Abstract
The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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3T3 Cells
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ADP Ribose Transferases / pharmacology
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Actin Cytoskeleton / drug effects
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Actin Cytoskeleton / metabolism
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Amides / pharmacology
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Animals
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Botulinum Toxins*
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Carrier Proteins / genetics
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Carrier Proteins / metabolism*
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Crk-Associated Substrate Protein
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Enzyme Inhibitors / pharmacology
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Focal Adhesions / drug effects
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Focal Adhesions / metabolism
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Formins
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Gene Expression / physiology
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HeLa Cells
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Humans
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Intracellular Signaling Peptides and Proteins
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Lysophospholipids / pharmacology
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Mice
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Mutation / physiology
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Phosphoproteins / metabolism
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Phosphorylation
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Protein Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Proteins*
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Proto-Oncogene Proteins c-crk
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Proto-Oncogene Proteins*
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Pyridines / pharmacology
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Retinoblastoma-Like Protein p130
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Signal Transduction / physiology
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Tyrosine / metabolism
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rac GTP-Binding Proteins / metabolism*
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rho GTP-Binding Proteins / metabolism*
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rho-Associated Kinases
Substances
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Amides
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BCAR1 protein, human
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Bcar1 protein, mouse
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Carrier Proteins
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Crk-Associated Substrate Protein
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Diap1 protein, mouse
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Enzyme Inhibitors
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Formins
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Intracellular Signaling Peptides and Proteins
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Lysophospholipids
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Phosphoproteins
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Proteins
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-crk
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Pyridines
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Retinoblastoma-Like Protein p130
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Y 27632
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Tyrosine
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ADP Ribose Transferases
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exoenzyme C3, Clostridium botulinum
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Protein Kinases
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Protein Serine-Threonine Kinases
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rho-Associated Kinases
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Botulinum Toxins
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rac GTP-Binding Proteins
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rho GTP-Binding Proteins