ENDOCYTIC CONTROL OF CELL-AUTONOMOUS AND NON-CELL-AUTONOMOUS FUNCTIONS OF P53
type: CreativeWork
description: NUMB is a multifunctional adaptor protein historically recognized for its role in determining cell fate decisions through inhibition of NOTCH signaling, a process achieved via endocytic trafficking. Subsequently, NUMB has emerged as a significant tumor suppressor, particularly in the context of breast cancer, where it stabilizes the critical tumor suppressor protein p53 by inhibiting its targeting to proteasomal degradation by the E3 ubiquitin ligase MDM2. This role of NUMB is isoform-specific, mediated by alternatively spliced variants containing a short insert encoded by exon 3 (Ex3) – NUMB-1 and NUMB-2. Loss of expression of these NUMB isoforms, but not of isoforms lacking Ex3 (NUMB-3 and NUMB-4), is a frequent alteration in breast cancer that correlates with reduced p53 activity, increased chemoresistance, and poor clinical outcomes. Despite the importance of isoform-specific functions of NUMB to cancer, little is known about their differential biological roles in physiological or pathological contexts. To bridge this gap, we identified the interactomes of the four major mammalian NUMB isoforms using an affinity purification-mass spectrometry approach. Among these interactomes, the endocytic adaptor protein Sorting Nexin 9 (SNX9) was found to be the most abundant specific interactor of NUMB-1 and NUMB-2. We validated this interaction through in vitro binding, GST-pulldown and co-immunoprecipitation assays, which revealed that NUMB-1/2 and SNX9 participate in tripartite complex with p53. We conducted a systematic analysis of the molecular determinants mediating the formation of the NUMB-SNX9-p53 complex by in vitro binding assays with a series of recombinant full-length proteins and domain-specific fragments, alongside in vivo co-immunoprecipitation experiments with wild-type (WT) and mutant versions of the proteins. Our results revealed that formation of the NUMB-SNX9-p53 complex critically depends on the interaction of both NUMB-1/2 and SNX9 with acidic phospho…
abstract: NUMB is a multifunctional adaptor protein historically recognized for its role in determining cell fate decisions through inhibition of NOTCH signaling, a process achieved via endocytic trafficking. Subsequently, NUMB has emerged as a significant tumor suppressor, particularly in the context of breast cancer, where it stabilizes the critical tumor suppressor protein p53 by inhibiting its targeting to proteasomal degradation by the E3 ubiquitin ligase MDM2. This role of NUMB is isoform-specific, mediated by alternatively spliced variants containing a short insert encoded by exon 3 (Ex3) – NUMB-1 and NUMB-2. Loss of expression of these NUMB isoforms, but not of isoforms lacking Ex3 (NUMB-3 and NUMB-4), is a frequent alteration in breast cancer that correlates with reduced p53 activity, increased chemoresistance, and poor clinical outcomes. Despite the importance of isoform-specific functions of NUMB to cancer, little is known about their differential biological roles in physiological or pathological contexts. To bridge this gap, we identified the interactomes of the four major mammalian NUMB isoforms using an affinity purification-mass spectrometry approach. Among these interactomes, the endocytic adaptor protein Sorting Nexin 9 (SNX9) was found to be the most abundant specific interactor of NUMB-1 and NUMB-2. We validated this interaction through in vitro binding, GST-pulldown and co-immunoprecipitation assays, which revealed that NUMB-1/2 and SNX9 participate in tripartite complex with p53. We conducted a systematic analysis of the molecular determinants mediating the formation of the NUMB-SNX9-p53 complex by in vitro binding assays with a series of recombinant full-length proteins and domain-specific fragments, alongside in vivo co-immunoprecipitation experiments with wild-type (WT) and mutant versions of the proteins. Our results revealed that formation of the NUMB-SNX9-p53 complex critically depends on the interaction of both NUMB-1/2 and SNX9 with acidic phospho…
author: CACCIATORE, ROBERTA
language: eng
keywords: p53, Extracellular vesicle, NUMB, Endocytosi, SNX9, Settore MEDS-02/A - Patologia generale
date published: 2025-12-17
date modified: 2026-07-27T20:19:00Z
provider: Università degli Studi di Milano
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Università degli Studi di Milano

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property ID: Handle
value: https://hdl.handle.net/2434/1194318