Myosin light chain-kinase

Myosin light chain kinase is a Ser/Thr-type protein kinase involved in regulation of smooth muscle. The enzyme is also found in smooth muscle and platelets. This assay uses a synthetic substrate that is not radioactively labeled. 

The substrate, KKRPQRATSNVFS-NH2, and its phosphorylated derivative, are separated by chromatography at 40° C on a Q8 silica column (4.6 mm x 150 mm). The mobile phase contained 18% acetonitrile-0.1% trifluoroacetic-H20. The column eluate was monitored at 220 nm. 

The peptide substrate was phosphorylated in a reaction mixture (0.25 mL) containing 25 mA/ Tris-HCl (pH 7.5), 0.5 mg/mL bovine serum albumin, 4.0 mA/ MgCl2, 0.2 mA/ CaCl2, 2.6 nM calmodulin, 24 fiM peptide, 1.5 nA/ myosin light chain kinase, and 400 fiM ATP. The reaction was started by adding ATP. After 30 minutes at 28°C, the reaction was terminated by adding 0.1 mL of 10% acetic acid. Aliquots were directly applied to the HPLC column. The reaction was linear for up to 60 minutes. 

Myosin light chain kinase was purified from chicken gizzard smooth muscle. 

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In the presence of calcium, the primary contractile protein, myosin, is phosphorylated by the myosin light-chain kinase initiating the subsequent actin-activation of the myosin adenosine triphosphate activity and resulting in muscle contraction. Removal of calcium inactivates the kinase and allows the myosin light chain to dephosphorylate myosin which results in muscle relaxation. Therefore the general biochemical mechanism for the muscle contractile process is dependent on the avaUabUity of a sufficient intraceUular calcium concentration. 

Myosin light-chain kinase (MLCK) —ia[Pg.467]

Smooth muscle contractions are subject to the actions of hormones and related agents. As shown in Figure 17.32, binding of the hormone epinephrine to smooth muscle receptors activates an intracellular adenylyl cyclase reaction that produces cyclic AMP (cAMP). The cAMP serves to activate a protein kinase that phosphorylates the myosin light chain kinase. The phosphorylated MLCK has a lower affinity for the Ca -calmodulin complex and thus is physiologically inactive. Reversal of this inactivation occurs via myosin light chain kinase phosphatase. 

The ETa receptor activates G proteins of the Gq/n and G12/i3 family. The ETB receptor stimulates G proteins of the G and Gq/11 family. In endothelial cells, activation of the ETB receptor stimulates the release of NO and prostacyclin (PGI2) via pertussis toxin-sensitive G proteins. In smooth muscle cells, the activation of ETA receptors leads to an increase of intracellular calcium via pertussis toxin-insensitive G proteins of the Gq/11 family and to an activation of Rho proteins most likely via G proteins of the Gi2/i3 family. Increase of intracellular calcium results in a calmodulin-dependent activation of the myosin light chain kinase (MLCK, Fig. 2). MLCK phosphorylates the 20 kDa myosin light chain (MLC-20), which then stimulates actin-myosin interaction of vascular smooth muscle cells resulting in vasoconstriction. Since activated Rho... 

Smooth muscle myosin contains two myosin light chains. Phosphorylation of the regulatory light chain by myosin light chain kinase is a mandatory step to induce contraction. 

Jiang, H Rao, K., Halayko, A.J., Liu. X., Stephens, N.L. (1992). Ragweed sensitization-induced increase of myosin light chain kinase content in canine airway smooth muscle. Am. J. Respir. Cell. Mol. Biol. 7, 567-573. 

Of the several kinase activities which are important in smooth muscle, myosin light chain kinase, MLCK, is the one responsible for activation of the actin-myosin system to in vivo levels. MLCK is present in the other nonmuscle cell types which have the actin-myosin contractile system and all of these are probably activated in a manner similar to smooth muscle rather than by way of the Ca -troponin mechanism of striated muscle. MLCK from smooth muscle is about 130 kDa and is rather variable in shape. It is present in smooth muscle in 1-4 pM concentrations and binds with an equally high affinity to both myosin and actin. Thus, most MLCK molecules are bound to actin. Myosin light chain serine-19 is the primary target of smooth muscle myosin light chain kinase. 

Myosin Light Chain Kinase Is Activated by Calmodulin-4Ca + Then Phosphorylates the Light Chains... 

Smooth muscle sarcoplasm contains a myosin light chain kinase that is calcium-dependent. The Ca activation of myosin fight chain kinase requires binding of calmodulin-4Ca to its kinase subunit (Figure 49-14). 

Effect of protein-bound Ca TpC 4Ca antagonizes Tpl inhibition of F-actin-myosin interaction (allows F-actin activation of ATPase) Calmodulin 4Ca activates myosin light chain kinase that phosphorylates myosin p-light chain. The phosphorylated p-light chain no longer inhibits F-actin-myosin interaction (allows F-actin activation of ATPase). 

Protein phosphorylation Calmodulin kinase I ATI Elongation factor-2 kinase Phosphorylase kinase Myosin Light Chain kinase... 

GFP hopo ICBP IP3 Ln3+ mal memal MLCK nota oxine par pdta pmea py quin-2 green fluorescent protein hydroxypyridinon(at)e intestinal calcium-binding protein inositol 1,4,5-triphosphate a lanthanide(III) cation malonate methylmalonate myosin light chain kinase 1,4,7-triazacyclononane-l,4,7-triacetate 8- hydroxyquinoline pyridine-2-azo-4 -dimethylaniline propylene-1,2-diaminetetraacetate 9- [2-(phosphonomethoxy)ethyl] adenine pjrridine pjrridyl 8-amino-2- [(2-amino-5-methylphenoxy )methyl] -6-methoxyquinoline-ATJV -tetraacetate 2- [ [2-[his(carboxymethyl)amino]-5-methyl-phenoxy] methyl] -6-methoxy-8- [bis(carboxymethyl) amino] quinoline]... 

The remaining three types of CaMK are phosphorylase kinase, myosin light-chain kinase and CaMKIII. These each appear to phosphorylate fewer substrate proteins, and in some cases only one protein, under physiological conditions, and each may therefore mediate relatively fewer actions of Ca2+ in the nervous system. 

As mentioned above, the junctional SR is connected to sheets of perpendicular SR (Fig. 4), which extend from the PM through a peripheral cytoplasmic region with lower myofilament density into the myoplasm. It is proposed that during the active state of wave-like [Ca2+]j oscillations, Ca2+ taken up by the junctional SR is released by these perpendicular sheets near the calmodulin, which is tethered to the myosin light chain kinase (MLCK) of the thin filaments (M. Walsh, personal communication, 2001). This process would enhance the specificity and efficiency of Ca2+ regulation of contraction. 

Figure 11.9 (Left) The EF hand helix-loop-helix motif (centre) rat testes calmodulin. The globular domains each have two Ca2+-binding sites, indicated by white spheres, connected by a seven-turn a-helix (right) two views of the (Ca2+)4 fruit fly calmodulin in complex with its 26-residue target peptide from rabbit skeletal muscle myosin light chain kinase, ((left, centre) From Voet and Voet, 2004. Reproduced with permission from John Wiley Sons., Inc. and (right) Carafoli, 2002. Copyright (2002) National Academy of Sciences, USA.)...

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