STRUCTURE VS. FUNCTION IN MYOSIN LIGHT CHAIN KINASE

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The long-term goal of this project is to gain a better understanding of the physiology and biochemistry of smooth muscle contraction. Regulation of smooth muscle contraction is by the Ca2+-calmodulin-dependent enzyme myosin light chain kinase (MLCK) that phosphorylates the regulatory light chain of myosin. Phosphorylation is a prerequisite for actin activation of myosin ATPase and contraction. It has been proposed that MLCK contains an inhibitory region that is regulated by calmodulin-binding. MLCK also contains a catalytic region and an actin-binding region. The function of the carboxy-terminus (approximately 24 kDa) is unknown, but preliminary evidence suggests that this portion is expressed independent of MLCK. The isolation of a partial cDNA for this enzyme makes it possible to use molecular biology techniques to further knowledge in this area by defining the relationship between the structure of these domains and function. This cDNA is 60% complete and includes the carboxy terminus, but the sequence of the amino terminal end of the molecule is unknown. The specific aims, proposed are: 1) Establish a bacterial system for the expression of active and Ca2+-calmodulin-dependent enzyme using the partial cDNA; 2) Define the domains contained within the partial cDNA using site-directed and deletion mutagenesis; 3) Determine the full-length sequence for MLCK by isolation of cDNA clones that extend the 5'-end of the partial cDNA; and 4) Characterize a new acidic protein (24 kDa) that has been isolated from smooth muscle and is thought to be identical with the carboxy-terminus of MLCK. MLCK is a key regulatory component in smooth muscle and a clear understanding of its mechanism is vital to our appreciation of normal smooth muscle function. This is a prerequisite for treatment of abnormal smooth muscle behavior; an important example is vascular smooth muscle. These studies will help in the design of pharmacological agents for the treatment of abnormal function.