Reconstitution of the NF-kappa B system in Saccharomyces cerevisiae for isolation of effectors by phenotype modulation.
NF-kappa B is a ubiquitous transcription factor that contributes to the induction of many genes playing a central role in immune and inflammatory responses. The NF-kappa B proteins are subject to multiple regulatory influences including post-translational modifications such as phosphorylation and proteolytic processing. A very important component of this regulation is the control of their subcellular localization: cytoplasmic retention of NF-kappa B is achieved through interaction with I kappa B molecules. In response to extracellular signals, these molecules undergo degradation, NF-kappa B translocates to the nucleus and activates its target genes. To investigate novel proteins involved in this dynamic response, we have reconstituted the NF-kappa B/I kappa Beta system in the yeast Saccharomyces cerevisiae. We have successively introduced p65, the main transcriptional activator of the NF-kappa B family, which leads to the activation of two reporter genes controlled by kappa B sites, and the I kappa B alpha inhibitory protein, which abolishes this activation. By transforming such a yeast strain with a cDNA library we have performed a genetic screen for cDNAs encoding proteins capable of either dissociating the p65/I kappa B alpha complex or directly transactivating the expression of the reporter genes. The efficiency of our screen was demonstrated by the isolation of a cDNA encoding the p105 precursor of the p50 subunit of NF-kappa B. We also used this system to test stimuli known to activate signalling pathways in yeast, in order to investigate whether the related mammalian cascades might be involved in NF-kappa B activation. We showed that yeast endogenous kinase cascades activated by pheromone, hypo- or hyperosmotic shock cannot modulate NF-kappa B activity in our system, and that the p38 human MAP kinase does not act directly on the p65/I kappa B alpha complex.
Epinat
J C
JC
Unité de Biologie Moléculaire de l'Expression Génique, UMR 0321 CNRS Institut Pasteur, Paris, France.
Whiteside
S T
ST
Rice
N R
NR
Israël
A
A
D018076:DNA, Complementary / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D015723:Gene Library; D017930:Genes, Reporter; D006801:Humans; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D009997:Osmotic Pressure; D010675:Pheromones / Q000494:pharmacology; D010770:Phosphotransferases / Q000378:metabolism; D010957:Plasmids; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D015398:Signal Transduction; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D001616:beta-Galactosidase / Q000378:metabolism
D018076:DNA, Complementary; D016328:NF-kappa B; D010675:Pheromones; D010770:Phosphotransferases; D001616:beta-Galactosidase; D005690:Galactose