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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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Rho- and Rho-kinase–dependent phosphorylation of MBS at Ser-854 in MDCK cells. (A) TPA- and HGF-induced phosphorylation of MBS at Ser-854. Serum-deprived MDCK cells were stimulated with 200 nM TPA, 50 pM HGF for 15 min or 5 mM dibutyryl cAMP (cAMP) for 30 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panel) and anti-pnMBS Ab (lower panel). Arrowhead indicates the position of MBS phosphorylated at Ser-854. A minor band with apparent relative molecular mass of 90 kD may be a degradation product of MBS. (B) Time course of the phosphorylation of MBS at Ser-854 in the TPA- and HGF-stimulated MDCK cells. Serum-deprived MDCK cells were stimulated with 200 nM TPA (a) or 50 pM HGF (b) for 3, 5, 15, 30, 60, or 120 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The amount of MBS phosphorylated at Ser-854 was quantitatively determined by scanning densitometry. The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab at 0 min was set at 100 arbitrary units. The values shown are means ± SE of triplicates. (C) Inhibition of the TPA- and HGF-induced MBS phosphorylation by C3 or Rho-kinase inhibitors. Nonpretreated (2), 50 or 100 μg/ml C3-pretreated (3 and 4), 1 or 10 μM of HA1077-pretreated (5 and 6), or 1 or 10 μM of Y-32885–pretreated (7 and 8) serum-deprived MDCK cells were stimulated with (2–8) or without (1) 200 nM TPA (a) or 50 pM HGF (b) for 15 min and the lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab in the nonstimulated cells was set at 100 arbitrary units. The values shown are means ± SE of triplicates.
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Figure 3: Rho- and Rho-kinase–dependent phosphorylation of MBS at Ser-854 in MDCK cells. (A) TPA- and HGF-induced phosphorylation of MBS at Ser-854. Serum-deprived MDCK cells were stimulated with 200 nM TPA, 50 pM HGF for 15 min or 5 mM dibutyryl cAMP (cAMP) for 30 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panel) and anti-pnMBS Ab (lower panel). Arrowhead indicates the position of MBS phosphorylated at Ser-854. A minor band with apparent relative molecular mass of 90 kD may be a degradation product of MBS. (B) Time course of the phosphorylation of MBS at Ser-854 in the TPA- and HGF-stimulated MDCK cells. Serum-deprived MDCK cells were stimulated with 200 nM TPA (a) or 50 pM HGF (b) for 3, 5, 15, 30, 60, or 120 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The amount of MBS phosphorylated at Ser-854 was quantitatively determined by scanning densitometry. The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab at 0 min was set at 100 arbitrary units. The values shown are means ± SE of triplicates. (C) Inhibition of the TPA- and HGF-induced MBS phosphorylation by C3 or Rho-kinase inhibitors. Nonpretreated (2), 50 or 100 μg/ml C3-pretreated (3 and 4), 1 or 10 μM of HA1077-pretreated (5 and 6), or 1 or 10 μM of Y-32885–pretreated (7 and 8) serum-deprived MDCK cells were stimulated with (2–8) or without (1) 200 nM TPA (a) or 50 pM HGF (b) for 15 min and the lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab in the nonstimulated cells was set at 100 arbitrary units. The values shown are means ± SE of triplicates.

Mentions: Rho/Rho-kinase are implicated in HGF- and phorbol ester–induced membrane ruffling in MDCK and KB epithelial cells (Nishiyama et al. 1994; Takaishi et al. 1995; Fukata et al. 1999). We examined whether MBS was phosphorylated at Ser-854 via the Rho/Rho-kinase pathway in vivo. When total cell lysate of nonstimulated MDCK cells was immunoblotted with anti-pS854 Ab, MBS phosphorylated at Ser-854 was weakly detected. The addition of TPA enhanced the phosphorylation of MBS at Ser-854 (Fig. 3 A). Similar results were obtained when the cells were stimulated with HGF. A minor band with apparent relative molecular mass of 90 kD was also detected. This minor band may be a degradation product of MBS, because the immunoreactive band detected by anti-MBS Ab was found at the same position (data not shown). After TPA stimulation, the phosphorylation level of MBS at Ser-854 elevated within 3 min, reached maximum at ∼30 min, and was sustained for at least 2 h (Fig. 3 B). The maximal phosphorylation level of MBS at Ser-854 was about fivefold the basal level. The stoichiometries of phosphorylation at Ser-854 were ∼0.04 at the basal level and ∼0.20 at the maximal level, respectively. Similar results were obtained when the cells were stimulated with HGF, although the level of MBS phosphorylation induced by HGF was slightly lower than that induced by TPA (Fig. 3 B). We also confirmed that the addition of dibutyryl cAMP did not induce the phosphorylation of MBS at Ser-854 (Fig. 3 A), whereas it induced the phosphorylation of cAMP-response element binding protein (CREB) at Ser-133 (data not shown). These results indicate that MBS is phosphorylated during the action of TPA and HGF in MDCK cells. We furthermore examined whether MBS was phosphorylated at Ser-854 via the Rho/Rho-kinase pathway during the action of TPA and HGF in MDCK cells (Fig. 3 C). Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with endogenous Rho functions, inhibited the focal adhesion formation in MDCK cells (∼50% inhibition by incubation of 100 μg/ml of C3; data not shown). Under the conditions, the TPA-induced MBS phosphorylation was inhibited to a similar extent (Fig. 3 C). A similar inhibition was observed when the cells were pretreated with HA1077 or Y-32885, both of which are inhibitors of Rho-kinase (Fig. 3 C; Uehata et al. 1997).


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)

Rho- and Rho-kinase–dependent phosphorylation of MBS at Ser-854 in MDCK cells. (A) TPA- and HGF-induced phosphorylation of MBS at Ser-854. Serum-deprived MDCK cells were stimulated with 200 nM TPA, 50 pM HGF for 15 min or 5 mM dibutyryl cAMP (cAMP) for 30 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panel) and anti-pnMBS Ab (lower panel). Arrowhead indicates the position of MBS phosphorylated at Ser-854. A minor band with apparent relative molecular mass of 90 kD may be a degradation product of MBS. (B) Time course of the phosphorylation of MBS at Ser-854 in the TPA- and HGF-stimulated MDCK cells. Serum-deprived MDCK cells were stimulated with 200 nM TPA (a) or 50 pM HGF (b) for 3, 5, 15, 30, 60, or 120 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The amount of MBS phosphorylated at Ser-854 was quantitatively determined by scanning densitometry. The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab at 0 min was set at 100 arbitrary units. The values shown are means ± SE of triplicates. (C) Inhibition of the TPA- and HGF-induced MBS phosphorylation by C3 or Rho-kinase inhibitors. Nonpretreated (2), 50 or 100 μg/ml C3-pretreated (3 and 4), 1 or 10 μM of HA1077-pretreated (5 and 6), or 1 or 10 μM of Y-32885–pretreated (7 and 8) serum-deprived MDCK cells were stimulated with (2–8) or without (1) 200 nM TPA (a) or 50 pM HGF (b) for 15 min and the lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab in the nonstimulated cells was set at 100 arbitrary units. The values shown are means ± SE of triplicates.
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Figure 3: Rho- and Rho-kinase–dependent phosphorylation of MBS at Ser-854 in MDCK cells. (A) TPA- and HGF-induced phosphorylation of MBS at Ser-854. Serum-deprived MDCK cells were stimulated with 200 nM TPA, 50 pM HGF for 15 min or 5 mM dibutyryl cAMP (cAMP) for 30 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panel) and anti-pnMBS Ab (lower panel). Arrowhead indicates the position of MBS phosphorylated at Ser-854. A minor band with apparent relative molecular mass of 90 kD may be a degradation product of MBS. (B) Time course of the phosphorylation of MBS at Ser-854 in the TPA- and HGF-stimulated MDCK cells. Serum-deprived MDCK cells were stimulated with 200 nM TPA (a) or 50 pM HGF (b) for 3, 5, 15, 30, 60, or 120 min, and the whole-cell lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The amount of MBS phosphorylated at Ser-854 was quantitatively determined by scanning densitometry. The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab at 0 min was set at 100 arbitrary units. The values shown are means ± SE of triplicates. (C) Inhibition of the TPA- and HGF-induced MBS phosphorylation by C3 or Rho-kinase inhibitors. Nonpretreated (2), 50 or 100 μg/ml C3-pretreated (3 and 4), 1 or 10 μM of HA1077-pretreated (5 and 6), or 1 or 10 μM of Y-32885–pretreated (7 and 8) serum-deprived MDCK cells were stimulated with (2–8) or without (1) 200 nM TPA (a) or 50 pM HGF (b) for 15 min and the lysates were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels) and anti-pnMBS Ab (lower panels). The densities of the immunoreactive bands with anti-pS854 Ab were normalized by that of total MBS. The mean density of the immunoreactive bands with anti-pS854 Ab in the nonstimulated cells was set at 100 arbitrary units. The values shown are means ± SE of triplicates.
Mentions: Rho/Rho-kinase are implicated in HGF- and phorbol ester–induced membrane ruffling in MDCK and KB epithelial cells (Nishiyama et al. 1994; Takaishi et al. 1995; Fukata et al. 1999). We examined whether MBS was phosphorylated at Ser-854 via the Rho/Rho-kinase pathway in vivo. When total cell lysate of nonstimulated MDCK cells was immunoblotted with anti-pS854 Ab, MBS phosphorylated at Ser-854 was weakly detected. The addition of TPA enhanced the phosphorylation of MBS at Ser-854 (Fig. 3 A). Similar results were obtained when the cells were stimulated with HGF. A minor band with apparent relative molecular mass of 90 kD was also detected. This minor band may be a degradation product of MBS, because the immunoreactive band detected by anti-MBS Ab was found at the same position (data not shown). After TPA stimulation, the phosphorylation level of MBS at Ser-854 elevated within 3 min, reached maximum at ∼30 min, and was sustained for at least 2 h (Fig. 3 B). The maximal phosphorylation level of MBS at Ser-854 was about fivefold the basal level. The stoichiometries of phosphorylation at Ser-854 were ∼0.04 at the basal level and ∼0.20 at the maximal level, respectively. Similar results were obtained when the cells were stimulated with HGF, although the level of MBS phosphorylation induced by HGF was slightly lower than that induced by TPA (Fig. 3 B). We also confirmed that the addition of dibutyryl cAMP did not induce the phosphorylation of MBS at Ser-854 (Fig. 3 A), whereas it induced the phosphorylation of cAMP-response element binding protein (CREB) at Ser-133 (data not shown). These results indicate that MBS is phosphorylated during the action of TPA and HGF in MDCK cells. We furthermore examined whether MBS was phosphorylated at Ser-854 via the Rho/Rho-kinase pathway during the action of TPA and HGF in MDCK cells (Fig. 3 C). Botulinum C3 ADP-ribosyltransferase (C3), which is thought to interfere with endogenous Rho functions, inhibited the focal adhesion formation in MDCK cells (∼50% inhibition by incubation of 100 μg/ml of C3; data not shown). Under the conditions, the TPA-induced MBS phosphorylation was inhibited to a similar extent (Fig. 3 C). A similar inhibition was observed when the cells were pretreated with HA1077 or Y-32885, both of which are inhibitors of Rho-kinase (Fig. 3 C; Uehata et al. 1997).

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
Related in: MedlinePlus