Post-transcriptional Down-regulation of ROCKI/Rho-kinase through an MEK-dependent Pathway Leads to Cytoskeleton Disruption in Ras-transformed Fibroblasts
| Autor: | David M. Helfman, Geraldine Pawlak |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2002 |
| Předmět: |
MAPK/ERK pathway
Stress fiber Transcription Genetic MAP Kinase Signaling System Pyridines Morpholines Down-Regulation Fluorescent Antibody Technique Biology Oncogene Protein p21(ras) Protein Serine-Threonine Kinases Transfection Actin cytoskeleton organization Article Gene Expression Regulation Enzymologic Adherens junction Nitriles Butadienes Animals Enzyme Inhibitors Molecular Biology Rho-associated protein kinase Cell Line Transformed Flavonoids Mitogen-Activated Protein Kinase Kinases rho-Associated Kinases Intracellular Signaling Peptides and Proteins Cell Biology Cofilin Fibroblasts Actin cytoskeleton Amides Cell biology Rats Actin Cytoskeleton Chromones Calcium-Calmodulin-Dependent Protein Kinases |
| Popis: | Transformation by oncogenic Ras profoundly alters actin cytoskeleton organization. We investigated Ras-dependent signaling pathways involved in cytoskeleton disruption by transfecting normal rat kidney (NRK) cells with different Ras mutants. RasV12S35, a mutant known to activate specifically the Raf/MAPK pathway, led to stress fiber and focal contact disruption, whereas the adherens junctions remained intact. Next, we found that pharmacological inhibition of MEK was sufficient to restore the cytoskeletal defects of ras-transformed NRK cells, including assembly of stress fibers and focal contacts, but it did not induce reorganization of the cell-cell junctions. Investigating the mechanism underlying this phenotypic reversion, we found that the sustained MAPK signaling resulting from Ras-transformation down-regulated the expression of ROCKI and Rho-kinase, two-Rho effectors required for stress fiber formation, at the post-transcriptional level. On MEK inhibition, ROCKI/Rho-kinase expression and cofilin phosphorylation were increased, demonstrating that the Rho-kinase/LIM-kinase/cofilin pathway was functionally restored. Finally, using dominant negative or constitutively active mutants, we demonstrated that expression of ROCKI/Rho-kinase was both necessary and sufficient to promote cytoskeleton reorganization in NRK/ras cells. These findings further establish the Ras/MAPK pathway as the critical pathway involved in cytoskeleton disruption during Ras-transformation, and they suggest a new mechanism, involving alteration in ROCKI/Rho-kinase expression, by which oncogenic Ras can specifically target the actin-based cytoskeleton and achieve morphological transformation of the cells. |
| Databáze: | OpenAIRE |
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