Myosin Heavy Chain Phosphorylation Research Articles

Release of myosin II from the membrane-cytoskeleton of Dictyostelium discoideum mediated by heavy-chain phosphorylation at the foci within the cortical actin network.

Membrane-cytoskeletons were prepared from Dictyostelium amebas, and networks of actin and myosin II filaments were visualized on the exposed cytoplasmic surfaces of the cell membranes by fluorescence staining (Yumura, S., and T. Kitanishi-Yumura. 1990. Cell Struct. Funct. 15:355-364). Addition of ATP caused contraction of the cytoskeleton with aggregation of part of actin into several foci within the network, but most of myosin II was released via the foci. However, in the presence of 10 mM MgCl2, which stabilized myosin II filaments, myosin II remained at the foci. Ultrastructural examination revealed that, after contraction, only traces of monomeric myosin II remained at the foci. By contrast, myosin II filaments remained in the foci in the presence of 10 mM MgCl2. These observations suggest that myosin II was released not in a filamentous form but in a monomeric form. Using [gamma 32P]ATP, we found that the heavy chains of myosin II released from membrane-cytoskeletons were phosphorylated, and this phosphorylation resulted in disassembly of myosin filaments. Using ITP (a substrate for myosin II ATPase) and/or ATP gamma S (a substrate for myosin II heavy-chain kinase [MHCK]), we demonstrated that phosphorylation of myosin heavy chains occurred at the foci within the actin network, a result that suggests that MHCK was localized at the foci. These results together indicate that, during contraction, the heavy chains of myosin II that have moved toward the foci within the actin network are phosphorylated by a specific MHCK, with the resultant disassembly of filaments which are finally released from membrane-cytoskeletons. This series of reactions could represent the mechanism for the relocation of myosin II from the cortical region to the endoplasm.

Analysis of plasma membrane protein changes in Dictyostelium discoideum during concanavalin A induced receptor redistribution using two‐dimensional gel electrophoresis

The 127 major polypeptides obtained from the purified plasma membrane of Dictyostelium discoideum were examined using two-dimensional gel electrophoresis and a microcomputer-based videodensitometer. Plasma membrane proteins were analyzed at four discrete stages of concanavalin A induced cell surface capping; (i) the cell surface in the absence of ligand (unbound), (ii) the surface immediately after ligand binding (bound), (iii) the cell surface after receptors had patched (patched) and (iv) the cell surface after receptors had capped (capped). Plasma membranes were obtained at various stages of capping by using a colloidal silica density perturbation technique which immediately immobilized the proteins, preserving their lateral distribution in the bilayer during the isolation. Proteins were characterized with respect to post-translational modification changes resulting from the capping process as well as changes in their association with the plasma membrane fraction. Posttranslational changes of plasma membrane proteins, such as phosphorylation, methylation and proteolytic cleavage, were not observed during the four stages of capping. Myosin heavy chain phosphorylation, however, decreased almost twofold during patching and capping. Actin, which is known to colocalize directly underneath capped receptors did not appear to be recruited to the cap from the cytoplasm.

Myosin Heavy Chain Kinase from Developed Dictyostelium cells: Purification and Characterization

Abstract We purified to homogeneity the Dictyostelium discoideum myosin heavy chain kinase that is implicated in the heavy chain phosphorylation increases that occur during chemotaxis. The kinase is initially found in the insoluble fraction of developed cells. The major purification step was achieved by affinity chromatography using a tail fragment of Dictyostelium myosin (LMM58) expressed in Escherichia coli (De Lozanne, A., Berlot, C. H., Leinwand, L. A., and Spudich, J. A. (1988) J. Cell Biol. 105, 2990-3005). The kinase has an apparent molecular weight of 84,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent native molecular weight by gel filtration is 240,000. The kinase catalyzes phosphorylation of myosin heavy chain or LMM58 with similar kinetics, and the extent of phosphorylation for both is 4 mol of phosphate/mol. With both substrates the Vmax is about 18 mumol/min/mg and the Km is 15 microM. The myosin heavy chain kinase is specific to Dictyostelium myosin heavy chain, and the phosphorylated amino acid is threonine. The kinase undergoes autophosphorylation. Each mole of kinase subunit incorporates about 20 mol of phosphates. Phosphorylation of myosin by this kinase inhibits myosin thick filament formation, suggesting that the kinase plays a role in the regulation of myosin assembly.

Evaluation of myosin light chain phosphorylation in isolated pancreatic acini.

The role of contractile proteins in secretory granule exocytosis was evaluated by determining whether myosin light chain phosphorylation was altered during stimulation of secretion in mouse pancreatic acini. Acinar myosin was purified by extraction into isosmotic sucrose solution containing 40 mM pyrophosphate followed by ammonium sulfate precipitation and Sepharose 4B-CL chromatography. Myosin was eluted as a single peak of K+-EDTA ATPase activity and was purified over 2,000-fold to a final ATPase specific activity of 0.96 mumol.min-1.mg protein-1. Three major myosin subunits of apparent Mr of 200,000, 20,000, and 17,000 were present in the purified myosin preparation. A fourth protein of Mr 21,000 was also present. Purification of myosin from 32P-labeled acini revealed the Mr 200,000, 21,000, and 20,000 proteins to be heavily labeled. The effect of cholecystokinin octapeptide (CCK-8) on myosin phosphorylation was studied after isolation of myosin from 32P-labeled acinar lysates by immunoprecipitation. Treatment of acini for 1-10 min with a concentration of CCK-8 that gives a maximal secretory response caused a 25-40% increase in light chain labeling. Treatment with a supramaximal CCK-8 concentration produced a 50-80% increase in light chain labeling. Phosphorylation of myosin heavy chain was not significantly affected by secretagogue treatment. These results indicate that stimulation of pancreatic acinar secretion is accompanied by an increase in myosin light chain phosphorylation.

Brush border myosin heavy chain phosphorylation is regulated by calcium and calmodulin

Myosin from chicken intestinal brush borders is phosphorylated on its heavy chains at threonine by a kinase isolated from brush borders. In contrast to other heavy chain kinases, the brush border kinase activity is dependent on calcium and calmodulin. The partially purified preparation also phosphorylated myosin on its light chains at serine, but in a calmodulin-independent manner. Phosphorylation of the light chains in the absence of calmodulin or both heavy and light chains in the presence of calmodulin activated its actinactivated ATPase activity about 10-fold, to about 50 nmol min per mg.

9] Isolation and characterization of calmodulin-dependent myosin heavy chain kinase from intestinal brush border

This chapter describes methods to identify myosin heavy chain kinase activity in crude extracts of brush borders from chicken intestinal epithelial cells. A procedure for isolation of the myosin heavy chain kinase is described, and it shows that it is completely dependent on calcium and calmodulin for activity. No mechanism for regulation of myosin heavy chain kinase was previously reported in any cell type. Phosphorylation of myosin on its heavy chains, in addition to the well-established light-chain-phosphorylation mechanism, may regulate actomyosin-based contractile events in nonmuscle cells. Myosin heavy chain phosphorylation is shown to regulate actomyosin ATPase activity and myosin filament assembly in vitro in several invertebrates (Acanthamoeba, Dictyostelium, and Physarum). Heavy chain phosphorylation is demonstrated in several vertebrate cell types as well (fibroblasts, macrophages, lymphocytes, leukemic myeloblasts, and brain). Isolated myosin heavy chain kinase is completely dependent on calcium and calmodulin for its activity. The total dependence of the myosin heavy chain kinase activity in the brush border extract on calcium indicates that no calcium-independent heavy chain kinase is present in brush borders. The brush border kinase phosphorylates a threonine residue located very near the tip of the tail of myosin. This region of the molecule is phosphorylated in myosins from other cell types.

Chapter 11 Isolation of the Actin Cytoskeleton from Amoeboid Cells of Dictyostelium

This chapter describes methods for the isolation of the actin cytoskeleton of Dictyostelium using sea urchin eggs. Those studies involved the isolation of a low-speed sedimentable actin fraction from unfertilized and fertilized sea urchin eggs to study changes in assembly states of the actin on fertilization. A number of similar procedures for isolation of cytoskeletal preparations from Dictyostelium are developed by other investigators to study cytoskeleton-associated proteins. The use of isolated cortices for ultrastructural localization of proteins with gold probes is described. A detergent-insoluble membrane fragment associated with the filamentous actin matrix isolated from Dictyostelium cells has been used to identify a group of integral membrane proteins that have the capacity to bind actin. A pool of folate receptors on vegetative cells that are associated with the detergent-insoluble cytoskeleton is identified. The association of the receptor with the cytoskeleton is maintained for up to 12 hours of development. It is shown that the amount of myosin in a Triton-resistant low-speed pellet of developed Dictyostelium cells increases significantly as the cell responds to the chemoattractant cyclic adenosine monophosphate (AMP). It is also shown that the kinase activity responsible for a chemoattractant-elicited increase in myosin heavy-chain phosphorylation is in the detergent insoluble pellet. Data presented suggest that the changes in the composition and organization of the isolated cytoskeleton can reflect physiological activities of the cell. The isolated cytoskeleton should be useful as a starting material for studying the modulation of proteins that are regulated during motile events.

Regulation of phosphorylation of myosin heavy chain during the chemotactic response of Dictyostelium cells.

Chemotactic stimulation of the cellular slime mould Dictyostelium discoideum by cyclic AMP increases the incorporation of 32P into a 210-kDa polypeptide. This was determined in lysates prepared from stimulated and nonstimulated cells. Earlier results suggesting that the 210-kDa polypeptide corresponds to myosin heavy chains [7] have now been confirmed. We have investigated the sequence of events following chemotactic stimulation that lead to the change of myosin heavy-chain phosphorylation. 1. Chemotactic stimulation did not activate myosin heavy chain kinase, but seemed to elicit the transient accumulation of dephosphorylated myosin heavy chains. Thus, the inefficient incorporation of 32P into myosin heavy chains in lysates of control cells seems to be due to the lack of dephosphorylated myosin heavy chains. 2. Using membranes of aggregation-competent cells, we found that incorporation of 32P into myosin heavy chains was inhibited by 0.1-1 mM Ca2+. The effect of calcium seems to be mediated by endogeneous calmodulin and was due to the inhibition of myosin kinase activity rather than to the activation of myosin phosphatase. 3. By the addition of calmodulin, the inhibition of myosin heavy-chain phosphorylation was further enhanced. 4. Folic acid, an attractant of undifferentiated cells, also caused enhanced incorporation of 32P into myosin heavy chains, as determined in cell lysates. 5. The amount of 32P incorporated in response to cyclic AMP into myosin heavy chains increased during differentiation to the aggregation-competent stage. Only a small fraction of the cell surface receptors had to be activated in order to elicit a maximal reaction. Our results suggest that a phosphorylated form of myosin heavy chains prevails at the onset of the chemotactic response and that dephosphorylation begins within 5-10 s at 23 degrees C.