title: Myosin light chain kinase structure function analysis using bacterial expression.
abstract: A 40-kDa fragment of chicken smooth muscle myosin light chain kinase was produced and partially purified from a bacterial expression system. This fragment exhibits calmodulin binding and substrate phosphorylation properties similar to those of the isolated chicken gizzard enzyme. A series of 3'-deletion mutants was prepared and used to produce proteins with the same NH2 terminus but with COOH termini varying over 180 amino acids. Results show that truncation of the enzyme at Ser-512 (based on the amino acid numbering system described for the partial cDNA clone by Guerriero, V., Jr., Russo, M. A., Olson, N. J., Putkey, J. A., and Means, A. R. (1986) Biochemistry 25, 8372-8381) does not alter calmodulin binding, calmodulin regulation, or enzymatic properties. Removal of an additional 5 residues from the COOH terminus completely inhibits calmodulin binding and results in an inactive kinase that can be fully activated by limited proteolysis. Site specific mutations within these 5 residues demonstrate that Gly-508 and Arg-509 are independently involved in calmodulin-dependent binding and activation of myosin light chain kinase. Truncation of the enzyme at residues within the protein kinase catalytic domain results in inactive protein that cannot be activated by proteolysis.
authors.affiliation: Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030.
authors.forename: I C
authors.initials: IC
authors.lastname: Bagchi
authors.forename: B E
authors.initials: BE
authors.lastname: Kemp
authors.forename: A R
authors.initials: AR
authors.lastname: Means
mesh_terms: D000818:Animals; D002147:Calmodulin / Q000378:metabolism; D002645:Chickens; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005822:Genetic Vectors; D005895:Gizzard, Avian / Q000201:enzymology; D008970:Molecular Weight; D009130:Muscle, Smooth / Q000201:enzymology*; D009154:Mutation; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids
chemical_list: D002147:Calmodulin; D004247:DNA; D009219:Myosin-Light-Chain Kinase