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.

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

GTP gamma S-dependent regulation of smooth muscle contractile elements

1992 ◽  
Vol 262 (2) ◽  
pp. C405-C410 ◽  
Author(s):  
Y. Kubota ◽  
M. Nomura ◽  
K. E. Kamm ◽  
M. C. Mumby ◽  
J. T. Stull
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Protein Phosphatase ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Activity ◽  
Tracheal Smooth Muscle ◽  
Proportional Increase ◽  
Gamma S ◽  
Muscle Contractile

Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) increases the sensitivity of the contractile response to activation by Ca2+ in permeabilized tracheal smooth muscle. Increased tension was associated with a proportional increase in myosin light chain phosphorylation. The site of phosphorylation was determined to be serine-19, which corresponds to the site rapidly phosphorylated by myosin light chain kinase. GTP gamma S did not affect the contraction induced by the protein phosphatase inhibitor okadaic acid but did enhance contraction produced by Ca(2+)-independent myosin light chain kinase. In tracheal homogenates Ca(2+)-dependent myosin light chain kinase activity was not affected by GTP gamma S; however, dephosphorylation of 32P-labeled heavy meromyosin by phosphatase was inhibited. Thus GTP gamma S may increase the Ca2+ sensitivity of contractile elements in tracheal smooth muscle by inhibition of protein phosphatase activity toward myosin light chain.

scholarly journals Calcium-independent contraction in lysed cell models of teleost retinal cones: activation by unregulated myosin light chain kinase or high magnesium and loss of cAMP inhibition.

1987 ◽  
Vol 105 (1) ◽  
pp. 397-402 ◽  
Author(s):  
B Burnside ◽  
N Ackland
Keyword(s):  
Smooth Muscle ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Myosin Phosphorylation ◽  
Calmodulin Binding ◽  
Dependent Protein ◽  
Myosin Interaction ◽  
High Concentrations

The retinal cones of teleost fish contract at dawn and elongate at dusk. We have previously reported that we can selectively induce detergent-lysed models of cones to undergo either reactivated contraction or reactivated elongation, with rates and morphology comparable to those observed in vivo. Reactivated contraction is ATP dependent, activated by Ca2+, and inhibited by cAMP. In addition, reactivated cone contraction exhibits several properties that suggest that myosin phosphorylation plays a role in mediating Ca2+-activation (Porrello, K., and B. Burnside, 1984, J. Cell Biol., 98:2230-2238). We report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with trypsin-digested, unregulated myosin light chain kinase (MLCK) obtained from smooth muscle. This observation provides further evidence that MLCK plays a role in regulating cone contraction. We also report here that lysed cone models can be induced to contract in the absence of Ca2+ by incubation with high concentrations of MgCl2 (10-20 mM). Mg2+-induced reactivated contraction is supported by inosine triphosphate (ITP) just as well as by ATP. Because ITP will not serve as a substrate for MLCK, this finding suggests that Mg2+-activation of contraction does not require myosin phosphorylation. Although Ca2+-induced contraction is completely blocked by cAMP at concentrations less than 10 microM, cAMP has no effect on cone contraction activated by unregulated MLCK or by high Mg2+ in the absence of Ca2+. Because trypsin digestion of MLCK cleaves off not only the Ca2+/calmodulin-binding site but also the site phosphorylated by cAMP-dependent protein kinase, and because Mg2+ activation of cone contraction circumvents MLCK action altogether, both these observations would be expected if cAMP inhibits reactivated cone contraction by catalyzing the phosphorylation of MLCK and thus reducing its affinity for Ca2+, as has been described for smooth muscle. Together our results suggest that in lysed cone models, myosin phosphorylation is sufficient for activating cone contraction, even in the absence of other Ca2+-mediated events, that cAMP inhibition of contraction is mediated by cAMP-dependent phosphorylation of MLCK, and that 10-20 mM Mg2+ can activate actin-myosin interaction to produce contraction in the absence of myosin phosphorylation.

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