Abstract
We describe an approach to selectively activate a kinase in a specific protein complex or at a specific subcellular location within living cells and within minutes. This reveals the effects of specific kinase pathways without time for genetic compensation. The new technique, dubbed rapamycin-regulated targeted activation of pathways (RapRTAP), was used to dissect the role of Src kinase interactions with FAK and p130Cas in cell motility and morphodynamics. The overall effects of Src activation on cell morphology and adhesion dynamics were first quantified, without restricting effector access. Subsets of Src-induced behaviors were then attributed to specific interactions between Src and the two downstream proteins. Activation of Src in the cytoplasm versus at the cell membrane also produced distinct phenotypes. The conserved nature of the kinase site modified for RapRTAP indicates that the technique can be applied to many kinases.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Cell Adhesion / drug effects
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Cell Membrane / enzymology
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Cell Membrane / ultrastructure
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Cell Movement / drug effects*
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Cell Movement / genetics
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Cell Proliferation / drug effects
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Crk-Associated Substrate Protein / genetics
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Crk-Associated Substrate Protein / metabolism
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Cytoplasm / enzymology
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Cytoplasm / ultrastructure
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Focal Adhesion Kinase 1 / genetics
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Focal Adhesion Kinase 1 / metabolism
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Genes, src / drug effects*
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Humans
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Microscopy, Fluorescence
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Phenotype
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Protein Kinases / drug effects*
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Pseudopodia / drug effects
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Pseudopodia / ultrastructure
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Signal Transduction / drug effects*
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Subcellular Fractions / metabolism
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Subcellular Fractions / ultrastructure
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Tacrolimus Binding Proteins / genetics
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Tacrolimus Binding Proteins / metabolism
Substances
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BCAR1 protein, human
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Crk-Associated Substrate Protein
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Protein Kinases
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Focal Adhesion Kinase 1
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PTK2 protein, human
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Tacrolimus Binding Proteins