scholarly journals Association of the Myosin-binding Subunit of Myosin Phosphatase and Moesin: Dual Regulation of Moesin Phosphorylation by Rho-associated Kinase and Myosin Phosphatase

1998 ◽  
Vol 141 (2) ◽  
pp. 409-418 ◽  
Author(s):  
Yuko Fukata ◽  
Kazushi Kimura ◽  
Noriko Oshiro ◽  
Hideyuki Saya ◽  
Yoshiharu Matsuura ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Phosphatase Activity ◽  
Mdck Cells ◽  
Rho Kinase ◽  
Small Gtpase ◽  
Myosin Phosphatase ◽  
Membrane Ruffling ◽  
Amino Terminal ◽  
Myosin Binding ◽  
Binding Subunit

The small GTPase Rho is believed to regulate the actin cytoskeleton and cell adhesion through its specific targets. We previously identified the Rho targets: protein kinase N, Rho-associated kinase (Rho- kinase), and the myosin-binding subunit (MBS) of myosin phosphatase. We found that in MDCK epithelial cells, MBS accumulated at the tetradecanoylphorbol-13-acetate (TPA)-induced membrane ruffling area, where moesin, a member of the ERM (ezrin, radixin, and moesin) family, was localized. Neither membrane ruffling nor an accumulation of moesin and MBS at the free-end plasma membrane was induced when MDCK cells were stimulated with TPA after the microinjection of C3, which ADP-ribosylates and inactivates Rho. MBS was colocalized with moesin at the cell–cell contact sites in MDCK cells. We also found that moesin was coimmunoprecipitated with MBS from MDCK cells. Recombinant MBS interacted with the amino-terminal domains of moesin and ezrin. Myosin phosphatase composed of the catalytic subunit and MBS showed phosphatase activity toward moesin, which was phosphorylated by Rho-kinase. The phosphatase activity was inhibited when MBS was phosphorylated by Rho-kinase. These results suggest that MBS is recruited with moesin to the plasma membrane and that myosin phosphatase and Rho-kinase regulate the phosphorylation state of moesin downstream of Rho.

scholarly journals Phosphorylation of Myosin-Binding Subunit (Mbs) of Myosin Phosphatase by Rho-Kinase in Vivo

1999 ◽  
Vol 147 (5) ◽  
pp. 1023-1038 ◽  
Author(s):  
Yoji Kawano ◽  
Yuko Fukata ◽  
Noriko Oshiro ◽  
Mutsuki Amano ◽  
Toshikazu Nakamura ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Rho Kinase ◽  
Small Gtpase ◽  
Stress Fibers ◽  
Myosin Phosphatase ◽  
Phosphorylation State ◽  
Membrane Ruffling ◽  
Myosin Binding ◽  
Binding Subunit

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.

scholarly journals Regulation of Myosin Phosphatase Through Phosphorylation of the Myosin-Binding Subunit in Platelet Activation

Blood ◽  
1997 ◽  
Vol 90 (10) ◽  
pp. 3936-3942 ◽  
Author(s):  
Keiji Nakai ◽  
Yoshinori Suzuki ◽  
Hisakazu Kihira ◽  
Hideo Wada ◽  
Masanori Fujioka ◽  
...  
Keyword(s):  
Protein Phosphatase ◽  
Rho Kinase ◽  
Human Platelets ◽  
Myosin Phosphatase ◽  
Phosphatase Treatment ◽  
Myosin Binding ◽  
Muscle Myosin ◽  
Protein Phosphatase Type ◽  
Putative Mechanism ◽  
Binding Subunit

Abstract Human platelets were found to contain myosin phosphatase consisting of a 38-kD catalytic subunit of protein phosphatase type 1δ, a 130-kD myosin-binding subunit (MBS) and a 20-kD subunit, all of which cross-reacted with antibodies against these subunits of smooth muscle myosin phosphatase. Anti-MBS antibody coimmunoprecipitated RhoA and Rho-kinase of human platelets. Platelets MBS is a substrate for Rho-kinase and phosphorylation of MBS decreases the activity of myosin phosphatase. Treatment of intact platelets with 9,11-epithio-11,12-methano-thromboxane A2 led to a dramatic increase in phosphorylation of MBS and a significant decrease in the activity of myosin phosphatase. These findings suggest a putative mechanism for agonist-induced regulation of myosin phosphatase activity in platelets.

scholarly journals Phosphorylation of the Myosin-binding Subunit of Myosin Phosphatase by Raf-1 and Inhibition of Phosphatase Activity

2001 ◽  
Vol 277 (4) ◽  
pp. 3053-3059 ◽  
Author(s):  
Constantinos G. Broustas ◽  
Nicholas Grammatikakis ◽  
Masumi Eto ◽  
Paul Dent ◽  
David L. Brautigan ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Myosin Phosphatase ◽  
Myosin Binding ◽  
Binding Subunit

Myosin Binding Subunit of Smooth Muscle Myosin Phosphatase at the Cell-Cell Adhesion Sites in MDCK Cells

1997 ◽  
Vol 230 (3) ◽  
pp. 552-556 ◽  
Author(s):  
Naoyuki Inagaki ◽  
Miwako Nishizawa ◽  
Masaaki Ito ◽  
Masaki Fujioka ◽  
Takeshi Nakano ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Adhesion ◽  
Mdck Cells ◽  
Smooth Muscle Myosin ◽  
Myosin Phosphatase ◽  
Adhesion Sites ◽  
Myosin Binding ◽  
Muscle Myosin ◽  
Binding Subunit ◽  
Cell Cell

Agonist-Induced Regulation of Myosin Phosphatase Activity in Human Platelets Through Activation of Rho-Kinase

Blood ◽  
1999 ◽  
Vol 93 (10) ◽  
pp. 3408-3417 ◽  
Author(s):  
Yoshinori Suzuki ◽  
Masatoshi Yamamoto ◽  
Hideo Wada ◽  
Masaaki Ito ◽  
Takeshi Nakano ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Kinase Inhibitors ◽  
Rho Kinase ◽  
Human Platelets ◽  
Myosin Phosphatase ◽  
Rho Kinase Inhibitors ◽  
Atp Secretion ◽  
The Common ◽  
Myosin Binding ◽  
Mlc Phosphorylation

Human platelets contained about 15 times lower amounts of Rho-kinase than Ca2+/calmodulin-dependent myosin light chain (MLC) kinase. Anti–myosin-binding subunit (MBS) antibody coimmunoprecipitated Rho-kinase of human platelets, and addition of GTPγS-RhoA stimulated phosphorylation of the 130-kD MBS of myosin phosphatase and consequently inactivated myosin phosphatase. Two kinds of selective Rho-kinase inhibitors, HA1077 and Y-27632, reduced both GTPγS-RhoA–dependent MBS phosphorylation and inactivation of the phosphatase activity. Activation of human platelets with thrombin, a stable thromboxane A2 analog STA2, epinephrine, and serotonin resulted in an increase in MBS phosphorylation, and the agonist-induced MBS phosphorylation was prevented by pretreatment with the respective receptor antagonist. HA1077 and Y-27632 inhibited MBS phosphorylation in platelets stimulated with these agonists. These compounds also blocked agonist-induced inactivation of myosin phosphatase in intact platelets. In addition, HA1077 and Y-27632 inhibited 20-kD MLC phosphorylation at Ser19 and ATP secretion of platelets stimulated with STA2, thrombin (0.05 U/mL), and simultaneous addition of serotonin and epinephrine, whereas these compounds did not affect MLC phosphorylation or ATP secretion when platelets were stimulated with more than 0.1 U/mL thrombin. Thus, activation of Rho-kinase and the resultant phosphorylation of MBS is likely to be the common pathway for platelet activation induced by various agonists. These results also suggest that Rho-kinase–mediated MLC phosphorylation contributes to a greater extent to the platelet secretion induced by relatively weak agonists.

RhoA- and PKC-α-mediated phosphorylation of MYPT and its association with HSP27 in colonic smooth muscle cells.

2006 ◽  
Vol 290 (1) ◽  
pp. G83-G95 ◽  
Author(s):  
Suresh B. Patil ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Phosphatase Activity ◽  
Cross Talk ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Selective Inhibition ◽  
Particulate Fraction ◽  
Myosin Phosphatase ◽  
Sustained Phosphorylation

Agonist-induced activation of the RhoA/Rho kinase (ROCK) pathway results in inhibition of myosin phosphatase and maintenance of myosin light chain (MLC20) phosphorylation. We have shown that RhoA/ROCKII translocates and associates with heat shock protein (HSP)27 in the particulate fraction. We hypothesize that inhibition of the 130-kDa regulatory myosin-binding subunit (MYPT) requires its association with HSP27 in the particulate fraction. Furthermore, it is not certain whether regulation of MYPT by CPI-17 or by ROCKII is due to cross talk between RhoA and PKC-α. Presently, we examined the cross talk between RhoA and PKC-α in the regulation of MYPT phosphorylation in rabbit colon smooth muscle cells. Acetylcholine induced 1) sustained phosphorylation of PKC-α, CPI-17, and MYPT; 2) an increase in the association of phospho-MYPT with HSP27 in the particulate fraction; 3) a decrease in myosin phosphatase activity (66.21 ± 3.52 and 42.19 ± 3.85%nM/ml lysate at 30 s and 4 min); and 4) an increase in PKC activity (298.12 ± 46.60% and 290.59 ± 22.07% at 30 s and 4 min). Inhibition of RhoA/ROCKII by Y-27632 inhibited phosphorylation of MYPT and its association with HSP27. Both Y27632 and a negative dominant construct of RhoA inhibited phosphorylation of MYPT and CPI-17. Inhibition of PKCs or calphostin C or selective inhibition of PKC-α by negative dominant constructs inhibited phosphorylation of MYPT and CPI-17. The results suggest that 1) acetylcholine induces activation of both RhoA and/or PKC-α pathways, suggesting cross talk between RhoA and PKC-α resulting in phosphorylation of MYPT, inhibition of myosin phosphatase activity, and maintenance of MLC phosphorylation; and 2) phosphorylated MYPT is associated with HSP27 and translocated to the particulate fraction, suggesting a scaffolding role for HSP27 in mediating the association of the complex MYPT/RhoA-ROCKII. Thus both pathways (PKC and RhoA) converge on the regulation of myosin phosphatase activities and modulate sustained phosphorylation of MLC20.

Membrane ruffling and macropinocytosis in A431 cells require cholesterol

2002 ◽  
Vol 115 (14) ◽  
pp. 2953-2962 ◽  
Author(s):  
Stine Grimmer ◽  
Bo van Deurs ◽  
Kirsten Sandvig
Keyword(s):  
Plasma Membrane ◽  
Phorbol Ester ◽  
Small Gtpase ◽  
Cholesterol Depletion ◽  
Cell Periphery ◽  
Filamentous Actin ◽  
A431 Cells ◽  
Coated Pits ◽  
Actin Reorganization ◽  
Membrane Ruffling

Cholesterol is important for the formation of caveolea and deeply invaginated clathrin-coated pits. We have now investigated whether formation of macropinosomes is dependent on the presence of cholesterol in the plasma membrane. Macropinocytosis in A431 cells was induced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, a potent activator of protein kinase C (PKC). When cells were pretreated with methyl-β-cyclodextrin to extract cholesterol, the phorbol ester was unable to induce the increased endocytosis of ricin otherwise seen, although PKC could still be activated. Electron microscopy revealed that extraction of cholesterol inhibited the formation of membrane ruffles and macropinosomes at the plasma membrane. Furthermore, cholesterol depletion inhibited the phorbol ester-induced reorganization of filamentous actin at the cell periphery, a prerequisite for the formation of membrane ruffles that close into macropinosomes. Under normal conditions the small GTPase Rac1 is activated by the phorbol ester and subsequently localized to the plasma membrane, where it induces the reorganization of actin filaments required for formation of membrane ruffles. Cholesterol depletion did not inhibit the activation of Rac1. However,confocal microscopy showed that extraction of cholesterol prevented the phorbol ester-stimulated localization of Rac1 to the plasma membrane. Thus,our results demonstrate that cholesterol is required for the membrane localization of activated Rac1, actin reorganization, membrane ruffling and macropinocytosis.

scholarly journals Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells

2000 ◽  
Vol 278 (1) ◽  
pp. C57-C65 ◽  
Author(s):  
Hiromitsu Nagumo ◽  
Yasuharu Sasaki ◽  
Yoshitaka Ono ◽  
Hiroyuki Okamoto ◽  
Minoru Seto ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Phosphatase Activity ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Dependent Manner ◽  
Myosin Phosphatase ◽  
Rho Kinase Inhibitor

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca2+-induced 20-kDa myosin light chain (MLC20) phosphorylation due to abrogating Rho-mediated inhibition of MLC20dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5′- O-(3-thiotriphosphate) (GTPγS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPγS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC20phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.

scholarly journals Drosophila PATJ supports adherens junction stability by modulating Myosin light chain activity

2012 ◽  
Vol 199 (4) ◽  
pp. 685-698 ◽  
Author(s):  
Arnab Sen ◽  
Zsanett Nagy-Zsvér-Vadas ◽  
Michael P. Krahn
Keyword(s):  
Epithelial Cells ◽  
Myosin Light Chain ◽  
Adherens Junction ◽  
Myosin Phosphatase ◽  
Junction Protein ◽  
Discs Large ◽  
Myosin Binding ◽  
The Stability ◽  
Key Events ◽  
Binding Subunit

The assembly and consolidation of the adherens junctions (AJs) are key events in the establishment of an intact epithelium. However, AJs are further modified to obtain flexibility for cell migration and morphogenetic movements. Intact AJs in turn are a prerequisite for the establishment and maintenance of apical–basal polarity in epithelial cells. In this study, we report that the conserved PDZ (PSD95, Discs large, ZO-1) domain–containing protein PATJ (Pals1-associated tight junction protein) was not per se crucial for the maintenance of apical–basal polarity in Drosophila melanogaster epithelial cells but rather regulated Myosin localization and phosphorylation. PATJ directly bound to the Myosin-binding subunit of Myosin phosphatase and decreased Myosin dephosphorylation, resulting in activated Myosin. Thereby, PATJ supports the stability of the Zonula Adherens. Notably, weakening of AJ in a PATJ mutant epithelium led first to a loss of Myosin from the AJ, subsequently to a disassembly of the AJ, and finally, to a loss of apical–basal polarity and disruption of the tissue.

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