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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 Ser-854–phosphorylated MBS in the TPA- or HGF-stimulated MDCK cells. (A) Distribution of Ser-854–phosphorylated MBS. Serum-deprived MDCK cells were stimulated with 200 nM TPA (c, d, and h) or 50 pM HGF (e and f) for 15 min. MDCK cells were stained with anti-pS854 Ab (a, c, and e) or TRITC-phalloidin (b, d, and f). MDCK cells were also stained with anti-pS854 Ab absorbed with a 100-fold amount of antigen phosphopeptide (g and h). Arrowheads indicate the induced membrane ruffling. (B) Distribution of phosphorylated and total MBS. Serum-deprived MDCK cells were stimulated with 200 nM TPA (e–h) or 50 pM HGF (i–l) for 15 min. MDCK cells were doubly stained with anti-pS854 Ab (a, e, and i) and anti-mMBS Ab (b, f, and j). The merged (c, g, and k) and ratio (phosphorylated MBS/MBS; d, h, and l) images are shown. (C) Subcellular distribution of phosphorylated MBS. Non- (−) and TPA- (+) stimulated MDCK cells were separated into nuclear (nucleus), cytoplasmic (cytoplasm), and membrane (membrane) fractions, and the fractions were immunoblotted with anti-pS854 Ab (upper panel) and anti-pnMBS Ab (lower panel). These results are representative of three independent experiments. Bars, 10 μm.
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Figure 4: Distribution of Ser-854–phosphorylated MBS in the TPA- or HGF-stimulated MDCK cells. (A) Distribution of Ser-854–phosphorylated MBS. Serum-deprived MDCK cells were stimulated with 200 nM TPA (c, d, and h) or 50 pM HGF (e and f) for 15 min. MDCK cells were stained with anti-pS854 Ab (a, c, and e) or TRITC-phalloidin (b, d, and f). MDCK cells were also stained with anti-pS854 Ab absorbed with a 100-fold amount of antigen phosphopeptide (g and h). Arrowheads indicate the induced membrane ruffling. (B) Distribution of phosphorylated and total MBS. Serum-deprived MDCK cells were stimulated with 200 nM TPA (e–h) or 50 pM HGF (i–l) for 15 min. MDCK cells were doubly stained with anti-pS854 Ab (a, e, and i) and anti-mMBS Ab (b, f, and j). The merged (c, g, and k) and ratio (phosphorylated MBS/MBS; d, h, and l) images are shown. (C) Subcellular distribution of phosphorylated MBS. Non- (−) and TPA- (+) stimulated MDCK cells were separated into nuclear (nucleus), cytoplasmic (cytoplasm), and membrane (membrane) fractions, and the fractions were immunoblotted with anti-pS854 Ab (upper panel) and anti-pnMBS Ab (lower panel). These results are representative of three independent experiments. Bars, 10 μm.

Mentions: MDCK cells were fixed with 3.0% formaldehyde in PBS for 20 min and treated with PBS containing 0.2% Triton X-100 for 10 min on ice or acetone for 10 min at −20°C (for anti-pp2b Ab). REF52 cells were fixed with 3.0% formaldehyde in PBS for 10 min and treated with PBS containing 0.2% Triton X-100 for 10 min at room temperature. After being washed with PBS three times, the cells were incubated with anti-pS854 Ab, anti-pnMBS Ab, anti-pT558 Ab, anti-ERM Ab, anti-pp2b Ab, or anti-MLC Ab at 4°C overnight, TM71 for 2 h at room temperature, or anti-mMBS Ab for 1 h at room temperature. The cells were washed with PBS three times, then incubated with fluorescein isothiocyanate (FITC)-conjugated anti–rabbit or –mouse Ig Ab, Texas red–conjugated anti–rabbit, –mouse, or –rat Ig Ab for 1 h at room temperature or tetramethylrhodamine B isothiocyanate (TRITC)-phalloidin for 30 min at room temperature. DNAs were stained with 1 μg/ml of bisbenzimide Hoechst for 3 min at room temperature. Fluorescently labeled cells were examined with a Zeiss LSM510 (Carl Zeiss). In some experiments, fluorescent images (Fig. 4 and Fig. 5) were taken with PXL cooled CCD camera (Photometrics) with DeltaVision processing software (Applied Precision Inc.). Exposure time was adjusted to obtain FITC and Texas red or rhodamine images with roughly equal intensities in nonstimulated MDCK cells (see Fig. 4 A, a and b, and B, a and b, respectively; checked by histogram analysis). Under the same condition, the images of TPA- or HGF-stimulated MDCK cells were taken. The images of Fig. 4 A and 5 A, or Fig. 4 B and 5 B were treated with same establishment of contrast and brightness, respectively. Grayscale FITC and Texas red images were converted into green and red images, respectively, and then merged to synthesize RGB color images. A ratio image was created using an Image-Pro image processing system (Media Cybernetics). A grayscale image of anti-pS854 Ab was divided by a corresponding grayscale image of anti-mMBS Ab, and the resultant image was multiplied by 50.


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 Ser-854–phosphorylated MBS in the TPA- or HGF-stimulated MDCK cells. (A) Distribution of Ser-854–phosphorylated MBS. Serum-deprived MDCK cells were stimulated with 200 nM TPA (c, d, and h) or 50 pM HGF (e and f) for 15 min. MDCK cells were stained with anti-pS854 Ab (a, c, and e) or TRITC-phalloidin (b, d, and f). MDCK cells were also stained with anti-pS854 Ab absorbed with a 100-fold amount of antigen phosphopeptide (g and h). Arrowheads indicate the induced membrane ruffling. (B) Distribution of phosphorylated and total MBS. Serum-deprived MDCK cells were stimulated with 200 nM TPA (e–h) or 50 pM HGF (i–l) for 15 min. MDCK cells were doubly stained with anti-pS854 Ab (a, e, and i) and anti-mMBS Ab (b, f, and j). The merged (c, g, and k) and ratio (phosphorylated MBS/MBS; d, h, and l) images are shown. (C) Subcellular distribution of phosphorylated MBS. Non- (−) and TPA- (+) stimulated MDCK cells were separated into nuclear (nucleus), cytoplasmic (cytoplasm), and membrane (membrane) fractions, and the fractions were immunoblotted with anti-pS854 Ab (upper panel) and anti-pnMBS Ab (lower panel). These results are representative of three independent experiments. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 4: Distribution of Ser-854–phosphorylated MBS in the TPA- or HGF-stimulated MDCK cells. (A) Distribution of Ser-854–phosphorylated MBS. Serum-deprived MDCK cells were stimulated with 200 nM TPA (c, d, and h) or 50 pM HGF (e and f) for 15 min. MDCK cells were stained with anti-pS854 Ab (a, c, and e) or TRITC-phalloidin (b, d, and f). MDCK cells were also stained with anti-pS854 Ab absorbed with a 100-fold amount of antigen phosphopeptide (g and h). Arrowheads indicate the induced membrane ruffling. (B) Distribution of phosphorylated and total MBS. Serum-deprived MDCK cells were stimulated with 200 nM TPA (e–h) or 50 pM HGF (i–l) for 15 min. MDCK cells were doubly stained with anti-pS854 Ab (a, e, and i) and anti-mMBS Ab (b, f, and j). The merged (c, g, and k) and ratio (phosphorylated MBS/MBS; d, h, and l) images are shown. (C) Subcellular distribution of phosphorylated MBS. Non- (−) and TPA- (+) stimulated MDCK cells were separated into nuclear (nucleus), cytoplasmic (cytoplasm), and membrane (membrane) fractions, and the fractions were immunoblotted with anti-pS854 Ab (upper panel) and anti-pnMBS Ab (lower panel). These results are representative of three independent experiments. Bars, 10 μm.
Mentions: MDCK cells were fixed with 3.0% formaldehyde in PBS for 20 min and treated with PBS containing 0.2% Triton X-100 for 10 min on ice or acetone for 10 min at −20°C (for anti-pp2b Ab). REF52 cells were fixed with 3.0% formaldehyde in PBS for 10 min and treated with PBS containing 0.2% Triton X-100 for 10 min at room temperature. After being washed with PBS three times, the cells were incubated with anti-pS854 Ab, anti-pnMBS Ab, anti-pT558 Ab, anti-ERM Ab, anti-pp2b Ab, or anti-MLC Ab at 4°C overnight, TM71 for 2 h at room temperature, or anti-mMBS Ab for 1 h at room temperature. The cells were washed with PBS three times, then incubated with fluorescein isothiocyanate (FITC)-conjugated anti–rabbit or –mouse Ig Ab, Texas red–conjugated anti–rabbit, –mouse, or –rat Ig Ab for 1 h at room temperature or tetramethylrhodamine B isothiocyanate (TRITC)-phalloidin for 30 min at room temperature. DNAs were stained with 1 μg/ml of bisbenzimide Hoechst for 3 min at room temperature. Fluorescently labeled cells were examined with a Zeiss LSM510 (Carl Zeiss). In some experiments, fluorescent images (Fig. 4 and Fig. 5) were taken with PXL cooled CCD camera (Photometrics) with DeltaVision processing software (Applied Precision Inc.). Exposure time was adjusted to obtain FITC and Texas red or rhodamine images with roughly equal intensities in nonstimulated MDCK cells (see Fig. 4 A, a and b, and B, a and b, respectively; checked by histogram analysis). Under the same condition, the images of TPA- or HGF-stimulated MDCK cells were taken. The images of Fig. 4 A and 5 A, or Fig. 4 B and 5 B were treated with same establishment of contrast and brightness, respectively. Grayscale FITC and Texas red images were converted into green and red images, respectively, and then merged to synthesize RGB color images. A ratio image was created using an Image-Pro image processing system (Media Cybernetics). A grayscale image of anti-pS854 Ab was divided by a corresponding grayscale image of anti-mMBS Ab, and the resultant image was multiplied by 50.

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