Construction, Expression And Functional Research Of Calmodulin-binding Site Mutant Of Myosin Light Chain Kinase

Objective: Myosin light chain kinase (MLCK) is a key enzyme that regulates smooth muscle contraction. It phosphorylates the 20KD light chain of smooth muscle myosin (MLC20) in the presence of Ca²⁺ and calmodulin (Ca²⁺/CaM) resulting increasing of myosin Mg²⁺-ATPase activities, which initiates the ATP-dependent interaction between myosin and actin, and then induces smooth muscle contraction. This is a main regulatory pathway in smooth muscle contraction. As a multifunctional regulatory protein, in addition to its kinase activity, MLCK also has the non-kinase activities which contain the actin binding activity and the myosin binding activity. It was reported that these non-kinase activities also play a role on smooth muscle contraction. In order to further examine the non-kinase activities of MLCK, we engineeredΔCaM/MLCK that was devoid of kinase activitiy by site-directed mutagenesis in the ^1002-1022CaM-binding domain and expressed in E.coli. Researching the effects ofΔCaM/MLCK on myosin Mg²⁺-ATPase activities by Malachite Green method and the effects on interaction between myosin and actin in in vitro motility assay, the results suggest that the non-kinase activities of MLCK play a role on smooth muscle contraction.Methods: Designed two mutant primes through full length recombinant MLCK cDNA, and site-directed mutagenesis by PCR reaction. Recombinant MLCK mutant was expressed in E.coli, and then purified by affinity chromatography and gel filtration. It was confirmed the recombinant MLCK mutant could be expressed and purified by SDS-PAGE. We examined the level of phosphorylation of myosin light chain by Glycerol-PAGE, the effects ofΔCaM/MLCK on myosin Mg²⁺-ATPase activities by Malachite Green method and the effects of Ca²⁺/CaM on the binding activities between recombinant MLCKs and actin by protein binding assay. We also measured the effects of recombinant MLCKs on the velocity of actin filaments by in vitro motility assay.Results: The sequence analysis showed that the recombinant ^1002-1022CaM-binding site MLCK mutant (ΔCaM/MLCK) was successfully obtained by site mutagenesis. TheΔCaM/MLCK could be expressed in E.coli and purified by CaM- Sepharose 4B and Superose 6 HR purification system. Myosin light chain (MLC20) could be phosphorylated by the wild type of recombinant MLCK in Glycerol-PAGE with a dose-dependent manner. However, theΔCaM/MLCK lost the ability to phosphorylate myosin light chain. The Mg²⁺-ATPase activity assay results showed that theΔCaM/MLCK could increase the unphosphorylated myosin Mg²⁺-ATPase but decrease the phosphorylated myosin Mg²⁺-ATPase in the absence of Ca²⁺/CaM. The binding assay results showed that both WT/MLCK andΔCaM/MLCK had the binding activities to actin. In the presence of Ca²⁺/CaM, the binding of WT/MLCK to actin was antagonized partially(p<0.05), however, the effect on the binding ofΔCaM/MLCK to actin was not obvious. In vitro motility assay, the results showed that both WT/MLCK andΔCaM/MLCK inhibited the actin filaments movement in the absence of Ca²⁺/CaM(p<0.01); in the presence of Ca²⁺/CaM, the actin filaments movement was not inhibited with increase of WT/MLCK but was inhibited obviously with increase ofΔCaM/MLCK(p<0.01).Conclusions: 1. The recombinant ^1002-1022CaM-binding site mutant MLCK(ΔCaM /MLCK) was obtained by site mutagenesis and expressed in E.coli quantifiably as a soluble form;ΔCaM/MLCK could be purified by affinity chromatography and gel filtration. 2.ΔCaM/MLCK lost the ability to phosphorylate 20KD myosin light chain. 3. The Mg²⁺-ATPase activity assay results suggest that MLCK has non-kinase activity that increases unphosphorylated myosin Mg²⁺-ATPase activity and decreases phosphorylated myosin Mg²⁺-ATPase activity in the absence of Ca²⁺/CaM. 4. The binding assay results suggest that Ca²⁺/CaM affect the binding activity of MLCK to actin by binding to ^1002-1022CaM-binding site of MLCK. 5. In vitro motility assay results suggest that Ca²⁺/CaM affect the binding activity of MLCK to actin by ^1002-1022CaM-binding site of MLCK, which then affect the interaction between actin and myosin.