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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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Specificity of phosphorylation site-specific antibody (anti-pS854 Ab). (A) Recognition of MBS phosphorylated at Ser-854. A total of 200 fmol of GST-MBS-CT (CT: 758–1032 aa) containing the indicated amounts of GST-MBS-CT phosphorylated by GST-CAT and 200 fmol of GST-MBS-CTS854A, T855A (AA) and GST-MBS-NT (NT: 1–763 aa) phosphorylated by GST-CAT were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels), anti-pS854 Ab absorbed with a 100-fold amount of antigen phosphopeptide (middle panels) or anti-pnMBS or pcMBS Ab (lower panels). (B) Specific recognition of MBS phosphorylated by Rho-kinase. A total of 4 pmol of full-length MBS (1–1032 aa) phosphorylated by GST-CAT (CAT) or PKC was resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panel) or anti-pnMBS Ab (lower panel) or by autoradiography (middle panel). The arrowhead indicates the position of MBS phosphorylated at Ser-854. These results are representative of three independent experiments.
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Figure 2: Specificity of phosphorylation site-specific antibody (anti-pS854 Ab). (A) Recognition of MBS phosphorylated at Ser-854. A total of 200 fmol of GST-MBS-CT (CT: 758–1032 aa) containing the indicated amounts of GST-MBS-CT phosphorylated by GST-CAT and 200 fmol of GST-MBS-CTS854A, T855A (AA) and GST-MBS-NT (NT: 1–763 aa) phosphorylated by GST-CAT were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels), anti-pS854 Ab absorbed with a 100-fold amount of antigen phosphopeptide (middle panels) or anti-pnMBS or pcMBS Ab (lower panels). (B) Specific recognition of MBS phosphorylated by Rho-kinase. A total of 4 pmol of full-length MBS (1–1032 aa) phosphorylated by GST-CAT (CAT) or PKC was resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panel) or anti-pnMBS Ab (lower panel) or by autoradiography (middle panel). The arrowhead indicates the position of MBS phosphorylated at Ser-854. These results are representative of three independent experiments.

Mentions: To investigate how the phosphorylation of MBS by Rho-kinase is regulated in vivo, we prepared the site- and phosphorylation state-specific antibody for MBS. As shown in Table , Rho-kinase phosphorylated multiple sites of MBS in vitro. Thr-697 was one of the major sites of phosphorylation of MBS by Rho-kinase in vitro. However, we can not distinguish the phosphorylation of Thr-697 by Rho-kinase and the endogenous kinase in vivo as described above. Ser-854, which was also one of the major sites of phosphorylation of MBS by Rho-kinase in vitro, is the phosphorylation site specific to Rho-kinase among known MBS kinases in vitro (We confirmed that PKC did not phosphorylate MBS at Ser-854: see below). The phosphorylation at Ser-854 can serve as a pertinent indicator to study MBS phosphorylation by Rho-kinase in vivo. Thus, we prepared the rabbit polyclonal antibody (anti-pS854 Ab), raised against the synthetic phosphopeptide (CEKRRphoshoS854TGVSF). The specificity of anti-pS854 Ab was examined by immunoblot analysis. Equal amounts of GST-MBS-CT with various ratios between phosphorylated and unphosphorylated forms were loaded on the gel. GST-MBS-CT phosphorylated by GST-CAT in vitro was specifically detected by anti-pS854 Ab in a dose-dependent manner (Fig. 2 A). The binding was neutralized by preincubation of the antibody with the antigen phosphopeptide. We also confirmed that anti-pS854 Ab recognized neither GST-MBS-CTS854A, T855A (substitution of residues by Ala), GST-MBS-NT phosphorylated by GST-CAT (Fig. 2 A) nor full-length MBS phosphorylated by PKC (Fig. 2 B). These results indicate that anti-pS854 Ab specifically recognizes MBS phosphorylated at Ser-854 by Rho-kinase.


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)

Specificity of phosphorylation site-specific antibody (anti-pS854 Ab). (A) Recognition of MBS phosphorylated at Ser-854. A total of 200 fmol of GST-MBS-CT (CT: 758–1032 aa) containing the indicated amounts of GST-MBS-CT phosphorylated by GST-CAT and 200 fmol of GST-MBS-CTS854A, T855A (AA) and GST-MBS-NT (NT: 1–763 aa) phosphorylated by GST-CAT were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels), anti-pS854 Ab absorbed with a 100-fold amount of antigen phosphopeptide (middle panels) or anti-pnMBS or pcMBS Ab (lower panels). (B) Specific recognition of MBS phosphorylated by Rho-kinase. A total of 4 pmol of full-length MBS (1–1032 aa) phosphorylated by GST-CAT (CAT) or PKC was resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panel) or anti-pnMBS Ab (lower panel) or by autoradiography (middle panel). The arrowhead indicates the position of MBS phosphorylated at Ser-854. These results are representative of three independent experiments.
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Related In: Results  -  Collection

Show All Figures
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Figure 2: Specificity of phosphorylation site-specific antibody (anti-pS854 Ab). (A) Recognition of MBS phosphorylated at Ser-854. A total of 200 fmol of GST-MBS-CT (CT: 758–1032 aa) containing the indicated amounts of GST-MBS-CT phosphorylated by GST-CAT and 200 fmol of GST-MBS-CTS854A, T855A (AA) and GST-MBS-NT (NT: 1–763 aa) phosphorylated by GST-CAT were resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panels), anti-pS854 Ab absorbed with a 100-fold amount of antigen phosphopeptide (middle panels) or anti-pnMBS or pcMBS Ab (lower panels). (B) Specific recognition of MBS phosphorylated by Rho-kinase. A total of 4 pmol of full-length MBS (1–1032 aa) phosphorylated by GST-CAT (CAT) or PKC was resolved by SDS-PAGE followed by immunoblotting with anti-pS854 Ab (upper panel) or anti-pnMBS Ab (lower panel) or by autoradiography (middle panel). The arrowhead indicates the position of MBS phosphorylated at Ser-854. These results are representative of three independent experiments.
Mentions: To investigate how the phosphorylation of MBS by Rho-kinase is regulated in vivo, we prepared the site- and phosphorylation state-specific antibody for MBS. As shown in Table , Rho-kinase phosphorylated multiple sites of MBS in vitro. Thr-697 was one of the major sites of phosphorylation of MBS by Rho-kinase in vitro. However, we can not distinguish the phosphorylation of Thr-697 by Rho-kinase and the endogenous kinase in vivo as described above. Ser-854, which was also one of the major sites of phosphorylation of MBS by Rho-kinase in vitro, is the phosphorylation site specific to Rho-kinase among known MBS kinases in vitro (We confirmed that PKC did not phosphorylate MBS at Ser-854: see below). The phosphorylation at Ser-854 can serve as a pertinent indicator to study MBS phosphorylation by Rho-kinase in vivo. Thus, we prepared the rabbit polyclonal antibody (anti-pS854 Ab), raised against the synthetic phosphopeptide (CEKRRphoshoS854TGVSF). The specificity of anti-pS854 Ab was examined by immunoblot analysis. Equal amounts of GST-MBS-CT with various ratios between phosphorylated and unphosphorylated forms were loaded on the gel. GST-MBS-CT phosphorylated by GST-CAT in vitro was specifically detected by anti-pS854 Ab in a dose-dependent manner (Fig. 2 A). The binding was neutralized by preincubation of the antibody with the antigen phosphopeptide. We also confirmed that anti-pS854 Ab recognized neither GST-MBS-CTS854A, T855A (substitution of residues by Ala), GST-MBS-NT phosphorylated by GST-CAT (Fig. 2 A) nor full-length MBS phosphorylated by PKC (Fig. 2 B). These results indicate that anti-pS854 Ab specifically recognizes MBS phosphorylated at Ser-854 by Rho-kinase.

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