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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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Distribution of phosphorylated MBS and ERM family proteins during cytokinesis in MDCK cells. (A) Accumulation of phosphorylated MBS and ERM family proteins at the cleavage furrow. MDCK cells in late anaphase (a, d, g, j, m, and p), telophase (b, e, h, k, n, and q) or cytokinesis (c, f, i, l, o, and r; indicated by arrowheads) stained with anti-pS854 Ab (a–c), anti-pp2b Ab (g–i), or anti-pT558 Ab (m–o) are shown. DNAs were stained with bisbenzimide Hoechst (d–f, j–l, and p–r). (B) Specific localization of phosphorylated Rho-kinase substrates during cytokinesis. MDCK cells in cytokinesis doubly stained with TM71 (b, e, h, k, and n) and anti-pnMBS Ab (a), anti-pS854 Ab (d), anti-ERM Ab (g), anti-pT558 Ab (j), and anti-pT445 Ab (m). Merged images are shown (c, f, i, l, and o). (C) Elevation of phosphorylation of MBS at Ser-854 during cytokinesis. Total cell lysates were prepared from interphase cells (I), early mitotic cells (metaphase, M) and cells at different stages of cell division (time in minutes after the release of mitotic arrest), and immunoblotted with anti-pS854 Ab (upper panel) or anti-pnMBS Ab (lower panel). These results are representative of three independent experiments. Bars, 10 μm.
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Figure 7: Distribution of phosphorylated MBS and ERM family proteins during cytokinesis in MDCK cells. (A) Accumulation of phosphorylated MBS and ERM family proteins at the cleavage furrow. MDCK cells in late anaphase (a, d, g, j, m, and p), telophase (b, e, h, k, n, and q) or cytokinesis (c, f, i, l, o, and r; indicated by arrowheads) stained with anti-pS854 Ab (a–c), anti-pp2b Ab (g–i), or anti-pT558 Ab (m–o) are shown. DNAs were stained with bisbenzimide Hoechst (d–f, j–l, and p–r). (B) Specific localization of phosphorylated Rho-kinase substrates during cytokinesis. MDCK cells in cytokinesis doubly stained with TM71 (b, e, h, k, and n) and anti-pnMBS Ab (a), anti-pS854 Ab (d), anti-ERM Ab (g), anti-pT558 Ab (j), and anti-pT445 Ab (m). Merged images are shown (c, f, i, l, and o). (C) Elevation of phosphorylation of MBS at Ser-854 during cytokinesis. Total cell lysates were prepared from interphase cells (I), early mitotic cells (metaphase, M) and cells at different stages of cell division (time in minutes after the release of mitotic arrest), and immunoblotted with anti-pS854 Ab (upper panel) or anti-pnMBS Ab (lower panel). These results are representative of three independent experiments. Bars, 10 μm.

Mentions: It has been shown that ERM family proteins and MLC phosphorylated at Ser-19 highly accumulate at the cleavage furrow during cytokinesis (Sato et al. 1991; Matsumura et al. 1998). In a recent study, we have found that Rho-kinase also highly and circumferentially accumulates at the cleavage furrow in various cell lines (Kosako et al. 1999), and that dominant negative Rho-kinase inhibits the progress of cytokinesis (Yasui et al. 1998). Here we examined the distribution of phosphorylated MBS during the different mitotic stages of MDCK cells. Phosphorylated MBS was enriched at the mid zone between the daughter chromosomes in late anaphase and at the cleavage furrow in telophase (Fig. 7 A, a and b). Phosphorylated MBS persisted at the mid body until the end of cytokinesis (Fig. 7 A, c). Next, we compared the distribution of phosphorylated MLC and ERM family proteins to that of phosphorylated MBS during different mitotic stages of MDCK cells. The staining patterns of phosphorylated MLC were spatially and temporally similar to that of phosphorylated MBS in dividing cells (Fig. 7 A, g–i). Phosphorylated ERM family proteins accumulated in the microvilli-like structures in the cell body at all stages as described (Oshiro et al. 1998), and highly and circumferentially accumulated around the mid zone in late anaphase, and the cleavage furrow in telophase. The staining patterns of phosphorylated ERM family proteins were also similar to that of phosphorylated MBS, but phosphorylated ERM family proteins did not persist at the mid body until the end of cytokinesis (Fig. 7 A, m–o). Vimentin is the most widely expressed intermediate filament protein, which is phosphorylated by Rho-kinase at Ser-71 (Goto et al. 1998). Using TM71, which recognizes the phosphorylation of vimentin at Ser-71, vimentin is shown to be specifically phosphorylated at the cleavage furrow whereas total vimentin is diffusely localized throughout the cytoplasm (Yasui et al. 1998). Although phosphorylated vimentin, MBS and ERM family proteins accumulated around the cleavage furrow, they were not completely colocalized (Fig. 7 B, f and l). Phosphorylated adducin, which is one of the Rho-kinase substrates, was diffusely localized throughout the cytoplasm (Fig. 7 B, m). It should be noted that total MBS was diffusely localized throughout the cytoplasm, but not accumulated at the cleavage furrow (Fig. 7 B, a). In contrast, phosphorylated MBS strongly accumulated at the cleavage furrow (Fig. 7 B, d), indicating that MBS was phosphorylated specifically at the cleavage furrow. Total ERM family proteins was diffusely localized throughout the cytoplasm, and at the microvilli and cleavage furrow (Fig. 7 B, g), whereas phosphorylated ERM family proteins accumulated at the microvilli and cleavage furrow preferentially (Fig. 7 B, j). To further confirm that the phosphorylation level of MBS at Ser-854 elevates during the cytokinesis, immunoblot analysis of synchronized MDCK cells lysates was carried out (Fig. 7 C). By release from mitotic arrest by nocodazole, early mitotic cells synchronistically entered into later stages of cell division, and at 30 min after release of mitotic arrest, ∼40% of the cells were in late anaphase, telophase or cytokinesis. At later phases, the cells were in post mitotic spreading (60–180 min) as described (data not shown; Matsumura et al. 1998). The immunoreactivity of anti-pS854 Ab was increased at 30 min after release of mitotic arrest as compared with that in interphase and early mitotic cells (Fig. 7 C). This increment of phosphorylation level of MBS was reversed at 60 min.


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)

Distribution of phosphorylated MBS and ERM family proteins during cytokinesis in MDCK cells. (A) Accumulation of phosphorylated MBS and ERM family proteins at the cleavage furrow. MDCK cells in late anaphase (a, d, g, j, m, and p), telophase (b, e, h, k, n, and q) or cytokinesis (c, f, i, l, o, and r; indicated by arrowheads) stained with anti-pS854 Ab (a–c), anti-pp2b Ab (g–i), or anti-pT558 Ab (m–o) are shown. DNAs were stained with bisbenzimide Hoechst (d–f, j–l, and p–r). (B) Specific localization of phosphorylated Rho-kinase substrates during cytokinesis. MDCK cells in cytokinesis doubly stained with TM71 (b, e, h, k, and n) and anti-pnMBS Ab (a), anti-pS854 Ab (d), anti-ERM Ab (g), anti-pT558 Ab (j), and anti-pT445 Ab (m). Merged images are shown (c, f, i, l, and o). (C) Elevation of phosphorylation of MBS at Ser-854 during cytokinesis. Total cell lysates were prepared from interphase cells (I), early mitotic cells (metaphase, M) and cells at different stages of cell division (time in minutes after the release of mitotic arrest), and immunoblotted with anti-pS854 Ab (upper panel) or anti-pnMBS Ab (lower panel). These results are representative of three independent experiments. Bars, 10 μm.
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Figure 7: Distribution of phosphorylated MBS and ERM family proteins during cytokinesis in MDCK cells. (A) Accumulation of phosphorylated MBS and ERM family proteins at the cleavage furrow. MDCK cells in late anaphase (a, d, g, j, m, and p), telophase (b, e, h, k, n, and q) or cytokinesis (c, f, i, l, o, and r; indicated by arrowheads) stained with anti-pS854 Ab (a–c), anti-pp2b Ab (g–i), or anti-pT558 Ab (m–o) are shown. DNAs were stained with bisbenzimide Hoechst (d–f, j–l, and p–r). (B) Specific localization of phosphorylated Rho-kinase substrates during cytokinesis. MDCK cells in cytokinesis doubly stained with TM71 (b, e, h, k, and n) and anti-pnMBS Ab (a), anti-pS854 Ab (d), anti-ERM Ab (g), anti-pT558 Ab (j), and anti-pT445 Ab (m). Merged images are shown (c, f, i, l, and o). (C) Elevation of phosphorylation of MBS at Ser-854 during cytokinesis. Total cell lysates were prepared from interphase cells (I), early mitotic cells (metaphase, M) and cells at different stages of cell division (time in minutes after the release of mitotic arrest), and immunoblotted with anti-pS854 Ab (upper panel) or anti-pnMBS Ab (lower panel). These results are representative of three independent experiments. Bars, 10 μm.
Mentions: It has been shown that ERM family proteins and MLC phosphorylated at Ser-19 highly accumulate at the cleavage furrow during cytokinesis (Sato et al. 1991; Matsumura et al. 1998). In a recent study, we have found that Rho-kinase also highly and circumferentially accumulates at the cleavage furrow in various cell lines (Kosako et al. 1999), and that dominant negative Rho-kinase inhibits the progress of cytokinesis (Yasui et al. 1998). Here we examined the distribution of phosphorylated MBS during the different mitotic stages of MDCK cells. Phosphorylated MBS was enriched at the mid zone between the daughter chromosomes in late anaphase and at the cleavage furrow in telophase (Fig. 7 A, a and b). Phosphorylated MBS persisted at the mid body until the end of cytokinesis (Fig. 7 A, c). Next, we compared the distribution of phosphorylated MLC and ERM family proteins to that of phosphorylated MBS during different mitotic stages of MDCK cells. The staining patterns of phosphorylated MLC were spatially and temporally similar to that of phosphorylated MBS in dividing cells (Fig. 7 A, g–i). Phosphorylated ERM family proteins accumulated in the microvilli-like structures in the cell body at all stages as described (Oshiro et al. 1998), and highly and circumferentially accumulated around the mid zone in late anaphase, and the cleavage furrow in telophase. The staining patterns of phosphorylated ERM family proteins were also similar to that of phosphorylated MBS, but phosphorylated ERM family proteins did not persist at the mid body until the end of cytokinesis (Fig. 7 A, m–o). Vimentin is the most widely expressed intermediate filament protein, which is phosphorylated by Rho-kinase at Ser-71 (Goto et al. 1998). Using TM71, which recognizes the phosphorylation of vimentin at Ser-71, vimentin is shown to be specifically phosphorylated at the cleavage furrow whereas total vimentin is diffusely localized throughout the cytoplasm (Yasui et al. 1998). Although phosphorylated vimentin, MBS and ERM family proteins accumulated around the cleavage furrow, they were not completely colocalized (Fig. 7 B, f and l). Phosphorylated adducin, which is one of the Rho-kinase substrates, was diffusely localized throughout the cytoplasm (Fig. 7 B, m). It should be noted that total MBS was diffusely localized throughout the cytoplasm, but not accumulated at the cleavage furrow (Fig. 7 B, a). In contrast, phosphorylated MBS strongly accumulated at the cleavage furrow (Fig. 7 B, d), indicating that MBS was phosphorylated specifically at the cleavage furrow. Total ERM family proteins was diffusely localized throughout the cytoplasm, and at the microvilli and cleavage furrow (Fig. 7 B, g), whereas phosphorylated ERM family proteins accumulated at the microvilli and cleavage furrow preferentially (Fig. 7 B, j). To further confirm that the phosphorylation level of MBS at Ser-854 elevates during the cytokinesis, immunoblot analysis of synchronized MDCK cells lysates was carried out (Fig. 7 C). By release from mitotic arrest by nocodazole, early mitotic cells synchronistically entered into later stages of cell division, and at 30 min after release of mitotic arrest, ∼40% of the cells were in late anaphase, telophase or cytokinesis. At later phases, the cells were in post mitotic spreading (60–180 min) as described (data not shown; Matsumura et al. 1998). The immunoreactivity of anti-pS854 Ab was increased at 30 min after release of mitotic arrest as compared with that in interphase and early mitotic cells (Fig. 7 C). This increment of phosphorylation level of MBS was reversed at 60 min.

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