ABSTRACT
ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11(Cul)). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11(Cul) fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11(Cul) mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.
Subject(s)
Neurons/metabolism , Receptors, Notch/metabolism , Signal Transduction , Suppressor of Cytokine Signaling Proteins/physiology , Zebrafish Proteins/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Binding Sites/genetics , Cell Proliferation , Cullin Proteins/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Immunoblotting , In Situ Hybridization , Intracellular Signaling Peptides and Proteins , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Confocal , Mutation , Neurons/cytology , Receptors, Notch/genetics , Reverse Transcriptase Polymerase Chain Reaction , Suppressor of Cytokine Signaling Proteins/genetics , Suppressor of Cytokine Signaling Proteins/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
In canonical Delta-Notch signalling, expression of Delta activates Notch in neighbouring cells, leading to downregulation of Delta in these cells. This process of lateral inhibition results in selection of either Delta-signalling cells or Notch-signalling cells. Here we show that d-Asb11 is an important mediator of this lateral inhibition. In zebrafish embryos, morpholino oligonucleotide (MO)-mediated knockdown of d-Asb11 caused repression of specific Delta-Notch elements and their transcriptional targets, whereas these were induced when d-Asb11 was misexpressed. d-Asb11 also activated legitimate Notch reporters cell-non-autonomously in vitro and in vivo when co-expressed with a Notch reporter. However, it repressed Notch reporters when expressed in Delta-expressing cells. Consistent with these results, d-Asb11 was able to specifically ubiquitylate and degrade DeltaA both in vitro and in vivo. We conclude that d-Asb11 is a component in the regulation of Delta-Notch signalling, important in fine-tuning the lateral inhibition gradients between DeltaA and Notch through a cell non-autonomous mechanism.
