The ebi gene of Drosophila melanogaster has been implicated in diverse signalling pathways, cellular functions and developmental processes. However, a thorough genetic analysis of this gene has been lacking and the true extent of its biological roles is unclear. Here, we characterize eleven ebi mutations and find that ebi has a novel role in promoting growth of the wing imaginal disc: viable combinations of mutant alleles give rise to adults with small wings. Wing discs with reduced EBI levels are correspondingly small and exhibit down-regulation of Notch target genes. Furthermore, we show that EBI colocalizes on polytene chromosomes with Smrter (SMR), a transcriptional corepressor, and Suppressor of Hairless (SU(H)), the primary transcription factor involved in Notch signalling. Interestingly, the mammalian orthologs of ebi, transducin β-like 1 (TBL1) and TBL-related 1 (TBLR1), function as corepressor/coactivator exchange factors and are required for transcriptional activation of Notch target genes. We hypothesize that EBI acts to activate (de-repress) transcription of Notch target genes important for Drosophila wing growth by functioning as a corepressor/coactivator exchange factor for SU(H).

Class I phosphoinositide 3-kinases (PI(3)Ks) are activated through associated adaptor molecules in response to G protein-coupled and tyrosine kinase receptor signalling1. They contain Ras-binding domains (RBDs) and can also be activated through direct association with active GTP-bound Ras2,3,4,5,6,7,8,9,10. The ability of Ras to activate PI(3)K has been established in vitro and by overexpression analysis, but its relevance for normal PI(3)K function in vivo is unknown. The Drosophila class I PI(3)K, Dp110, is activated by nutrient-responsive insulin signalling and modulates growth, oogenesis and metabolism11,12,13,14,15. To investigate the importance of Ras-mediated PI(3)K activation for normal PI(3)K function, we replaced Dp110 with Dp110RBD, which is unable to bind to Ras but otherwise biochemically normal. We found that Ras-mediated Dp110 regulation is dispensable for viability. However, egg production, which requires large amounts of growth, is dramatically lowered in Dp110RBD flies. Furthermore, insulin cannot maximally activate PI(3)K signalling in Dp110RBD imaginal discs and Dp110RBD flies are small. Thus, Dp110 integrates inputs from its phosphotyrosine-binding adaptor and Ras to achieve maximal PI(3)K signalling in specific biological situations.

In the fruit fly Drosophila melanogaster, the insulin and ecdysone signaling pathways have long been known to regulate growth and developmental timing, respectively. Recent findings reveal that crosstalk between these pathways allows coordination of growth and developmental timing and thus determines final body size.

Mutations in APC or in β-catenin, which are common in colon cancer, lead to constitutive activation of β-catenin/Tcf-dependent signaling. α-Catenin is also found in some colon cancer cell nuclei, and loss of its expression correlates with increased β-catenin/Tcf transcriptional activity. Moreover, targeted expression of α-catenin in the nucleus inhibits β-catenin/Tcf-dependent transcription. Thus, an understanding of the regulation of α-catenin localization could provide insight into the control of β-catenin signaling. While the β-catenin/Tcf complex can promote nuclear import of α-catenin, the mechanism for its nuclear export is not known. We found that leptomycin B (LMB) inhibited nuclear export of GFP-α-catenin in HCT116 colon cancer cells, suggesting that α-catenin localization is regulated by CRM-1-dependent nuclear export. We identified two putative nuclear export signals in a domain of α-catenin that overlaps with the β-catenin binding domain. Using a nuclear export assay, we determined that one of these (NES1) is a weak LMB-insensitive NES, whereas the other (NES2) is strong and LMB-sensitive. Mutations in either NES reduced nuclear export of α-catenin in HCT116 cells. In addition, mutations in NES1, but not NES2, reduced binding of α-catenin to β-catenin and impaired the ability of α-catenin to repress β-catenin/Tcf-dependent transcription. Therefore, NES1 is required both for repression of β-catenin signaling and for nuclear export, while NES2 is required only for nuclear export.

GABA(C) receptors contain rho subunits and mediate feedback inhibition from retinal amacrine cells to bipolar cells. We previously identified the cytoskeletal protein MAP1B as a rho1 subunit anchoring protein. Here, we analyze the structural basis and functional significance of the MAP1B-rho1 interaction. Twelve amino acids at the C terminus of the large intracellular loop of rho1 (and also rho2) are sufficient for interaction with MAP1B. Disruption of the MAP1B-rho interaction in bipolar cells in retinal slices decreased the EC(50) of their GABA(C) receptors, doubling the receptors' current at low GABA concentrations without affecting their maximum current at high concentrations. Thus, anchoring to the cytoskeleton lowers the sensitivity of GABA(C) receptors and provides a likely site for functional modulation of GABA(C) receptor-mediated inhibition.