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Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo.

Kawano Y, Fukata Y, Oshiro N, Amano M, Nakamura T, Ito M, Matsumura F, Inagaki M, Kaibuchi K - J. Cell Biol. (1999)

Bottom Line: Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro.During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased.Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.

ABSTRACT
Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. Here we identified the sites of phosphorylation of MBS by Rho-kinase as Thr-697, Ser-854 and several residues, and prepared antibody that specifically recognized MBS phosphorylated at Ser-854. We found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induced the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration were induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 were localized in the leading edge and posterior region. Phosphorylated MBS was localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupted stress fibers and weakened the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.

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Colocalization of phosphorylated MBS, F-actin and phosphorylated MLC in REF52 fibroblasts. (A) Localization of phosphorylated MBS. REF52 cells were doubly stained with TRITC-phalloidin (b and d) and anti-pS854 Ab (a) or anti-pp2b Ab (c). (B) Distribution of phosphorylated and total MBS in REF52 cells. REF52 cells were doubly stained with anti-pS854 Ab (a) and anti-mMBS Ab (b). The merged (c) and ratio (phosphorylated MBS/MBS; d) images are shown. (C) Inhibition of phosphorylation of MBS by C3 or dominant negative Rho-kinase. REF52 cells were microinjected with MBP (5.0 mg/ml) (a–d), C3 (0.1 mg/ml) (e–h), C3 plus GTPγS·RhoAI41 (0.4 mg/ml) (i–l), or MBP-RB/PH(TT) (5.0 mg/ml) (m–p). REF52 cells were stained with TRITC-phalloidin (a, e, i, and m), anti-pS854 Ab (b, f, j, and n), anti-pnMBS Ab (c, g, k, and o), and anti-pp2b Ab (d, h, l, and p). The arrowheads indicate the injected cells. Bars, 10 μm.
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Figure 6: Colocalization of phosphorylated MBS, F-actin and phosphorylated MLC in REF52 fibroblasts. (A) Localization of phosphorylated MBS. REF52 cells were doubly stained with TRITC-phalloidin (b and d) and anti-pS854 Ab (a) or anti-pp2b Ab (c). (B) Distribution of phosphorylated and total MBS in REF52 cells. REF52 cells were doubly stained with anti-pS854 Ab (a) and anti-mMBS Ab (b). The merged (c) and ratio (phosphorylated MBS/MBS; d) images are shown. (C) Inhibition of phosphorylation of MBS by C3 or dominant negative Rho-kinase. REF52 cells were microinjected with MBP (5.0 mg/ml) (a–d), C3 (0.1 mg/ml) (e–h), C3 plus GTPγS·RhoAI41 (0.4 mg/ml) (i–l), or MBP-RB/PH(TT) (5.0 mg/ml) (m–p). REF52 cells were stained with TRITC-phalloidin (a, e, i, and m), anti-pS854 Ab (b, f, j, and n), anti-pnMBS Ab (c, g, k, and o), and anti-pp2b Ab (d, h, l, and p). The arrowheads indicate the injected cells. Bars, 10 μm.

Mentions: We further investigated the subcellular distribution of phosphorylated MBS, F-actin, and phosphorylated MLC in REF52 fibroblasts. REF52 cells grown in 10% fetal bovine serum have thick actin stress fibers and vinculin-containing focal adhesions, and show a filamentous periodical pattern of myosin on stress fibers. We have previously shown that total MBS is localized on stress fibers (Inagaki, N., et al. 1997). Phosphorylated MBS was localized on stress fibers, cortical actin filaments, and in the nucleus in REF52 cells (Fig. 6 A, a). The double-immunostaining by TRITC-phalloidin and anti-pS854 Ab (Fig. 6 A, a and b) or anti-pp2b Ab (Fig. 6 A, c and d) demonstrated coexistence of phosphorylated MBS, F-actin and phosphorylated MLC on stress fibers and cortical actin filaments. The intracellular localization of phosphorylated MBS is consistent with in vitro binding of MBS and myosin (Alessi et al. 1992; Shimizu et al. 1994). There was the difference in localization of phosphorylated MBS between REF52 and MDCK cells. REF52 cells highly developed thick stress fibers (Fig. 6 A, b), whereas serum-starved MDCK cells had a few thin stress fibers (Fig. 4 A, b). Thus, phosphorylated MBS might not be detected in stress fibers in MDCK cells. Next, we compared the distribution of phosphorylated and total MBS. The distribution of phosphorylated MBS was similar to that of total MBS (Fig. 6 B, c). The ratio (phosphorylated MBS/MBS) image showed that the phosphorylation level of MBS was high on stress fiber and in nucleus (Fig. 6 B, d).


Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo.

Kawano Y, Fukata Y, Oshiro N, Amano M, Nakamura T, Ito M, Matsumura F, Inagaki M, Kaibuchi K - J. Cell Biol. (1999)

Colocalization of phosphorylated MBS, F-actin and phosphorylated MLC in REF52 fibroblasts. (A) Localization of phosphorylated MBS. REF52 cells were doubly stained with TRITC-phalloidin (b and d) and anti-pS854 Ab (a) or anti-pp2b Ab (c). (B) Distribution of phosphorylated and total MBS in REF52 cells. REF52 cells were doubly stained with anti-pS854 Ab (a) and anti-mMBS Ab (b). The merged (c) and ratio (phosphorylated MBS/MBS; d) images are shown. (C) Inhibition of phosphorylation of MBS by C3 or dominant negative Rho-kinase. REF52 cells were microinjected with MBP (5.0 mg/ml) (a–d), C3 (0.1 mg/ml) (e–h), C3 plus GTPγS·RhoAI41 (0.4 mg/ml) (i–l), or MBP-RB/PH(TT) (5.0 mg/ml) (m–p). REF52 cells were stained with TRITC-phalloidin (a, e, i, and m), anti-pS854 Ab (b, f, j, and n), anti-pnMBS Ab (c, g, k, and o), and anti-pp2b Ab (d, h, l, and p). The arrowheads indicate the injected cells. Bars, 10 μm.
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Figure 6: Colocalization of phosphorylated MBS, F-actin and phosphorylated MLC in REF52 fibroblasts. (A) Localization of phosphorylated MBS. REF52 cells were doubly stained with TRITC-phalloidin (b and d) and anti-pS854 Ab (a) or anti-pp2b Ab (c). (B) Distribution of phosphorylated and total MBS in REF52 cells. REF52 cells were doubly stained with anti-pS854 Ab (a) and anti-mMBS Ab (b). The merged (c) and ratio (phosphorylated MBS/MBS; d) images are shown. (C) Inhibition of phosphorylation of MBS by C3 or dominant negative Rho-kinase. REF52 cells were microinjected with MBP (5.0 mg/ml) (a–d), C3 (0.1 mg/ml) (e–h), C3 plus GTPγS·RhoAI41 (0.4 mg/ml) (i–l), or MBP-RB/PH(TT) (5.0 mg/ml) (m–p). REF52 cells were stained with TRITC-phalloidin (a, e, i, and m), anti-pS854 Ab (b, f, j, and n), anti-pnMBS Ab (c, g, k, and o), and anti-pp2b Ab (d, h, l, and p). The arrowheads indicate the injected cells. Bars, 10 μm.
Mentions: We further investigated the subcellular distribution of phosphorylated MBS, F-actin, and phosphorylated MLC in REF52 fibroblasts. REF52 cells grown in 10% fetal bovine serum have thick actin stress fibers and vinculin-containing focal adhesions, and show a filamentous periodical pattern of myosin on stress fibers. We have previously shown that total MBS is localized on stress fibers (Inagaki, N., et al. 1997). Phosphorylated MBS was localized on stress fibers, cortical actin filaments, and in the nucleus in REF52 cells (Fig. 6 A, a). The double-immunostaining by TRITC-phalloidin and anti-pS854 Ab (Fig. 6 A, a and b) or anti-pp2b Ab (Fig. 6 A, c and d) demonstrated coexistence of phosphorylated MBS, F-actin and phosphorylated MLC on stress fibers and cortical actin filaments. The intracellular localization of phosphorylated MBS is consistent with in vitro binding of MBS and myosin (Alessi et al. 1992; Shimizu et al. 1994). There was the difference in localization of phosphorylated MBS between REF52 and MDCK cells. REF52 cells highly developed thick stress fibers (Fig. 6 A, b), whereas serum-starved MDCK cells had a few thin stress fibers (Fig. 4 A, b). Thus, phosphorylated MBS might not be detected in stress fibers in MDCK cells. Next, we compared the distribution of phosphorylated and total MBS. The distribution of phosphorylated MBS was similar to that of total MBS (Fig. 6 B, c). The ratio (phosphorylated MBS/MBS) image showed that the phosphorylation level of MBS was high on stress fiber and in nucleus (Fig. 6 B, d).

Bottom Line: Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro.During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased.Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.

View Article: PubMed Central - PubMed

Affiliation: Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0101, Japan.

ABSTRACT
Rho-associated kinase (Rho-kinase), which is activated by the small GTPase Rho, phosphorylates myosin-binding subunit (MBS) of myosin phosphatase and thereby inactivates the phosphatase activity in vitro. Rho-kinase is thought to regulate the phosphorylation state of the substrates including myosin light chain (MLC), ERM (ezrin/radixin/moesin) family proteins and adducin by their direct phosphorylation and by the inactivation of myosin phosphatase. Here we identified the sites of phosphorylation of MBS by Rho-kinase as Thr-697, Ser-854 and several residues, and prepared antibody that specifically recognized MBS phosphorylated at Ser-854. We found by use of this antibody that the stimulation of MDCK epithelial cells with tetradecanoylphorbol-13-acetate (TPA) or hepatocyte growth factor (HGF) induced the phosphorylation of MBS at Ser-854 under the conditions in which membrane ruffling and cell migration were induced. Pretreatment of the cells with Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with Rho functions, or Rho-kinase inhibitors inhibited the TPA- or HGF-induced MBS phosphorylation. The TPA stimulation enhanced the immunoreactivity of phosphorylated MBS in the cytoplasm and membrane ruffling area of MDCK cells. In migrating MDCK cells, phosphorylated MBS as well as phosphorylated MLC at Ser-19 were localized in the leading edge and posterior region. Phosphorylated MBS was localized on actin stress fibers in REF52 fibroblasts. The microinjection of C3 or dominant negative Rho-kinase disrupted stress fibers and weakened the accumulation of phosphorylated MBS in REF52 cells. During cytokinesis, phosphorylated MBS, MLC and ERM family proteins accumulated at the cleavage furrow, and the phosphorylation level of MBS at Ser-854 was increased. Taken together, these results indicate that MBS is phosphorylated by Rho-kinase downstream of Rho in vivo, and suggest that myosin phosphatase and Rho-kinase spatiotemporally regulate the phosphorylation state of Rho-kinase substrates including MLC and ERM family proteins in vivo in a cooperative manner.

Show MeSH