title: Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle.
abstract: Smooth muscle myosin (SMM) light chain kinase (MLCK) phosphorylates SMM, thereby activating the ATPase activity required for muscle contraction. The abundance of active MLCK, which is tightly associated with the contractile apparatus, is low relative to that of SMM. SMM phosphorylation is rapid despite the low ratio of MLCK to SMM, raising the question of how one MLCK rapidly phosphorylates many SMM molecules. We used total internal reflection fluorescence microscopy to monitor single molecules of streptavidin-coated quantum dot-labeled MLCK interacting with purified actin, actin bundles, and stress fibers of smooth muscle cells. Surprisingly, MLCK and the N-terminal 75 residues of MLCK (N75) moved on actin bundles and stress fibers of smooth muscle cell cytoskeletons by a random one-dimensional (1-D) diffusion mechanism. Although diffusion of proteins along microtubules and oligonucleotides has been observed previously, this is the first characterization to our knowledge of a protein diffusing in a sustained manner along actin. By measuring the frequency of motion, we found that MLCK motion is permitted only if acto-myosin and MLCK-myosin interactions are weak. From these data, diffusion coefficients, and other kinetic and geometric considerations relating to the contractile apparatus, we suggest that 1-D diffusion of MLCK along actin (a) ensures that diffusion is not rate limiting for phosphorylation, (b) allows MLCK to locate to areas in which myosin is not yet phosphorylated, and (c) allows MLCK to avoid getting "stuck" on myosins that have already been phosphorylated. Diffusion of MLCK along actin filaments may be an important mechanism for enhancing the rate of SMM phosphorylation in smooth muscle.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557.
authors.forename: Feng
authors.initials: F
authors.lastname: Hong
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557.
authors.forename: Richard K
authors.initials: RK
authors.lastname: Brizendine
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Carter
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557.
authors.forename: Diego B
authors.initials: DB
authors.lastname: Alcala
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557.
authors.forename: Avery E
authors.initials: AE
authors.lastname: Brown
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Chattin
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Haldeman
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary Faculty of Medicine, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557.
authors.forename: Kevin C
authors.initials: KC
authors.lastname: Facemyer
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557.
authors.forename: Josh E
authors.initials: JE
authors.lastname: Baker
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 99557 cremo@unr.edu.
authors.forename: Christine R
authors.initials: CR
authors.lastname: Cremo
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D002645:Chickens; D004058:Diffusion; D006801:Humans; D009130:Muscle, Smooth / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010766:Phosphorylation; D045663:Quantum Dots; D051381:Rats
chemical_list: D000199:Actins; D009219:Myosin-Light-Chain Kinase