scholarly journals Upregulation of In Situ Myosin Light Chain Kinase Activity In Fetal Compared to Adult Ovine Carotid Arteries

2008 ◽  
Vol 22 (S1) ◽  
Author(s):  
Elisha R Injeti ◽  
Gerhart Graupner ◽  
William J Pearce
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Carotid Arteries ◽  
Kinase Activity

scholarly journals Maximal stimulation-induced in situ myosin light chain kinase activity is upregulated in fetal compared with adult ovine carotid arteries

2008 ◽  
Vol 295 (6) ◽  
pp. H2289-H2298 ◽  
Author(s):  
Elisha R. Injeti ◽  
Renan J. Sandoval ◽  
James M. Williams ◽  
Alexander V. Smolensky ◽  
Lincoln E. Ford ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Vascular Reactivity ◽  
Carotid Arteries ◽  
Thick Filament ◽  
Calcium Sensitivity ◽  
Cytosolic Calcium ◽  
Age Related

Postnatal decreases in vascular reactivity involve decreases in the thick filament component of myofilament calcium sensitivity, which is measured as the relationship between cytosolic calcium concentration and myosin light chain (MLC20) phosphorylation. The present study tests the hypothesis that downregulation of thick filament reactivity is due to downregulation of myosin light chain kinase (MLCK) activity in adult compared with fetal arteries. Total MLCK activity, calculated as %MLC20 phosphorylated per second in intact arteries during optimal inhibition of myosin light chain phosphatase activity, was significantly less in adult (6.56 ± 0.29%) than in fetal preparations (7.39 ± 0.53%). In situ MLC20 concentrations (μM) in adult (198 ± 28) and fetal arteries (236 ± 44) did not differ significantly. In situ MLCK concentrations (μM), however, were significantly greater in adult (8.21 ± 0.59) than in fetal arteries (1.83 ± 0.13). In situ MLCK activities (ng MLC20 phosphorylated·s−1·ng MLCK−1) were significantly less in adult (0.26 ± 0.01) than in fetal arteries (1.52 ± 0.11). In contrast, MLCK activities in adult (15.8 ± 1.5) and fetal artery homogenates (17.3 ± 1.3) were not significantly different. When in situ fractional activation was calculated, adult values (1.72 ± 0.17%) were significantly less than fetal values (9.08 ± 0.83%). Together, these results indicate that decreased thick filament reactivity in adult compared with fetal ovine carotid arteries is due at least in part to greater MLCK activity in fetal arteries, which in turn cannot be explained by differences in MLCK, MLC20, or calmodulin concentrations. Instead, this difference appears to involve age-related differences in fractional activation of the MLCK enzyme.

Regulation of Myosin Light Chain Kinase Activity in Smooth Muscle

1995 ◽  
pp. 139-158 ◽  
Author(s):  
Kristine E. Kamm ◽  
Katherine Luby-Phelps ◽  
Malu G. Tansey ◽  
Patricia J. Gallagher ◽  
James T. Stull
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity

Regulation of myosin light chain kinase: kinetic mechanism, autophosphorylation, and cooperative activation by Ca2+ and calmodulin

1994 ◽  
Vol 72 (11) ◽  
pp. 1368-1376 ◽  
Author(s):  
Apolinary Sobieszek
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Kinetic Mechanism ◽  
Binding Stoichiometry ◽  
Myosin Interaction ◽  
Cooperative Activation ◽  
Fold Excess

Phosphorylation of the regulatory light chain of myosin catalyzed by myosin light-chain kinase (MLCK) is the key reaction in the regulation of actin–myosin interaction in smooth muscle. It is shown that this reaction is of an ordered type, whereby kinase first binds ATP and then the light chain, and following phosphate transfer, the phosphorylated light chain is released before ADP. The MLCK also phosphorylates itself, and this intramolecular autophosphorylation is Ca2+ and calmodulin (CaM) dependent. It has, however, no pronounced effect on the kinase activity or on its affinity for Ca2+ and CaM. With the aim of understanding the cooperativity of MLCK activation, the activity of the kinase was systematically measured as a function of different ligands involved. In these measurements the isolated light chain and intact filamentous myosin, as well as native actomyosin, were used as substrates. The activation of the kinase by Ca2+ was positively cooperative but only at relatively low CaM levels. The activation by CaM (at saturating Ca2+ levels) was also cooperative, even though noncooperative activation would be expected from the established 1:1 binding stoichiometry between CaM and the kinase. This cooperativity was shown to result from time-dependent changes in the MLCK that take place during incubation with Ca2+ and CaM before addition of ATP in phosphorylation assays. As a result, activity of the kinase as a function of its concentration at constant CaM was biphasic: there was optimum activity at a ratio of 1:1 CaM to kinase and almost complete inhibition of the activity at a three- to six-fold excess of the kinase over CaM. The modification required 10–15 min preincubation (with Ca2+ and CaM) and could be explained by a dimerization of the kinase, demonstrated by the use of a zero-length cross-linker.Key words: kinetic mechanism, autophosphorylation, calcium and calmodulin activation, cooperativity, myosin light chain kinase, smooth muscle.

scholarly journals Activation of ROCK and MLCK tunes regional stress fiber formation and mechanics via preferential myosin light chain phosphorylation

2017 ◽  
Vol 28 (26) ◽  
pp. 3832-3843 ◽  
Author(s):  
Elena Kassianidou ◽  
Jasmine H. Hughes ◽  
Sanjay Kumar
Keyword(s):  
Light Chain ◽  
Viscoelastic Properties ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Myosin Regulatory Light Chain ◽  
Regional Stress ◽  
Stress Fiber Formation

The assembly and mechanics of actomyosin stress fibers (SFs) depend on myosin regulatory light chain (RLC) phosphorylation, which is driven by myosin light chain kinase (MLCK) and Rho-associated kinase (ROCK). Although previous work suggests that MLCK and ROCK control distinct pools of cellular SFs, it remains unclear how these kinases differ in their regulation of RLC phosphorylation or how phosphorylation influences individual SF mechanics. Here, we combine genetic approaches with biophysical tools to explore relationships between kinase activity, RLC phosphorylation, SF localization, and SF mechanics. We show that graded MLCK overexpression increases RLC monophosphorylation (p-RLC) in a graded manner and that this p-RLC localizes to peripheral SFs. Conversely, graded ROCK overexpression preferentially increases RLC diphosphorylation (pp-RLC), with pp-RLC localizing to central SFs. Interrogation of single SFs with subcellular laser ablation reveals that MLCK and ROCK quantitatively regulate the viscoelastic properties of peripheral and central SFs, respectively. The effects of MLCK and ROCK on single-SF mechanics may be correspondingly phenocopied by overexpression of mono- and diphosphomimetic RLC mutants. Our results point to a model in which MLCK and ROCK regulate peripheral and central SF viscoelastic properties through mono- and diphosphorylation of RLC, offering new quantitative connections between kinase activity, RLC phosphorylation, and SF viscoelasticity.

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