Essential Role for the d-Asb11 cul5 Box Domain for Proper Notch Signaling and Neural Cell Fate Decisions In Vivo

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 (Asb11Cul). 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, asb11Cul 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, Asb11Cul 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.

The SOCS box—Adapting proteins for ubiquitination and proteasomal degradation

“…Regulation is mediated by the ubiquitination of DeltaA, a mechanism that relies on the d-Asb11 SOCS box and influences the neural progenitor compartment of zebrafish embryos (67,68). These findings were later validated by the isolation of a naturally occurring d-Asb11 SOCS box mutant, with impaired expression of Notch target genes (69).…”

Silencing of the Fibroblast growth factor 21 gene is an underlying cause of acinar cell injury in mice lacking MIST1

“…From our previous work, we identified decreased expression of Asb11 (Ankyrin-SOCS binding 11) an essential component of canonical delta-Notch signaling (8). Overexpression of Asb11 in neuronal tissue maintains progenitor populations in the brain (7,45). If Asb11 also maintains pancreatic progenitor populations, then increased Asb11 in Mist1 Ϫ/Ϫ tissue would be consistent with the observation that Mist1 Ϫ/Ϫ acini are less differentiated and have greater potential to undergo acinar-to-duct cell metaplasia (ADM).…”

Notch Ligand Ubiquitylation: What Is It Good For?

“…Thus, overexpressed Neur ubiquitylates ligand, thereby stimulating ligand degradation and downregulation of Notch signaling, yet genetic studies suggest strongly that the effects of Neur overexpression do not reflect the normal functions of mammalian Neur ligases. Interestingly, studies in zebrafish have identified a structurally unrelated E3 ligase that targets DeltaA for Ub-based degradation, which is proposed to repress Notch signaling during lateral inhibition (Diks et al, 2006; Diks et al, 2008; Sartori da Silva et al, 2010). …”