Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site

The heavy chains and the 19-kDa and 20-kDa light chains of bovine brain myosin can by phosphorylated. To localise the site of heavy-chain phosphorylation, the myosin was initially subjected to digestion with chymotrypsin and papain under a variety of conditions and the fragments thus produced were identified. Irrespective of the ionic strength, i.e. whether the myosin was monomeric or filamentous, chymotryptic digestion produced two major fragments of 68 kDa and 140 kDa; the 140-kDa fragment was further digested by papain to yield a 120-kDa and a 23-kDa fragment. These fragments were characterised by (a) a gel overlay technique using 125I-labelled light chains, which showed that the 140-kDa and 23-kDa polypeptides contain the light-chain-binding sites; (b) using myosin photoaffinity labelled at the active site with [3H]UTP, which showed that the 68-kDa fragment contained the catalytic site, and (c) electron microscopy, using rotary shadowing and negative-staining techniques, which demonstrated that after chymotryptic digestion the myosin head remains attached to the tail whereas on papain digestion isolated heads and tails were observed. Thus the 120-kDa polypeptide derived from the 140-kDa fragment is the tail of the myosin, and the 68-kDa fragment containing the catalytic site and the 23-kDa fragment, with the light-chain-binding sites, form the head (S1) portion of the myosin. When [32P]-phosphorylated brain myosin was digested with chymotrypsin and papain it was shown that the heavy-chain phosphorylation site is located in a 5-kDa peptide at the C-terminal end of the heavy chain, i.e. the end of the myosin tail. Using hydrodynamic and electron microscopic techniques, no significant effect of either light-chain or heavy-chain phosphorylation on the stability of brain myosin filaments was observed, even in the presence of MgATP. Brain myosin filaments appear to be more stable than those of other non-muscle myosins. Light-chain phosphorylation did, however, have an effect on the conformation of brain myosin, for example in the presence of MgATP non-phosphorylated myosin molecules were induced to fold into a very compact folded state.     

Specific phosphorylation of threonine by the Dictyostelium myosin II heavy chain kinase family

Dictyostelium myosin II heavy chain kinase A (MHCK A), MHCK B, and MHCK C contain a novel type of protein kinase catalytic domain that displays no sequence identity to the catalytic domain present in conventional serine, threonine, and/or tyrosine protein kinases. Several proteins, including myelin basic protein, myosin regulatory light chain, caldesmon, and casein were phosphorylated by the bacterially expressed MHCK A, MHCK B, and MHCK C catalytic domains. Phosphoamino acid analyses of the proteins showed that 91 to 99% of the phosphate was incorporated into threonine with the remainder into serine. Acceptor amino acid specificity was further examined using a synthetic peptide library (MAXXXX(S/T)XXXXAKKK; where X is any amino acid except cysteine, tryptophan, serine, and threonine and position 7 contains serine and threonine in a 1.7:1 ratio). Phosphorylation of the peptide library with the three MHCK catalytic domains resulted in 97 to 99% of the phosphate being incorporated into threonine, while phosphorylation with a conventional serine/threonine protein kinase, the p21-activated kinase, resulted in 80% of the phosphate being incorporated into serine. The acceptor amino acid specificity of MHCK A was tested directly by substituting serine for threonine in a synthetic peptide and a glutathione S-transferase fusion peptide substrate. The serine-containing substrates were phosphorylated at a 25-fold lower rate than the threonine-containing substrates. The results indicate that the MHCKs are specific for the phosphorylation of threonine.     

Dictyostelium myosin: characterization of chymotryptic fragments and localization of the heavy-chain phosphorylation site

Chymotrypsin cleaves Dictyostelium myosin in half, splitting the heavy chain (210,000 daltons) into two fragments of 105,000 daltons each. One of the two major fragments is soluble at low ionic strength and has a native molecular weight of 130,000. As judged by SDS polyacrylamide gel electrophoresis, this soluble fragment consists of the two intact myosin light chains of 18,000 and 16,000 daltons and a 105,000-dalton polypeptide derived from the myosin heavy chain. The soluble fragment retains actin-activated ATPase activity and the ability to bind to actin in an ATP-dissociable fashion. The maximal velocity of the actin- activated ATPase activity of the soluble fragment is 80% of that of uncleaved myosin, although its apparent Km for actin is 12-fold greater than that of myosin. In addition to the major soluble 105,000-dalton fragment discussed above, chymotryptic cleavage of the Dictyostelium myosin also generates fragments that are insoluble at low ionic strength. The major insoluble fragment is 105,000 daltons on an SDS polyacrylamide gel and forms thick filaments that are devoid of myosin heads. A less prevalent insoluble fragment has a molecular weight of 83,000 and is probably a subfragment of the insoluble 105,000-dalton fragment. The heavy chain of myosin is phosphorylated in vivo and the phosphorylation site has been localized to the insoluble fragments, which derive from the tail portion of the myosin molecule.     

Effect of heavy chain phosphorylation on the polymerization and structure of Dictyostelium myosin filaments

In Dictyostelium amebas, myosin appears to be organized into filaments that relocalize during cell division and in response to stimulation by cAMP. To better understand the regulation of myosin assembly, we have studied the polymerization properties of purified Dictyostelium myosin. In 150 mM KCl, the myosin remained in the supernate following centrifugation at 100,000 g. Rotary shadowing showed that this soluble myosin was monomeric and that approximately 80% of the molecules had a single bend 98 nm from the head-tail junction. In very low concentrations of KCl (less than 10 mM) the Dictyostelium myosin was also soluble at 100,000 g. But rather than being monomeric, most of the molecules were associated into dimers or tetramers. At pH 7.5 in 50 mM KCl, dephosphorylated myosin polymerized into filaments whereas myosin phosphorylated to a level of 0.85 mol Pi/mol heavy chain failed to form filaments. The phosphorylated myosin could be induced to form filaments by lowering the pH or by increasing the magnesium concentration to 10 mM. The resulting filaments were bipolar, had blunt ends, and had a uniform length of approximately 0.43 micron. In contrast, filaments formed from fully dephosphorylated myosin were longer, had tapered ends, and aggregated to form very long, threadlike structures. The Dictyostelium myosin had a very low critical concentration for assembly of approximately 5 micrograms/ml, and this value did not appear to be affected by the level of heavy chain phosphorylation. The concentration of polymer at equilibrium, however, was significantly reduced, indicating that heavy chain phosphorylation inhibited the affinity of subunits for each other. Detailed assembly curves revealed that small changes in the concentration of KCl, magnesium, ATP, or H+ strongly influenced the degree of assembly. Thus, changes in both the intracellular milieu and the level of heavy chain phosphorylation may control the location and state of assembly of myosin in response to physiological stimuli.     

Intermolecular versus intramolecular interactions of Dictyostelium myosin: possible regulation by heavy chain phosphorylation

Dictyostelium myosin has been examined under conditions that reveal intramolecular and intermolecular interactions that may be important in the process of assembly and its regulation. Rotary shadowed myosin molecules exhibit primarily two configurations under these conditions: straight parallel dimers and folded monomers. All of the monomers bend in a specific region of the 1860-A-long tail that is 1200 A from the head-tail junction. Molecules in parallel dimers are staggered by 140 A, which is a periodicity in the packing of myosin molecules originally observed in native thick filaments of muscle. The most common region for interaction in the dimers is a segment of the tail about 200-A-long, extending from 900 to 1100 A from the head-tail junction. Parallel dimers form tetramers by way of antiparallel interactions in their tail regions with overlaps in multiples of 140 A. The folded configuration of the myosin molecules is promoted by phosphorylation of the heavy chain by Dictyostelium myosin heavy chain kinase. It appears that the bent monomers are excluded from filaments formed upon addition of salt while the dimeric molecules assemble. These results may provide the structural basis for primary steps in myosin filament assembly and its regulation by heavy chain phosphorylation.     

TRPM7 regulates myosin IIA filament stability and protein localization by heavy chain phosphorylation

Deregulation of myosin II-based contractility contributes to the pathogenesis of human diseases, such as cancer, which underscores the necessity for tight spatial and temporal control of myosin II activity. Recently, we demonstrated that activation of the mammalian α-kinase TRPM7 inhibits myosin II-based contractility in a Ca²⁺- and kinase-dependent manner. However, the molecular mechanism is poorly defined. Here, we demonstrate that TRPM7 phosphorylates the COOH-termini of both mouse and human myosin IIA heavy chains—the COOH-terminus being a region that is critical for filament stability. Phosphorylated residues were mapped to Thr1800, Ser1803 and Ser1808. Mutation of these residues to alanine and that to aspartic acid lead to an increase and a decrease, respectively, in myosin IIA incorporation into the actomyosin cytoskeleton and accordingly affect subcellular localization. In conclusion, our data demonstrate that TRPM7 regulates myosin IIA filament stability and localization by phosphorylating a short stretch of amino acids within the α-helical tail of the myosin IIA heavy chain.     

Cell motility and chemotaxis in Dictyostelium amebae lacking myosin heavy chain

Dictyostelium amebae have been engineered by homologous recombination of a truncated copy of the myosin heavy chain gene (heavy meromyosin (HMM) cells) and by transformation with a vector encoding an antisense RNA to myosin heavy chain mRNA (mhcA cells) so that they lack native myosin heavy chain protein. In the former case, cells synthesize only the heavy meromyosin portion of the protein and in the latter case they synthesize negligible amounts of the protein. Surprisingly, it was demonstrated that both cell lines are viable and motile. In order to compare the motility of these cells with normal cells, the newly developed computer-assisted Dynamic Morphology System (DMS) was employed. The results demonstrate that the average HMM or mhcA ameba moves at a rate of translocation less than half that of normal cells. It is rounder and less polar than a normal cell, and exhibits a rate of cytoplasmic expansion and contraction roughly half that of normal cells. In a spatial gradient of cAMP, the average ameba of HMM or mhcA exhibits a chemotactic index of +0.10 or less, compared to the chemotactic index of +0.50 exhibited by normal cells. Finally, the initial area, rate of expansion, and final area of pseudopods are roughly half that of normal cells. The five fastest HMM amebae (out of 35 analyzed in detail) moved at an average rate of translocation equal to that of normal amebae, and exhibited an average chemotactic index of +0.34. In addition, the average rate of cytoplasmic flow in fast HMM cells was equal to that of the average normal ameba. However, fast HMM amebae still exhibited the same defects in pseudopod formation that were exhibited by the entire HMM cell population. These results suggest that myosin heavy chain is involved in the "fine tuning" and efficiency of pseudopod formation, but is not essential for the basic behavior of pseudopod expansion.     

Actin-activated Mg-ATPase activity of Dictyostelium myosin II. Effects of filament formation and heavy chain phosphorylation.

The actin-activated Mg-ATPase activities of unphosphorylated and heavy chain phosphorylated Dictyostelium myosin II and of a Dictyostelium myosin II heavy meromyosin (HMM) fragment were examined at different Mg2+ and KCl concentrations. The Mg-ATPase activity of HMM displayed a maximum rate, Vmax, of about 4.0/s and a Kapp (actin concentration required to achieve 1/2 Vmax) that increased from 8 to 300 microM as the KCl concentration increased from 0 to 120 mM. When assayed with greater than 5 mM Mg2+ and 0 mM KCl the unphosphorylated Dictyostelium myosin II yielded a Kapp of 0.25 microM and a Vmax of 2.8/s. At lower Mg2+ concentrations or with 50 mM KCl the data were not fit well by a single hyperbolic curve and Kapp increased to 25-100 microM. The increase in Kapp did not correlate with the loss of sedimentable filaments. At KCl concentrations above 100 mM Vmax increased to greater than 4/s. Heavy chain phosphorylated myosin (3.5 mol of phosphate/mol myosin) displayed a Vmax of about 5/s and a Kapp of 50 microM under all conditions tested. Thus, heavy chain phosphorylation inhibited the actin-activated Mg-ATPase activity of Dictyostelium myosin II in 5-10 mM Mg2+ and low ionic strength through an increase in Kapp.     

Developmental consequences of the lack of myosin heavy chain in Dictyostelium discoideum

Two different Dictyostelium discoideum cell lines that lack myosin heavy chain protein (MHC A) have been previously described. One cell line (mhcA) was created by antisense RNA inactivation of the endogenous mRNA and the other (HMM) by insertional mutagenesis of the endogenous myosin gene. The two cell lines show similar developmental defects; they are delayed in aggregation and become arrested at the mound stage. However, when cells that lack myosin heavy chain are mixed with wild-type cells, some of the mutant cells are capable of completing development to form mature spores. The pattern of expression of a number of developmentally regulated genes has been examined in both mutant cell lines. Although morphogenesis becomes aberrant before aggregation is completed, all of the markers that we have examined are expressed normally. These include genes expressed prior to aggregation as well as prespore genes expressed later in development. It appears that the signals necessary for cell-type differentiation are generated in the aborted structures formed by cells lacking MHC A. The mhcA cells have negligible amounts of MHC A protein while the HMM cells express normal amounts of a fragment of the myosin heavy chain protein similar to heavy meromyosin (HMM). The expression of myosin light chain was examined in these two cell lines. HMM cells accumulate normal amounts of the 18,000-D light chain, while the amount of light chain in mhcA cells is dramatically reduced. It is likely that the light chains assemble normally with the HMM fragment in HMM cells, while in cells lacking myosin heavy chain (mhcA) the light chains are unstable.     

Phosphorylation of the heavy chain of skeletal muscle myosin by casein kinase II: localization of the phosphorylation site to the amino terminus

While the heavy chain of rabbit skeletal muscle myosin is not phosphorylatable by casein kinase II, it turned out to be phosphorylatable after removal of all of the light chains. The phosphorylation site for the kinase was determined to be Ser-1 and/or Ser-2 at the amino terminus.     

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.     

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.     

Phosphorylation of threonine residues on cloned fragments of the Dictyostelium myosin heavy chain

A tail fragment of Dictyostelium discoideum myosin has been cloned and expressed as a fusion protein with the N-terminal region of MS-2 polymerase. The cloned fragment was phosphorylated with myosin heavy chain kinase II from aggregation-competent D. discoideum cells that specifically phosphorylate threonine residues on the myosin tail. Phosphopeptide maps showed the same site specificity of phosphorylation with the fusion protein as a substrate as with native myosin. An improved assay for the kinase was developed in which the fusion protein is precipitated with a monoclonal antibody that inhibits polymerization of the myosin tails without preventing their phosphorylation. Sites of phosphorylation were tentatively localized to a sequence in the C-terminal region of the heavy chain where four threonine residues are found.     

Chemoattractant-elicited increases in Dictyostelium myosin phosphorylation are due to changes in myosin localization and increases in kinase activity

We previously reported (Berlot, C. H., Spudich, J. A., and Devreotes, P. N. (1985) Cell 43, 307-314) that cAMP stimulation of chemotactically competent Dictyostelium amoebae causes transient increases in phosphorylation of the myosin heavy chain and 18,000-dalton light chain in vivo and in vitro. In this report we investigate the mechanisms involved in these changes in phosphorylation. In the case of heavy chain phosphorylation, the amount of substrate available for phosphorylation appears to be the major factor regulating the in vitro phosphorylation rate. Almost all heavy chain kinase activity is insoluble in Triton X-100, and the increase in the heavy chain phosphorylation rate in vitro parallels an increase in Triton insolubility of myosin. Changes in heavy chain phosphatase activity are not involved in the changes in the in vitro phosphorylation rate. In the case of light chain phosphorylation, increases in the vitro phosphorylation rate occur under conditions where the amount of substrate available for phosphorylation is constant and phosphatase activity is undetectable, implicating light chain kinase activation as the means of regulation. The specificity of the myosin kinases operating in vivo and in vitro was explored using phosphoamino acid and chymotryptic phosphopeptide analysis. The light chain is phosphorylated on serine both in vivo and in vitro, and phosphopeptide maps of the light chain phosphorylated in vivo and in vitro are indistinguishable. In the case of the heavy chain, both serine and threonine are phosphorylated in vivo and in vitro, although the cAMP-stimulated increases in phosphorylation occur primarily on threonine. Phosphopeptide maps of the heavy chain show that the peptides phosphorylated in vitro represent a major subset of those phosphorylated in vivo. The kinetics of the transient increases in myosin phosphorylation rates observed in vitro can be predicted quantitatively from the in vivo myosin phosphorylation data assuming that there is a constant phosphatase activity.     

Chemoattractant-elicited increases in myosin phosphorylation in Dictyostelium

Cyclic AMP stimulation of chemotactically competent Dictyostelium amebas labeled with [32P]orthophosphate transiently increases phosphorylation in the heavy chain and the 18,000 dalton light chain of myosin. Immediately before the increase, heavy chain phosphorylation transiently decreases. These phosphorylation changes also occur when cAMP-induced activation of adenylate cyclase is blocked by pretreatment of amebas with caffeine. The time course of these phosphorylation responses correlates with the shape changes induced in amebas exposed to a temporal increase in cAMP concentration. The dose dependence of the phosphorylation responses is the same as that previously determined for chemotaxis. The phosphorylation responses exhibit adaptation properties in common with those of the shape change response and chemotaxis. Increases in the rate of myosin heavy chain and light chain phosphorylation can be observed in vitro by stimulating unlabeled amebas with cAMP and then lysing the cells into a gamma-[32P]ATP-containing reaction mixture.     

Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells

In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.     

Determinants for substrate phosphorylation by Dictyostelium myosin II heavy chain kinases A and B and eukaryotic elongation factor-2 kinase

The alpha kinases are a widespread family of atypical protein kinases characterized by a novel type of catalytic domain. In this paper the peptide substrate recognition motifs for three alpha kinases, Dictyostelium discoideum myosin heavy chain kinase (MHCK) A and MHCK B and mammalian eukaryotic elongation factor-2 kinase (eEF-2K), were characterized by incorporating amino acid substitutions into a previously identified MHCK A peptide substrate (YAYDTRYRR) (Luo X. et al. (2001) J. Biol. Chem. 276, 17836-43). A lysine or arginine in the P+1 position on the C-terminal side of the phosphoacceptor threonine (P site) was found to be critical for peptide substrate recognition by MHCK A, MHCK B and eEF-2K. Phosphorylation by MHCK B was further enhanced 8-fold by a basic residue in the P+2 position whereas phosphorylation by MHCK A was enhanced 2- to 4-fold by basic residues in the P+2, P+3 and P+4 positions. eEF-2K required basic residues in both the P+1 and P+3 positions to recognize peptide substrates. eEF-2K, like MHCK A and MHCK B, exhibited a strong preference for threonine as the phosphoacceptor amino acid. In contrast, the Dictyostelium VwkA and mammalian TRPM7 alpha kinases phosphorylated both threonine and serine residues. The results, together with a phylogenetic analysis of the alpha kinase catalytic domain, support the view that the metazoan eEF-2Ks and the Dictyostelium MHCKs form a distinct subgroup of alpha kinases with conserved properties.