periodicity) in the first 17 residues, while increasing accessibility throughout the first 24 residues. We have identified a domain within RLC, the N-terminal phosphorylation domain, in which phosphorylation increases helical order, internal dynamics, and accessibility. These results support a model in which this disorder-to-order transition within the phosphorylation domain results in decreased head-head interactions, activating myosin in smooth muscle.     

Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction

Repetitive stimulation potentiates contractile tension of fast-twitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca(2+)/calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fast-twitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca(2+)/calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.     

Filament structure as an essential factor for regulation of Dictyostelium myosin by regulatory light chain phosphorylation

Phosphorylation of the regulatory light chain (RLC) activates the actin-dependent ATPase activity of Dictyostelium myosin II. To elucidate this regulatory mechanism, we characterized two mutant myosins, MyΔC1225 and MyΔC1528, which are truncated at Ala-1224 and Ser-1527, respectively. These mutant myosins do not contain the C-terminal assembly domain and thus are unable to form filaments. Their activities were only weakly regulated by RLC phosphorylation, suggesting that, unlike smooth muscle myosin, efficient regulation of Dictyostelium myosin II requires filament assembly. Consistent with this hypothesis, wild-type myosin progressively lost the regulation as its concentration in the assay mixture was decreased. Dephosphorylated RLC did not inhibit the activity when the concentration of myosin in the reaction mixture was very low. Furthermore, 3xAsp myosin, which does not assemble efficiently due to point mutations in the tail, also was less well regulated than the wild-type. We conclude that the activity in the monomer state is exempt from inhibition by the dephosphorylated RLC and that the complete regulatory switch is formed only in the filament structure. Interestingly, a chimeric myosin composed of Dictyostelium heavy meromyosin fused to chicken skeletal light meromyosin was not well regulated by RLC phosphorylation. This suggests that, in addition to filament assembly, some specific feature of the filament structure is required for efficient regulation.     

The motor domain and the regulatory domain of myosin solely dictate enzymatic activity and phosphorylation-dependent regulation, respectively

While the structures of skeletal and smooth muscle myosins are homologous, they differ functionally from each other in several respects, i.e., motor activities and regulation. To investigate the molecular basis for these differences, we have produced a skeletal/smooth chimeric myosin molecule and analyzed the motor activities and regulation of this myosin. The produced chimeric myosin is composed of the globular motor domain of skeletal muscle myosin (Met¹–Gly⁷⁷³) and the C-terminal long α-helix domain of myosin subfragment 1 as well as myosin subfragment 2 (Gly⁷⁷³–Ser¹¹⁰⁴) and light chains of smooth muscle myosin. Both the actin-activated ATPase activity and the actin-translocating activity of the chimeric myosin were completely regulated by light chain phosphorylation. On the other hand, the maximum actin-activated ATPase activity of the chimeric myosin was the same as skeletal myosin and thus much higher than smooth myosin. These results show that the C-terminal light chain-associated domain of myosin head solely confers regulation by light chain phosphorylation, whereas the motor domain determines the rate of ATP hydrolysis. This is the first report, to our knowledge, that directly determines the function of the two structurally separated domains in myosin head.     

Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry

Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in sarcomeric proteins including the myosin regulatory light chain (RLC). Two such FHC mutations, R58Q and N47K, located near the cationic binding site of the RLC, have been identified from population studies. To examine the molecular basis for the observed phenotypes, we exchanged endogenous RLC from native porcine cardiac myosin with recombinant human ventricular wild type (WT) or FHC mutant RLC and examined the ability of the reconstituted myosin to propel actin filament sliding using the in vitro motility assay. We find that, whereas the mutant myosins are indistinguishable from the controls (WT or native myosin) under unloaded conditions, both R58Q- and N47K-exchanged myosins show reductions in force and power output compared with WT or native myosin. We also show that the changes in loaded kinetics are a result of mutation-induced loss of myosin strain sensitivity of ADP affinity. We propose that the R58Q and N47K mutations alter the mechanical properties of the myosin neck region, leading to altered load-dependent kinetics that may explain the observed mutant-induced FHC phenotypes.     

Cloning and characterization of a vertebrate cellular myosin regulatory light chain complementary DNA

We have isolated two series of complementary DNAs (cDNAs) from a chicken gizzard cDNA library encoding two isoforms of phosphorylatable myosin regulatory light chain (RLC). One of the cDNAs encodes a previously isolated smooth muscle myosin RLC (also referred to as LC20-A); the other encodes a protein that shares 92% homology with the LC20-A isoform. The phosphorylatable threonine and serine residues at positions 18 and 19 of the two myosin RLC sequences are conserved. The two cDNAs are 81% homologous at the nucleotide level over the coding region; the 5' and 3' untranslated regions are divergent. Most of the DNA nonhomology in the coding region does not affect the protein sequence, indicating strong evolutionary conservation pressure to maintain the myosin RLC structure. Northern blot analysis using 3' untranslated region probes reveals restrictive tissue specific expression of one myosin RLC isoform (LC20-A) in smooth muscle tissue and not in other tissues examined. In contrast, the novel myosin RLC isoform messenger RNA (mRNA) is uniformly expressed in all smooth and nonmuscle tissues examined and is designated as cellular myosin RLC for this reason. Our results indicate that cellular and smooth muscle myosin RLC isoforms are distinct and are encoded by separate genes. This report describes the cloning of a novel vertebrate cellular myosin RLC mRNA that differs from previously characterized smooth muscle RLC isoform mRNAs in both primary sequence and expression pattern.     

Myosin light chain kinase inhibitors can block invasion and adhesion of human pancreatic cancer cell lines.

INTRODUCTION: Invasion and metastasis of pancreatic cancer (PC) require cell motility and adhesion, which depend on the activity of cytoskeleton. A cytoskeletal component indispensable for these processes is myosin II, the cytoplasmic analogue of smooth and skeletal muscle myosin. AIMS AND METHODOLOGY: Because the activity of myosin II is accelerated by phosphorylation of myosin II on its regulatory light chain (RLC) by myosin light chain kinase (MLCK), we used two specific MLCK inhibitors, ML-7 and ML-9, for suppression of motility and adhesion of PC cell lines. RESULTS: Both drugs were potent inhibitors, as measured by in vitro motility assay and adhesion assay. When treated with the same concentration of ML-7, the PC cells were rounded up, and the number of stress fibers was reduced markedly. The in vitro migration and adhesion of PC cells were inhibited by ML-7 and ML-9 in a dose-dependent manner, supporting a specific and competitive inhibition of MLCK by these drugs. The inhibition occurred at nontoxic concentrations. CONCLUSIONS: These results highlight the importance of myosin II in the invasion and metastasis of PC cells and suggest the possibility that blocking of myosin II activity by a specific MLCK inhibitor may be a therapeutic strategy for preventing the invasion and metastasis of PC.     

Regulatory light chain mutations associated with cardiomyopathy affect myosin mechanics and kinetics

The myosin regulatory light chain (RLC) wraps around the alpha-helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis, with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K show reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation.     

Effect of phosphorylation of smooth muscle myosin on actin activation and Ca2+ regulation.

A 35--70% ammonium sulfate fraction of smooth muscle actomyosin was prepared from guinea pig vas deferens. This fraction also contains a smooth muscle myosin kinase and a phosphatase that phosphorylates and dephosphorylates, respectively, the 20,000-dalton light chain of smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin. Phosphorylated and dephosphorylated smooth muscle myosin were purified from this ammonium sulfate fraction by gel filtration, which also separated the kinase and the phosphatase from the myosin. Purified phosphorylated and dephosphorylated myosin have identical stained patterns after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. They also have similar ATPase activities measured in 0.5 M KCl in the presence of K+-EDTA and Ca2+. However, the actin-activated myosin ATPase activity is markedly increased after phosphorylation. Moreover, the actin-activated ATPase activity of phosphorylated myosin is inhibited by the removal of Ca2+ in the absence of any added regulatory proteins. Dephosphorylation of myosin results in a decrease in the actin-activated ATPase activity. Skeletal muscle tropomyosin markedly increased the actin-activated ATPase activity of phosphorylated but not dephosphorylated myosin in the presence, but not in the absence, of Ca2+.     

Slow cycling of unphosphorylated myosin is inhibited by calponin, thus keeping smooth muscle relaxed

A key unanswered question in smooth muscle biology is whether phosphorylation of the myosin regulatory light chain (RLC) is sufficient for regulation of contraction, or if thin-filament-based regulatory systems also contribute to this process. To address this issue, the endogenous RLC was extracted from single smooth muscle cells and replaced with either a thiophosphorylated RLC or a mutant RLC (T18A/S19A) that cannot be phosphorylated by myosin light chain kinase. The actin-binding protein calponin was also extracted. Following photolysis of caged ATP, cells without calponin that contained a nonphosphorylatable RLC shortened at 30% of the velocity and produced 65% of the isometric force of cells reconstituted with the thiophosphorylated RLC. The contraction of cells reconstituted with nonphosphorylatable RLC was, however, specifically suppressed in cells that contained calponin. These results indicate that calponin is required to maintain cells in a relaxed state, and that in the absence of this inhibition, dephosphorylated cross-bridges can slowly cycle and generate force. These findings thus provide a possible framework for understanding the development of latch contraction, a widely studied but poorly understood feature of smooth muscle.     

Alteration of cross-bridge kinetics by myosin light chain phosphorylation in rabbit skeletal muscle: implications for regulation of actin-myosin interaction.

Myosin light chain phosphorylation in permeable skeletal muscle fibers increases isometric force and the rate of force production at submaximal levels of calcium activation; myosin light chain phosphorylation may underlie the increased rate and extent of force production associated with isometric twitch potentiation in intact fibers. To understand the mechanism by which myosin light chain phosphorylation manifests these effects, we have measured isometric force, isometric stiffness, rate of isometric force redevelopment after isotonic shortening, and isometric ATPase activity in permeabilized rabbit psoas muscle fibers. These measurements were made in the presence and absence of myosin light chain phosphorylation over a range of calcium concentrations that caused various levels of activation. The results were analyzed with a two-state cross-bridge cycle model as suggested by Brenner [Brenner, B. (1988) Proc. Natl. Acad. Sci. USA 85, 3265-3269]. The results indicate that myosin light chain phosphorylation exerts its effect on force generation and the isometric rate of force redevelopment in striated muscle through a single mechanism, namely, by increasing the rate constant describing the transition from non-force-generating cross-bridges to force-generating states (fapp). gapp, the reverse rate constant, is unaffected by phosphorylation as are the number of cycling cross-bridges. Since both calcium and myosin light chain phosphorylation increase fapp, the possibility is considered that modulation of fapp may represent a general mechanism for regulating force in actin-myosin systems.     

Crystal structures of human cardiac beta-myosin II S2-Delta provide insight into the functional role of the S2 subfragment

Myosin II is the major component of the muscle thick filament. It consists of two N-terminal S1 subfragments ("heads") connected to a long dimeric coiled-coil rod. The rod is in itself twofold symmetric, but in the filament, the two heads point away from the filament surface and are therefore not equivalent. This breaking of symmetry requires the initial section of the rod, subfragment 2 (S2), to be relatively flexible. S2 is an important functional element, involved in various mechanisms by which the activity of smooth and striated muscle is regulated. We have determined crystal structures of the 126 N-terminal residues of S2 from human cardiac beta-myosin II (S2-Delta), of both WT and the disease-associated E924K mutant. S2-Delta is a straight parallel dimeric coiled coil, but the N terminus of one chain is disordered in WT-S2-Delta due to crystal contacts, indicative of unstable local structure. Bulky noncanonical side chains pack into a/d positions of S2-Delta's N terminus, leading to defined local asymmetry and axial stagger, which could induce nonequivalence of the S1 subfragments. Additionally, S2 possesses a conserved charge distribution with three prominent rings of negative potential within S2-Delta, the first of which may provide a binding interface for the "blocked head" of smooth muscle myosin in the OFF state. The observation that many disease-associated mutations affect the second negatively charged ring further suggests that charge interactions play an important role in regulation of cardiac muscle activity through myosin-binding protein C.     

Myosin phosphorylation decreases the ATPase activity of cardiac myofibrils

Our previous work showed that myosin phosphorylation decreased the ATPase activity of skeletal muscle myofibrils that were lightly fixed with glutaraldehyde. The fixation process prevented sarcomere shortening and destruction of the ordered filament array upon the addition of ATP. We have now extended these results to myofibrils prepared from hearts of rabbits, dogs and rats. Myofibrils were phosphorylated by incubation with myosin light chain kinase, calmodulin and either ATP-gamma s or ATP, for 15 minutes at 25 degrees C. The extent of myosin light chain phosphorylation was 50% to 80%. The ATPase activity of unphosphorylated myofibrils was not altered by reaction with 0.01% glutaraldehyde for 5 minutes at 0 degrees C, and the ATPase activity of unfixed myofibrils was not changed by phosphorylation. However, phosphorylation decreased the ATPase activity of fixed myofibrils by 50%. The effect on myocardial myofibrillar ATPase activity of phosphorylation was similar in the three animal species. These results suggest that in both skeletal and cardiac muscle, myosin phosphorylation decreases the rate of cross-bridge cycling resulting in decreased energy expenditure. It also appears that the effect of myosin light chain phosphorylation on ATPase activity requires an ordered myofilament structure.     

Calcium-sensitive regulation of actin-myosin interactions in baby hamster kidney (BHK-21) cells.

A fraction has been obtained from baby hamster kidney (BHK-21) cells that will stimulate the actin-moderated ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity of both BHK-21 myosin and gizzard smooth muscle myosin. This activation is associated with the specific phosphorylation of the myosin 20,000-dalton light chain. The BHK-21 myosin light chain kinase preparation contains a major protein of approximately 105,000 molecular weight as determined by sodium dodecyl sulfate gel electrophoresis. Both the actin activation and phosphorylation events require the presence of Ca2+ and the so-called modulator or calcium-dependent regulator protein that has been isolated from smooth muscle, brain, and other tissues. On the basis of these results we propose that this kinase system constitutes a Ca2+-dependent regulatory mechanism for myosin-actin interactions in nonmuscle mammalian cells.     

Caldesmon and a 20-kDa actin-binding fragment of caldesmon inhibit tension development in skinned gizzard muscle fiber bundles.

Caldesmon is known to inhibit actin-activated myosin ATPase activity in solution, to inhibit force production when added to skeletal muscle fibers, and to alter actin movement in the in vitro cell motility assay. It is less clear that caldesmon can inhibit contraction in smooth muscle cells in which caldesmon is abundant. We now show that caldesmon and its 20-kDa actin-binding fragment are able to inhibit force in chemically skinned gizzard fiber bundles, which are activated by a constitutively active myosin light-chain kinase in the presence and absence of okadaic acid. This inhibitory effect is reversed by high concentrations of Ca2+ and calmodulin. Therefore, caldesmon may act by increasing the level of myosin phosphorylation required to obtain full activation. Our results also suggest that caldesmon does not act to maintain force in smooth muscle by cross-linking myosin with actin since competition of binding of caldesmon with myosin does not cause a reduction in tension.     

Myosin Light Chain Kinase Is Involved in the Mechanism of Gastrointestinal Dysfunction in Diabetic Rats

Background  

It is well established that smooth muscle contractility is regulated by an elevation of cytosolic Ca²⁺ via myosin light chain phosphorylation, which is activated by myosin light chain kinase (MLCK). Recently, MLCK has been demonstrated to play an important role in smooth muscle contraction and normal gastrointestinal motility.     

Scallop striated and smooth muscle myosin heavy-chain isoforms are produced by alternative RNA splicing from a single gene.

We report here that the catch and striated adductor muscle myosin heavy-chain (MHC) isoforms of scallop (Argopecten irradians, previously Aequipecten irradians) are generated by alternative RNA splicing from a single gene. Scallop catch muscle cDNA and genomic DNA were amplified by PCR using primers based on the previously sequenced scallop striated muscle MHC cDNA. Mapping of the exon/intron borders and sequencing of a full-length catch muscle MHC in overlapping fragments revealed that the 24-kb gene encodes the MHC polypeptide in 27 exons and that four sets of tandem exon pairs are alternatively spliced into a striated and a catch MHC isoform. An additional alternative exon was identified in catch cDNA and is apparently spliced into a minor MHC isoform. The striated muscle-specific isoform is not expressed in other tissues, whereas the catch-type isoforms were also detected in various smooth muscles, but not in the striated one. Of the alternative exons, exons 5 and 6 encode part of the ATP-binding region and the 25-kDa/50-kDa proteolytic junction; exon 13 encodes part of one of the actin-binding regions and extends to the active site; exon 20 encodes the middle of the rod hinge region; exon 26 in the striated-specific sequence starts with the stop codon, whereas the catch-specific exon codes for an additional 10 residues. Differences between the alternative exons presumably determine the lower ATPase activity of smooth muscle myosin, contribute to the different structure of the striated and smooth muscle thick filaments, and may also be important for the molecular mechanism of the catch phenomenon.