Okadaic acid uncouples myosin light chain phosphorylation and tension in smooth muscle

Tracheal smooth muscle precontracted with carbachol relaxes upon the addition of 3 μM okadaic add. Although cytosolic Ca²⁺ concentrations decrease, myosin light chain remains highly phosphorylated (50%). In smooth muscle treated with carbachol alone or carbachol plus okadaic acid ³²P is incorporated into a single peptide on myosin light chain which corresponds to the site phosphorylated by myosin light chain kinase. Treatment with okadaic acid alone does not result in myosin light chain phosphorylation or tension development. These results suggest that a cellular mechanism other than myosin light chain phosphorylation can regulate contractile tension.     

Chemical energy usage and myosin light chain phosphorylation in mammalian smooth muscle

Experiments have been done to determine the relationships among active force output, average rate of high-energy phosphate utilization, and the degree of phosphorylation of the 20,000-dalton myosin light chain in the rabbit tenia coli at 18 C. During an isometric tetanus at l0 the degree of light chain phosphorylation increases to a maximum of 30-40% before maximum force is developed, and then phosphorylation slowly decreases while active force is maintained. During the period when there is a small decrease in degree of phosphorylation, the average rate of chemical energy usage falls by fourfold. In contrast, when the calcium concentration of the bathing medium is lowered from 1.9 to 1.0 mM a very large decrease in degree of phosphorylation is associated with only a small decrease in both energy usage and active force. At lower calcium levels both force and chemical energy usage decrease proportionately with little further decrease in degree of phosphorylation. We conclude that under isometric conditions there is no consistent relationship between degree of myosin light chain phosphorylation and the rate of cross-bridge cycling as measured by the rate of high-energy phosphate usage in this mammalian smooth muscle.     

A cAMP-dependent regulatory protein for RLC-a myosin kinase catalyzing the phosphorylation of scallop smooth muscle myosin light chain

A cAMP-dependent regulatory protein which modulates the phosphorylation of scallop myosin regulatory light chain-a (RLC-a) by RLC-a myosin kinase (aMK) (Sohma, H. & Morita, F. (1986) J. Biochem. 100, 1155-1163) was purified from the scallop smooth muscle. RLC-a is abundant in the opaque portion of scallop smooth muscle, one of the catch muscles. The regulatory protein for aMK was purified by employing successively DEAE Toyopearl ion exchange chromatography, Sepharose 4B-8(6-aminohexylamino)cAMP affinity chromatography, and Sephadex G 100 gel filtration. The molecular mass of the regulatory protein was 41 kDa, based on the mobility in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. With increasing amounts of the regulatory protein, the aMK activity decreased, and complete inhibition was observed at the concentration of twice that of aMK. The aMK activity inhibited by the regulatory protein was restored by the addition of cAMP. These results suggest that aMK is similar to a catalytic subunit of cAMP-dependent protein kinase, and the protein reported here is similar to its regulatory subunit. aMK may exist as an inactive form, as a combination with this regulatory protein, in vivo and be deinhibited by an increase in the intracellular concentration of cAMP. We discuss a possible correlation between the phosphorylation of RLC-a in myosin catalyzed by aMK and the catch state of the opaque portion of scallop smooth muscle.     

Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle

We tested the hypothesis that increases in force at a given cytosolic Ca(2+) concentration (i.e., Ca(2+) sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in alpha-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 microM Ca(2+) in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 microM) plus GTP (1 microM) significantly shifted the Ca(2+)-force relationship curve to the left (EC(50): 0.39 +/- 0.06 to 0.078 +/- 0.006 microM Ca(2+)) and significantly increased the maximum force (104.4 +/- 4.8 to 120.2 +/- 2.8%; n = 6 observations). The Ca(2+)-rMLC phosphorylation relationship curve was also shifted to the left (EC(50): 1.26 +/- 0.57 to 0.13 +/- 0.04 microM Ca(2+)) and upward (maximum rMLC phosphorylation: 70.9 +/- 7.9 to 88.5 +/- 5. 1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca(2+) concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca(2+) sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.     

Stretch-induced myosin light chain phosphorylation and stretch-release-induced tension development in arterial smooth muscle

Stretching arteries from resting length to 1.7 times the resting length increased myosin light chain phosphorylation from 40 to 70% in a graded fashion, reaching a plateau at 1.6 times the resting length. When the fully stretched arteries were released, active tension developed without any exogenous stimulating agent. This stretch-release-induced tension approached the same magnitude as that of the control K+-induced tension. Stretch-induced phosphorylation and the subsequent tension development upon release of stretch were prevented by incubating the arteries in physiological salt solutions containing ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or chlorpromazine. The inhibition produced by EGTA was reversible. Stretch-induced phosphorylation decreased as a function of time, regardless of whether stretch was maintained, or slackened slowly, or released quickly. While tension developed upon release of stretch, light chain phosphorylation simultaneously decreased. As tension reached and maintained its maximal value, phosphorylation continued to decrease. Thus, light chain phosphorylation is necessary for activation of arterial muscle contraction, but it need not be maintained during tension development or maintenance.     

Influence of smooth muscle myosin conformation on myosin light chain kinase binding and on phosphorylation

Conventional smooth muscle myosin preparations contain a tightly bound myosin light chain kinase activity, which is incompletely removed by gel filtration at high ionic strength. We show here that by contrast, this kinase activity is released, together with calmodulin, under conditions in which myosin is in the folded configuration. The conformation-related release of kinase occurred for dephosphorylated myosin in both the presence and absence of ATP and Ca2+. Binding of kinase to extended phosphorylated myosin was relatively weaker than to dephosphorylated myosin, but was nonetheless detected. The kinetic consequences of this binding behaviour were determined by measuring initial myosin phosphorylation rates as a function of KCl concentration. Rate optima occurred at 60 mM KCl and 300 mM KCl, conditions favouring respectively stable filaments and stable extended monomers. Phosphorylation of the folded monomer was uniformly slow at low KCl concentrations. The folded myosin monomer is thus a relatively poor substrate for the kinase, and is therefore unlikely to represent an analog of the relaxed crossbridge configuration in myosin filaments.     

Functional role of the C-terminal domain of smooth muscle myosin light chain kinase on the phosphorylation of smooth muscle myosin

Smooth muscle myosin light chain kinase (MLCK) is known to bind to thin filaments and myosin filaments. Telokin, an independently expressed protein with an identical amino acid sequence to that of the C-terminal domain of MLCK, has been shown to bind to unphosphorylated smooth muscle myosin. Thus, the functional significance of the C-terminal domain and the molecular morphology of MLCK were examined in detail. The C-terminal domain was removed from MLCK by alpha-chymotryptic digestion, and the activity of the digested MLCK was measured using myosin or the isolated 20-kDa light chain (LC20) as a substrate. The results showed that the digestion increased K(m) for myosin 3-fold whereas it did not change the value for LC20. In addition, telokin inhibited the phosphorylation of myosin by MLCK by increasing K(m) but only slightly increased K(m) for LC20. Electron microscopy indicated that MLCK was an elongated molecule but was flexible so as to form folded conformations. MLCK was crosslinked to unphosphorylated heavy meromyosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the absence of Ca(2+)/calmodulin (CaM), and electron microscopic observation of the products revealed that the MLCK molecule bound to the head-tail junction of heavy meromyosin. These results suggest that MLCK binds to the head-tail junction of unphosphorylated myosin through its C-terminal domain, where LC20 can be promptly phosphorylated through its catalytic domain following the Ca(2+)/CaM-dependent activation.     

Role of the essential light chain in the activation of smooth muscle myosin by regulatory light chain phosphorylation

The activity of smooth and non-muscle myosin II is regulated by phosphorylation of the regulatory light chain (RLC) at serine 19. The dephosphorylated state of full-length monomeric myosin is characterized by an asymmetric intramolecular head–head interaction that completely inhibits the ATPase activity, accompanied by a hairpin fold of the tail, which prevents filament assembly. Phosphorylation of serine 19 disrupts these head–head interactions by an unknown mechanism. Computational modeling (Tama et al., 2005. J. Mol. Biol. 345, 837–854) suggested that formation of the inhibited state is characterized by both torsional and bending motions about the myosin heavy chain (HC) at a location between the RLC and the essential light chain (ELC). Therefore, altering relative motions between the ELC and the RLC at this locus might disrupt the inhibited state. Based on this hypothesis we have derived an atomic model for the phosphorylated state of the smooth muscle myosin light chain domain (LCD). This model predicts a set of specific interactions between the N-terminal residues of the RLC with both the myosin HC and the ELC. Site directed mutagenesis was used to show that interactions between the phosphorylated N-terminus of the RLC and helix-A of the ELC are required for phosphorylation to activate smooth muscle myosin.     

Neuropeptide Y stimulation of myosin light chain phosphorylation in cultured aortic smooth muscle cells

Neuropeptide Y (NPY) is released from an extensive network of postganglionic sympathetic perivascular neurons. NPY has been shown to affect vascular tone postsynaptically by 1) directly stimulating contraction; 2) inhibiting vasorelaxation; and 3) potentiating contraction elicited by exogenous vasoconstrictors. The molecular mechanisms mediating these effects of NPY are undefined. Therefore, we examined the possibility that NPY could stimulate smooth muscle contraction through myosin light chain phosphorylation in cultured porcine aortic smooth muscle cells. NPY (100 nM) caused a rapid, transient increase in myosin light chain (MLC) phosphorylation, an important regulatory event in the initiation of smooth muscle contraction. NPY-stimulated MLC phosphorylation was prevented by preincubation of cells with pertussis toxin and was independent of extracellular Ca2+. In parallel studies, NPY alone had no detectable effect on cellular cAMP or cGMP content; however, NPY potently inhibited forskolin-stimulated cAMP accumulation (IC50 = 0.03 nM) through a pertussis toxin-sensitive pathway. NPY had no detectable effect on basal phosphoinositide hydrolysis or protein kinase C activation but enhanced angiotensin II-stimulated production of inositol phosphates and activation of protein kinase C. These results indicate that NPY-stimulated MLC phosphorylation can occur in the absence of detectable changes in cAMP content, cGMP content, inositol phosphate production, or protein kinase C activation; however, the interactions between NPY and other vasoactive agents may be mediated by the indirect effects of NPY on adenylate cyclase activity and phosphoinositide hydrolysis.     

Stretch-induced phosphorylation of the 20,000-dalton light chain of myosin in arterial smooth muscle

Stretching to 1.7 times the resting length of porcine carotid arteries reversibly prevents active tension development by K+ or norepinephrine stimulation. The 20,000-dalton light chain of myosin was maximally phosphorylated in the stretched noncontracting muscles, equal to that in the nonstretched contracting muscles challenged with K+ or norepinephrine. These results show that the contractile event is not a prerequisite for phosphorylation. Furthermore, stretching alone also induced maximal light chain phosphorylation even in the absence of K+ or norepinephrine. The stretch-induced light chain phosphorylation was not affected by exhaustive washing of the muscle with Ca2+-free physiological salt solution, treatment of the muscle with verapamil, or by a short exposure to ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Prolonged EGTA treatment abolished the stretch-induced light chain phosphorylation. All evidence suggests that upon stretch, Ca2+ is released from intracellular sources and this Ca2+ activates the myosin light chain kinase producing phosphorylation of the light chain.     

Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot–labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto–myosin and MLCK–myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting “stuck” on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.     

Biochemistry of smooth muscle myosin light chain kinase

The smooth muscle isoform of myosin light chain kinase (MLCK) is a Ca²⁺-calmodulin-activated kinase that is found in many tissues. It is particularly important for regulating smooth muscle contraction by phosphorylation of myosin. This review summarizes selected aspects of recent biochemical work on MLCK that pertains to its function in smooth muscle. In general, the focus of the review is on new findings, unresolved issues, and areas with the potential for high physiological significance that need further study. The review includes a concise summary of the structure, substrates, and enzyme activity, followed by a discussion of the factors that may limit the effective activity of MLCK in the muscle. The interactions of each of the many domains of MLCK with the proteins of the contractile apparatus, and the multi-domain interactions of MLCK that may control its behaviors in the cell are summarized. Finally, new in vitro approaches to studying the mechanism of phosphorylation of myosin are introduced.     

Calmodulin-linked equilibria in smooth muscle myosin light chain kinase

Competition experiments using 9-anthroylcholine, a fluorescent dye that undergoes calmodulin-dependent binding by smooth muscle myosin light chain kinase [Malencik, D. A., Anderson, S. R., Bohnert, J. L., & Shalitin, Y. S. (1982) Biochemistry 21, 4031], demonstrate a strongly stabilizing interaction between the adenosine 5'-triphosphate and myosin light chain binding sites operating within the enzyme-calmodulin complex but probably not in the free enzyme. The interactions in the latter case may be even slightly destabilizing. The fluorescence enhancement in solutions containing 5.0 microM each of the enzyme and calmodulin is directly proportional to the maximum possible concentration of bound calcium on the basis of four calcium binding sites. Evidently, all four calcium binding sites of calmodulin contribute about equally to the enhanced binding of 9-anthroylcholine by the enzyme. Fluorescence titrations on solutions containing 1.0 microM enzyme plus calmodulin yield a Hill coefficient of 1.2 and K = 0.35 +/- 0.08 microM calcium. Three proteolytic fragments of smooth muscle myosin light chain kinase, apparent products of endogenous proteolysis, were isolated and characterized. All three possess calmodulin-dependent catalytic activity. Their interactions with 9-anthroylcholine, in both the presence and absence of calmodulin, are similar to those of the native enzyme. However, the stabilities of their complexes with calmodulin vary. The corresponding dissociation constants range from 2.8 nM for the native enzyme and 8.5 nM for the 96K fragment to approximately 15 nM for the 68K and 90K fragments [0.20 N KCl, 50 mM 3-(N-morpholino)propanesulfonic acid, and 1 mM CaCl2, pH 7.3, 25 degrees C]. A coupled fluorometric assay, modified from a spectrophotometric assay for adenosine cyclic 3',5'-phosphate dependent protein kinase [Cook, P. F., Neville, M. E., Vrana, K. E., Hartl, F. T., & Roskoski, R. (1982) Biochemistry 21, 5794], has provided the first continuous recordings of myosin light chain kinase phosphotransferase activity. The results show that smooth muscle myosin light chain kinase is a responsive enzyme, whose activity adjusts rapidly to changes in solution conditions.     

Angiotensin II stimulates phosphorylation of the myosin light chain in cultured vascular smooth muscle cells

The vasoactive peptide angiotensin II stimulates phosphorylation of myosin light chain in 32P-labeled confluent cultures of vascular smooth muscle cells derived from rat mesenteric arteries. Myosin light chain was identified and its 32P-phosphorylation level quantitated following selective immunoprecipitation with an antiserum raised against purified human uterine smooth muscle myosin. Following exposure to 0.1 nM angiotensin II, phosphorylation of the light chain peaked at 4 min and then slowly decreased. The stimulation of light chain phosphorylation at 4 min is half-maximal at approximately 0.2 mM angiotensin II; the maximal response is approximately 210% of the unstimulated level. Basal myosin light chain phosphorylation was markedly reduced by incubation of cells with dibutyryl cyclic AMP or the calmodulin-inhibitor chlorpromazine. These data suggest that angiotensin II-mediated contraction in intact blood vessels involves phosphorylation of the myosin light chain, and that phosphorylation is inhibited by a cAMP-mediated process and may be calmodulin-dependent.     

Concanavalin A- and fetal-calf-serum-induced rounding and myosin light chain phosphorylation in cultured smooth muscle cells

Rabbit aorta smooth muscle cells (SMC) in long-term culture retracted in less than 10 min in response to a sequential order of stimulations by concanavalin A (Con A) and fetal calf serum (FCS). With additional continuous stimulation by FCS, the SMC took on a circular shape and were anchored to the substrate by retraction fibrils. This rounding was observed only when the cells were sequentially stimulated by Con A and FCS. Depletion of intracellular Ca2+ stores by the addition of EGTA and Ca2+ ionophores inhibited the rounding. Transient phosphorylation of MLC20 was observed in the initial stage during the SMC rounding. The extent of monophosphorylated MLC20 increased for up to 5 min to a maximal value of 49%. The diphosphorylated form reached a maximal value of 29% within 2 min; then both forms of MLC20 decreased. The process of the SMC rounding was inhibited by antimycin A or cytochalasins, in a dose-dependent manner, findings which suggested a dependency on both metabolic energy and actin-containing microfilaments. The smooth-muscle-relaxing agent, HA1077, also inhibited the process of SMC rounding. These observations suggest that a cellular contractile process might be involved in rounding of SMC.