NUMB Promotes Notch Signaling Via Regulating NOTCH Receptor Ubiquitination

Notch signaling is a conserved signaling pathway,participating in lots of biological processes like cell proliferation,stem maintenance and differentiation.The activation of Notch signaling pathway leads to two proteolytic cleavage events in the Notch receptor,releasing the Notch Intracellular Domain(NICD),which subsequently translocates to the nucleus and cooperates the DNA-binding protein to promote transcription.Numb which serves as an endogenous cell fate determinant,makes the daughter cells different after asymmetric cell division via segregating to only one daughter cell.In Drosophila,d-Numb functions through inhibiting Notch signaling.However,the relationship between Numb and Notch is controversial in vertebrate.During corticogenesis of mouse brain,gain or loss of Numb causes phenotypes similar to gain or loss of Notch.These similarities indicate Numb maintains self-renewal properties of neural progenitor cells,but the molecular mechanism is an enigma.Here,we constructed stable cell lines of Notch signaling,which allowed us to elucidate the effect of Numb on Notch signaling.In N1 ICD stably expressing HeLa cells,overexpression of NUMB protein can inhibit the degradation of N1 ICD,while knockdown of NUMB by shRNA leads to the reduction of N1 ICD,this result clarified that NUMB stabilizes N1 ICD.However,NUMB overexpression do not influence the expression of Notch ligands such as DLL1,JAG1 and JAG2,indicating NUMB regulates Notch signaling via Notch receptor.Dual-luciferase assay showed NUMB can enhance the effect of N1ICD-mediated transcription activation,and NUMB overexpression can promote the endogenous Notch signaling in 293 T or H4 cells.These results proved that NUMB is a positive regulator of Notch signaling.Co-IP(Co-Immunoprecipitation)experiment showed NUMB interacted with N1 ICD,but overexpression of PTB(phosphotyrosine binding)or CTD(C-terminal domain)deletion mutant do not influence the function of NUMB promoting N1 ICD,and these mutants can still bind N1 ICD.However,the deletion of AB(ACBD3 binding)domain causes the loss functions of NUMB,which can not interact with N1 ICD and stabilize N1 ICD.Cycloheximide treatment verified that N1 ICD is an unstable,short half-life molecule,and MG-132 treatment can improve the N1 ICD expression level obviously,showing N1 ICD is degraded by the Ubiquitin-Proteasome pathway.MDM2 as an E3 ubiquitin ligase can bind N1 ICD and stabilize N1 ICD.However,NUMB stabilizes N1 ICD by inhibiting its ubiquitination,indicating NUMB regulates the stability of N1 ICD via additional pathway.We screened the Deubiquitinase Library and identified that BAP1 can bind N1 ICD and inhibit its degradation.BAP1 interacted with the PRR(proline-rich)domain of NUMB,and knockdown of BAP1 can rescue the phenotype of NUMB overexpression.This result clarified NUMB can stabilizes N1 ICD via BAP1.Interestingly,BAP1 stabilizes N1 ICD by a deubiquitinase-independent way,it interferes the activity of BRCA1-BARD1 complex.At last,we utilized the in utero electroporation(IUE)technique to overexpression BAP1 in vivo during the development of mouse neocortex.BAP1 overexpression can inhibit the neurogenesis,which is similar to the phenotype of N1 ICD overexpression,while siencing BAP1 in vivo promotes the neurogenesis.Moreover,knockdown of BAP1 in neurosphere culture leads to the impaired self-renewal of NPCs and inhibition of Notch signaling.These results confirmed that BAP1 maintain the stemness of neural progenitor cells via promoting Notch signaling.In general,this paper elucidates a new mechanism of Numb as a positive regulator of Notch signaling,which not only provide an explanation of Numb's function in cortical development,but also leading to a greater in-depth understanding of the ubiquitination modification of Notch signaling...