Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse

Isotani, Eiji; Zhi, Gang; Lau, Kim S.; Huang, Jian; Mizuno, Yusuke; Persechini, Anthony; Geguchadze, Ramaz; Kamm, Kristine E.; Stull, James T.

doi:10.1073/pnas.0308742101

Cited by 119 publications

(164 citation statements)

References 30 publications

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“…It is also possible that the small amount of RLC phosphorylated in MLCK SMKO tissues in response to ACh is due to trace amounts of residual MLCK because only a small fraction (<20%) of the kinase is normally activated during a maximal contraction response. 16 The physiological importance of MLCK is strongly supported by the robust gut dysmotility phenotype in MLCK SMKO mice. The depletion of MLCK in other types of smooth muscle (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…16 The phosphorylated RLC bands were verified by anti-phospho-myosin light chain [pSer 19 ] antibody (data not shown, Sigma). The percent phosphorylated RLC was determined by quantification with Jieda 801 Image Analysis system 3.3.2: Jiangsu JEDA Science-Technology Development Co., Ltd, Nanjing, China.…”

Section: Measurement Of Myosin Regulatory Light Chain Phosphorylationmentioning

confidence: 98%

“…16 Briefly, tissue samples were collected and frozen quickly in 10% trichloroacetic acid and 10 mM dithiothreitol in acetone precooled to a slush at −80°C. After homogenizing thoroughly, the sample pellet was washed three times with ether for 5 minutes each and dried to remove residual ether.…”

Section: Western Blot Analysismentioning

confidence: 99%

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Myosin Light Chain Kinase Is Central to Smooth Muscle Contraction and Required for Gastrointestinal Motility in Mice

et al. 2008

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Background & Aims-Smooth muscle is essential for maintaining homeostasis for many body functions and provides adaptive responses to stresses imposed by pathological disorders. Identified cell signaling networks have defined many potential mechanisms for initiating smooth muscle contraction with or without myosin regulatory light chain (RLC) phosphorylation by myosin light chain kinase (MLCK). We generate tamoxifen-inducible and smooth muscle-specific MLCK knockout (KO) mice and provide direct loss-of-function evidence that shows the primary importance of MLCK in phasic smooth muscle contractions.

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Section: Discussionmentioning

confidence: 99%

Section: Measurement Of Myosin Regulatory Light Chain Phosphorylationmentioning

confidence: 98%

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Myosin Light Chain Kinase Is Central to Smooth Muscle Contraction and Required for Gastrointestinal Motility in Mice

et al. 2008

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“…For analysis of skeletal and smooth muscle MLCK expression, tissues were collected and homogenized in SDS sample buffer (18). Lysate protein concentration was measured by bicinchoninic acid (BCA) protein assay reagent, and 40 g of protein was loaded per well for 7.5% SDS͞PAGE, followed by transfer to nitrocellulose membranes.…”

Section: Methodsmentioning

confidence: 99%

Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction

Zhi

Ryder

Huang

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Repetitive stimulation potentiates contractile tension of fasttwitch skeletal muscle. We examined the role of myosin regulatory light chain (RLC) phosphorylation in this physiological response by ablating Ca 2؉ ͞calmodulin-dependent skeletal muscle myosin light chain kinase (MLCK) gene expression. Western blot and quantitative-PCR showed that MLCK is expressed predominantly in fasttwitch skeletal muscle fibers with insignificant amounts in heart and smooth muscle. In contrast, smooth muscle MLCK had a more ubiquitous tissue distribution, with the greatest expression observed in smooth muscle tissue. Ablation of the MYLK2 gene in mice resulted in loss of skeletal muscle MLCK expression, with no change in smooth muscle MLCK expression. In isolated fast-twitch skeletal muscles from these knockout mice, there was no significant increase in RLC phosphorylation in response to repetitive electrical stimulation. Furthermore, isometric twitch-tension potentiation after a brief tetanus (posttetanic twitch potentiation) or low-frequency twitch potentiation (staircase) was attenuated relative to responses in muscles from wild-type mice. Interestingly, the site of phosphorylation of the small amount of monophosphorylated RLC in the knockout mice was the same site phosphorylated by MLCK, indicating a potential alternative signaling pathway affecting contractile potentiation. Loss of skeletal muscle MLCK expression had no effect on cardiac RLC phosphorylation. These results identify myosin light chain phosphorylation by the dedicated skeletal muscle Ca 2؉ ͞calmodulin-dependent MLCK as a primary biochemical mechanism for tension potentiation due to repetitive stimulation in fast-twitch skeletal muscle.calcium ͉ calmodulin ͉ twitch S keletal muscle contraction depends on a voltage-driven conformational change in the L-type Ca 2ϩ channel in the transverse tubule that triggers Ca 2ϩ release from the sarcoplasmic reticulum through the intracellular ryanodine receptor (1, 2). The Ca 2ϩ binds to troponin in thin filaments, thereby allowing myosin cross bridges to bind actin and generate muscle tension (3). However, muscle contractions involve more complex mechanisms that affect performance. Since Ranke noted in 1865 (4) that, with stimuli uniform in strength the later twitch contractions were stronger than the first, there has been considerable interest in identifying the mechanisms involved in isometric twitch potentiation during trains of stimuli at low frequency (staircase) or after a tetanus (posttetanic potentiation). Considerations have been given to changes in compliance of the series elastic elements, to activation of more fibers within a muscle, to increased Ca 2ϩ release within a single fiber to activate fully the contractile proteins, and to changes in excitation-contraction coupling processes (5-8).Ca 2ϩ released during muscle contraction can also activate the dedicated protein kinase Ca 2ϩ ͞calmodulin-dependent skeletal muscle myosin light chain kinase (skMLCK) to initiate myosin regulatory light chain (RLC) phosphorylat...

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“…The free Ca 2ϩ /calmodulin concentration is typically Ͻ1% of the total cellular calmodulin (16 -19). The free Ca 2ϩ /calmodulin in smooth, but not skeletal, muscle is limiting for full activation of a high affinity target such as MLCK (11,20 …”

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Myosin Regulatory Light Chain Phosphorylation Attenuates Cardiac Hypertrophy

Huang

Shelton

Richardson

et al. 2008

Journal of Biological Chemistry

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Myosin, the mechanochemical transducer of cardiomyocytes, is modulated by phosphorylation. Its motor domain contains an actin-binding surface and ATP catalytic pocket that tapers to an ␣-helical neck connected to the rod region responsible for self-assembly into thick filaments. Two small polypeptides, the essential light chain and the RLC, 2 wrap around each ␣-helical neck region.Phosphorylation of RLC in striated muscles potentiates the force and speed of contractions that are dependent on Ca 2ϩ binding to troponin on actin-containing thin filaments (1-3). Both skeletal and cardiac muscle RLC phosphorylation in sarcomeres cause a leftward shift in the myofilament force-pCa relationship, showing an increase in Ca 2ϩ sensitivity and an increase in the rate of weakly attached cross-bridges entering the contractile cycle (1, 4 -8). In heart muscle, a spatial gradient of cRLC phosphorylation across the ventricle with decreasing cRLC phosphorylation from apex to base may facilitate torsion during contraction (7, 9). Transgenic mice expressing a nonphosphorylatable, ventricular cRLC show suppressed hemodynamic performance (10), consistent with demonstrated effects of RLC phosphorylation in skeletal muscle (2, 11).Cardiomyocytes also contain a cytoplasmic, nonmuscle myosin IIB with its RLC (nRLC) where phosphorylation increases actin-activated ATPase activity. Myosin IIB is diffusely distributed in the cytoplasm during development and then localizes to Z-lines and intercalated discs after birth (12, 13). Ablation of cytoplasmic myosin IIB expression results in sarcomere disarray and aberrant development of the heart, leading to heart failure and death (14). RLCs are phosphorylated by dedicated Ca 2ϩ /calmodulin-dependent MLCKs (15). skMLCK was reported to be in cardiac muscle where a mutation in the kinase from a young patient with hypertrophic myocardium was associated with an increased V max value. Thus, it was suggested that development of compensatory hypertrophy results from an increase in cRLC phosphorylation (7,9). In the current study, we tested this hypothesis by generating transgenic mice overexpressing skMLCK in cardiomyocytes. skMLCK is a dedicated protein kinase that phosphorylates RLCs from striated as well as smooth muscle and nonmuscle cells (15). We also examined adaptations to exercise conditioning and ␤-adrenergic stimulation in these transgenic animals, two stresses that induce cardiac hypertrophy.The availability of free Ca 2ϩ /calmodulin for activation of its cellular targets may be limiting in cells. Total calmodulin ranges from 5 to 40 M in different kinds of cells where it affects protein structures and enzyme activities as well as gene expression (16). The free Ca 2ϩ /calmodulin concentration is typically Ͻ1% of the total cellular calmodulin (16 -19). The free Ca 2ϩ /calmodulin in smooth, but not skeletal, muscle is limiting for full activation of a high affinity target such as MLCK (11,20

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Real-time evaluation of myosin light chain kinase activation in smooth muscle tissues from a transgenic calmodulin-biosensor mouse

Cited by 119 publications

References 30 publications

Myosin Light Chain Kinase Is Central to Smooth Muscle Contraction and Required for Gastrointestinal Motility in Mice

Myosin Light Chain Kinase Is Central to Smooth Muscle Contraction and Required for Gastrointestinal Motility in Mice

Myosin light chain kinase and myosin phosphorylation effect frequency-dependent potentiation of skeletal muscle contraction

Myosin Regulatory Light Chain Phosphorylation Attenuates Cardiac Hypertrophy

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