Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo

Egelhoff, Thomas; Lee, Randall J.; Spudich, James A.

doi:10.1016/0092-8674(93)80077-r

Cited by 262 publications

(347 citation statements)

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“…creased myosin II assembly levels, consistent with the idea that MHCK A phosphorylates MHC in vivo, driving filament disassembly (Kolman et al, 1996). Earlier studies also demonstrated that mutation of the three mapped MHCK target sites in the myosin tail, creating a nonphosphorylatable "3x ALA" mutant myosin II, resulted in gross overassembly of myosin in the cytoskeletal ghost assay (Egelhoff et al, 1993), due to inability of any MHCKs to phosphorylate these target sites in the myosin tail. To assess whether VwkA might act in a similar manner, we isolated cytoskeletal ghosts and evaluated myosin II assembly levels via SDS-PAGE, Coomassie staining, and densitometry.…”

Section: Vwka Involvement In the Regulation Of Myosin II Expression Asupporting

confidence: 55%

Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

Betapudi

Mason

Licate

et al. 2005

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We have identified a new protein kinase in Dictyostelium discoideum that carries the same conserved class of "␣-kinase" catalytic domain as reported previously in myosin heavy chain kinases (MHCKs) in this amoeba but that has a completely novel domain organization. The protein contains an N-terminal von Willebrand factor A (vWFA)-like motif and is therefore named VwkA. Manipulation of VwkA expression level via high copy number plasmids (VwkA ؉؉ cells) or gene disruption (vwkA null cells) results in an array of cellular defects, including impaired growth and multinucleation in suspension culture, impaired development, and alterations in myosin II abundance and assembly. Despite sequence similarity to MHCKs, the purified protein failed to phosphorylate myosin II in vitro. Autophosphorylation activity, however, was enhanced by calcium/calmodulin, and the enzyme can be precipitated from cellular lysates with calmodulin-agarose, suggesting that VwkA may directly bind calmodulin. VwkA is cytosolic in distribution but enriched on the membranes of the contractile vacuole and Golgi-like structures in the cell. We propose that VwkA likely acts indirectly to influence myosin II abundance and assembly behavior and possibly has broader roles than previously characterized ␣ kinases in this organism, which all seem to be MHCKs. INTRODUCTIONNearly all aspects of cell life are regulated by protein phosphorylation involving a large number of biochemical reactions and distinct signaling pathways. Existence of ϳ500 genes encoding various protein kinases in the human genome further underscores their importance in cell life (Manning et al., 2002). Most of these conventional protein kinases belong to serine/threonine/tyrosine, a kinase superfamily whose members carry a conserved catalytic domain with recognizable sequence similarity in spite of having different targets in the cell (Hardie, 1994;Hanks and Hunter, 1995). In recent years, however, biochemical and molecular approaches have led to the identification of more divergent classes of eukaryotic protein kinases carrying a catalytic domain sequence with no sequence similarity with conventional protein kinase catalytic domains (Manning et al., 2002). One major class of unconventional protein kinases was first identified via representatives of myosin heavy chain kinases (MHCKs) in Dictyostelium discoideum (Futey et al., 1995;Côté et al., 1997) and via identification of mammalian eEF-2 kinase (eEF-2K) as an unconventional protein kinase (Ryazanov et al., 1997). This novel, conserved group of unconventional eukaryotic protein kinases has been termed the ␣-kinase family (Ryazanov et al., 1999;Drennan and Ryazanov, 2004).Molecular cloning and biochemical characterization studies demonstrated that at least three Dictyostelium ␣-kinases genes encode enzymes that can specifically phosphorylate myosin II heavy chain (MHC) in vitro and in vivo, and were thus named as MHC kinase A (MHCK A), MHC kinase B (MHCK B) and MHC kinase C (MHCK C) (Futey et al., 1995;Clancy et al., 1997;Luo et al., 200...

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Section: Vwka Involvement In the Regulation Of Myosin II Expression Asupporting

confidence: 55%

Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

Betapudi

Mason

Licate

et al. 2005

MBoC

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“…In Dictyostelium, filament assembly and the recruitment of myosin II to the contractile ring require the phosphorylation of three threonine residues at the C terminus (Egelhoff et al, 1991;Lee et al, 1994;Sabry et al, 1997;Shu et al, 1999) by at least two myosin II heavy chain kinases Spudich, 1989, 1992;Kolman et al, 1996). Mutation of all three sites (Egelhoff et al, 1993) or, indeed, just one of them (Nock et al 2000), abolishes myosin function. In fission yeast also, a single charge change abolishes the assembly of Myo2 into the CAR.…”

Section: Discussionmentioning

confidence: 99%

Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

Mulvihill

Barretto

Hyams

2001

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Myo2 truncations fused to green fluorescent protein (GFP) defined a C-terminal domain essential for the localization of Myo2 to the cytokinetic actin ring (CAR). The localization domain contained two predicted phosphorylation sites. Mutation of serine 1518 to alanine (S1518A) abolished Myo2 localization, whereas Myo2 with a glutamic acid at this position (S1518E) localized to the CAR. GFP-Myo2 formed rings in the septation initiation kinase (SIN) mutant cdc7-24 at 25°C but not at 36°C. GFP-Myo2S1518E rings persisted at 36°C incdc7-24 but not in another SIN kinase mutant,sid2-250. To further examine the relationship between Myo2 and the SIN pathway, the chromosomal copy ofmyo2 + was fused to GFP (strainmyo2-gc). Myo2 ring formation was abolished in the double mutants myo2-gc cdc7.24 and myo2-gc sid2-250 at the restrictive temperature. In contrast, activation of the SIN pathway in the double mutant myo2-gc cdc16-116 resulted in the formation of Myo2 rings which subsequently collapsed at 36°C. We conclude that the SIN pathway that controls septation in fission yeast also regulates Myo2 ring formation and contraction. Cdc7 and Sid2 are involved in ring formation, in the case of Cdc7 by phosphorylation of a single serine residue in the Myo2 tail. Other kinases and/or phosphatases may control ring contraction.

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“…In addition, in vitro studies strongly suggest that MHC phosphorylation plays an important role in the regulation of myosin II filament formation (7)(8)(9)(10). The importance of MHC phosphorylation in the regulation of myosin II in vivo was demonstrated by Egelhoff et al (11). They found that elimination of the MHC phosphorylation sites allows in vivo contractile activity, but this myosin II shows substantial overassembly.…”

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confidence: 90%

“…They found that elimination of the MHC phosphorylation sites allows in vivo contractile activity, but this myosin II shows substantial overassembly. Mimicking the negative charge state of phosphorylated myosin II eliminates filament formation in vitro and renders the myosin II unable to drive any tested contractile event in vivo (11).…”

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Dictyostelium Myosin II Is Regulated during Chemotaxis by a Novel Protein Kinase C

Abu-Elneel

Karchi

Ravid

1996

Journal of Biological Chemistry

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When cells of Dictyostelium are starved, they acquire the ability to bind cAMP to specific cell surface receptors and to respond to this signal by chemotaxis. The process of chemotaxis involves phosphorylation and reorganization of myosin II (1-5). In response to cAMP stimulation, myosin II, which exists as thick filaments, translocates to the cortex (5). This translocation is correlated with a transient increase in the rate of myosin II heavy chain (MHC) 1 as well as light chain phosphorylation (3, 4, 6). In addition, in vitro studies strongly suggest that MHC phosphorylation plays an important role in the regulation of myosin II filament formation (7-10). The importance of MHC phosphorylation in the regulation of myosin II in vivo was demonstrated by Egelhoff et al. (11). They found that elimination of the MHC phosphorylation sites allows in vivo contractile activity, but this myosin II shows substantial overassembly. Mimicking the negative charge state of phosphorylated myosin II eliminates filament formation in vitro and renders the myosin II unable to drive any tested contractile event in vivo (11).We previously reported the isolation of a MHC-specific PKC (MHC-PKC) from Dictyostelium that phosphorylates Dictyostelium MHC specifically and is homologous to ␣, ␤, and ␥ subtypes of mammalian PKC (9, 12). This kinase, which is membrane-associated and is expressed during Dictyostelium development, was implicated in the increase in MHC phosphorylation during chemotaxis in this species (9). In vitro phosphorylation of MHC by MHC-PKC results in inhibition of myosin II thick filament formation (9), by inducing the formation of a bent monomer of myosin II whose assembly domain is tied up in an intramolecular interaction that precludes intermolecular interaction, which is necessary for thick filament formation (10). The findings that MHC-PKC is a member of the PKC family and that it regulates myosin II assembly suggest a link between the extracellular chemotactic signal and subsequent intracellular events.A considerable amount of information is now available regarding the regulation of myosin II by MHC phosphorylation, and a picture is beginning to emerge in which the molecular changes in myosin II that are involved in MHC phosphorylation are related to cAMP-induced directed cell movement. Nevertheless, the molecular mechanism by which a cAMP signal is transmitted to myosin II, resulting in myosin II localization and chemotaxis, remains unclear. In an attempt to throw some light on this issue, we studied the role of MHC-PKC in vivo by eliminating and by overexpressing the MHC-PKC protein in Dictyostelium cells. Analysis of these cell lines allowed us to address directly the role of MHC-PKC in vivo and consequently the role of MHC phosphorylation in the regulation of myosin II. The results presented here indicate that MHC-PKC is a key modulator in the regulation of myosin II localization in response to the chemoattractant, cAMP.

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Dictyostelium myosin heavy chain phosphorylation sites regulate myosin filament assembly and localization in vivo

Cited by 262 publications

References 36 publications

Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

Identification and Characterization of a Novel α-Kinase with a von Willebrand Factor A-like Motif Localized to the Contractile Vacuole and Golgi Complex inDictyostelium discoideum

Localization of Fission Yeast Type II Myosin, Myo2, to the Cytokinetic Actin Ring Is Regulated by Phosphorylation of a C-Terminal Coiled-Coil Domain and Requires a Functional Septation Initiation Network

Dictyostelium Myosin II Is Regulated during Chemotaxis by a Novel Protein Kinase C

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