scholarly journals Inhibiting Myosin Light Chain Kinase Induces Apoptosis In Vitro and In Vivo

2005 ◽  
Vol 25 (14) ◽  
pp. 6259-6266 ◽  
Author(s):  
Fabeha Fazal ◽  
Lianzhi Gu ◽  
Ivanna Ihnatovych ◽  
YooJeong Han ◽  
WenYang Hu ◽  
...  
Keyword(s):  
Cell Death ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Apoptotic Cells ◽  
Caspase Activation ◽  
Inhibitory Antibody ◽  
Necrosis Factor Alpha

ABSTRACT Previous short-term studies have correlated an increase in the phosphorylation of the 20-kDa light chain of myosin II (MLC20) with blebbing in apoptotic cells. We have found that this increase in MLC20 phosphorylation is rapidly followed by MLC20 dephosphorylation when cells are stimulated with various apoptotic agents. MLC20 dephosphorylation is not a consequence of apoptosis because MLC20 dephosphorylation precedes caspase activation when cells are stimulated with a proapoptotic agent or when myosin light chain kinase (MLCK) is inhibited pharmacologically or by microinjecting an inhibitory antibody to MLCK. Moreover, blocking caspase activation increased cell survival when MLCK is inhibited or when cells are treated with tumor necrosis factor alpha. Depolymerizing actin filaments or detaching cells, processes that destabilize the cytoskeleton, or inhibiting myosin ATPase activity also resulted in MLC20 dephosphorylation and cell death. In vivo experiments showed that inhibiting MLCK increased the number of apoptotic cells and retarded the growth of mammary cancer cells in mice. Thus, MLC20 dephosphorylation occurs during physiological cell death and prolonged MLC20 dephosphorylation can trigger apoptosis.

The Regulation of Myosin Light Chain Kinase by Ca++ and Calmodulin in Vitro and in Vivo

1982 ◽  
pp. 219-238 ◽  
Author(s):  
J. T. Stull ◽  
D. K. Blumenthal ◽  
B. R. Botterman ◽  
G. A. Klug ◽  
D. R. Manning ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain

scholarly journals CHD1L prevents lipopolysaccharide-induced hepatocellular carcinomar cell death by activating hnRNP A2/B1-nmMYLK axis

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Guangliang Wang ◽  
Xiaofeng Zhang ◽  
Wei Cheng ◽  
Yanxuan Mo ◽  
Juan Chen ◽  
...  
Keyword(s):  
Cell Death ◽  
Tumor Cell ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Driver Gene ◽  
Caspase 8 ◽  
Mechanism Analysis ◽  
Tumor Cell Death

AbstractChromodomain helicase/ATPase DNA-binding protein 1-like gene (CHD1L) has been characterized to be a driver gene in hepatocellular carcinoma (HCC). However, the intrinsic connections between CHD1L and intestinal dysbacteriosis-related inflammation reaction in HCC progression remain incompletely understood. In this study, a specific correlation between CHD1L and nonmuscle isoform of myosin light chain kinase (nmMLCK/nmMYLK), a newly identified molecule associated NF-κB signaling transduction, was disclosed in HCC. CHD1L promotes nmMYLK expression and prevents lipopolysaccharide (LPS) induced tumor cell death. In vitro experiment demonstrated that overexpressed nmMYLK is essential for CHD1L to maintain HCC cell alive, while knocking down nmMYLK significantly attenuate the oncogenic roles of CHD1L. Mechanism analysis revealed that nmMYLK can prevent Caspase-8 from combining with MyD88, an important linker of TLRs signaling pathway, while, knocking down nmMYLK facilitate the MyD88 combines with Caspase-8 and lead to the proteolytic cascade of Caspase as well as the consequent cell apoptosis. Mechanism analysis showed that CHD1L promotes the nmMYLK expression potentially through upregulating the heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) expression, which can bind to myosin light chain kinase (MYLK) pre-mRNA and lead to the regnant translation of nmMYLK. In summary, this work characterizes a previously unknown role of CHD1L in preventing LPS-induced tumor cell death through activating hnRNP A2/B1-nmMYLK axis. Further inhibition of CHD1L and its downstream signaling could be a novel promising strategy in HCC treatment.

scholarly journals Viable “Haemophilus somnus” Induces Myosin Light-Chain Kinase-Dependent Decrease in Brain Endothelial Cell Monolayer Resistance

2007 ◽  
Vol 75 (9) ◽  
pp. 4572-4581 ◽  
Author(s):  
E. Behling-Kelly ◽  
David McClenahan ◽  
K. S. Kim ◽  
C. J. Czuprynski
Keyword(s):  
Endothelial Cell ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Cell Monolayer ◽  
Brain Endothelial Cell ◽  
Endothelial Cell Monolayer ◽  
Necrosis Factor Alpha ◽  
Haemophilus Somnus

ABSTRACT “Haemophilus somnus” causes thrombotic meningoencephalitis in cattle. Our laboratory has previously reported that H. somnus has the ability to adhere to, but not invade, bovine brain endothelial cells (BBEC) in vitro. The goal of this study was to determine if H. somnus alters brain endothelial cell monolayer integrity in vitro, in a manner that would be expected to contribute to inflammation of the central nervous system (CNS). Monolayer integrity was monitored by measuring transendothelial electrical resistance (TEER) and albumin flux. BBEC incubated with H. somnus underwent rapid cytoskeletal rearrangement, significant increases in albumin flux, and reductions in TEER. Decreased monolayer TEER was preceded by phosphorylation of the myosin regulatory light chain and was partially dependent on tumor necrosis factor alpha and myosin light-chain kinase but not interleukin-1β. Neither heat-killed H. somnus, formalin-fixed H. somnus, nor purified lipooligosaccharide altered monolayer integrity within a 2-h incubation period, whereas conditioned medium from H. somnus-treated BBEC caused a modest reduction in TEER. The data from this study support the hypothesis that viable H. somnus alters integrity of the blood-brain barrier by promoting contraction of BBEC and increasing paracellular permeability of the CNS vasculature.

Phosphorylation of caldesmon by myosin light chain kinase increases its binding affinity for phosphorylated myosin filaments

2010 ◽  
Vol 391 (9) ◽  
Author(s):  
Apolinary Sobieszek ◽  
Bettina Sarg ◽  
Herbert Lindner ◽  
Chun Y. Seow
Keyword(s):  
Binding Affinity ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Muscle Activation ◽  
Smooth Muscle Contraction ◽  
Actomyosin Interaction ◽  
Myosin Filaments

Abstract Phosphorylation of myosin by myosin light chain kinase (MLCK) is essential for smooth muscle contraction. In this study we show that caldesmon (CaD) is also phosphorylated in vitro by MLCK. The phosphorylation is calcium- and calmodulin (CaM)-dependent and requires a MLCK concentration close to that found in vivo. On average, approximately 2 mol P i per mol of CaD are incorporated at Thr-626 and Thr-693, with additional partial phosphorylation at Ser-658 and Ser-702. The phosphorylation rate for CaD is 20- to 50-fold slower than that for filamentous myosin; faster relative rates were obtained with CaD added to purified actomyosin or myosin preparations containing endogenous MLCK/CaM complex. Addition of CaM also augmented CaD phosphorylation. We further demonstrate that [32P] labeled CaD binds much more readily to phosphorylated filamentous myosin than to unphosphorylated myosin. For actomyosin, CaD binding affinity doubles after myosin phosphorylation, without a significant change in binding stoichiometry (approx. one CaD per myosin molecule). Unphosphorylated CaD is ineffective in competing with the phosphorylated protein for the binding site(s) on myosin filaments. The ATPase activity of reconstituted actomyosin is inhibited by unphosphorylated CaD, and this inhibition was removed by CaD phosphorylation. Our results suggest that CaD phosphorylation plays a role in modifying actomyosin interaction in vivo, particularly during prolonged muscle activation.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Stroke ◽  
2021 ◽  
Vol 52 (Suppl_1) ◽  
Author(s):  
Francisco J Gonzalez-Gonzalez ◽  
Perike Srikanth ◽  
Andrielle E Capote ◽  
Alsina Katherina M ◽  
Benjamin Levin ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Molecular Mechanisms ◽  
Contractile Function ◽  
Right Atrial ◽  
Western Blots

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

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