Purification of myosin from Ehrlich ascites tumour cells (phosphorylation of its light chain and heavy chain)

1. The myosin molecule from Ehrlich ascites tumour cells consists of heavy chains of about 200 kDa and three species of light chains of 20, 19 and 15 kDa. 2. The heavy chain can be phosphorylated in vitro either by endogenous Ca2+-independent kinase or by casein kinase II. 3. The 20 and 19 kDa light chains can be phosphorylated either by an endogenous kinase or by myosin light chain kinase from chicken gizzard. 4. The Ca2+-ATPase activity of the purified myosin was 0.3 mumol/min mg protein. The Mg2+-ATPase activity was activated 14-fold by actin upon the light chain phosphorylation.     

Correlation of enzymatic properties and conformation of bovine erythrocyte myosin

Myosin was purified from bovine erythrocytes by chromatography on DEAE-cellulose, Sepharose CL-4B, hydroxylapatite, and DEAE-5PW. The yield was about 200 micrograms/L of packed cells. From SDS-polyacrylamide gels, the purity was estimated to be greater than 95%. The bovine erythrocyte myosin is composed of heavy chains of 200 kDa and light chains of 20 and 17 kDa, in a molar stoichiometry of 1. Myosin was also purified from human erythrocytes by the same method. The molecular weights of two light chains were 26K and 19.5K which confirmed the earlier reports [Fowler, V. M., Davis, J. Q., & Bennet, V. (1985) J. Cell Biol. 100, 47-55; Wong, A. J., Kiehart, D. P., & Pollard, T.D. (1985) J. Biol. Chem. 260, 46-49]. Phosphorylation by gizzard myosin light chain kinase, to a level of 1 mol of phosphate/mol of 20-kDa light chain, increased actin-activated ATPase, and the extent of activation was dependent on the MgCl2 concentration. Both Ca2+-ATPase and Mg2+-ATPase activities were dependent on KCl concentration and markedly decreased below 0.3 M KCl. Mg2+-ATPase of phosphorylated myosin, while more resistant to decreasing ionic strength, was also decreased below 0.2 M KCl. These results are similar to those obtained with smooth muscle myosin and suggest that the 10S-6S transition occurs. In confirmation of this, gel filtration, viscosity, and electron microscopy (rotary shadowing) show that erythrocyte myosin forms extended and folded conformations in high and low salt, respectively. It is proposed that each conformation is characterized by distinct enzymatic properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent

A method is described for the preparation of partially and fully phosphorylated chicken gizzard myosin. When fully phosphorylated it possessed an actin-activated Mg2+-ATPase of similar specific activity to that of mammalian skeletal muscle myosin. The Mg2+-ATPase activity of these preparations was related in a non-linear fashion to increasing phosphorylation of the P light chain. When P light chain phosphorylation occurred during enzymic assay the Mg2+-ATPase activity remained constant. Fully phosphorylated preparations of gizzard myosin possessed an actin-activated Mg2+-ATPase that was not Ca2+-sensitive, whereas the Mg2+-ATPase of partially phosphorylated myosin preparations was Ca2+-sensitive.     

Purification and characterization of a myosin heavy chain kinase from Dictyostelium discoideum

A Dictyostelium discoideum myosin heavy chain kinase has been purified 14,000-fold to near homogeneity. The enzyme has a Mr = 130,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and greater than 700,000 as determined by gel filtration on Bio-Gel A-1.5m. The enzyme has a specific activity of 1 mumol/min X mg when assayed at a Dictyostelium myosin concentration of 0.3 mg/ml. A maximum of 2 mol of phosphate/mol of myosin is incorporated by the kinase, and the phosphorylated amino acid is threonine. Phosphate is incorporated only into the myosin heavy chains, not into the light chains. The actin-activated Mg2+-ATPase of Dictyostelium myosin is inhibited 70-80% following maximal phosphorylation with the kinase. The myosin heavy chain kinase requires 1-2 mM Mg2+ for activity and is most active at pH 7.0-7.5. The activity of the enzyme is not significantly altered by the presence of Ca2+, Ca2+ and calmodulin, EGTA, cAMP, or cGMP. When incubated with Mg2+ and ATP, phosphate is incorporated into the myosin heavy chain kinase, perhaps by autophosphorylation.     

Filament formation and actin-activated ATPase activity are abolished by proteolytic removal of a small peptide from the tip of the tail of the heavy chain of Acanthamoeba myosin II

Actin-activated Mg2+-ATPase activity of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of three serine residues located at the carboxyl-terminal end of each of the two 185,000-Da heavy chains; the phosphorylated molecule has full Ca2+-ATPase activity but no actin-activated Mg2+-ATPase activity. Under controlled conditions, chymotrypsin removes a small peptide containing all three phosphorylation sites from the ends of the myosin II heavy chains producing a molecule with heavy chains of 175,000 Da and undigested light chains. The length of the myosin II tail decreased from 89 to 76 nm. Chymotrypsin-cleaved myosin II has complete Ca2+-ATPase activity but no actin-activated Mg2+-ATPase activity under standard assay conditions and binds to F-actin as well as undigested myosin II in the absence, but not in the presence, of MgATP. In the presence of MgCl2, undigested myosin II forms biopolar filaments but chymotrypsin-cleaved myosin II forms only parallel (monopolar) dimers, as assessed by analytical ultra-centrifugation and rotary shadow electron microscopy. We conclude that the short segment very near the end of the myosin II tail that contains the three phosphorylatable serines is necessary for the formation of biopolar filaments and, probably as a consequence of filament formation, for the high-affinity binding of myosin II to F-actin in the presence of ATP and the actin-activated Mg2+-ATPase activity of native myosin II. This supports our previous conclusion that actin-activated Mg2+-ATPase of native myosin II is expressed only when the enzyme is in bipolar filaments with the proper conformation as determined by the state of phosphorylation of the heavy chains.     

Cooperative dependence of the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II on the extent of filament phosphorylation

The actin-activated Mg2+-ATPase of myosin II from Acanthamoeba castellanii is regulated by phosphorylation of 3 serine residues at the tip of the tail of each of its two heavy chains; only dephosphorylated myosin II is active, whereas the phosphorylated and dephosphorylated forms have identical Ca2+-ATPase activities and Mg2+-ATPase activities in the absence of F-actin. We have now chemically modified phosphorylated and dephosphorylated myosin II with N-ethylmaleimide (NEM). The modification occurred principally at a single site within the NH2-terminal 73,000 Da of the globular head of the heavy chain. NEM-myosin II bound to F-actin and formed filaments normally, but the Ca2+- and Mg2+-ATPase activities of phosphorylated and dephosphorylated myosin II and the actin-activated Mg2+-ATPase activity of NEM-dephosphorylated myosin II were inhibited. Only filamentous myosin II has actin-activated Mg2+-ATPase activity. Native phosphorylated myosin II acquired actin-activated Mg2+-ATPase activity when it was co-polymerized with NEM-inactivated dephosphorylated myosin II, and the increase in its activity was cooperatively dependent on the fraction of NEM-dephosphorylated myosin II in the filaments. From this result, we conclude that the specific activity of each molecule within a filament is independent of its own state of phosphorylation, but is highly cooperatively dependent upon the state of phosphorylation of the filament as a whole. This enables the actin-activated Mg2+-ATPase activity of myosin II filaments to respond rapidly and extensively to small changes in the level of their phosphorylation.     

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.     

Acanthamoeba cofactor protein is a heavy chain kinase required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I.

We have purified a cofactor protein previously shown (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697) to be required for actin activation of the Mg2+-ATPase activity of Acanthamoeba myosin I. The purified cofactor protein is a novel myosin kinase that phosphorylates the single heavy chain, but neither of the two light chains, of Acanthamoeba myosin I. Phosphorylation of Acanthamoeba myosin I by the purified cofactor protein requires ATP and Mg2+ but is Ca2+-independent. The Mg2+-ATPase activity of phosphorylated Acanthamoeba myosin I is highly activated by F-actin in the absence of cofactor protein. Actin-activated Mg2+-ATPase activity is lost when phosphorylated Acanthamoeba myosin I is dephosphorylated by platelet phosphatase. Phosphorylation and dephosphorylation have no effect on the (K+,EDTA)-ATPase and Ca2+-ATPase activities of Acanthamoeba myosin I. These results show that cofactor protein is an Acanthamoeba myosin I heavy chain kinase and that phosphorylation of the heavy chain of this myosin is required for actin activation of its Mg2+-ATPase activity.     

Purification of smooth-muscle myosin free of calmodulin and myosin light-chain kinase. Susceptibility to oxidation.

Smooth-muscle myosin purified as described by Persechini & Hartshorne [(1983) Biochemistry 22, 470-476] contains trace amounts of calmodulin and myosin light-chain kinase, which can be removed by Ca2+-dependent hydrophobic-interaction chromatography followed by calmodulin-Sepharose affinity chromatography. The resultant column-purified myosin exhibits properties similar to those of the non-purified myosin, e.g. actin activation of the Mg2+-ATPase requires Ca2+/calmodulin-dependent phosphorylation of the two 20 kDa light chains. However, unlike the non-purified myosin, the column-purified myosin undergoes a time-dependent transition to a form which no longer requires phosphorylation for actin activation of the myosin Mg2+-ATPase. This transition is identified as a time-dependent change in conformation of the column-purified myosin from a 10 S to 6 S form and is caused by slow oxidation of the column-purified myosin, since it could be prevented by storage under N2 and reversed by 5 mM-dithiothreitol.     

Regulation of the actin-activated ATPase activity of Acanthamoeba myosin II by copolymerization with phosphorylated and dephosphorylated peptides derived from the carboxyl-terminal end of the heavy chain

Myosin II from Acanthamoeba castellanii is a conventional myosin composed of two heavy chains and two pairs of light chains. The amino-terminal approximately 90 kDa of each heavy chain form a globular head that contains the ATPase site and an ATP-sensitive actin-binding site. The carboxyl-terminal approximately 80 kDa of both heavy chains interact to form a coiled coil, helical rod (through which the molecules self-associate into bipolar filaments) ending in a short nonhelical tailpiece. Phosphorylation of 3 serine residues at the tip of the tail (at positions 11, 16, and 21 from the carboxyl terminus) inactivates the actin-activated Mg2(+)-ATPase activity of myosin II filaments. Previous work had indicated that the activity of each myosin II molecule in a filament reflects the global state of phosphorylation of the filament rather than the phosphorylation state of the molecule itself. We have now purified the approximately 28-kDa carboxyl-terminal region of the heavy chain lacking the last two phosphorylation sites, and we have shown that this peptide copolymerizes with and regulates the actin-activated Mg2(+)-ATPase activities of native dephosphorylated and phosphorylated myosin II. It can be concluded from these studies that the biologically relevant enzymatic activity of myosin II is regulated by a phosphorylation-dependent conformational change in the myosin filaments.     

Ontogenic differentiation of pig atrial and ventricular myosin

Myosin was isolated from pig atrial and ventricular myocardium during postnatal development and Ca2+-ATPase was determined and myosin light chains were analysed by electrophoresis in sodium dodecylsulfate polyacrylamide gel. During ontogenesis ATPase activity of ventricular myosin remains virtually unchanged, whereas that of atrial myosin increases. The patterns of myosin light chains of atrial and ventricular myosin differ from each other, but the individual pattern remains unchanged during the development.     

Requirement of phosphorylation of Physarum myosin heavy chain for thick filament formation, actin activation of Mg2+-ATPase activity, and Ca2+-inhibitory superprecipitation

In an attempt to elucidate the Ca2+-regulated mechanism of motility in Physarum plasmodia, we improved the preparation method for myosin B and pure myosin. The obtained results are as follows: 1. We obtained two types of myosin B which are distinguishable from each other with respect to their sensitivity to Ca2+. The inactive type of myosin B had low superprecipitation activities both in the presence and in the absence of Ca2+. The active type showed very high superprecipitation activity in EGTA, and the activity was conspicuously inhibited by Ca2+. The active type was converted into the inactive type by treatment with potato acid phosphatase. Also the inactive type or the phosphatase-treated active type was converted into the active type upon reacting with ATP-gamma-S. 2. In the reaction with ATP-gamma-S, only the myosin HC of myosin B was phosphorylated. The phosphorylation was independent of Ca2+ and calmodulin, and the extent was about 1 mol/mol HC. 3. The Ca2+ sensitivity in the superprecipitation of the active type was not decreased by adding an excess amount of F-actin. Besides, the actin-activated Mg2+-ATPase activity of purified phosphorylated myosin was not Ca2+-sensitive. Therefore, presence of a Ca2+-dependent inhibitory factor(s) that could bind to myosin was suggested. 4. The Mg2+-ATPase activity of purified phosphorylated myosin was 7-8 times enhanced by F-actin, but that of dephosphorylated myosin was hardly activated at all. 5. In a gel filtration in 0.5 M KCl, phosphorylated myosin was eluted behind dephosphorylated myosin. Electron microscopy applying the rotary-shadow method showed significant difference in flexibility in the tail between phosphorylated and dephosphorylated myosin molecules. 6. In 40 mM KCl and 5-10 mM MgCl2, phosphorylated myosin formed thick filaments, but dephosphorylated myosin did not, whether there was ATP or not. The above results clearly show that the phosphorylation of myosin HC is indispensable to ATP-induced superprecipitation, the actin-activated Mg2+-ATPase activity, and the formation of thick filaments of myosin. A myosin-linked factor(s) that inhibits an actin-myosin interaction in a Ca2+-dependent manner may exist.     

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.     

Myosin from human erythrocytes

We have purified myosin from human erythrocytes using methods similar to that for other cytoplasmic myosins with a yield of about 500 micrograms/100 ml of packed cells. It consists of a 200-kDa heavy chain and light chains of 26- and 19.5 kDa and therefore differs from the isozyme in platelets which has light chains of 20- and 15 kDa. At low ionic strength, the myosin forms short bipolar filaments like those of platelet myosin. Eight of eight monoclonal antibodies to platelet myosin also bind to erythrocyte myosin. Like most myosins, it has a high ATPase activity in the presence of Ca2+ or EDTA, but is inhibited by Mg2+. Myosin light-chain kinase transfers 1 phosphate from ATP to the 20-kDa light chain, and this stimulates the actin-activated ATPase. Thus, myosin may play a role in shape changes in the erythrocytes.     

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.     

Antigen-induced secretion of histamine and the phosphorylation of myosin by protein kinase C in rat basophilic leukemia cells

IgE-mediated stimulation of rat basophilic leukemia (RBL-2H3) cells results in the secretion of histamine. Myosin immunoprecipitated from these cells shows an increase in the amount of radioactive phosphate incorporated into its heavy (200 kDa) and light (20 kDa) chains. In unstimulated cells two-dimensional mapping of tryptic peptides of the myosin light chain reveals one phosphopeptide containing the serine residue phosphorylated by myosin light chain kinase. Following stimulation a second phosphopeptide appears containing a serine residue phosphorylated by protein kinase C. Tryptic phosphopeptide maps derived from myosin heavy chains show that unstimulated cells contain three major phosphopeptides. Following stimulation a new tryptic phosphopeptide appears containing a serine site phosphorylated by protein kinase C. The stoichiometry of phosphorylation of the myosin light and heavy chains was determined before and after antigenic stimulation. Before stimulation, myosin light chains contained 0.4 mol of phosphate/mol of light chain all confined to a serine not phosphorylated by protein kinase C. Cells that secreted 44% of their total histamine in 10 min exhibited an increase in phosphate content at sites phosphorylated by protein kinase C from 0 mol of phosphate/mol of myosin subunit to 0.7 mol of phosphate/mol of light chain and to 1 mol of phosphate/mol of heavy chain. When RBL-2H3 cells were made permeable with streptolysin O they still showed a qualitatively similar pattern of secretion and phosphorylation. Our results show that the time course of histamine secretion from stimulated RBL-2H3 cells parallels that of myosin heavy and light chain phosphorylation by protein kinase C.     

Regulation of Drosophila myosin ATPase activity by phosphorylation of myosin light chains--II. Flightless mfd- fly

1. The Ca2(+)-activated and Mg2+ actin-activated myosin ATPase activities of flightless mfd- mutant Drosophila flight muscle myosin were one-half and one-third of those of the wild-type fly muscle myosin, respectively. 2. In the two-dimensional gel electrophoresis, the spots corresponding to phosphorylated myosin light chains, Lfl and Ltl, were hardly detected in mfd- mutant myosin. 3. These results support not only the conclusion that phosphorylation of myosin light chains regulates Drosophila myosin ATPase activity but also the assumption that the phosphorylation of myosin light chains is directly involved in flight function of the Drosophila fly.     

Inhibition of conformational change in smooth muscle myosin by a monoclonal antibody against the 17-kDa light chain

Monoclonal antibodies against gizzard smooth muscle myosin were generated and characterized. One of these antibodies, designated MM-2, recognized the 17-kDa light chain and modulated the ATPase activities and hydrodynamic properties of smooth muscle myosin. Rotary shadowing electron microscopy showed that MM-2 binds 51 (+/- 25) A from the head-rod junction. The depression of Ca2+- and Mg2+-ATPase activities of myosin and Ca2+-ATPase activity of heavy meromyosin at low KCl concentration were abolished by MM-2. Viscosity measurement indicated that MM-2 inhibits the transition of 6 S myosin to 10 S myosin. While the rate of the production of subfragment-1 by papain proteolysis of 6 S myosin was inhibited by MM-2, the rate of proteolysis of the heavy chain of 10 S myosin was enhanced by MM-2 and reached the same rate as that of 6 S myosin plus MM-2. These results suggest that MM-2 inhibits the formation of 10 S myosin by binding to the 17-kDa light chain which is localized at the head-neck region of the myosin molecule. MM-2 increased the Vmax of actin-activated Mg2+-ATPase activities of both dephosphorylated myosin and dephosphorylated heavy meromyosin about 10- and 20-fold, respectively. MM-2 also activated the actin-activated Mg2+-ATPase activity of phosphorylated myosin at a low MgCl2 concentration and thus abolished the Mg2+-dependence of acto phosphorylated myosin ATPase activity. These results suggest that MM-2 inhibits the formation of 10 S myosin, and this results in the activation of actin-activated Mg2+-ATPase activity even in the absence of phosphorylation.     

Myosin from pancreatic acinar carcinoma cells. Isolation, characterization and demonstration of heavy- and light-chain phosphorylation.

Myosin has been identified in a variety of non-muscle cells, and is believed to play a role in maintenance of cell shape, locomotion, cytokinesis, exocytosis and other cellular functions. In this paper we describe the purification of myosin from a pancreatic acinar-cell carcinoma of the rat which forms solid tumours, but retains many differentiated functions. The purified myosin was composed of a 200,000 Da heavy chain and two or three classes of light chains. Electron-microscopic examination of rotary-shadowed preparations revealed that individual molecules had two globular heads and a long tail measuring approx. 149 nm. The myosin was soluble in high-salt buffers and became sedimentable as the ionic strength was lowered. Examination of negative-stained preparations showed that this sedimentable myosin consisted of short, bipolar, thick filaments which had a strong tendency to aggregate in a head-to-head manner. The ATPase activity of the purified myosin was stimulated by EDTA or Ca2+, but not by Mg2+. In low ionic strength the Mg2+-dependent ATPase activity was activated by muscle f-actin. The pancreatic myosin bound to actin and could be dissociated by the addition of MgATP. Myosin purified from cells cultured in media containing [32P]Pi was phosphorylated on one of the light chains as well as the heavy chain. Thus pancreatic acinar cells contain a typical non-muscle myosin, and the subunits of this molecule are subject to post-translational modification by phosphorylation.     

Purification and characterization of a third isoform of myosin I from Acanthamoeba castellanii

A third isoform of myosin I has been isolated from Acanthamoeba and designated myosin IC. Peptide maps and immunoassays indicate that myosin IC is not a modified form of myosin IA, IB, or II. However, myosin IC has most of the distinctive properties of a myosin I. It is a globular protein of native Mr approximately 162,000, apparently composed of a single 130-kDa heavy chain and a pair of 14-kDa light chains. It is soluble in MgATP at low ionic strength, conditions favoring filament assembly by myosin II. Myosin IC has high Ca2+- and (K+,EDTA)-ATPase activities. Its low Mg2+-ATPase activity is stimulated to a maximum rate of 20 s-1 by the addition of F-actin if its heavy chain has been phosphorylated by myosin I heavy chain kinase. The dependence of the Mg2+-ATPase activity of myosin IC on F-actin concentration is triphasic; and, at fixed concentrations of F-action, this activity increases cooperatively as the concentration of myosin IC is increased. These unusual kinetics were first demonstrated for myosins IA and IB and shown to be due to the presence of two actin-binding sites on each heavy chain which enable those myosins I to cross-link actin filaments. Myosin IC is also capable of cross-linking F-actin, which, together with the kinetics of its actin-activated Mg2+-ATPase activity, suggests that it, like myosins IA and IB, possesses two independent actin-binding domains.