Kaempferol inhibits myosin light chain kinase

Kaempferol, 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, was found to inhibit bovine aorta myosin light chain kinase with a Ki of 0.3-0.5 microM. It was found to be competitive with ATP and non-competitive with isolated myosin light chains. The specificity of this inhibitor was studied relative to protein kinase C and cAMP dependent protein kinase (IC50 = 15 microM and 150 microM, respectively). It appears not to interact strongly with calmodulin binding proteins, such as Ca2+-calmodulin dependent phosphodiesterase (IC50 = 45 microM), and had little effect on actin-activated myosin subfragment-1 ATPase activity (IC50 greater than 100 microM) or smooth muscle phosphatase activities (IC50 greater than 100 microM).     

Properties of filament-bound myosin light chain kinase

Myosin light chain kinase binds to actin-containing filaments from cells with a greater affinity than to F-actin. However, it is not known if this binding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells. Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca2+-dependent phosphorylation of myosin regulatory light chain and contraction of stress fibers. Thus, dissociation of myosin light chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.     

Mitosis-specific phosphorylation of myosin light chain kinase

Cell cytosol preparations from mitotic HeLa cells exhibit a kinase activity that phosphorylates myosin light chain kinase (MLCK). This MLCK kinase activity is apparently distinct from the known MLCK kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, Ca(2+)-activated phospholipid-dependent protein kinase, or Ca(2+)-calmodulin-dependent protein kinase II, based on the following criteria. First, the MLCK kinase activity of mitotic cells does not respond to a variety of characteristic activators or inhibitors of these known kinases. Second, one- and two-dimensional peptide maps have revealed that the site of phosphorylation by the MLCK kinase of mitotic cells differs from those by these known kinases. The mitotic MLCK kinase phosphorylates MLCK at a threonine residue at a ratio of up to 1 mol of phosphate/mol of chicken gizzard MLCK. The MLCK kinase is mitosis-specific because mitotic cell extracts show much higher phosphorylation activity than nonmitotic cell extracts.     

Diphosphorylation of platelet myosin by myosin light chain kinase.

Recently, one of the authors (K.I.) and other investigators reported that myosin light chain (MLC) of smooth muscle (gizzard, arterial and tracheal) was diphosphorylated by myosin light chain kinase (MLCK) and that diphosphorylated myosin showed a marked increase in the actin-activated myosin ATPase activity in vitro and ex vivo. In this study, we prepared myosin, actin, tropomyosin (human platelet), MLCK (chicken gizzard) and calmodulin (bovine brain) and demonstrated diphosphorylation of MLC of platelet by MLCK in vitro. Our results are as follows. (1) Platelet MLC was diphosphorylated by a relatively high concentration (greater than 20 micrograms/ml) of MLCK in vitro. As a result of diphosphorylation, the actin-activated myosin ATPase activity was increased 3 to 4-fold as compared to the monophosphorylation. (2) Both di- and monophosphorylation reactions showed similar Ca2+, KCl, MgCl2-dependence. Maximal reaction was seen at [Ca2+] greater than 10(-6) M, 60 mM KCl and 2 mM MgCl2. This condition was physiological in activated platelets. (3) Di- and monophosphorylated myosin showed similar Ca2+, KCl-dependence of ATPase activity but distinct MgCl2-dependence. Diphosphorylated myosin showed maximal ATPase activity at 2 mM MgCl2 and monophosphorylated myosin showed a maximum at 10 mM MgCl2. (4) The addition of tropomyosin stimulated actin-activated ATPase activity in both di- and monophosphorylated myosin to the same degree. (5) ML-9, a relatively specific inhibitor of MLCK, inhibited the aggregation of human platelets induced by thrombin ex vivo in a dose-dependent manner. Moreover, this drug also partially inhibited both di- and monophosphorylation reactions and actin-activated ATPase activity. On the other hand, H-7, a synthetic inhibitor of protein kinase C, had little effect on the aggregation of human platelets induced by thrombin ex vivo. From these results, we conclude that diphosphorylation of platelet myosin by MLCK may play an important role in activated platelets in vivo.     

Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.     

Effects of relaxin on rat uterine myosin light chain kinase activity and myosin light chain phosphorylation

Isometrically suspended uteri from estrogen-primed rats were stimulated with prostaglandin F2 alpha and then exposed to relaxin. Relaxin-dependent decreases in the ratio of phosphorylated to total myosin light chains (MLC) and in MLC kinase activity, measured in the presence of 0.5 mg/ml of uterine myosin and the absence and presence of Ca2+-calmodulin (CaM), were observed. The time-course and concentration-response of these biochemical effects of relaxin paralleled the hormone-induced inhibition of uterine contractile activity. Relaxin treatment resulted in a change in the requirements of MLC kinase for Ca2+, CaM, and myosin. Titrations of MLC kinase activity showed a shift in K50 values for Ca2+ from 82 to 260 nM and for CaM from 2.2 to 25 nM in extracts from control and relaxin-treated tissues, respectively. The myosin Km values of MLC kinase from control and relaxin-treated tissues were 0.33 and 0.71 mg/ml, respectively. Under optimal assay conditions (100 microM Ca2+, 1 microM CaM, and 1.2 mg/ml of myosin) the activities of MLC kinase in both extracts were identical, regardless of hormone concentration or exposure time. These data suggest that relaxin-treatment results in a change in the affinity of MLC kinase for its substrate and modulator and that relaxin inhibits uterine contractile activity by a mechanism which involves a decrease in MLC kinase activity and, in turn, a decrease in phosphorylation of the 20,000-dalton light chains of myosin.     

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.     

Peptidase-resistant peptide inhibitors of myosin light chain kinase

Myosin light chain kinase (MLCK) is a key regulator of various forms of cellular mobility, in particular, endothelial and epithelial permeability. The membrane-penetrative peptide (H-RKKYKYRRK-NH₂, L-PIK) is one of the potential MLCK inhibitors for use in humans. Five analogs of L-PIK were synthesized by the solid phase method of peptide synthesis using Fmoc technology. According to ¹H NMR, these analogs exhibited increased stability towards degradation in blood plasma. One of the synthesized peptides, L-[MeArg¹]PIK, inhibited MLCK activity in vitro, and the inhibition efficacy of L-[MeArg¹]PIK was equal to that of L-PIK. The inhibitory effect of the other analogs was lower than that of L-PIK. The L-PIK analog that consisted of D-amino acids was the least active. Thus, we demonstrated the possibility of creating an effective peptide inhibitor of MLCK with increased stability against biodegradation. Such a peptide inhibitor is a promising compound for further pharmacological studies.     

Inhibition of myosin light chain kinase by amiloride

Phosphorylation of regulatory light chain (LC20) by myosin light chain kinase (MLCK) has been thought to play an important role in both smooth muscle contraction and several functions of vertebrate non-muscle cells. Amiloride, a frequently used Na+/H+ exchange inhibitor, potently inhibited phosphorylation of LC20 by MLCK. The inhibition was non-competitive with respect to myosin but competitive with ATP (Ki = 0.95 microM), suggesting that amiloride may act as an ATP analogue. Amiloride also inhibited the tension development of ether-treated gizzard fibers which were lacking in Na+/H+ antiport, even in the presence of ATP regenerating system. Thus, it must be reminded that amiloride cannot be used as a specific inhibitor of Na+/H+ exchange, and that the inhibition of myosin phosphorylation by amiloride should be taken into consideration in studying the role of Na+/H+ antiport in the cellular function.     

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.     

Myosin light chain kinase transference induces myosin light chain activation and endothelial hyperpermeability

The actomyosincomplex is the major cytoskeletal component that controls cellcontraction. In this study, we investigated the effects of actomyosininteraction on endothelial barrier function and gap formation.Activated myosin light chain kinase (MLCK) protein was transferred intocoronary venular endothelial cell (CVEC) monolayers. Uptake of theactivated protein resulted in a significant shift in myosin light chain(MLC) from an unphosphorylated to a diphosphorylated form. In addition,MLCK induced a hyperpermeability response of the monolayer as measuredby albumin transendothelial flux. Microscopic examination ofMLCK-treated CVECs revealed widespread gap formation in the monolayer,loss of peripheral -catenin, and increases in actin stress fibers.Inhibition of all of the above responses by a specific MLCK inhibitorsuggests they are the direct result of exogenously added MLCK. Thesedata suggest that activation of MLCK in CVECs causes phosphorylation ofMLC and contraction of CVECs, resulting in gap formation andconcomitant increases in permeability. This study uses a noveltechnique to measure the effects of an activated kinase on both itssubstrate and cellular morphology and function through directtransference into endothelial cells.

     

Purification of myosin light chain kinase from Limulus muscle

It has previously been shown that the regulatory light chains of myosin from Limulus, the horseshoe crab, can be phosphorylated either by purified turkey gizzard smooth muscle myosin light chain (MLC) kinase or by a crude kinase fraction prepared from Limulus muscle [Sellers, J. R. (1981) J. Biol. Chem. 256, 9274-9278]. This phosphorylation was shown to be associated with a 20-fold increase in the actin-activated MgATPase activity of the myosin. We have now purified the Ca2+-calmodulin-dependent MLC kinase from Limulus muscle to near homogeneity by using a combination of low ionic strength extraction, ammonium sulfate fractionation, and chromatography on Sephacryl S-300 and DEAE-Sephacel. The final purification was achieved by affinity chromatography on a calmodulin-Sepharose 4B column. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed 95% of the protein to be comprised of a doublet with Mr = 39000 and 37000. Electrophoresis of the kinase fraction under nondenaturing conditions resulted in a partial separation of the two major bands and demonstrated that each had catalytic activity. An SDS-polyacrylamide gel overlayed with 125I-calmodulin demonstrated that both the Mr 39K and the Mr 37K proteins bind calmodulin. Neither of the bands could be phosphorylated by the catalytic subunit of cAMP-dependent protein kinase. With Limulus myosin light chains as a substrate, the Vmax was 15.4 mumol min-1 mg-1, and the Km was 15.6 microM. The KD for calmodulin was determined to be 6 nM. The enzyme did not phosphorylate histones, casein, actin, or tropomyosin.     

Limited proteolysis of smooth muscle myosin light chain kinase

Myosin light chain kinase plays a central role in the regulation of smooth muscle contraction. The activity of this enzyme is controlled by protein-protein interaction (the Ca2+-dependent binding of calmodulin) and by phosphorylation catalyzed by cAMP-dependent protein kinase. The effects of these two regulatory mechanisms on the conformation of myosin light chain kinase and the locations of the phosphorylation sites, the calmodulin-binding site, and the active site have been probed by limited proteolysis. Phosphorylated and nonphosphorylated myosin light chain kinases were subjected to limited digestion by four proteases having different peptide bond specificities (trypsin, chymotrypsin, Staphylococcus aureus V8 protease, and thrombin), both in the presence and in the absence of bound calmodulin. The digests were compared in terms of gel electrophoretic pattern, distribution of phosphorylation sites, and Ca2+ dependence of kinase activity. A 24 500-dalton chymotryptic peptide containing both sites of phosphorylation was purified and tentatively identified as the amino-terminal peptide. The following conclusions can be drawn: neither phosphorylation nor calmodulin binding induces dramatic changes in the conformation of the kinase; the kinase contains two regions that are particularly susceptible to proteolytic cleavage, one located approximately 25 000 daltons from the amino terminus and the other near the center of the molecule; the two phosphorylation sites are located within 24 500 (probably 17 500) daltons of the amino terminus; the active site is located close to the center of the molecule; the calmodulin-binding site is located in the amino-terminal half of the molecule, between the sites of phosphorylation and the active site, and this region is very susceptible to cleavage by trypsin.     

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.     

Regulatory mechanism of Dictyostelium myosin light chain kinase A

In this study, we examined the activation mechanism of Dictyostelium myosin light chain kinase A (MLCK-A) using constitutively active Ca2+/calmodulin-dependent protein kinase kinase as a surrogate MLCK-A kinase. MLCK-A was phosphorylated at Thr166 by constitutively active Ca2+/calmodulin-dependent protein kinase kinase, resulting in an approximately 140-fold increase in catalytic activity, using intact Dictyostelium myosin II. Recombinant Dictyostelium myosin II regulatory light chain and Kemptamide were also readily phosphorylated by activated MLCK-A. Mass spectrometry analysis revealed that MLCK-A expressed by Escherichia coli was autophosphorylated at Thr289 and that, subsequent to Thr166 phosphorylation, MLCK-A also underwent a slow rate of autophosphorylation at multiple Ser residues. Using site-directed mutagenesis, we show that autophosphorylation at Thr289 is required for efficient phosphorylation and activation by an upstream kinase. By performing enzyme kinetics analysis on a series of MLCK-A truncation mutants, we found that residues 283-288 function as an autoinhibitory domain and that autoinhibition is fully relieved by Thr166 phosphorylation. Simple removal of this region resulted in a significant increase in the kcat of MLCK-A; however, it did not generate maximum enzymatic activity. Together with the results of our kinetic analysis of the enzymes, these findings demonstrate that Thr166 phosphorylation of MLCK-A by an upstream kinase subsequent to autophosphorylation at Thr289 results in generation of maximum MLCK-A activity through both release of an autoinhibitory domain from its catalytic core and a further increase (15-19-fold) in the kcat of the enzyme.     

Selective binding of L-thyroxine by myosin light chain kinase

L-Thyroxine selectively inhibited Ca2+-calmodulin-activated myosin light chain kinases (MLC kinase) purified from rabbit skeletal muscle, chicken gizzard smooth muscle, bovine thyroid gland, and human platelet with similar Ki values (Ki = 2.5 microM). A detailed analysis of L-thyroxine inhibition of smooth muscle myosin light chain kinase activation was undertaken in order to determine the effect of L-thyroxine on the stoichiometries of Ca2+, calmodulin, and the enzyme in the activation process. The kinetic data indicated that L-thyroxine does not interact with calmodulin but, instead, through direct association with the enzyme, inhibits the binding of the Ca2+-calmodulin complex to MLC kinase. L-[125I]Thyroxine gel overlay revealed that the 95-kDa fragment of chicken gizzard MLC kinase digested by chymotrypsin and all the fragments of 110, 94, 70, and 43 kDa produced by Staphylococcus aureus V8 protease digestion which contain the calmodulin binding domain retain L-[125I]thyroxine binding activity, whereas smaller peptides were not radioactive. Since MLC kinase is phosphorylated by cAMP-dependent protein kinase (2 mol of phosphate/mol of MLC kinase), the effect of L-thyroxine on the phosphorylation of MLC kinase also was examined. L-Thyroxine binding did not inhibit the phosphorylation of MLC kinase and, moreover, reversed the inhibition of phosphorylation obtained with the calmodulin-enzyme complex. These observations support the suggestion that L-thyroxine binds at or near the calmodulin-binding site of MLC kinase. L-Thyroxine may serve as a different type of pharmacological tool for elucidating the biological significance of MLC kinase-mediated reactions.     

Role of myosin light chain kinase in muscle contraction

In resting striated muscles of the rabbit muscle in vivo, the phosphorylatable light chain is partially phosphorylated. Tetanic stimulation increased the level of phosphorylation more rapidly in fast twitch than in slow twitch muscle. In both types of muscle the rate of dephosphorylation was relatively slow. In rabbit fast twitch muscles, phosphorylation levels persisted significantly above the resting value for some time after posttetanic potentiation had disappeared. The role of myosin light chain kinase in modulating contractile response in striated muscle is uncertain. In vertebrate smooth muscle the role of myosin phosphorylation appears to be different from that in striated muscle despite the general similarity of the actomyosin system in both tissues. Although phosphorylation in vitro increases the Mg2+ -ATPase of actomyosin, a number of features imply that a somewhat complex relationship exists between the level of phosphorylation and the actin activation of the Mg2+ -ATPase in vertebrate smooth muscle. Contrary to many earlier reports, preparations of smooth muscle actomyosin can be obtained with Mg2+ -ATPase activities comparable to those of actomyosin from skeletal muscle. Preliminary evidence is presented that suggests that phosphorylation changes the Ca2+ sensitivity of the Mg2+ -ATPase of smooth muscle actomyosin.     

Autophosphorylation of skeletal muscle myosin light chain kinase.

Ca2+/calmodulin-dependent myosin light chain kinase phosphorylates the regulatory light chain of myosin. Rabbit skeletal muscle myosin light chain kinase also catalyzes a Ca2+/calmodulin-dependent autophosphorylation with a rapid rate of incorporation of 1 mol of 32P/mol of kinase and a slower rate of incorporation up to 1.52 mol of 32P/mol. Autophosphorylation was inhibited by a peptide substrate that has a low Km value for myosin light chain kinase. Autophosphorylation at both rates was concentration-independent, indicating an intramolecular mechanism. There were no significant changes in catalytic properties toward light chain and MgATP substrates or in calmodulin activation properties upon autophosphorylation. After digestion with V8 protease, phosphopeptides were purified and sequenced. Two phosphorylation sites were identified, Ser 160 and Ser 234, with the former associated with the rapid rate of phosphorylation. Both sites are located amino terminal of the catalytic domain. These results indicate that the extended "tail" region of the enzyme can fold into the active site of the kinase.     

Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.     

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.