Dictyostelium myosin light chain kinase. Purification and characterization

A Dictyostelium myosin light chain kinase has been purified approximately 15,000-fold to near homogeneity. The purified kinase is a single polypeptide of approximately 34 kDa that phosphorylates only the 18-kDa Dictyostelium myosin regulatory light chain and itself among substrates tested. The enzyme was purified largely by ammonium sulfate fractionation and hydrophobic (butyl) interaction chromatography. Analysis using polyclonal antibodies raised against the purified 34-kDa protein confirms that this protein is responsible for myosin light chain kinase activity. Protein microsequence of the 34-kDa protein reveals conserved protein kinase sequences. The purified Dictyostelium myosin light chain kinase exhibits a Km for Dictyostelium myosin of 4 microM and a Vmax of 8 nmol/min/mg. Unlike other characterized myosin light chain kinases, this enzyme is not regulated by calcium/calmodulin. Western blot analysis demonstrates that the purified kinase is not a proteolytic fragment that has lost calcium/calmodulin regulation. The Dictyostelium myosin light chain kinase activity is not directly regulated by cyclic nucleotides. However, this kinase undergoes an intramolecular autophosphorylation that activates the enzyme.     

Domain characterization of rabbit skeletal muscle myosin light chain kinase.

Myosin light chain kinase can be divided into three distinct structural domains, an amino-terminal "tail," of unknown function, a central catalytic core and a carboxy-terminal calmodulin-binding regulatory region. We have used a combination of deletion mutagenesis and monoclonal antibody epitope mapping to define these domains more closely. A 2.95-kilobase cDNA has been isolated that includes the entire coding sequence of rabbit skeletal muscle myosin light chain kinase (607 amino acids). This cDNA, expressed in COS cells encoded a Ca2+/calmodulin-dependent myosin light chain kinase with a specific activity similar to that of the enzyme purified from rabbit skeletal muscle. Serial carboxy-terminal deletions of the regulatory and catalytic domains were constructed and expressed in COS cells. The truncated kinases had no detectable myosin light chain kinase activity. Monoclonal antibodies which inhibit the activity of the enzyme competitively with respect to myosin light chain were found to bind between residues 235-319 and 165-173, amino-terminal of the previously defined catalytic core. Thus, residues that are either involved in substrate binding or in close proximity to a light chain binding site may be located more amino-terminal than the previously defined catalytic core.     

Molecular characterization of a mammalian smooth muscle myosin light chain kinase.

A 5.6-kilobase cDNA clone has been isolated which includes the entire coding region for the myosin light chain kinase from rabbit uterine tissue. This cDNA, expressed in COS cells, encodes a Ca2+/calmodulin-dependent protein kinase with catalytic properties similar to other purified smooth muscle myosin light chain kinases. A module (TLKPVGNIKPAE), repeated sequentially 15 times, has been identified near the N terminus of this smooth muscle kinase. It is not present in chicken gizzard or rabbit skeletal muscle myosin light chain kinases. This repeat module and a subrepeat (K P A/V) are similar in amino acid content to repeated motifs present in other proteins, some of which have been shown to associate with chromatin structures. Immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to compare myosin light chain kinase present in rabbit, bovine, and chicken smooth and nonmuscle tissues, showed that within each species both tissue types have myosin light chain kinases with indistinguishable molecular masses. These data suggest that myosin light chain kinases present in smooth and nonmuscle tissues are the same protein.     

Acidic residues comprise part of the myosin light chain-binding site on skeletal muscle myosin light chain kinase

Myosin light chain kinase is a Ca2+/calmodulin-dependent protein kinase which exhibits a very high degree of protein substrate specificity. The regulatory light chain of myosin is the only known physiological substrate of the enzyme. Based upon epitope mapping of monoclonal antibodies which inhibit kinase activity competitively with respect to the light chain substrate, residues 235-319 of the rabbit skeletal muscle kinase have been proposed to contain a light chain-binding site (Herring, B. P., Stull, J. T., and Gallagher, P. J. (1990) J. Biol. Chem. 265, 1724-1730). With the expression of a truncated kinase, we have further localized this putative binding site to residues 235-294. Mutation of acidic residues at positions 269 and 270 of the kinase resulted in a 10-fold increase in the Km value for the myosin light chain, with no significant change in the Vmax value. In contrast, altering a cluster of acidic amino acids at positions 261-263 had little effect on the Km value for the myosin light chain. These results suggest that residues 269 and 270 may be involved in protein-substrate binding. Interestingly, these residues, located amino-terminal of the homologous catalytic core (positions 302-539), are in a region which is highly conserved among myosin light chain kinases, but not other protein kinases. It is probable that the homologous catalytic core contains structural elements required for phosphotransferase activity. The catalytic domain of myosin light chain kinase would therefore include these conserved elements together with additional specific substrate-binding residues.     

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.     

Regulation of the Actin-Activated Mg-ATPase of Brain Myosin via Phosphorylation by the Brain Ca2+, Calmodulin-Dependent Protein Kinases

We have previously isolated two Ca2+, calmodulin-dependent protein kinases with molecular weights of 120,000 (120K enzyme) and 640,000 (640K enzyme), respectively, by gel filtration analysis from rat brain. Chicken gizzard myosin light-chain kinase and the 120K enzyme phosphorylated two light chains of brain myosin, whereas the 640K enzyme phosphorylated both the two light chains and the heavy chain. The phosphopeptides of the light chains digested by Staphylococcus aureus V8 protease were similar among chicken gizzard myosin light-chain kinase, the 120K enzyme, and the 640K enzyme. Only the seryl residue in the light chains and the heavy chain was phosphorylated by the enzymes. The phosphorylation of brain myosin by any of these enzymes led to an increase in actin-activated Mg-ATPase activity. The results suggest that brain myosin is regulated by brain Ca2+, calmodulin-dependent protein kinases in a similar but distinct mechanism in comparison with that of smooth muscle myosin.     

Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.     

Nonmuscle myosin phosphorylation sites for calcium-dependent and calcium-independent protein kinases

Thymus myosin, light chains and a synthetic peptide (S-S-K-R-A-K-A-K-T-T-K-K-R-P-Q-R-A-T-S-N-V-F-S) corresponding to the N-terminal sequence of smooth muscle myosin light chains were compared as substrates for calcium/calmodulin-dependent protein kinase (MLCK), calcium/phospholipid-dependent protein kinase (PKC), and a MgATP-activated protein kinase (H4PK) from lymphoid cells. All protein kinases catalyzed phosphorylation of the substrates although H4PK showed higher affinity for isolated light chains and the peptide. Phosphoamino acid analysis and analysis of thermolysin peptides established that PKC catalyzed phosphorylation of threonine-9 or 10. In addition, PKC and H4PK catalyzed phosphorylation at serine-19, the MLCK site. Collectively the data support the hypothesis that myosin filament assembly in nonmuscle cells may be regulated by a variety of calcium-dependent and calcium-independent protein kinases.     

Association of calmodulin with peptide analogues of the inhibitory region of the heat-stable protein inhibitor of adenosine cyclic 3',5'-phosphate dependent protein kinase

A 20-residue peptide analogue (IASGRTGRRNAIHDILVSSA) of the 8000-dalton heat-stable cAMP-dependent protein kinase inhibitor undergoes efficient calcium-dependent binding by calmodulin, with Kd approximately 70 nM when calcium is present. It is a potent inhibitor of smooth muscle myosin light chain kinase and of the calmodulin-dependent phosphatase activity of calcineurin. At concentrations above 3 microM, the peptide stimulates the basal activity of calcineurin. The native protein kinase inhibitor has no effect on the catalytic activity of myosin light chain kinase and is moderately inhibitory to both the calmodulin-dependent and -independent phosphatase activity of calcineurin. Competition experiments using excess concentrations of calcineurin and calmodulin suggest that the primary interaction of the native heat-stable inhibitor is with the catalytic subunit of protein kinase. Dansylcalmodulin exhibits only a weak interaction with the inhibitor. Observations on deletion peptides of the 20-residue analogue help to delineate the overlapping peptide binding specificities of the cAMP-dependent protein kinase [Scott, J. D., Glaccum, M. B., Fischer, E. H., & Krebs, E. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1613-1616] and calmodulin. In both cases, the most effectively bound peptides contain the RTGRR sequence.     

Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.

A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.     

Myosin light chain kinase structure function analysis using bacterial expression

A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.     

Regulation of Dictyostelium myosin I and II

Dictyostelium expresses 12 different myosins, including seven single-headed myosins I and one conventional two-headed myosin II. In this review we focus on the signaling pathways that regulate Dictyostelium myosin I and myosin II. Activation of myosin I is catalyzed by a Cdc42/Rac-stimulated myosin I heavy chain kinase that is a member of the p21-activated kinase (PAK) family. Evidence that myosin I is linked to the Arp2/3 complex suggests that pathways that regulate myosin I may also influence actin filament assembly. Myosin II activity is stimulated by a cGMP-activated myosin light chain kinase and inhibited by myosin heavy chain kinases (MHCKs) that block bipolar filament assembly. Known MHCKs include MHCK A and MHCK B, which have a novel type of kinase catalytic domain joined to a WD repeat domain, and MHC-protein kinase C (PKC), which contains both diacylglycerol kinase and PKC-related protein kinase catalytic domains. A Dictyostelium PAK (PAKa) acts indirectly to promote myosin II filament formation, suggesting that the MHCKs may be indirectly regulated by Rac GTPases.     

Myosin heavy chain kinase B participates in the regulation of myosin assembly into the cytoskeleton

Myosin II plays critical roles in events such as cytokinesis, chemotactic migration, and morphological changes during multicellular development. The amoeba Dictyostelium discoideum provides a simple system for the study of this contractile protein. In this system, myosin II filament assembly is regulated by myosin heavy chain (MHC) phosphorylation in the tail region of the molecule. Earlier studies identified an alpha-kinase, MHC kinase A (MHCK A), which phosphorylates three mapped threonine residues in the myosin tail, driving myosin disassembly. Using molecular and genomic approaches, we have identified a series of related kinases in Dictyostelium. The enzyme MHCK B shares with MHCK A a domain organization that includes a highly novel catalytic domain coupled to a carboxyl-terminal WD repeat domain. We have engineered, expressed, and purified a FLAG-tagged version of the novel kinase. In the present study, we report detailed biochemical and cellular studies documenting that MHCK B plays a physiological role in the control of Dictyostelium myosin II assembly and disassembly during the vegetative life of Dictyostelium amoebae. The presented data supports a model of multiple related MHCKs in this system, with different regulatory mechanisms and pathways controlling each enzyme.     

Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis

Site-directed mutagenesis of smooth muscle myosin light chain kinase was applied to define its autoinhibitory domain. Mutants were all initiated at Leu-447 but contained varying lengths of C-terminal sequence. Those containing the complete C-terminal sequence to Glu-972 possessed kinase activities that were calmodulin-dependent. Removal of the putative inhibitory domain by truncation to Thr-778 resulted in generation of a constitutively active (calmodulin-independent) species. Thus, the inhibitory domain lies to the C-terminal side of Thr-778. Truncation to Lys-793 and to Trp-800 also resulted in constitutively active mutants, although the specific activity of the latter was less than the other mutants. None of the truncated mutants bound calmodulin. For each mutant, the Km values with respect to ATP and to the 20,000-dalton light chain were similar to values obtained with the native enzyme. The presence of the inhibitory domain was detected by activation of kinase activity following limited proteolysis with trypsin. Using this procedure, it was determined that the inhibitory domain was manifest only in the mutant truncated to Trp-800 and was absent from that ending at Lys-793. These results indicate that a critical region of the inhibitory domain is contained within the sequence Tyr-794 to Trp-800. This region overlaps with the calmodulin-binding site for five residues. Our assignment of the inhibitory sequence is consistent with autoinhibition via a pseudosubstrate domain.     

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

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

The regulation of myosin II in Dictyostelium

Dictyostelium conventional myosin (myosin II) is an abundant protein that plays a role in various cellular processes such as cytokinesis, cell protrusion and development. This review will focus on the signal transduction pathways that regulate myosin II during cell movement. Myosin II appears to have two modes of action in Dictyostelium: local stabilization of the cytoskeleton by myosin filament association to the actin meshwork (structural mode) and force generation by contraction of actin filaments (motor mode). Some processes, such as cell movement under restrictive environment, require only the structural mode of myosin. However, cytokinesis in suspension and uropod retraction depend on motor activity as well. Myosin II can self-assemble into bipolar filaments. The formation of these filaments is negatively regulated by heavy chain phosphorylation through the action of a set of novel alpha kinases and is relatively well understood. However, only recently it has become clear that the formation of bipolar filaments and their translocation to the cortex are separate events. Translocation depends on filamentous actin, and is regulated by a cGMP pathway and possibly also by the cAMP phosphodiesterase RegA and the p21-activated kinase PAKa. Myosin motor activity is regulated by phosphorylation of the regulatory light chain through myosin light chain kinase A. Unlike conventional light chain kinases, this enzyme is not regulated by calcium but is activated by cGMP-induced phosphorylation via an upstream kinase and subsequent autophosphorylation.     

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.     

Phosphorylation of mammalian myosin light chain kinases by the catalytic subunit of cyclic AMP-dependent protein kinase and by cyclic GMP-dependent protein kinase

The phosphorylation of the calmodulin-dependent enzyme myosin light chain kinase, purified from bovine tracheal smooth muscle and human blood platelets, by the catalytic subunit of cAMP-dependent protein kinase and by cGMP-dependent protein kinase was investigated. When myosin light chain kinase which has calmodulin bound is phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, 1 mol of phosphate is incorporated per mol of tracheal myosin light chain kinase or platelet myosin light chain kinase, with no effect on the catalytic activity. Phosphorylation when calmodulin is not bound results in the incorporation of 2 mol of phosphate and significantly decreases the activity. The decrease in myosin light chain kinase activity is due to a 5 to 7-fold increase in the amount of calmodulin required for half-maximal activation of both tracheal and platelet myosin light chain kinase. In contrast to the results with the catalytic subunit of cAMP-dependent protein kinase, cGMP-dependent protein kinase cannot phosphorylate tracheal myosin light chain kinase in the presence of bound calmodulin. When calmodulin is not bound to tracheal myosin light chain kinase, cGMP-dependent protein kinase phosphorylates only one site, and this phosphorylation has no effect on myosin light chain kinase activity. On the other hand, cGMP-dependent protein kinase incorporates phosphate into two sites in platelet myosin light chain kinase when calmodulin is not bound. The sites phosphorylated by the two cyclic nucleotide-dependent protein kinases were compared by two-dimensional peptide mapping following extensive tryptic digestion of the phosphorylated myosin light chain kinases. With respect to the tracheal myosin light chain kinase, the single site phosphorylated by cGMP-dependent protein kinase when calmodulin is not bound appears to be the same site phosphorylated in the tracheal enzyme by the catalytic subunit of cAMP-dependent protein kinase when calmodulin is bound. With respect to the platelet myosin light chain kinase, the additional site that was phosphorylated by cGMP-dependent protein kinase when calmodulin was not bound was different from that phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.     

Identification of a calmodulin-dependent protein kinase in the cardiac cytosol, which phosphorylates phospholamban in the sarcoplasmic reticulum

A multifunctional calmodulin-dependent protein kinase in the canine cardiac cytosol was purified to near homogeneity. The purified enzyme inactivated glycogen synthase by means of phosphorylation. The enzyme also phosphorylated phospholamban and several other proteins. In view of its physicochemical properties and substrate specificity, the enzyme differed from myosin light chain kinase and phosphorylase kinase, and was considered to belong to a class of similar calmodulin-dependent protein kinases from brain, liver, and skeletal muscle. The results suggest that the enzyme mediates multiple Ca2+-dependent functions in the heart.     

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.