Essential role for the d-Asb11 cul5 Box domain for proper notch signaling and neural cell fate decisions in vivo.
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.
Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands.
Maria A
MA
Sartori da Silva
Jin-Ming
JM
Tee
Judith
J
Paridaen
Anke
A
Brouwers
Vincent
V
Runtuwene
Danica
D
Zivkovic
Sander H
SH
Diks
Daniele
D
Guardavaccaro
Maikel P
MP
Peppelenbosch
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D049109:Cell Proliferation; D044842:Cullin Proteins / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting; D017403:In Situ Hybridization; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051880:Receptors, Notch / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D050826:Suppressor of Cytokine Signaling Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D051792:Basic Helix-Loop-Helix Proteins; D044842:Cullin Proteins; D049452:Green Fluorescent Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D051880:Receptors, Notch; D050826:Suppressor of Cytokine Signaling Proteins; D029961:Zebrafish Proteins; C514282:Asb11 protein, zebrafish; C064045:delta protein; C119433:her4.1 protein, zebrafish