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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL043651-01
Application #
3362366
Study Section
Physiology Study Section (PHY)
Project Start
1990-04-01
Project End
1995-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Arizona
Department
Type
Schools of Earth Sciences/Natur
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
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Obara, K; Nikcevic, G; Pestic, L et al. (1995) Fibroblast contractility without an increase in basal myosin light chain phosphorylation in wild type cells and cells expressing the catalytic domain of myosin light chain kinase. J Biol Chem 270:18734-7
Matsushima, S; Huang, Y P; Dudas, C V et al. (1994) Mutants of smooth muscle myosin light chain kinase at tryptophan 800. Biochem Biophys Res Commun 202:1329-36
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Ito, M; Guerriero Jr, V; Chen, X M et al. (1991) Definition of the inhibitory domain of smooth muscle myosin light chain kinase by site-directed mutagenesis. Biochemistry 30:3498-503