Abstract
The cell fate determinant Numb influences developmental decisions by antagonizing the Notch signaling pathway. However, the underlying molecular mechanism of this inhibition is poorly understood. Here we report that the mammalian Numb protein promotes the ubiquitination of membrane-bound Notch1 receptor. Furthermore, Numb expression resulted in the degradation of the Notch intracellular domain following activation, which correlated with a loss of Notch-dependent transcriptional activation of the Hes1 promoter as measured by a Hes1 luciferase reporter assay. The phosphotyrosine-binding (PTB) domain of Numb was required for both Notch1 ubiquitination and down-regulation of Notch1 nuclear activity. Numb-mediated ubiquitination of Notch1 was not dependent on the PEST region, which was previously shown to mediate Sel10-dependent ubiquitination of Notch in the nucleus, suggesting a distinct E3 ubiquitin ligase is involved. In agreement we demonstrate that Numb interacts with the cytosolic HECT domain-containing E3 ligase Itch and that Numb and Itch act cooperatively to promote ubiquitination of membrane-tethered Notch1. These results suggest that Numb recruits components of the ubiquitination machinery to the Notch receptor thereby facilitating Notch1 ubiquitination at the membrane, which in turn promotes degradation of the intracellular domain circumventing its nuclear translocation and downstream activation of Notch1 target genes.
Highlights
In Drosophila, the phosphotyrosine-binding (PTB)1 domaincontaining protein Numb is an intrinsic regulator of binary cell fate decisions during peripheral and central nervous system development as well as muscle cell differentiation [1,2,3,4]
Given that Numb has recently been shown to interact with several E3 ubiquitin ligases and is implicated as a regulator of receptor endocytosis, we examined whether Numb might affect Notch1 receptor down-regulation by ubiquitination
The effect of Numb expression on the ubiquitination of a constitutively active membranetethered Notch1 mutant, ⌬EC-Notch consisting of the transmembrane domain and the entire intracellular domain of
Summary
In Drosophila, the phosphotyrosine-binding (PTB) domaincontaining protein Numb is an intrinsic regulator of binary cell fate decisions during peripheral and central nervous system development as well as muscle cell differentiation [1,2,3,4]. Genetic evidence in Drosophila indicates that Numb may influence cell fate by negatively regulating the Notch signaling pathway [3, 7, 8]. Notch signaling plays an important role in cellular differentiation, proliferation, and apoptotic events at all stages of development, functioning as an essential communication mechanism to direct cell fate selection of neighboring cells Notch is first processed in the trans-Golgi network by a furin-like convertase into two distinct fragments that interact to form a functional heterodimeric receptor on the cell surface [23, 24] In this heterodimeric form, Notch is able to bind transmembrane ligands of the DSL (Delta/Serrate/Lag2) family presented on neighboring cells. While ubiquitin often targets proteins for degradation by the proteasome, it can serve as a signal for receptor internalization and trafficking to multivesicular bodies and the lysosome [31, 34, 35]
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