Disease relevance of MYLK

• ML-9 will aid in determining pathophysiological functions of MLCK of increased vascular contractility in hypertension [1].

High impact information on MYLK

• Recently we found that a specific inhibitor of myosin light chain kinase (MLCK), wortmannin, inhibits Ca(2+)-induced catecholamine release from digitonin-permeabilized chromaffin cells, suggesting an implication of MLCK in the mechanisms of Ca(2+)-induced exocytosis (Imaizumi et al., 1992b) [2].
• The Ca2+ concentration required for half-maximal phosphorylation was 500 nM for MLCK and 250 nM for myosin light chain [3].
• Myosin light chain kinase (MLCK) is phosphorylated in contracting smooth muscle [3].
• It is postulated that basic residues within the inhibitory region of myosin light chain kinase (MLCK) bind acidic residues within the catalytic core to maintain the kinase in an inactive form [4].
• In chemically skinned smooth muscles of the rabbit mesenteric artery, ML-9 inhibited the Ca2+-independent contraction provoked by application of trypsin-treated MLCK [5].

Biological context of MYLK

• Comparisons with the draft of the human genome revealed that the sequence and structure of MYLK are conserved in mammals [6].
• These results suggest that MLCK plays an essential role in ATP-dependent priming of Ca(2+)-induced exocytosis in chromaffin cells [2].
• In both cases, the ACT had fallen back to baseline approximately 10-30 ms before maximal binding of CaM to MLCK or SR Ca2+/ATPase activation occurred and binding and enzyme activation persisted long after the Ca transient had subsided [7].
• Differential effect of MLC kinase in TNF-alpha-induced endothelial cell apoptosis and barrier dysfunction [8].
• Both TNFalpha-induced increase in NFkappaB dependent transactivation measured by NFkappaB luciferase reporter assay (approximately fivefold) and nuclear translocation of NFkappaB were significantly inhibited by MLCK-selective inhibitors, KT5926 (60% inhibition of luciferase activity) and ML7 (50% decrease) [9].

Anatomical context of MYLK

• We investigated the effects of a newly synthesized compound, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), a myosin light chain kinase (MLCK) inhibitor of superprecipitation of actomyosin, isometric tension development, and phosphorylation of the 20,000-Da myosin light chain (LC20) in vascular smooth muscle [5].
• We have recently demonstrated that thrombin-induced actin reorganization in bovine pulmonary artery endothelial cells (BPAEC) requires activation of both myosin light chain kinase (MLCK) and protein kinase C (PKC) [10].
• These alterations occurred prior to actin translocation to the cytoskeleton and are prevented by inhibition of either MLCK or PKC [10].
• Bovine brain MLCK phosphorylates a serine residue in the isolated turkey gizzard myosin light chain (MLC), with a specific activity of 1.8 mumol/min per mg of enzyme [11].
• A 0.6 ms hw ACT was sufficient for the N-terminal of CaM to transiently bind approximately 60% of myosin light chain kinase (MLCK), while a 1.8 ms hw ACT produced approximately 22% transient activation of the sarcoplasmic reticulum (SR) Ca2+/ATPase [7].

Associations of MYLK with chemical compounds

• TA-calmodulin and calmodulin selectively modified by 1-N, N-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl-C1) at Lys75 (dansyl-calmodulin) were characterized as activators of cyclic AMP phosphodiesterase (PDE) and inhibitors of MLCK [12].
• Furthermore, inhibition either of the release of Ca(2+) from internal stores or of the activation of myosin light-chain kinase (MLCK) prevented gel contraction in response to TGF-beta1 [13].
• SDS/polyacrylamide-gel electrophoresis of purified MLCK reveals the presence of a major protein band with an apparent Mr of 152000, which is capable of binding 125I-calmodulin in a Ca2+-dependent manner [11].
• The H2O2-induced actin recruitment was significantly inhibited by the calmodulin antagonists, W7 and TFP, but not by the MLCK inhibitor, KT5926, nor the PKC inhibitors, H7 and calphostin C [10].
• MLCK activity was determined by measuring the rate of formation of one of the reaction products, ADP, in a coupled enzymatic assay by continuous fluorimetric monitoring of NADH removal in 100 microM CaCl2 at ionic strength 0.15 M, pH 7.0 and 21 degreesC [12].

Other interactions of MYLK

• Since thrombin-induced MLC phosphorylation is also attenuated by these inhibitors, MARCKS may be involved in MLC kinase activation and subsequent BPAEC contraction [14].
• The increases in MLC phosphorylation were attributed to decreases in myosin-specific phosphatase activity without significant increases in MLC kinase activity and were attenuated by paclitaxel or by several strategies (C3 exotoxin, toxin B, Rho kinase inhibition) to inhibit Rho GTPase [15].

Analytical, diagnostic and therapeutic context of MYLK

• Western blotting indicated expression of the non-muscle and smooth muscle isoforms of MLCK [16].
• RESULTS: RT-PCR showed expression of MLCK, PAR-1, Rho kinase-1, and RhoA [16].
• Expression of myosin light chain (MLC) kinase (MLCK), ZO-1, claudin-5, and phosphorylated MLC, occludin and claudin-5 were determined by immunoprecipitation and Western blot [17].
• 1) Taking myosin light chain kinase (MLCK) activity as the index, bovine extract was fractionated by the use of anion-exchange chromatography, cation-exchange chromatography, and calmodulin affinity chromatography [18].

References