RESUMO
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.
Assuntos
Movimento Celular/efeitos dos fármacos , Genes src/efeitos dos fármacos , Proteínas Quinases/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Membrana Celular/ultraestrutura , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Proteína Substrato Associada a Crk/genética , Proteína Substrato Associada a Crk/metabolismo , Citoplasma/enzimologia , Citoplasma/ultraestrutura , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Humanos , Microscopia de Fluorescência , Fenótipo , Pseudópodes/efeitos dos fármacos , Pseudópodes/ultraestrutura , Frações Subcelulares/metabolismo , Frações Subcelulares/ultraestrutura , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
BACKGROUND: In both Drosophila and the mouse, the zinc finger transcription factor Snail is required for mesoderm formation; its vertebrate paralog Slug (Snai2) appears to be required for neural crest formation in the chick and the clawed frog Xenopus laevis. Both Slug and Snail act to induce epithelial to mesenchymal transition (EMT) and to suppress apoptosis. METHODOLOGY & PRINCIPLE FINDINGS: Morpholino-based loss of function studies indicate that Slug is required for the normal expression of both mesodermal and neural crest markers in X. laevis. Both phenotypes are rescued by injection of RNA encoding the anti-apoptotic protein Bcl-xL; Bcl-xL's effects are dependent upon IkappaB kinase-mediated activation of the bipartite transcription factor NF-kappaB. NF-kappaB, in turn, directly up-regulates levels of Slug and Snail RNAs. Slug indirectly up-regulates levels of RNAs encoding the NF-kappaB subunit proteins RelA, Rel2, and Rel3, and directly down-regulates levels of the pro-apopotic Caspase-9 RNA. CONCLUSIONS/SIGNIFICANCE: These studies reveal a Slug/Snail-NF-kappaB regulatory circuit, analogous to that present in the early Drosophila embryo, active during mesodermal formation in Xenopus. This is a regulatory interaction of significance both in development and in the course of inflammatory and metastatic disease.
