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Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle

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 desp...

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Autores principales: Hong, Feng, Brizendine, Richard K., Carter, Michael S., Alcala, Diego B., Brown, Avery E., Chattin, Amy M., Haldeman, Brian D., Walsh, Michael P., Facemyer, Kevin C., Baker, Josh E., Cremo, Christine R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Rockefeller University Press 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586593/
https://www.ncbi.nlm.nih.gov/pubmed/26415568
http://dx.doi.org/10.1085/jgp.201511483
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author Hong, Feng
Brizendine, Richard K.
Carter, Michael S.
Alcala, Diego B.
Brown, Avery E.
Chattin, Amy M.
Haldeman, Brian D.
Walsh, Michael P.
Facemyer, Kevin C.
Baker, Josh E.
Cremo, Christine R.
author_facet Hong, Feng
Brizendine, Richard K.
Carter, Michael S.
Alcala, Diego B.
Brown, Avery E.
Chattin, Amy M.
Haldeman, Brian D.
Walsh, Michael P.
Facemyer, Kevin C.
Baker, Josh E.
Cremo, Christine R.
author_sort Hong, Feng
collection PubMed
description 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.
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spelling pubmed-45865932016-04-01 Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle Hong, Feng Brizendine, Richard K. Carter, Michael S. Alcala, Diego B. Brown, Avery E. Chattin, Amy M. Haldeman, Brian D. Walsh, Michael P. Facemyer, Kevin C. Baker, Josh E. Cremo, Christine R. J Gen Physiol Research Articles 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. The Rockefeller University Press 2015-10 /pmc/articles/PMC4586593/ /pubmed/26415568 http://dx.doi.org/10.1085/jgp.201511483 Text en © 2015 Hong et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
spellingShingle Research Articles
Hong, Feng
Brizendine, Richard K.
Carter, Michael S.
Alcala, Diego B.
Brown, Avery E.
Chattin, Amy M.
Haldeman, Brian D.
Walsh, Michael P.
Facemyer, Kevin C.
Baker, Josh E.
Cremo, Christine R.
Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle
title Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle
title_full Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle
title_fullStr Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle
title_full_unstemmed Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle
title_short Diffusion of myosin light chain kinase on actin: A mechanism to enhance myosin phosphorylation rates in smooth muscle
title_sort diffusion of myosin light chain kinase on actin: a mechanism to enhance myosin phosphorylation rates in smooth muscle
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586593/
https://www.ncbi.nlm.nih.gov/pubmed/26415568
http://dx.doi.org/10.1085/jgp.201511483
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