MLCK Activity Research Articles

Activation of calcineurin and smooth muscle myosin light chain kinase by Met-to-Leu mutants of calmodulin.

The effects of replacement of each of the individual Met in calmodulin (CaM) with Leu on the activation of two CaM target enzymes [smooth muscle myosin light chain kinase (smMLCK) and calcineurin (CN)] were investigated. The KD and Pmax (percentage maximal activation) values for activation of both enzymes by M76L-CaM were indistinguishable from wild-type (wt)-CaM, which is consistent with the location of Met-76 in the central linker that is not involved in target protein interaction. The other eight Met in CaM are exposed in the hydrophobic surfaces that are involved in target-enzymes binding, and in general equivalent effects are observed for substitutions of Leu for Met residues in homologous positions in the two CaM domains. However, the importance of the interaction of specific Met residues with the target enzyme depends on the particular enzyme. Leu substitution at Met-36 or Met-109 reduced the affinity of MLCK for the mutant and the maximal activation of CN. MLCK had a higher KD for M51L-CaM whereas M124L-CaM activated the kinase to only 68% of maximal activity induced by wt-CaM; these mutants were indistinguishable from wt-CaM in activation of CN. M71L- and M144L-CaMs behaved like wt-CaM in activation of MLCK, but activated the phosphatase to only about 80% of maximal activity induced by wt-CAM. M72L-CaM exhibited an increased affinity for MLCK compared to wt-CaM and slightly decreased maximal activation, whereas M145L-CaM exhibited maximal activation significantly greater than that due to wt-CaM; these mutants behaved like wt-CaM with respect to CN activation. Finally, a mutant CaM in which all four C-terminal Met were replaced by Leu (M4-CT-L4-CaM) had similar affinities for MLCK and CN as wt-CaM but maximal activation of these enzymes by this mutant was only 60-70% of that achieved with wt-CaM. These results imply that, in addition to removing the autoinhibitory domain from the active site of the target enzyme, CaM must induce a conformational change in the active site itself.

Biochemical and pharmacological studies with KT7692 and LY294002 on the role of phosphatidylinositol 3-kinase in Fc epsilon RI-mediated signal transduction.

Wortmannin inhibited phosphatidylinositol 3-kinase (P13-kinase) and Fc epsilon RI-mediated histamine secretion in RBL-2H3 cells to a similar degree, with IC50 values of 3 and 2 nM, respectively. Although P13-kinase is an acknowledged regulator of intracellular trafficking and secretion, wortmannin has proved to be a difficult drug to use in assessing the role of P13-kinase because it inhibits another important enzyme, myosin light-chain kinase (MLCK; IC50 = 200 nM). In the present study we synthesized a unique derivative of wortmannin, O-acetyl-delta 16-wortmannin-17-ol (KT7692), that has an inhibitory potency against PI3-kinase one-hundredth that of wortmannin, but retains a similar potency to wortmannin against MLCK. Histamine secretion was influenced 100-fold more by wortmannin than by KT7692.2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), a structurally different PI3-kinase inhibitor from wortmannin, inhibited PI3-kinase with an IC50 of 2 microM but had little effect on MLCK activity in this concentration range. LY294002 also inhibited histamine secretion in RBL-2H3 cells with an IC50 of 5 microM. These results provide further evidence that PI3-kinase is involved in the signal transduction pathway responsible for histamine secretion after stimulation of Fc epsilon RI. Furthermore KT7692 in combination with wortmannin and LY294002 would be a powerful tool for clarifying the involvement of PI3-kinase as distinct from that of MLCK in signal transduction systems of various cellular responses.

Autophosphorylation of smooth muscle myosin light chain kinase at its regulatory domain.

Autophosphorylation of smooth muscle myosin light chain kinase was initially reported by Foyt et al. [Foyt, H. L., & Means, A. R. (1985) J. Cyclic Nucleotide Protein Phosphorylation Res. 260, 8978-8983], however, the effects of autophosphorylation on the kinase activity as well as the location of the sites have not been elucidated. Here we demonstrate that MLCK is autophosphorylated at three sites, Thr 803, Ser 815, and Ser 823, and this phosphorylation alters MLCK activity. Two phosphorylation sites are located in the regulatory domain of the kinase, the threonine site toward the autoinhibitory region and the serine site (Ser 815) in close proximity to the calmodulin anchoring site. The autophosphorylation was significantly inhibited by the binding of calmodulin. The autophosphorylation at Thr 803 is an intramolecular process, and the alignment of the basic amino acid residues nearby Thr 803 was highly homologous to the phosphorylation site of myosin light chain, suggesting that the regulatory site is in close proximity to the catalytic site in the three-dimensional structure. The phosphorylation at the threonine site activated the calmodulin-independent activity while the phosphorylation at the serine site inhibited the calmodulin-dependent activity due to a decrease in the affinity for calmodulin. This finding shows another example of the activation of calmodulin-dependent kinases by autophosphorylation at its autoinhibitory region and provides a new clue for understanding the calmodulin/MLCK signalling pathway.

A Protein-Bound Polysaccharide from Basidiomycetes Enhances Myosin Phosphorylation but Inhibits Myosin ATPase Activity: Studies with a Crude Actomyosin Preparation of Chicken Gizzard Smooth Muscle1

Chicken gizzard actomyosin, containing the calmodulin-myosin light chain kinase (MLCK) system, was incubated in the presence of various concentrations of PSK, a protein-bound polysaccharide from Basidiomycetes, together with Ca2+ and Mg-ATP. The phosphorylation of myosin was enhanced half-maximally by 10-4 g/ml of PSK. However, a similar concentration of PSK reduced the Mg-ATPase activity of the actomyosin. The former was brought about through stimulation of the MLCK activity and the latter through inhibition of the myosin ATPase activity.

Tracheal Smooth Muscle

Contraction of tracheal smooth muscle requires the binding of Ca2+ to calmodulin, which then binds to and activates MLCK. The Ca2+-calmodulin-MLCK complex catalyzes the phosphorylation of myosin, which causes contraction by stimulating actin-activated Mg2+-ATPase activity of myosin. Myosin phosphorylation appears to be a transient event that is responsible for a high velocity of shortening. The mechanism responsible for maintenance of isometric force is unknown, although a second Ca2+-dependent mechanism with a greater sensitivity to Ca2+ than the activation of MLCK has been hypothesized. Force would be maintained through the slow cycling of nonphosphorylated cross-bridges or a small population of phosphorylated cross-bridges. Tracheal smooth muscle utilizes both extracellular and intracellular pools of Ca2+ for contraction. Moreover, the membrane channels through which extracellular Ca2+ passes have been subdivided into potential-dependent channels (PDCs) and receptor-operated channels (ROCs) independent of membrane potential. The relative extent to which extracellular and intracellular sources of Ca2+ as well as PDCs and ROCs are utilized depends on the agonist used for contraction, its concentration, and the type and location of the smooth muscle being investigated. Calcium antagonists such as verapamil and nifedipine, which reportedly block PDCs but not ROCs, are much better inhibitors of tracheal smooth muscle contractions induced by serotonin than those induced by acetylcholine, histamine, and leukotriene D4, indicating an effect of these latter three agents on ROCs. Relaxation of tracheal smooth muscle following stimulation of beta-adrenergic receptors most likely results from an increase in cAMP that stimulates a cAMP-dependent protein kinase to catalyze a protein phosphorylation that leads to relaxation by decreasing the intracellular concentration of Ca2+. The primary mechanisms whereby cAMP is thought to reduce intracellular Ca2+ to effect relaxation include: activation of a calmodulin-sensitive Ca2+ ATPase in the plasma and sarcoplasmic reticulum membranes, and extrusion of Ca2+ by a Na+-Ca2+ exchange mechanism coupled to Na+-K+-ATPase in the cell membrane. A more controversial mechanism for relaxation that bypasses Ca2+ might involve the dephosphorylation of myosin. Leukotrienes are released by various stimuli, including immunologic challenge, and have been considered as important mediators of bronchoconstriction in allergic asthma.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of the calmodulin-binding domain of skeletal muscle myosin light chain kinase.

In the course of determining the primary structure of rabbit skeletal muscle myosin light chain kinase (MLCK; ATP:protein phosphotransferase, EC 2.7.1.37) a peptide fragment was obtained that appears to represent the calmodulin-binding domain of this enzyme. Low concentrations of the peptide inhibited calmodulin activation of MLCK (Ki congruent to 1 nM). The peptide was not associated with a catalytically active, calmodulin-independent form of MLCK that was obtained by limited proteolysis. The peptide is 27 residues in length and represents the carboxyl terminus of MLCK. The sequence of the peptide shows no significant homology with any known protein sequence. The peptide contains one tryptophanyl residue and a high percentage of basic and hydrophobic residues, but no acidic or prolyl residues. Much of the sequence has a high probability of forming alpha helix. A chemically synthesized peptide has been prepared to study the interactions of the peptide and calmodulin in more detail. The intrinsic tryptophan fluorescence of the synthetic peptide shows a significant enhancement (approximately equal to 45%) in the presence of Ca2+ and calmodulin; fluorescence enhancement is maximal at a peptide:calmodulin stoichiometry of 1:1. Calmodulin-Sepharose affinity chromatography in the presence of 2 M urea indicates that the interaction of peptide and calmodulin is Ca2+-dependent. The results of these studies indicate that the catalytic and calmodulin-binding domains of MLCK represent distinct and separable regions of the protein. In addition, the results provide a basis for future studies of the molecular and evolutionary details of calmodulin-dependent enzyme regulation.