[3] Purification and characterization of myosin II heavy chain kinase A from Dictyostelium (original) (raw)
Related papers
The Journal of cell biology, 1987
We have partially purified myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) from Dictyostelium discoideum. MLCK was purified 4,700-fold with a yield of approximately 1 mg from 350 g of cells. The enzyme is very acidic as suggested by its tight binding to DEAE. Dictyostelium MLCK has an apparent native molecular mass on HPLC G3000SW of approximately 30,000 D. Mg2+ is required for enzyme activity. Ca2+ inhibits activity and this inhibition is not relieved by calmodulin. cAMP or cGMP have no effect on enzyme activity. Dictyostelium MLCK is very specific for the 18,000-D light chain of Dictyostelium myosin and does not phosphorylate the light chain of several other myosins tested. Myosin purified from log-phase amebas of Dictyostelium has approximately 0.3 mol Pi/mol 18,000-D light chain as assayed by glycerol-urea gel electrophoresis. Dictyostelium MLCK can phosphorylate this myosin to a stoichiometry approaching 1 mol Pi/mol 18,000-D light chain. MLCP, which ...
Myosin heavy chain kinase from developed Dictyostelium cells. Purification and characterization
The Journal of biological chemistry, 1989
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 Dict...
Myosin Heavy Chain Kinase from Developed Dictyostelium cells
Journal of Biological Chemistry, 1989
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
Biochemistry, 1990
One of the major sites phosphorylated on the Dictyostelium myosin I1 heavy chain by the Dictyostelium myosin I1 heavy-chain kinase A (MHCK A) is Thr-2029. Two synthetic peptides based on the sequence of the Dictyostelium myosin I1 heavy chain around Thr-2029 have been synthesized: MH-1 (residues 2020-2035; RKKFGESEKTKTKEFL-amide) and MH-2 (residues 2024-2035). Both peptides are substrates for M H C K A and are phosphorylated to a level of 1 mol of phosphate/mol. Tryptic digests indicate that the peptides are phosphorylated on the threonine corresponding to Thr-2029. When assays are initiated by the addition of M H C K A, the rate of phosphate incorporation into the peptides increases 'This work was supported by grants from the Medical Research Council of Canada. J.G. and G.P.C. are recipients of Medical Research Council of Canada scholarships. *Queen's University. University of Toronto.
Journal of Biological Chemistry, 1997
Myosin II heavy chain (MHC)-specific protein kinase C (MHC-PKC) isolated from the ameba, Dictyostelium discoideum, regulates myosin II assembly and localization in response to the chemoattractant cAMP. cAMP stimulation of Dictyostelium cells leads to translocation of MHC-PKC from the cytosol to the membrane fraction, as well as causing an increase in both MHC-PKC phosphorylation and its kinase activity. MHC-PKC undergoes autophosphorylation with each mole of kinase incorporating about 20 mol of phosphate. The MHC-PKC autophosphorylation sites are thought to be located within a domain at the COOH-terminal region of MHC-PKC that contains a cluster of 21 serine and threonine residues. Here we report that deletion of this domain abolished the ability of the enzyme to undergo autophosphorylation in vitro. Furthermore, after this deletion, cAMP-dependent autophosphorylation of MHC-PKC as well as cAMP-dependent increases in kinase activity and subcellular localization were also abolished. These results provide evidence for the role of autophosphorylation in the regulation of MHC-PKC and indicate that this MHC-PKC autophosphorylation is required for the kinase activation in response to cAMP and for subcellular localization.
Proceedings of the National Academy of Sciences, 1992
A cDNA clone corresponding to the Dictyostelium myosin heavy chain kinase (MHCK) gene was isolated using antibodies specific to the purified enzyme. Sequence analysis of the cDNA revealed that the Dictyostelium MHCK possesses all of the domains characteristic of members of the protein kinase C family. The amino-terminal region of the MHCK contains the cysteine-rich motif with an internal duplication that is present in all known protein kinase C species. This domain precedes sequences that are highly homologous to protein kinase catalytic domains. The carboxyl-terminal region contains a cluster of 23 serine and threonine residues that may represent the autophosphorylation domain of the Dictyostelium MHCK. These results, along with previous studies that indicate that this enzyme has very restrictive substrate specificity, incorporates approximately 20 mol of phosphate per mol of kinase through an autophosphorylation reaction, and is expressed only during development, suggest that the ...
Febs Letters, 1990
The target sites of soluble myosin heavy chain kinases partially purified from growth phase or aggregation competent cells of Dictyostefium discoideum were identified by the usiz of normal and mutated fragments of the myosin heavy chain. The kinases from both developmental stages phosphorylated two previously established threonine residues, as well as an additional one. The newly identified site is located within the putative core region of the coiled-coil formed by the myosin tail. A lysine following the phosphorylated threonine residue is the only common feature of the sequences around these sites. The kinases, which specifically phosphorylate threonine residues in wild-type myosin, did accept serine if it was in the right structural context.
BMC Research Notes, 2010
Background: Heavy chain phosphorylation plays a central role in regulating myosin II bipolar filament assembly in Dictyostelium, as well as in higher eukaryotic nonmuscle cells. Our previous work has demonstrated that the WDrepeat domain of Dictyostelium myosin II heavy chain kinase B (MHCK-B), unlike its counterpart in MHCK-A, is not absolutely required for targeting of the kinase to phosphorylate MHC. Thus, we tested the hypothesis that an asparagine-rich and structurally disordered region that is unique to MHCK-B can by itself function in substrate targeting. Findings: Biochemical assays comparing the activities of full-length MHCK-B, a truncation lacking only the WDrepeat domain (B-Δ-WD), and a truncation lacking both the N-rich region and the WD-repeat domain (B-Δ-N-WD) revealed that the N-rich region targets MHCK-B to phosphorylate MHC in a manner that leads to bipolar filament disassembly. This targeting is physiologically relevant since cellular over-expression of the B-Δ-WD truncation, but not the B-Δ-N-WD truncation, leads to dramatically reduced levels of myosin II filament assembly and associated defects in cytokinesis and multicellular development.
Journal of Biological Chemistry
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 abo...
Journal of Cell Science, 2012
Behavioral analyses of the deletion mutants of the four known myosin II heavy chain (Mhc) kinases of D. discoideum revealed that all played a minor role in the efficiency of basic cell motility, but none played a role in chemotaxis in a spatial gradient of cAMP generated in vitro. However, each of the two kinases MhckA and MhckC, was essential for chemotaxis in a spatial gradient of Ca2+, shear induced directed movement, and reorientation in the front of waves of cAMP during natural aggregation. The mutant phenotypes of mhckA− and mhckC− were highly similar to that of the Ca2+ channel/receptor mutant iplA− and the myosin II phosphorylation mutant 3XALA, which produces constitutively unphosphorylated myosin II. These results demonstrate that IplA, MhckA and MhckC play a selective role in chemotaxis in a spatial gradient of Ca2+, but not cAMP and suggest that Ca2+ chemotaxis plays a role in the orientation of cells in the front of cAMP waves during natural aggregation.