Myosin light chain kinase (MLCK) catalyzes the phosphorylation of the 20 kDa regulatory light chain of myosin. In vascular smooth muscle, MLCK plays a critical regulatory role in the final common pathway through which intracellular Ca2+ transients produce initiation of contraction. Previous work with chicken smooth muscle and rabbit skeletal muscle MLCK have shown that within the calmodulin binding region is a region with similarity to the phosphorylated region of the 20 kDa light chain. In the absence of the Ca2+-calmodulin complex, MLCK is inactive because a portion of the calmodulin binding region specifically interacts with the protein substrate binding site. Synthetic peptides modeled on this inhibitory """"""""pseudosubstrate"""""""" region of MLCK effectively prevent the phosphorylation of myosin light chain in a competitive fashion. Pharmacologic agents modeled on the pseudosubstrate-MLCK active site interaction may be specific inhibitors of MLCK. This proposal represents the initial phases of work directed toward the goal of developing such agents. Western blots have confirmed the feasibility of isolating a human MLCK cDNA from a human aorta library using anti-chicken MLCK antibodies. Construction of this library in lambda gt11 is in progress. A cDNA encoding human vascular smooth muscle MLCK will be cloned and sequenced. An active, calmodulin regulated fragment will be subcloned into a bacteria expression vector, expressed, and purified. Synthetic peptides, deletion mutagenesis and site specific mutagenesis will be used to precisely define the calmodulin binding and pseudosubstrate domains. If the crystal structure of this bacterially expressed fragment is determined, that information will be used to design further mutagenesis experiments. These studies will lead to a more complete understanding of the interaction of MLCK and the Ca2+-calmodulin complex, and may lead to the development of a new class of drugs for treatment of disease states characterized by increased vascular resistance.
| Su, Z; Fan, D; George, S E (1994) Role of domain 3 of calmodulin in activation of calmodulin-stimulated phosphodiesterase and smooth muscle myosin light chain kinase. J Biol Chem 269:16761-5 |
