Abstract
Force-initiated signal transduction can occur either via membrane-based ionic mechanisms or through changes in cytoskeletal-matrix linkages. We report here the stretch-dependent binding of cytoplasmic proteins to Triton X-100 cytoskeletons of L-929 cells grown on collagen-coated silicone. Triton X-100-insoluble cytoskeletons were stretched by 10% and incubated with biotinylated cytoplasmic proteins. Analysis with two-dimensional gel electrophoresis showed stretch-dependent binding of more than 10 cytoplasmic protein spots. Bound cytoplasmic proteins were purified by a photocleavable biotin tag and stretch-dependent binding of paxillin, focal adhesion kinase, and p130Cas was found, whereas the binding of vinculin was unchanged and actin binding decreased with stretch. Paxillin binding upon stretch was morphologically and biochemically similar in vitro and in vivo, that is, enhanced in the periphery and inhibited by the tyrosine phosphatase inhibitor, phenylarsine oxide. Thus, we suggest that transduction of matrix forces occurs through force-dependent conformation changes in the integrated cytoskeleton.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Arsenicals / pharmacology
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Cell Line
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Crk-Associated Substrate Protein
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Cytoskeletal Proteins / metabolism
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Cytoskeleton / drug effects
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Cytoskeleton / metabolism*
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Cytoskeleton / physiology
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Detergents / pharmacology*
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Enzyme Inhibitors / pharmacology
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Focal Adhesion Kinase 1
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Focal Adhesion Protein-Tyrosine Kinases
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Genistein / pharmacology
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Humans
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L Cells
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Mice
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Octoxynol / pharmacology*
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Paxillin
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Phosphoproteins / metabolism
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Protein Serine-Threonine Kinases*
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Protein Tyrosine Phosphatases / antagonists & inhibitors
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Protein-Tyrosine Kinases / antagonists & inhibitors
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Protein-Tyrosine Kinases / metabolism
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Proteins*
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-akt
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Retinoblastoma-Like Protein p130
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Signal Transduction*
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Stress, Mechanical
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Vinculin / metabolism
Substances
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Arsenicals
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BCAR1 protein, human
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Bcar1 protein, mouse
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Crk-Associated Substrate Protein
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Cytoskeletal Proteins
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Detergents
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Enzyme Inhibitors
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PXN protein, human
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Paxillin
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Phosphoproteins
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Proteins
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Proto-Oncogene Proteins
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Pxn protein, mouse
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Retinoblastoma-Like Protein p130
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oxophenylarsine
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Vinculin
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Octoxynol
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Genistein
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Protein-Tyrosine Kinases
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Focal Adhesion Kinase 1
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Focal Adhesion Protein-Tyrosine Kinases
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PTK2 protein, human
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Ptk2 protein, mouse
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Protein Serine-Threonine Kinases
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Proto-Oncogene Proteins c-akt
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Protein Tyrosine Phosphatases