Purification and characterization of a calmodulin-dependent myosin heavy chain kinase from intestinal brush border

A calcium- and calmodulin-dependent kinase that represents the majority of the myosin heavy chain kinase activity in chicken intestinal brush borders has been highly purified. The purification steps include gel filtration, high performance chromatography on anion and cation exchangers, and affinity chromatography on calmodulin-Sepharose. The purified kinase consists of a single major, apparently autophosphorylatable polypeptide of 50,000 daltons. The Stokes radius (68 A) and sedimentation coefficient (17.5 S) indicate that it has a molecular weight of approximately 490,000. The kinase catalyzed the incorporation of a maximum of 0.8 mol of phosphate/mol of heavy chain, and essentially no phosphate was incorporated into the light chains. This kinase is distinct from other myosin kinases, but has a number of properties in common with the type II calmodulin-dependent protein kinases.     

Regulation in vitro of brush border myosin by light chain phosphorylation

Myosin was purified from chicken brush border cells to greater than 95% homogeneity and in a predominantly non-phosphorylated state. The effects of light chain phosphorylation by a Ca2+-calmodulin-dependent myosin light chain kinase on the conformational, enzymatic and filament assembly properties of this myosin were investigated. The actin-activated MgATPase activity of the non-phosphorylated myosin was low, and upon light chain phosphorylation an eight- to ninefold increase in this activity was observed, which was further potentiated by tropomyosin. Light chain phosphorylation was shown to control the assembly and disassembly of brush border myosin filaments. For example, turbidity measurements and electron microscopy demonstrated that MgATP disassembled non-phosphorylated myosin filaments; the disassembled myosin could reassemble when the light chains were phosphorylated, and could be disassembled again by dephosphorylating the light chains with phosphatase. In the electron microscope, the disassembled non-phosphorylated myosin molecules appeared in a folded conformation, and they were extended when phosphorylated. Proteolytic digestion was used to probe further the conformation of these folded and extended molecules, and their subunit organizations were characterized by a gel overlay technique. Quantitative analysis further demonstrated that light chain phosphorylation alters dramatically the monomer/polymer equilibrium of brush border myosin, shifting it towards filament formation. Comparison of analogous data for myosin from gizzard and thymus shows that each myosin has distinct solubility properties.     

Phosphorylation of synthetic peptides by skeletal muscle myosin light chain kinases

Substrate determinants for rabbit and chicken skeletal muscle myosin light chain kinases were examined with synthetic peptides. Both skeletal muscle myosin light chain kinases had similar phosphorylation kinetics with synthetic peptide substrates. Average kinetic constants for skeletal muscle myosin light chain heptadecapeptide, (formula; see text) where S(P) is phosphoserine, were Km, 2.3 microM and Vmax, 0.9 mumol/min/mg of enzyme. Km values were 122 and 162 microM for skeletal muscle peptides containing A-A for basic residues at positions 2-3 and 6-7, respectively. Average kinetic constants for smooth muscle myosin light chain peptide, (formula; see text), were Km, 1.4 microM and Vmax 27 mumol/min/mg of enzyme. Average Km values for the smooth muscle peptide, residues 11-23, were 10 microM which increased 6- and 11-fold with substitutions of alanine at residues 12 and 13, respectively. Vmax values decreased and Km values increased markedly by substitution of residue 16 with glutamate in the 11-23 smooth muscle tridecapeptide. Basic residues located 3 and 6-7 residues toward the NH2 terminus from phosphoserine in smooth muscle myosin light chain and 6-8 and 10-11 residues toward the NH2 terminus from phosphoserine in skeletal muscle myosin light chain appear to be important substrate determinants for skeletal muscle myosin light chain kinases. These properties are different from myosin light chain kinase from smooth muscle.     

Phosphorylation of smooth myosin light chain kinase by smooth muscle Ca2+/calmodulin-dependent multifunctional protein kinase

Smooth muscle myosin light chain kinase (MLC kinase) was phosphorylated by smooth muscle calmodulin-dependent protein kinase II (CaM protein kinase II). When MLC kinase was free from calmodulin, two sites were phosphorylated. The phosphorylation at the one site was much faster than the other site; however, the phosphorylation at the first site was completely blocked by calmodulin binding to MLC kinase. Phosphorylation of MLC kinase by CaM protein kinase II increased the dissociation constant of MLC kinase for calmodulin about 10 times without changing the Vmax. The location of the phosphorylation sites was identified by isolating and sequencing the tryptic phosphopeptides of MLC kinase. The preferred site was identified as serine 512 and the second site as serine 525. These sites are the same as the sites phosphorylated by cAMP-dependent protein kinase.     

Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells.

Stimulation of tracheal smooth muscle cells in culture with ionomycin resulted in a rapid increase in cytosolic free Ca2+ concentration ([Ca2+]i) and an increase in both myosin light chain kinase and myosin light chain phosphorylation. These responses were markedly inhibited in the absence of extracellular Ca2+. Pretreatment of cells with 1-[N-O-bis(5-isoquinolinesulfonyl)-N- methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a specific inhibitor of the multifunctional calmodulin-dependent protein kinase II (CaM kinase II), did not affect the increase in [Ca2+]i but inhibited ionomycin-induced phosphorylation of myosin light chain kinase at the regulatory site near the calmodulin-binding domain. KN-62 inhibited CaM kinase II activity toward purified myosin light chain kinase. Phosphorylation of myosin light chain kinase decreased its sensitivity to activation by Ca2+ in cell lysates. Pretreatment of cells with KN-62 prevented this desensitization to Ca2+ and potentiated myosin light chain phosphorylation. We propose that the Ca(2+)-dependent phosphorylation of myosin light chain kinase by CaM kinase II decreases the Ca2+ sensitivity of myosin light chain phosphorylation in smooth muscle.     

Phosphorylation of brush border myosin at threonine on its 20 kDa light chains by a calmodulin-independent kinase activates its ATPase

A calmodulin-independent kinase isolated from chicken intestinal brush border phosphorylates brush border myosin mainly at an apparently single threonine on its 20 kDa light chains. Phosphorylation to 1.9 mol phosphate/mol myosin activated the myosin actin-activated ATPase about 12-fold, to about 100 nmol/min per mg. Brush border myosin ATPase can thus be activated by phosphorylation either at threonine, by calmodulin-independent kinase, or at serine, by calmodulin-dependent myosin light chain kinase, as previously shown [(1987) FEBS Lett. 223, 262-266].     

Phosphorylation of myosin light chain by protease activated kinase I

Protease activated kinase I from rabbit reticulocytes has been shown to phosphorylate the P-light chain of myosin light chains isolated from rabbit skeletal muscle. The enzyme is not activated by Ca²⁺ and calmodulin or phospholipids. Protease activated kinase I is not inhibited by trifluoperazine at concentrations up to 200 μM or by the antibody to the Ca²⁺, calmodulin-dependent myosin light chain kinase from rabbit skeletal muscle. Two-dimensional peptide mapping of chymotryptic digests of myosin P-light chain show the site phosphorylated by the protease activated kinase is different from that phosphorylated by the Ca²⁺, calmodulin-dependent myosin light chain kinase.     

Substrate specificity of myosin light chain kinases.

Skeletal muscle myosin light chain kinase can phosphorylate myosin light chains isolated from skeletal or smooth muscle. In contrast, smooth muscle myosin light chain kinase specifically phosphorylates light chains isolated from smooth muscle. In this study, we have identified residues within the rabbit smooth and skeletal muscle myosin light chain kinases which may interact with the basic residues that are important substrate determinants in the light chains. Mutation of aspartic acid 270 amino-terminal of the catalytic core of the skeletal muscle myosin light chain kinase increased the Km value for both smooth and skeletal muscle light chains. Although deletions of the analogous region of the smooth muscle myosin light chain kinase (residues 663-678) markedly increased the Km value for light chain, mutation of any single acidic residue within this region did not have a similar effect. Mutation of single residues within the catalytic core of the skeletal muscle (E377 and E421) and smooth muscle (E777 and E821) myosin light chain kinases increased Km values for the smooth muscle light chain at least 35- and 100-fold, respectively. It is proposed that these residues may form ionic interactions with the arginine that is 3 residues amino-terminal of the phosphorylatable serine in the smooth muscle light chain.     

Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase

The 20,000-dalton light chain of turkey gizzard myosin is phosphorylated at two sites. Dual phosphorylation is observed when both intact myosin and isolated light chains are used as substrates. Phosphorylation of the second site is not observed at higher ionic strength (e.g. 0.35 M KCl). The first phosphorylation site (serine 19) is phosphorylated preferentially to the second site. The latter is phosphorylated more slowly than the first site, and its phosphorylation requires relatively high concentrations of myosin light chain kinase. It is suggested that myosin light chain kinase catalyzes the phosphorylation of both sites on the light chain, and several reasons are cited that make it unlikely that a contaminant kinase is involved. The second phosphorylation site is a threonine residue. Based on the results of limited proteolysis of the light chain, it is concluded that the threonine residue is close to serine 19, and possible locations are threonines 9, 10, and 18. At all concentrations of MgCl2, phosphorylation of the second site markedly increases the actin-activated ATPase activity of myosin and accelerates the superprecipitation response of myosin plus actin.     

WD repeat domains target dictyostelium myosin heavy chain kinases by binding directly to myosin filaments

Myosin heavy chain kinase (MHCK) A phosphorylates mapped sites at the C-terminal tail of Dictyostelium myosin II heavy chain, driving disassembly of myosin filaments both in vitro and in vivo. MHCK A is organized into three functional domains that include an N-terminal coiled-coil region, a central kinase catalytic domain unrelated to conventional protein kinases, and a WD repeat domain at the C terminus. MHCK B is a homologue of MHCK A that possesses structurally related catalytic and WD repeat domains. In the current study, we explored the role of the WD repeat domains in defining the activities of both MHCK A and MHCK B using recombinant bacterially expressed truncations of these kinases either with or without their WD repeat domains. We demonstrate that substrate targeting is a conserved function of the WD repeat domains of both MHCK A and MHCK B and that this targeting is specific for Dictyostelium myosin II filaments. We also show that the mechanism of targeting involves direct binding of the WD repeat domains to the myosin substrate. To our knowledge, this is the first report of WD repeat domains physically targeting attached kinase domains to their substrates. The examples presented here may serve as a paradigm for enzyme targeting in other systems.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody     

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.     

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.     

Phosphorylation of myosin light chain and the actin-activated ATPase activity of adrenal medullary myosin

Myosin light chain kinase was partially purified from bovine adrenal medulla. A polypeptide of Mr 165,000 dalton was identified as kinase by using anti-gizzard myosin light chain kinase IgG on immunoreplica. Phosphorylation of medullary myosin was Ca2+- and calmodulin-dependent. The phosphorylated myosin was showed to enhance the actin-activated Mg2+-ATPase activity. In contrast, the myosin ATPase activity was dramatically decreased by dephosphorylation of myosin.     

Insulin receptor capping and its correlation with calmodulin-dependent myosin light chain kinase

Both fluorescence microscopy and fluorometric analysis techniques have been used to characterize insulin receptor capping in IM-9 human lymphoblastoid cells. Morphologically, insulin caps appear similar to lectin or antiimmunoglobulin-induced caps displaying a preferential accumulation of actin, myosin, and actin-binding protein directly underneath the cap structure. Using the fluorescent calcium indicator quin2 we have detected no change in the calcium activity following insulin stimulation. However, in the presence of a number of calmodulin inhibitors, such as W-5, W-7, W-12, and trifluoperazine (TFP), insulin capping is significantly inhibited, which implies that a calmodulin-regulated process is involved. Using double immunofluorescence microscopy, we have found that the calmodulin-dependent myosin light chain kinase (MLCK) is concentrated directly beneath insulin caps. Upon treatment with trifluoperazine (TFP), the redistribution of both MLCK and insulin receptors are inhibited concomitantly. Our data indicate that the calmodulin-dependent myosin light chain kinase may be directly responsible for the activation of actomyosin-mediated contractility during insulin receptor capping.     

Phosphorylation of a second site for myosin light chain kinase on platelet myosin

The 20,000-dalton light chain of bovine platelet myosin is phosphorylated at two sites by myosin light chain kinase. The first and second phosphorylation sites are at a serine and a threonine residue, respectively. The location of the phosphorylation sites was determined by using limited proteolysis. The N-terminal sequence of the 17,000-dalton tryptic fragment of platelet myosin 20,000-dalton light chain was found to be identical with that of gizzard 20,000-dalton light chain from Ala-17 to Phe-33. On the basis of these results and the distribution of 32P among the proteolytic fragments, it was concluded that serine-19 and threonine-18 were the two phosphorylation sites. Phosphorylation at the threonine residue markedly increases the actin-activated ATPase activity of myosin. It was found that platelet myosin forms 10S and 6S conformations and its Mg2+-ATPase activity parallels the transition from the 6S to the 10S conformation. The conformational transition was influenced by phosphorylation at both sites, and the phosphorylation at the threonine residue further shifted the equilibrium toward the 6S conformation. The phosphorylation at the threonine residue also induced thick filament formation in the presence of ATP. These results suggest that the phosphorylation at the threonine residue as well as at the serine residue may play an important role in the contractility of nonmuscle cells.