Purification and Identification of Myosin Heavy Chain Kinase from Bovine Brain

Murakami, Noriko; Matsumura, Sueo; Kumon, Akira

doi:10.1093/oxfordjournals.jbchem.a134654

Cited by 45 publications

(14 citation statements)

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“…All three cell lines showed an increase in NMHC-IIB phosphorylation after EGF stimulation, yet the EGF-dependent NMHC-IIB phosphorylation in PKC␥ DN cells was 60% lower than that of control cells ( Figure 7A). These results indicate that PKC␥ is involved in EGF-dependent NMHC-IIB phosphorylation in (Murakami et al, 1984(Murakami et al, , 1998Straussman et al, 2001) may also phosphorylate NMHC-IIB. Another possibility is that the expressed PKC␥ DN did not inhibit completely the activity of the endogenous PKC␥.…”

mentioning

confidence: 75%

“…These results indicate that PKC␥ is involved in EGF-dependent NMHC-IIB phosphorylation in vivo. The incomplete inhibition of EGF-dependent NMHC-IIB phosphorylation in PKC␥ DN cells may indicate that PKC␥ is not the only kinase that phosphorylates NMHC-IIB, other kinases like CKII and members of the PKC family other than PKC␥ (Murakami et al, 1984(Murakami et al, , 1998Straussman et al, 2001) may also phosphorylate NMHC-IIB. Another possibility is that the expressed PKC␥ DN did not inhibit completely the activity of the endogenous PKC␥.…”

Section: The Effect Of Pkc␥ Overexpression On the Actomyosin Cytoskelmentioning

confidence: 99%

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Protein Kinase Cγ Regulates Myosin IIB Phosphorylation, Cellular Localization, and Filament Assembly

Rosenberg

Ravid

2006

MBoC

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Nonmuscle myosin II is an important component of the cytoskeleton, playing a major role in cell motility and chemotaxis. We have previously demonstrated that, on stimulation with epidermal growth factor (EGF), nonmuscle myosin heavy chain II-B (NMHC-IIB) undergoes a transient phosphorylation correlating with its cellular localization. We also showed that members of the PKC family are involved in this phosphorylation. Here we demonstrate that of the two conventional PKC isoforms expressed by prostate cancer cells, PKC␤II and PKC␥, PKC␥ directly phosphorylates NMHC-IIB. Overexpression of wild-type and kinase dead dominant negative PKC␥ result in both altered NMHC-IIB phosphorylation and subcellular localization. We have also mapped the phosphorylation sites of PKC␥ on NMHC-IIB. Conversion of the PKC␥ phosphorylation sites to alanine residues, reduces the EGF-dependent NMHC-IIB phosphorylation. Aspartate substitution of these sites reduces NMHC-IIB localization into cytoskeleton. These results indicate that PKC␥ regulates NMHC-IIB phosphorylation and cellular localization in response to EGF stimulation. INTRODUCTIONDirected cell migration is a complex process; it requires numerous factors to coordinate the functioning of the cell cytoskeleton so that the cell achieves the necessary polarization for chemotaxis (Lauffenburger and Horwitz, 1996;Mitchison and Cramer, 1996). One of the cytoskeletal proteins that plays an important role in chemotaxis is nonmuscle myosin II (Spudich, 1989;Chung et al., 2001;Postma et al., 2004). In vertebrates, there are at least three nonmuscle myosin II heavy chain (NMHC) genes that encode separate isoforms of the heavy chain: NMHC-IIA, NMHC-IIB, and NMHC-IIC (Katsuragawa et al., 1989;Kawamoto and Adelstein, 1991;Golomb et al., 2004). These myosin II isoforms differ in their heavy chain sequences (Takahashi et al., 1992;Golomb et al., 2004), in the kinetics of ATPase activity (Kelley et al., 1996;Kovacs et al., 2003;Rosenfeld et al., 2003;Wang et al., 2003;De La Cruz and Ostap, 2004;Kim et al., 2005) and in their subcellular localization in various cells types (Maupin et al., 1994;Kelley et al., 1996;Kolega, 1998Kolega, , 2003Bao et al., 2005;Kim et al., 2005). Myosin II is a hexamer composed of two heavy chains of ϳ200 kDa and two pairs of 17-and 20-kDa light chains, and it is regulated by light chain and heavy chain phosphorylation (Tan et al., 1992;Bresnick, 1999;de la Roche and Cote, 2001). Phosphorylation of the regulatory light chains regulates the actin-activated ATPase activity and filament assembly (Bresnick, 1999). Phosphorylation of myosin II heavy chains in the slime mold Dictyostelium discoideum inhibits filament assembly (Kuczmarski and Spudich, 1980;Pasternak et al., 1989;Liang et al., 1999). In addition, a number of myosin II heavy chain kinases (NMHCKs), purified from Dictyostelium cells, regulate the assembly properties of myosin II heavy chains in vivo (de la Roche and Cote, 2001). However, the mechanisms controlling myosin II heavy chain phosphorylation in mammalian ...

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mentioning

confidence: 75%

Section: The Effect Of Pkc␥ Overexpression On the Actomyosin Cytoskelmentioning

confidence: 99%

Protein Kinase Cγ Regulates Myosin IIB Phosphorylation, Cellular Localization, and Filament Assembly

Rosenberg

Ravid

2006

MBoC

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“…Myosin heavy-chain phosphorylation has been shown to occur in a number of nonmuscle myosins, including those from Acanthamoeba (Maruta & Korn 1977;Collins & Korn 1980Hammer et al 1983; for review, see Korn et al 1987), Dictyostelium (Kuczmarski & Spudich 1980;Kuczmarski 1986), Physarum (Ogihara et al 1983), macrophages (Trotter 1982), lymphocytes (Fechheimer & Cebra 1982), and brain cells (Matsamura et al 1982;Murakami et al 1984). For the Acanthamoeba myosin II and Dictyostelium myosin, this phosphorylation, which occurs on serine and threonine residues, inhibits both filament assembly and actin activation of myosin ATPase (Kuczmarski & Spudich 1980;Korn et al 1987;Peltz et al 1981;Pagh & Gerisch 1986).…”

Section: Cell Biology Of Myosinsmentioning

confidence: 99%

Myosin Structure and Function in Cell Motility

Warrick¹,

Spudich²

1987

Annu. Rev. Cell. Biol.

464

193

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“…This enzyme phosphorylates three sites on the tail portion of the two-headed Acanthamoeba myosin (myosin II) and the effect of phosphorylation is similar to that in Dictyostelium, namely Acanthamoeba myosin phosphorylation inhibits actin-activated ATPase and selfassembly (Collins et al, 1982). Murakami et al (1984) have reported that a bovine brain kinase is capable of phosphorylating heavy and light chains of brain myosin. This enzyme did not require Ca 2+-calmodulin or cAMP for activity.…”

Section: Discussionmentioning

confidence: 99%

Partial purification of two myosin heavy chain kinases fromDictyostelium discoideum

Kuczmarski

1986

J Muscle Res Cell Motil

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Myosin heavy chain kinase activity was identified in the high speed supernate of lysed Dictyostelium amoebae and was precipitated by 30-50% ammonium sulphate. In low ionic strength buffer, the activity bound tightly to a Cibacron Blue Sepharose column and eluted as a single peak with 1.0 M NaCl. Gel filtration chromatography resolved the kinase into two activities, each of which phosphorylated the tail portion of purified Dictyostelium myosin. One of these activities phosphorylated both serine and threonine residues of the heavy chain, while the other activity only phosphorylated threonine residues. Peptide mapping studies indicated that in vivo and in vitro phosphorylation sites were identical. The heavy chain kinases required Mg2+ for activity but were unaffected by Ca2+ or calmodulin. The heavy chain kinases did not phosphorylate Dictyostelium light chain, and also did not phosphorylate myosins from striated, smooth, or other nonmuscle sources.

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Purification and Identification of Myosin Heavy Chain Kinase from Bovine Brain

Cited by 45 publications

References 0 publications

Protein Kinase Cγ Regulates Myosin IIB Phosphorylation, Cellular Localization, and Filament Assembly

Protein Kinase Cγ Regulates Myosin IIB Phosphorylation, Cellular Localization, and Filament Assembly

Myosin Structure and Function in Cell Motility

Partial purification of two myosin heavy chain kinases fromDictyostelium discoideum

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