Expression And Characterization Of Full-length Smooth Muscle Myosin Light Chain Kinase And The ATP Binding Site Mutant

Objective: Smooth muscle myosin light chain kinase (MLCK), which plays a regulatory role in the contraction of smooth muscle, phosphorylates 20kDa myosin light chain (MLC20) by an Ca2+/calmodulin(CaM) dependent manner and initiates smooth muscle contraction. As a multifunctional regulatory protein, in addition to its kinase activity, MLCK also has the non-kinase activity such as the actin-binding activity at N-terminal region and myosin-binding activity at C-terminal region, etc. We hypothesed the non-kinase activity of MLCK may have some regulatory role in smooth muscle contraction and relaxation. For the analysis of the non-kinase activity and the relationship between kinase activity and non-kinase activity, the recombinant full length MLCK was expressed and purified quantifiably with the E.coli expression system. The ATP binding site site-directed mutants of MLCK which lost the kinase activity were acquired.The effect of recombinant full length MLCKs on myosin Mg2 +-ATPase activity and sliding velocity of actin filaments in vitro motility assay was studied which can be benefit to the study on the non-kinase regulatory role of MLCK.Methods: The pCold I expression vector containing the cDNA of full length MLCK was expressed in E.coli. Using SDS-PAGE to confirm recombinant full-length MLCKs and Glycerol-PAGE to confirm the phosphorylated myosin light chain. Using affinity chormatography and gel filtration to purify recombinant full-length MLCKs. Using marachite green method to examine the effect of recombinant full-length MLCKs on the phosphorylated and unphosphorylated myosin. Studying the effect of recombinant full-length MLCKs on the phosphorylated myosin in vitromotility assay.Results: The results showed that both the wild type of recombinant MLCK and the ATP binding site mutagenesis of MLCK(MLCK/â–³ATP) expressed in E.coli quantifiably with a large amount and exist as a soluble form. Purified MLCK showed one single band in SDS-PAGE using the CaM-Sepharose 4B and Superose 6 HR purification system. Glycerol -PAGE showed that the wild type of recombiant MLCK phosphorylated myosin light chain but MLCK/â–³ATP lost the ability to phosphorylate myosin light chain which related to its kianse activity. The effect of different concentration of MLCK on phosphorylated and unphosphorylated myosin was examined and showed that MLCK activated the unphosphorylated myosin, inhibited the phosphorylated myosin in Mg2 +-ATPase activity with a dose dependent manner and without Ca2+ . In the in vitro motility assay, sliding velocity of actin filaments was decreasing with the increasing of MLCK. Comparing the wild type of recombinant MLCK with MLCK/â–³ATP showed no difference between them on the ATPase activity assay(P>0.05). The inhibitory effect of MLCK/â–³ATP to the sliding velocity of actin filaments was stronger than the wild type of recombinant MLCK(P<0.05).Conclusion: 1. Both the wild type of recombinant MLCK and the ATP binding site mutagenesis of MLCK(MLCK/â–³ATP) expressed in E.coli quantifiably with a large amount and exist as a soluble form 2. SDS-PAGE analysis showed that the recombinant full length MLCKs can be purified by affinity chromatography and gel filtration. 3. The ATP binding site mutagenesis of MLCK(MLCK/â–³ATP) lost the ability to phosphorylate myosin light chain which related to its kinase activity. 4. Both the wild type of recombinant MLCK and MLCK/â–³ATP had an activated effect on unphosphorylated myosin and an inhibitory effect on phosphorylated myosin in Mg2+-ATPase activity assay and no difference between them(P>0.05). 5. In the in vitro motility assay, MLCKs reduced the sliding velocity of actin filaments and the inhibitory effect of MLCK/â–³ATP was stronger than the wild type of recombinant MLCK (P<0.05).