[3] Purification and characterization of myosin II heavy chain kinase A from Dictyostelium

Medley, Quintus G.; Lee, Sheu-Fen; Côté, Graham P.

doi:10.1016/0076-6879(91)96005-c

Cited by 15 publications

(6 citation statements)

References 18 publications

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“…Although MHCK-A has been extensively characterized at the biochemical level [18,22,25,31], only limited biochemical analysis has been performed with bacterially-expressed subdomains of MHCK-B and MHCK-C [17,18,22]. The current biochemical results provide strong support for the hypothesis that MHCK-C acts as a MHC kinase in vivo.…”

Section: Discussionsupporting

confidence: 62%

“…This 16-residue peptide corresponds to the phosphorylation target site at position 2029 of MHC. This peptide and the filter-binding assay used to measure its phosphorylation have been described previously [31]. All peptide phosphorylation studies were performed under conditions in which no more than 30% of the substrate was consumed to ensure linear reaction rates.…”

Section: Methodsmentioning

confidence: 99%

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Differential localization in cells of myosin II heavy chain kinases during cytokinesis and polarized migration

et al. 2002

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Background: Cortical myosin-II filaments in Dictyostelium discoideum display enrichment in the posterior of the cell during cell migration and in the cleavage furrow during cytokinesis. Filament assembly in turn is regulated by phosphorylation in the tail region of the myosin heavy chain (MHC). Early studies have revealed one enzyme, MHCK-A, which participates in filament assembly control, and two other structurally related enzymes, MHCK-B and -C. In this report we evaluate the biochemical properties of MHCK-C, and using fluorescence microscopy in living cells we examine the localization of GFP-labeled MHCK-A, -B, and -C in relation to GFP-myosin-II localization.

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Section: Discussionsupporting

confidence: 62%

Section: Methodsmentioning

confidence: 99%

Differential localization in cells of myosin II heavy chain kinases during cytokinesis and polarized migration

et al. 2002

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“…Additional studies are needed to reconcile these models. Physiological mechanisms regulating the Dictyostelium MHCK A [10, 44, 45] and MHCK B [46] have not yet been established. Further biochemical and cellular studies will be necessary to address the hypothesis that the MHC phosphatase identified here may undergo subcellular targeting to stimulate localized myosin assembly in vivo .…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of a Dictyostelium myosin II heavy‐chain phosphatase that promotes filament assembly

Murphy¹,

Egelhoff²

1999

European Journal of Biochemistry

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In Dictyostelium cells, myosin II is found as cytosolic nonassembled monomers and cytoskeletal bipolar filaments. It is thought that the phosphorylation state of three threonine residues in the tail of myosin II heavy chain regulates the molecular motor's assembly state and localization. Phosphorylation of the myosin heavy chain at threonine residues 1823, 1833 and 2029 is responsible for maintaining myosin in the nonassembled state, and subsequent dephosphorylation of these residues is a prerequisite for assembly into the cytoskeleton. We report here the characterization of myosin heavy-chain phosphatase activities in Dictyostelium utilizing myosin II phosphorylated by myosin heavy-chain kinase A as a substrate. One of the myosin heavy-chain phosphatase activities was identified as protein phosphatase 2A and the purified holoenzyme was composed of a 37-kDa catalytic subunit, a 65-kDa A subunit and a 55-kDa B subunit. The protein phosphatase 2A holoenzyme displays two orders of magnitude higher activity towards myosin phosphorylated on the heavy chains than it does towards myosin phosphorylated on the regulatory light chains, consistent with a role in the control of filament assembly. The purified myosin heavy-chain phosphatase activity promotes bipolar filament assembly in vitro via dephosphorylation of the myosin heavy chain. This system should provide a valuable model for studying the regulation and localization of protein phosphatase 2A in the context of cytoskeletal reorganization.Keywords: Dictyostelium; filament assembly; myosin II; phosphatase.In Dictyostelium cells, the conventional myosin, myosin II, is required for cytokinesis, cell-surface receptor capping, cortical tension maintenance, and multicellular development based upon the phenotypic defects observed in myosin null cells and upon other cellular analysis [1±6]. Myosin II function in vivo requires assembly into bipolar filaments, which can interact with the cortical actin cytoskeleton. Phosphorylation of the myosin heavy chain (MHC) plays a critical role in the regulation of filament assembly and localization of myosin in this system [7].Phosphorylation of three threonine residues at positions 1823, 1833, and 2029, located in the C-terminus of the myosin II coiled-coil heavy-chain tail, by the 130-kDa myosin heavy-chain kinase A (MHCK A) inhibits filament selfassembly in vitro and in vivo [8±10]. The inhibition of myosin II filament assembly by phosphorylation may be due to bending of the myosin tail which would sterically hinder selfassembly [11]. The functional importance of phosphorylation of these threonine sites has been demonstrated by expressing mutated forms of myosin II in Dictyostelium myosin II null cells [9]. In these studies, the three threonine residues were converted into alanine (3Â Ala) to abolish phosphorylation at these sites or into aspartate (3Â Asp) to mimic the phosphothreonine sites. It was observed that in vitro the 3Â Ala myosin formed filaments but the 3Â Asp myosin failed to. In addition, in vivo, myosin from 3Â Ala-...

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“…Myosin phosphorylation, MH1 peptide phosphorylation, and autophosphorylation assays were performed at 22°C in a water bath in kinase buffer (10 mM TES pH 7.0, 1 mM DTT, 2 mM MgCl 2 , and 0.5 mM [γ‐ 32 P]‐ATP with an specific activity of 400–800 Ci/mole). The MH1 peptide (RKKFGESEKTKTKEFL) described previously [Medley et al, 1991], corresponds to the mapped MHCK A target site in the myosin II heavy chain tail at residue 2029 (underlined threonine). The MH1 peptide was used as substrate for the study of kinetic parameters at the concentrations indicated in the corresponding figures.…”

Section: Experimental Methodsmentioning

confidence: 99%

“…Reactions using MH1 peptide were stopped by addition of EDTA to 20 mM final concentration. The amount of γ‐ 32 P incorporated into the peptide molecules was quantified by spotting reactions onto P‐81 filters (Whatman, Maidstone, England) as described [Medley et al, 1991]. Purified Dictyostelium myosin II (0.5 μM) was used to determine the stoichiometry of MHC phosphorylation.…”

Section: Experimental Methodsmentioning

confidence: 99%

Myosin heavy chain kinase B participates in the regulation of myosin assembly into the cytoskeleton

Rico

Egelhoff

2002

J of Cellular Biochemistry

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Myosin II plays critical roles in events such as cytokinesis, chemotactic migration, and morphological changes during multicellular development. The amoeba Dictyostelium discoideum provides a simple system for the study of this contractile protein. In this system, myosin II filament assembly is regulated by myosin heavy chain (MHC) phosphorylation in the tail region of the molecule. Earlier studies identified an alpha-kinase, MHC kinase A (MHCK A), which phosphorylates three mapped threonine residues in the myosin tail, driving myosin disassembly. Using molecular and genomic approaches, we have identified a series of related kinases in Dictyostelium. The enzyme MHCK B shares with MHCK A a domain organization that includes a highly novel catalytic domain coupled to a carboxyl-terminal WD repeat domain. We have engineered, expressed, and purified a FLAG-tagged version of the novel kinase. In the present study, we report detailed biochemical and cellular studies documenting that MHCK B plays a physiological role in the control of Dictyostelium myosin II assembly and disassembly during the vegetative life of Dictyostelium amoebae. The presented data supports a model of multiple related MHCKs in this system, with different regulatory mechanisms and pathways controlling each enzyme.

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[3] Purification and characterization of myosin II heavy chain kinase A from Dictyostelium

Cited by 15 publications

References 18 publications

Differential localization in cells of myosin II heavy chain kinases during cytokinesis and polarized migration

Differential localization in cells of myosin II heavy chain kinases during cytokinesis and polarized migration

Biochemical characterization of a Dictyostelium myosin II heavy‐chain phosphatase that promotes filament assembly

Myosin heavy chain kinase B participates in the regulation of myosin assembly into the cytoskeleton

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