Purification and characterization of a myosin I heavy chain kinase from Acanthamoeba castellanii

In previous work from this laboratory, a partially purified protein kinase from the soil amoeba Acanthamoeba castellanii was shown to phosphorylate the heavy chain of the two single-headed Acanthamoeba myosin isoenzymes, myosin IA and IB, resulting in a 10- to 20-fold increase in their actin-activated Mg2+-ATPase activities (Maruta, H., and Korn, E.D. (1977) J. Biol. Chem. 252, 8329-8332). A myosin I heavy chain kinase has now been purified to near homogeneity from Acanthamoeba by chromatography on DE-52 cellulose, phosphocellulose, and Procion red dye, followed by chromatography on histone-Sepharose. Myosin I heavy chain kinase contains a single polypeptide of 107,000 Da by electrophoretic analysis. Molecular sieve chromatography yields a Stokes radius of 4.1 nm, consistent with a molecular weight of 107,000 for a native protein with a frictional ratio of approximately 1.3:1. The kinase catalyzes the incorporation of 0.9 to 1.0 mol of phosphate into the heavy chain of both myosins IA and IB. Phosphoserine has been shown to be the phosphorylated amino acid in myosin IB. The kinase has highest specific activity toward myosin IA and IB, about 3-4 mumol of phosphate incorporated/min/mg (30 degrees C) at concentrations of myosin I that are well below saturating levels. The kinase also phosphorylates histone 2A, isolated smooth muscle light chains, and, to a very small extent, casein, but has no activity toward phosvitin or myosin II, a third Acanthamoeba myosin isoenzyme with a very different structure from myosin IA and IB. Myosin I heavy chain kinase requires Mg2+ but is not dependent on Ca2+, Ca2+/calmodulin, or cAMP for activity. The kinase undergoes an apparent autophosphorylation.     

Acanthamoeba myosin I heavy chain kinase is activated by phosphatidylserine-enhanced phosphorylation

The actin-activated Mg2(+)-ATPase activities of myosins I from Acanthamoeba castellanii are fully expressed only when a single amino acid on their heavy chain is phosphorylated by myosin I heavy chain kinase. Here we show that kinase isolated by a procedure designed to minimize its phosphorylation during purification can incorporate up to 7.5 mol of phosphate/mol of enzyme when incubated with ATP, possibly by autophosphorylation. The rate of phosphorylation is enhanced about 20-fold by phosphatidylserine but is unaffected by calcium ions. Phosphorylation increases the rate at which the kinase phosphorylates the regulatory site of myosin I by about 50-fold. These results suggest that (auto?)phosphorylation may regulate the activity of myosin I heavy chain kinase in vivo. The stimulation of kinase phosphorylation by phosphatidylserine (other phospholipids have not yet been tested) is of particular interest because myosin I has been shown to be tightly associated with membranes, especially the plasma membrane.     

Substrate specificity of Acanthamoeba myosin I heavy chain kinase as determined with synthetic peptides

Phosphorylation of a single threonine (myosin IA) or serine (myosins IB and IC) in the heavy chains of the Acanthamoeba myosin I isozymes is required for expression of their actin-activated Mg2(+)-ATPase activities. We now report that the synthetic peptide Gly-Arg-Gly-Arg-Ser-Ser-Val-Tyr-Ser, which corresponds to the phosphorylated region of Acanthamoeba myosin IC, is a good substrate for myosin I heavy chain kinase: Km = 54 microM, and Vmax = 15 mumols/min.mg. The same serine is phosphorylated as in the native substrate (residue 6 in the above sequence), and kinase activity with the synthetic peptide as substrate is also stimulated by phosphatidylserine-enhanced autophosphorylation of the kinase. These results indicate that all of the essential sequence determinants of kinase specificity are contained within this 9-residue peptide. With the peptide as substrate, we found that another acidic phospholipid, phosphatidylinositol, also enhances autophosphorylation of the kinase whereas the neutral phospholipids phosphatidylcholine and phosphatidylethanolamine do not. By comparing the Km and Vmax values for a series of synthetic peptide substrates, we established that 1 basic amino acid is essential on the NH2-terminal side of the phosphorylation site, and two are preferable, and that a tyrosine is essential 2 residues away on the COOH-terminal side. There is a slight preference for arginines over lysines. All of these local sequence specificity determinants are present in the three native substrates, Acanthamoeba myosins IA, IB, and IC, and in two Dictyostelium myosin I isozymes that are putative substrates for the kinase. Similar sequences do not occur in the myosins I from intestinal brush border, which is not a substrate for the Acanthamoeba kinase.     

Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.     

Quantification and localization of phosphorylated myosin I isoforms in Acanthamoeba castellanii

The actin-activated Mg(2+)-ATPase activities of the three myosin I isoforms in Acanthamoeba castellanii are significantly expressed only after phosphorylation of a single site in the myosin I heavy chain. Synthetic phosphorylated and unphosphorylated peptides corresponding to the phosphorylation site sequences, which differ for the three myosin I isoforms, were used to raise isoform-specific antibodies that recognized only the phosphorylated myosin I or the total myosin I isoform (phosphorylated and unphosphorylated), respectively. With these antisera, the amounts of total and phosphorylated isoform were quantified, the phosphomyosin I isoforms localized, and the compartmental distribution of the phosphomyosin isoforms determined. Myosin IA, which was almost entirely in the actin-rich cortex, was 70- 100% phosphorylated and particularly enriched under phagocytic cups. Myosins IB and IC were predominantly associated with plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmic myosins IB and IC, like cytoplasmic myosin IA, were mostly phosphorylated (60-100%). Moreover, phosphomyosin IB was concentrated in actively motile regions of the plasma membrane. More than 20-fold more phosphomyosin IC and 10-fold more F-actin were associated with the membranes of contracting contractile vacuoles (CV) than of filling CVs. As the total amount of CV-associated myosin IC remained constant, it must be phosphorylated at the start of CV contraction. These data extend previous proposals for the specific functions of myosin I isozymes in Acanthamoeba (Baines, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell Biol. 119: 1193-1203): phosphomyosin IA in phagocytosis, phosphomyosin IB in phagocytosis and pinocytosis, and phosphomyosin IC in contraction of the CV.     

Preparation of a phospholipid-insensitive, autophosphorylation-activated catalytic fragment of Acanthamoeba myosin I heavy chain kinase.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosin I depends on phosphorylation of its single heavy chain. The activity of the myosin I heavy chain kinase is increased about 50-fold by autophosphorylation, and the rate of kinase autophosphorylation is enhanced about 20-fold by acidic phospholipids independent of the presence of Ca2+ (Brzeska, H., Lynch, T. J., and Korn, E. D. (1990) J. Biol. Chem. 265, 3591-3594). In this paper, we show that chymotryptic digestion of the kinase produces a 54-kDa fragment which contains three to four of the approximately 11 original phosphorylation sites and whose activity is greatly stimulated by autophosphorylation. However, both the rate of autophosphorylation and the kinase activity of the 54-kDa fragment are independent of phospholipid and comparable to those of intact kinase in the presence of phospholipid. These data imply that the (probably NH2-terminal) region(s) removed by proteolysis is necessary for phospholipid-sensitive inhibition of autophosphorylation of sites residing within the (probably COOH-terminal) 54-kDa fragment. The 54-kDa fragment contains the catalytic site of the kinase as well as three to four sites whose phosphorylation is necessary for full expression of kinase activity. The middle region of the kinase molecule contains proline-rich regions that are similar to the COOH-terminal tail of the kinase substrate, Acanthamoeba myosin I.     

Calmodulin-binding and autoinhibitory domains of Acanthamoeba myosin I heavy chain kinase, a p21-activated kinase (PAK)

The sequence homology between Acanthamoeba myosin I heavy chain kinase (MIHCK) and other p21-activated kinases (PAKs) is relatively low, including only the catalytic domain and a short PAK N-terminal motif (PAN), and even these regions are not highly homologous. In this paper, we report the expression in insect cells of full-length, fully regulated Acanthamoeba MIHCK and further characterize the regulation of this PAK by Rac, calmodulin, and autoinhibition. We map the autoinhibitory region of MIHCK to its PAN region and show that the PAN region inhibits autophosphorylation and kinase activity of unphosphorylated full-length MIHCK and its expressed catalytic domain but has very little effect on either when they are phosphorylated. These properties are similar to those reported for mammalian PAK1. Unlike PAK1, MIHCK is activated by Rac only in the presence of phospholipid. However, peptides containing the PAN region of MIHCK bind Rac in the absence of lipid, and Rac binding reverses the inhibition of the MIHCK catalytic domain by PAN peptides. Our data suggest that a region N-terminal to PAN is required for optimal binding of Rac. Also unlike mammalian PAK, phospholipid stimulation of Acanthamoeba MIHCK and Dictyostelium MIHCK) (which is also a PAK) is inhibited by Ca(2+)-calmodulin. In contrast to Dictyostelium MIHCK, however, Ca(2+)-calmodulin also inhibits Rac-induced activity of Acanthamoeba MIHCK. The basic region N-terminal to PAN is essential for calmodulin binding.     

The isolated heavy chain of an Acanthamoeba myosin contains full enzymatic activity.

Acanthamoeba myosin IB is a single-headed enzyme containing one heavy chain of 125,000 daltons, one light chain of 27,000 daltons, and one light chain of 14,000 daltons. The 125,000- and 27,000-dalton polypeptides are consistently found in a molar ratio of 1:1. The content of the 14,000-dalton peptide is usually only 0.1 to 0.2, and always less than 0.5, relative to the other two chains and might be a contaminant or a degradation product of one of the other chains. The specific activities of the Ca2+-ATPase, (K+, EDTA)-ATPase, and (after phosphorylation of its heavy chain by a specific kinase) actin-activated Mg2+-ATPase of Acanthamoeba myosin IB are similar to those of rabbit skeletal muscle myosin. After treatment of the enzyme with 2 M LiCl, the 125,000-dalton heavy chain of Acanthamoeba myosin Ib can be obtained, by chromatography on Sephadex G-200, essentially free of the 14,000-dalton peptide and more than 90% free of the 27,000-dalton peptide. This isolated heavy chain has the same specific ATPase activities as the original enzyme. Therefore, the heavy chain of Acanthamoeba myosin IB contains the ATPase catalytic site, the actin-binding site, and the phosphorylation site and is fully active enzymatically in the absence of light chains.     

Purification from Dictyostelium discoideum of a low-molecular-weight myosin that resembles myosin I from Acanthamoeba castellanii

A low-molecular-weight myosin has been purified 1500-fold from extracts of Dictyostelium discoideum, based on the increase in K+,EDTA-ATPase specific activity. The purified enzyme resembles the single-headed, low-molecular-weight myosins IA and IB from Acanthamoeba castellanii, and differs from the conventional two-headed, high-molecular-weight myosin previously isolated from Dictyostelium, in several ways. It has higher K+,EDTA-ATPase activity than Ca2+-ATPase activity; it has a native molecular mass of about 150,000 and a single heavy chain of about 117,000; the 117,000-dalton heavy chain is phosphorylated by Acanthamoeba myosin I heavy chain kinase; phosphorylation of its heavy chain enhances its actin-activated Mg2+-ATPase activity; and the 117,000-dalton heavy chain reacts with antibodies raised against the heavy chain of Acanthamoeba myosin IA. None of these properties is shared by the low-molecular-weight active fragment that can be produced by chymotryptic digestion of conventional Dictyostelium myosin. We conclude that Dictyostelium contains an enzyme of the myosin I type previously isolated only from Acanthamoeba.     

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.     

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.     

Benzodiazepine binding to mitochondrial membranes of the amoeba Acanthamoeba castellanii and the yeast Saccharomyces cerevisiae

Benzodiazepine binding sites were studied in mitochondria of unicellular eukaryotes, the amoeba Acathamoeba castellanii and the yeast Saccharomyces cerevisiae, and also in rat liver mitochondria as a control. For that purpose we applied Ro5-4864, a well-known ligand of the mitochondrial benzodiazepine receptor (MBR) present in mammalian mitochondria. The levels of specific [(3)H]Ro5-4864 binding, the dissociation constant (K(D)) and the number of [(3)H]Ro5-4864 binding sites (B(max)) determined for fractions of the studied mitochondria indicate the presence of specific [(3)H]Ro5-4864 binding sites in the outer membrane of yeast and amoeba mitochondria as well as in yeast mitoplasts. Thus, A. castellanii and S. cerevisiae mitochondria, like rat liver mitochondria, contain proteins able to bind specifically [(3)H]Ro5-4864. Labeling of amoeba, yeast and rat liver mitochondria with [(3)H]Ro5-4864 revealed proteins identified as the voltage dependent anion selective channel (VDAC) in the outer membrane and adenine nucleotide translocase (ANT) in the inner membrane. Therefore, the specific MBR ligand binding is not confined only to mammalian mitochondria and is more widespread within the eukaryotic world. However, it can not be excluded that MBR ligand binding sites are exploited efficiently only by higher multicellular eukaryotes. Nevertheless, the MBR ligand binding sites in mitochondria of lower eukaryotes can be applied as useful models in studies on mammalian MBR.     

Isolation of a non-muscle myosin heavy chain gene from Acanthamoeba

We have isolated a non-muscle myosin heavy chain gene from Acanthamoeba castellanii using as a heterologous probe a sarcomeric myosin heavy chain gene from Caenorhabditis elegans. The amoeba genomic clone has been tentatively identified as containing a myosin II heavy chain gene based on hybridization to a 5300-nucleotide RNA species, hybrid selection of a mRNA encoding a 185-kDa polypeptide, specific immunoprecipitation of this polypeptide with antiserum to myosin II, and an exact match between the DNA sequence and a carboxyl-terminal myosin II peptide previously sequenced by protein chemical methods (C?té, G.P., Robinson, E.A., Appella, E., and Korn, E. D. (1984) J. Biol. Chem. 259, 12781-12787). We also sequenced a region of the gene whose deduced amino acid sequence shows strong homology with that region of muscle myosins which is thought to be involved in nucleotide binding. These results indicate that the amoeba genomic clone contains at least 90% of the coding information for the 185-kDa heavy chain polypeptide and that the bulk of the gene contains very little intron DNA. Genomic blots of amoeba DNA probed with a portion of this myosin gene indicate the presence of additional highly related sequences within the amoeba genome.     

Control of AMP deaminase 1binding to myosin heavy chain

AMP deaminase (AMPD) plays a central role in preserving theadenylate energy charge in myocytes following exercise and in producingintermediates for the citric acid cycle in muscle. Prior studies havedemonstrated that AMPD1 binds to myosin heavy chain (MHC)in vitro; binding to the myofibril varies with the state of musclecontraction in vivo, and binding of AMPD1 to MHC is required foractivation of this enzyme in myocytes. The present study has identifiedthree domains in AMPD1 that influence binding of this enzyme to MHCusing a cotransfection model that permits assessment of mutationsintroduced into the AMPD1 peptide. One domain that encompasses residues178-333 of this 727-amino acid peptide is essential for binding ofAMPD1 to MHC. This region of AMPD1 shares sequence similarity withseveral regions of titin, another MHC binding protein. Two additionaldomains regulate binding of this peptide to MHC in response tointracellular and extracellular signals. A nucleotide binding site,which is located at residues 660-674, controls binding of AMPD1 toMHC in response to changes in intracellular ATP concentration. Deletionanalyses demonstrate that the amino-terminal 65 residues of AMPD1 playa critical role in modulating the sensitivity to ATP-induced inhibitionof MHC binding. Alternative splicing of the AMPD1 gene product, which alters the sequence of residues 8-12, produces two AMPD1 isoforms that exhibit different MHC binding properties in the presence of ATP.These findings are discussed in the context of the various rolesproposed for AMPD in energy production in the myocyte.

     

Recruitment of a myosin heavy chain kinase to actin-rich protrusions in Dictyostelium

Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives the disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin-dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading-edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion.     

Characteristics of calmodulin binding to purified human lymphocyte plasma membranes

We have explored the role of calmodulin in plasma membrane-related phenomena in lymphocyte activation by measurement of [125I]calmodulin binding to highly purified plasma membrane of human peripheral blood lymphocytes. Calcium-dependent calmodulin binding to lymphocyte membrane was found to reach equilibrium within 5 min of incubation at 37 degrees C and to be saturable and specific. A single class of high affinity-binding sites was identified, with a dissociation constant (Kd) of 1 to 3 X 10(-8) M and a total binding capacity (Bt) of 1 to 2 pmol/mg membrane protein. The free calcium concentration necessary for half-maximal binding was 100 to 300 nM. This was strikingly similar to the cytoplasmic-free calcium activity [Ca2+]i measured by the Quin-2 fluorescence technique, particularly after stimulation with phytomitogens. Calmodulin binding was inhibitable by trifluoperazine (TFP), W-7, and chloropramazine, all of which are calmodulin antagonists. The concentration of TFP that caused 50% inhibition of lymphocyte proliferative responses to phytomitogens was found to be identical to the concentration of TFP which causes 50% inhibition of calmodulin binding to lymphocyte plasma membrane. SDS-polyacrylamide gel electrophoresis followed by gel overlay and autoradiography with iodinated calmodulin revealed five calcium-dependent, TFP-inhibitable, calmodulin-binding polypeptides.     

Identification of three phosphorylation sites on each heavy chain of Acanthamoeba myosin II

It has been previously demonstrated that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II is inhibited by phosphorylation of its two heavy chains (Collins, J. H., and Korn, E. D. (1980) J. Biol. Chem. 255, 8011-8014). In this paper, it is shown that a partially purified kinase preparation from Acanthamoeba catalyzes the incorporation of 3 mol of phosphate into each mole of myosin II heavy chain. Tryptic digestion of the 32P-myosin, followed by two-dimensional peptide mapping, indicates that two of the three sites phosphorylated by the kinase in vitro correspond to the two major phosphorylation sites on the myosin heavy chain in vivo. Phosphorylation of myosin II in vitro by the kinase fraction completely inhibits the actin-activated Mg2+-ATPase activity of myosin II. Myosin II can be isolated in a highly phosphorylated, enzymatically inactive form, then dephosphorylated to an active form, and finally rephosphorylated to an inactive form. The Acanthamoeba kinase fraction catalyzes the phosphorylation of all three sites on the heavy chain of myosin II at virtually the same rate. From a comparison of the decrease in actin-activated Mg2+-ATPase activity with the amount of phosphate incorporated into myosin II, and from the results obtained previously by dephosphorylating myosin II (Collins, J. H., and Korn, E. D., (1980) J. Biol. Chem. 255, 8011-8014), it can be inferred that two of the sites phosphorylated in vitro act in a synergistic manner to inhibit the actin-activated myosin II Mg2+-ATPase.     

Localization of myosin IC and myosin II in Acanthamoeba castellanii by indirect immunofluorescence and immunogold electron microscopy

Polyclonal antisera have been raised against purified Acanthamoeba myosin II and to a synthetic 26 amino acid peptide that corresponds in sequence to the phosphorylation site of Acanthamoeba myosin IC. These antisera are specific for their respective antigens as determined by immunoblotting after SDS-PAGE of total cell lysates. By using the antisera, localization studies were performed by indirect immunofluorescence and by immunogold electron microscopy. Myosin II occurred in the cell cytoplasm and appeared to be concentrated in the cortex. Immunogold cytochemistry revealed at high resolution that myosin II is organized into rodlike filaments approximately 200 nm long. The antibody raised against the myosin IC synthetic peptide recognized both the plasma membrane and the membrane of the contractile vacuole. The plasma membrane staining was labile to treatment with saponin suggesting an intimate association of the myosin IC with membrane phospholipids. Immunogold cytochemistry with the antimyosin IC synthetic peptide showed that the myosin IC is closely associated with the membrane bilayer.     

Effects of purified myosin light chain kinase on myosin light chain phosphorylation and catecholamine secretion in digitonin-permeabilized chromaffin cells

Many non-muscle cells including chromaffin cells contain actin and myosin. The 20,000 dalton light chain subunits of myosin can be phosphorylated by a Ca²⁺/calmodulin-dependent enzyme, myosin light chain kinase. In tissues other than striated muscle, light chain phosphorylation is required for actin-induced myosin ATPase activity. The possibility that actin and myosin are involved in catecholamine secretion was investigated by determining whether increased phosphorylation in the presence of [-³²P]ATP of myosin light chain by myosin light chain kinase enhances secretion from digitonin-treated chromaffin cells. In the absence of exogenous myosin light chain kinase, 1 M Ca²⁺ caused a 30–40% enhancement of the phosphorylation of a 20 kDa protein. This protein was identified on 2-dimensional gels as myosin light chain by its comigration with purified myosin light chain. Purified myosin light chain kinase (400 g/ml) in the presence of calmodulin (10 M) caused little or no enhancement of myosin light chain phosphorylation in the absence of Ca²⁺ in digitonin-treated cells. In the presence of 1 M Ca²⁺, myosin light chain kinase (400 g/ml) caused an approximately two-fold increase in myosin light chain phosphorylation in digitonin-treated cells in 5 min. The phosphorylation required permeabilization of the cells by digitonin and occurred within the cells rather than in the medium. Myosin light chain kinase-induced phosphorylation of myosin light chain was maximal at 1 M. Ca²⁺. Under identical conditions to those of the phosphorylation experiments, secretion was unaltered by myosin light chain kinase. The experiments indicate that the phosphorylation of myosin light chain by myosin light chain kinase is not a limiting factor in secretion in digitonin-treated chromaffin cells and suggest that the activation of myosin is not directly involved in secretion from the cells. The experiments also demonstrate the feasibility of investigation of effects of exogenously added proteins on secretion in digitonin-treated cells.Abbreviations EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - KGEPM solution containing potassium glutamate, EGTA, PIPES and MgCl₂ - NE norepinephrine - PIPES piperazine-N,-N-bis-(2-ethanesulfonic acid) - PSS physiological salt solution     

Dictyostelium myosin heavy chain kinase A regulates myosin localization during growth and development

Phosphorylation of the Dictyostelium myosin II heavy chain (MHC) has a key role in regulating myosin localization in vivo and drives filament disassembly in vitro. Previous molecular analysis of the Dictyostelium myosin II heavy chain kinase (MHCK A) gene has demonstrated that the catalytic domain of this enzyme is extremely novel, showing no significant similarity to the known classes of protein kinases (Futey, L. M., Q. G. Medley, G. P. Cote, and T. T. Egelhoff. 1995. J. Biol. Chem. 270:523-529). To address the physiological roles of this enzyme, we have analyzed the cellular consequences of MHCK A gene disruption (mhck A- cells) and MHCK A overexpression (MHCK A++ cells). The mhck A- cells are viable and competent for tested myosin-based contractile events, but display partial defects in myosin localization. Both growth phase and developed mhck A- cells show substantially reduced MHC kinase activity in crude lysates, as well as significant overassembly of myosin into the Triton-resistant cytoskeletal fractions. MHCK A++ cells display elevated levels of MHC kinase activity in crude extracts, and show reduced assembly of myosin into Triton-resistant cytoskeletal fractions. MHCK A++ cells show reduced growth rates in suspension, becoming large and multinucleated, and arrest at the mound stage during development. These results demonstrate that MHCK A functions in vivo as a protein kinase with physiological roles in regulating myosin II localization and assembly in Dictyostelium cells during both growth and developmental stages.