scholarly journals Cytotoxic Necrotizing Factor 1 of Escherichia coli Stimulates Rho/Rho-Kinase-Dependent Myosin Light-Chain Phosphorylation without Inactivating Myosin Light-Chain Phosphatase in Endothelial Cells

2003 ◽  
Vol 71 (9) ◽  
pp. 5188-5193 ◽  
Author(s):  
Markus Essler ◽  
Stefan Linder ◽  
Barbara Schell ◽  
Katharina Hüfner ◽  
Agnès Wiedemann ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Endothelial Cells ◽  
Phosphatase Activity ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Cytotoxic Necrotizing Factor ◽  
Cytotoxic Necrotizing Factor 1 ◽  
Mlc Phosphorylation ◽  
Stimulation Of

ABSTRACT Cytotoxic necrotizing factor 1 (CNF-1) is an exotoxin of Escherichia coli that constitutively activates the GTPases Rho, Rac, and CDC42. Stimulation of Rho was shown to enhance myosin light-chain (MLC) phosphorylation via Rho kinase-mediated inhibition of MLC phosphatase in endothelial cells. Here we report that 3 h after CNF stimulation of endothelial cells, RhoA was activated and MLC phosphorylation was increased in a Rho/Rho-kinase-dependent manner, but no decrease in MLC phosphatase activity could be detected. Despite continuous RhoA activation, MLC phosphatase activity was doubled after 24 h of CNF stimulation, and this coincided with decreased MLC phosphorylation and cell spreading. Rac was also activated at 3 to 24 h but did not contribute to MLC phosphorylation, and its amount gradually decreased in the CNF-stimulated cells. CDC42Hs was not activated above control values by CNF. These results suggest that CNF can induce specific decoupling (Rho kinase from MLC phosphatase) and deactivation events in Rho GTPase signaling, potentially reflecting cellular protection mechanisms against permanently active Rho GTPases.

scholarly journals Modulation of Myosin Light-Chain Phosphorylation by p21-Activated Kinase 1 in Escherichia coli Invasion of Human Brain Microvascular Endothelial Cells

2003 ◽  
Vol 71 (5) ◽  
pp. 2787-2797 ◽  
Author(s):  
Rajyalakshmi S. Rudrabhatla ◽  
Sunil K. Sukumaran ◽  
Gary M. Bokoch ◽  
Nemani V. Prasadarao
Keyword(s):  
Escherichia Coli ◽  
Endothelial Cells ◽  
Human Brain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Microvascular Endothelial Cells ◽  
Brain Microvascular Endothelial Cells ◽  
E Coli ◽  
P21 Activated Kinase ◽  
Mlc Phosphorylation

ABSTRACT Cytoskeletal dynamics, modulated by actin-myosin interactions, play an important role in Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC). Herein, we show that inhibitors of myosin function, butanedione monoxide and ML-7, significantly blocked the E. coli invasion of HBMEC. The invasive E. coli induces myosin light-chain (MLC) phosphorylation during the invasion process, which gets recruited to the site of actin condensation beneath the bacteria. We also show that invading E. coli downregulates the activity of p21-activated kinase 1 (PAK1), which is an upstream regulator of MLC kinase (MLCK). Overexpression of wild-type PAK1 and constitutively active PAK1 in HBMEC inhibits E. coli invasion significantly with a concomitant decrease in MLC phosphorylation. The inhibition of E. coli invasion by these PAK1 mutants is due to the absence of phospho-MLC at the actin condensation points. In contrast, the dominant-negative PAK1 shows no effect either on the invasion or on MLC phosphorylation or phospho-MLC recruitment to the actin focal points, suggesting that activated PAK1 inactivates MLCK. Taken together, these results suggest that E. coli invasion of HBMEC induces MLC phosphorylation by inhibiting the activity of PAK1 and the recruitment of phosphorylated MLC to the site of actin condensation beneath the bacteria for efficient internalization of E. coli into HBMEC.

scholarly journals Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl− cotransporter?

2005 ◽  
Vol 289 (1) ◽  
pp. C68-C81 ◽  
Author(s):  
Caterina Di Ciano-Oliveira ◽  
Monika Lodyga ◽  
Lingzhi Fan ◽  
Katalin Szászi ◽  
Hiroshi Hosoya ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Dominant Negative ◽  
Myosin Atpase ◽  
Osmotic Stimulation ◽  
Hypertonic Stimulation ◽  
Mlc Phosphorylation ◽  
The Impact ◽  
Permissive Role ◽  
Stimulation Of

Myosin light-chain (MLC) kinase (MLCK)-dependent increase in MLC phosphorylation has been proposed to be a key mediator of the hyperosmotic activation of the Na+-K+-2Cl− cotransporter (NKCC). To address this hypothesis and to assess whether MLC phosphorylation plays a signaling or permissive role in NKCC regulation, we used pharmacological and genetic means to manipulate MLCK, MLC phosphorylation, or myosin ATPase activity and followed the impact of these alterations on the hypertonic stimulation of NKCC in porcine kidney tubular LLC-PK1 epithelial cells. We found that the MLCK inhibitor ML-7 suppressed NKCC activity independently of MLC phosphorylation. Notably, ML-7 reduced both basal and hypertonically stimulated NKCC activity without influencing MLC phosphorylation under these conditions, and it inhibited NKCC activation by Cl− depletion, a treatment that did not increase MLC phosphorylation. Furthermore, prevention of the osmotically induced increase in MLC phosphorylation by viral induction of cells with a nonphosphorylatable, dominant negative MLC mutant (AA-MLC) did not affect the hypertonic activation of NKCC. Conversely, a constitutively active MLC mutant (DD-MLC) that mimics the diphosphorylated form neither stimulated isotonic nor potentiated hypertonic NKCC activity. Furthermore, a depolarization-induced increase in endogenous MLC phosphorylation failed to activate NKCC. However, complete abolition of basal MLC phosphorylation by K252a or the inhibition of myosin ATPase by blebbistatin significantly reduced the osmotic stimulation of NKCC without suppressing its basal or Cl− depletion-triggered activity. These results indicate that an increase in MLC phosphorylation is neither a sufficient nor a necessary signal to stimulate NKCC in tubular cells. However, basal myosin activity plays a permissive role in the optimal osmotic responsiveness of NKCC.

Contraction of myofibroblasts in granulation tissue is dependent on Rho/Rho kinase/myosin light chain phosphatase activity

2006 ◽  
Vol 14 (3) ◽  
pp. 313-320 ◽  
Author(s):  
James J. Tomasek ◽  
Melville B. Vaughan ◽  
Bradley P. Kropp ◽  
Giulio Gabbiani ◽  
Michael D. Martin ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Light Chain ◽  
Granulation Tissue ◽  
Myosin Light Chain ◽  
Rho Kinase ◽  
Myosin Light Chain Phosphatase

scholarly journals Androgens Induce Nongenomic Stimulation of Colonic Contractile Activity through Induction of Calcium Sensitization and Phosphorylation of LC20 and CPI-17

2010 ◽  
Vol 24 (5) ◽  
pp. 1007-1023 ◽  
Author(s):  
María C. González-Montelongo ◽  
Raquel Marín ◽  
Tomás Gómez ◽  
Jorge Marrero-Alonso ◽  
Mario Díaz
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Contractile Activity ◽  
Rho Kinase ◽  
Regulatory Subunit ◽  
Calcium Sensitization ◽  
Regulatory Myosin Light Chain ◽  
Rapid Activation ◽  
Stimulation Of

Abstract We show that androgens, testosterone and 5α-dihydrotestosterone (DHT), acutely (∼40 min) provoke the mechanical potentiation of spontaneous and agonist-induced contractile activity in mouse colonic longitudinal smooth muscle. The results using flutamide, finasteride, cycloheximide, and actinomycin D indicate that androgen-induced potentiation is dependent on androgen receptors, requires reduction of testosterone to DHT, and occurs independently of transcriptional and translational events. Using permeabilized colonic smooth muscle preparations, we could demonstrate that mechanical potentiation is entirely due to calcium sensitization of contractile machinery. In addition, DHT (10 nm) increased phosphorylation of both 20-kDa myosin light chain (LC20) [regulatory myosin light chain, (MLC)] and CPI-17 (an endogenous inhibitor of MLC phosphatase). Paralleling these findings, inhibition of Rho-associated Rho kinase (ROK) and/or protein kinase C (PKC) with, respectively, Y27632 and chelerythrine, prevented LC20 phosphorylation and abolished calcium sensitization. In addition, inhibition of ROK prevents CPI-17 phosphorylation, indicating that ROK is located upstream PKC-mediated CPI-17 modulation in the signalling cascade. Additionally, androgens induce a rapid activation of RhoA and its translocation to the plasma membrane to activate ROK. The results demonstrate that androgens induce sensitization of colonic smooth muscle to calcium through activation of ROK, which in turn, activates PKC to induce CPI-17 phosphorylation. Activation of this pathway induces a potent steady stimulation of LC20 by inhibiting MLC phosphatase and displacing the equilibrium of the regulatory subunit towards its phosphorylated state. This is the first demonstration that colonic smooth muscle is a physiological target for androgen hormones, and that androgens modulate force generation of smooth muscle contractile machinery through nongenomic calcium sensitization pathways.

scholarly journals Activation of G12/G13 Results in Shape Change and Rho/Rho-Kinase–mediated Myosin Light Chain Phosphorylation in Mouse Platelets

1999 ◽  
Vol 144 (4) ◽  
pp. 745-754 ◽  
Author(s):  
Birgit Klages ◽  
Ursula Brandt ◽  
Melvin I. Simon ◽  
Günter Schultz ◽  
Stefan Offermanns
Keyword(s):  
Light Chain ◽  
Tyrosine Kinases ◽  
Myosin Light Chain ◽  
Kinase Inhibitor ◽  
Shape Change ◽  
Rho Kinase ◽  
Α Subunit ◽  
Camp Analogue ◽  
Txa2 Receptor ◽  
Mlc Phosphorylation

Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents. Platelets lacking the α-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors. In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Gαq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change. TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72syk and stimulation of pp60c-src as well as in phosphorylation of myosin light chain (MLC) in Gαq-deficient platelets. Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Gαq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS. These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase–mediated regulation of MLC phosphorylation. We provide evidence that G12/G13-mediated Rho/Rho-kinase–dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change.

cAMP signaling regulates platelet myosin light chain (MLC) phosphorylation and shape change through targeting the RhoA-Rho kinase-MLC phosphatase signaling pathway

Blood ◽  
2013 ◽  
Vol 122 (20) ◽  
pp. 3533-3545 ◽  
Author(s):  
Ahmed Aburima ◽  
Katie S. Wraith ◽  
Zaher Raslan ◽  
Robert Law ◽  
Simbarashe Magwenzi ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Shape Change ◽  
Rho Kinase ◽  
Camp Signaling ◽  
Key Points ◽  
Rhoa Kinase ◽  
Mlc Phosphorylation ◽  
Kinase A ◽  
Platelet Shape

Key Points Protein kinase A (PKA) phosphorylates RhoA on serine188 to inhibit RhoA membrane translocation and RhoA kinase (ROCK) signaling. Inhibition of RhoA/ROCK2 promotes myosin light chain (MLC) phosphatase activity, which prevents the phosphorylation of MLC and platelet shape change.

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