Numb exon 9 inclusion regulates Integrinß5 surface expression and promotes breast cancer metastasis.

The endocytic adaptor protein Numb acts as a tumor suppressor through downregulation of oncogenic pathways in multiple cancer types. The identification of splicing alterations giving rise to changes in Numb protein isoform expression indicate that Numb also has tumor promoting activity, though the underlying mechanisms are unknown. Here we report that NUMB exon 9 inclusion, which results in production of a protein isoform with an additional 49 amino acids, is a feature of multiple cancer types including all subtypes of breast cancer and correlates with worse progression-free survival. Specific deletion of exon 9-included Numb isoforms (Exon9in) from breast cancer cells reduced cell growth and prevents spontaneous lung metastasis in a mouse model. Quantitative proteome profiling showed that loss of Exon9in causes downregulation of membrane receptors and adhesion molecules, as well as proteins involved in extracellular matrix organization and the epithelial-mesenchymal transition (EMT) state. In addition, exon 9 deletion caused remodeling of the endocytic network, decreased ITGß5 surface localization, cell spreading on vitronectin and downstream signaling to ERK and SRC. Together these observations suggest that Exon9in isoform expression disrupts the endocytic trafficking functions of Numb, resulting in increased surface expression of ITGß5 as well as other plasma membrane proteins to promote cell adhesion, EMT, and tumor metastasis.

Numb regulates post-endocytic trafficking and degradation of Notch1.

Notch is a transmembrane receptor that controls cell fate decisions during development and tissue homeostasis. Both activation and attenuation of the Notch signal are tightly regulated by endocytosis. The adaptor protein Numb acts as an inhibitor of Notch and is known to function within the intracellular trafficking pathways. However, a role for Numb in regulating Notch trafficking has not been defined. Here we show that mammalian Notch1 is constitutively internalized and trafficked to both recycling and late endosomal compartments, and we demonstrate that changes in Numb expression alter the dynamics of Notch1 trafficking. Overexpression of Numb promotes sorting of Notch1 through late endosomes for degradation, whereas depletion of Numb facilitates Notch1 recycling. Numb mutants that do not interact with the ubiquitin-protein isopeptide ligase, Itch, or that lack motifs important for interaction with endocytic proteins fail to promote Notch1 degradation. Our data suggest that Numb inhibits Notch1 activity by regulating post-endocytic sorting events that lead to Notch1 degradation.

Dynamic regulation of mammalian numb by G protein-coupled receptors and protein kinase C activation: Structural determinants of numb association with the cortical membrane.

The cell fate determinant Numb is a membrane-associated adaptor protein involved in both development and intracellular vesicular trafficking. It has a phosphotyrosine-binding (PTB) domain and COOH-terminal endocytic-binding motifs for alpha-adaptin and Eps15 homology domain-containing proteins. Four isoforms of Numb are expressed in vertebrates, two of which selectively associate with the cortical membrane. In this study, we have characterized a cortical pool of Numb that colocalizes with AP2 and Eps15 at substratum plasma membrane punctae and cortical membrane-associated vesicles. Green fluorescent protein (GFP)-tagged mutants of Numb were used to identify the structural determinants required for localization. In addition to the previously described association of the PTB domain with the plasma membrane, we show that the AP2-binding motifs facilitate the association of Numb with cortical membrane punctae and vesicles. We also show that agonist stimulation of G protein-coupled receptors (GPCRs) that are linked to phospholipase Cbeta and protein kinase C (PKC) activation causes redistribution of Numb from the cortical membrane to the cytosol. This effect is correlated with Numb phosphorylation and an increase in its Triton X-100 solubility. Live-imaging analysis of mutants identified two regions within Numb that are independently responsive to GPCR-mediated lipid hydrolysis and PKC activation: the PTB domain and a region encompassing at least three putative PKC phosphorylation sites. Our data indicate that membrane localization of Numb is dynamically regulated by GPCR-activated phospholipid hydrolysis and PKC-dependent phosphorylation events.

Proximity interactions of the ubiquitin ligase Mind bomb 1 reveal a role in regulation of epithelial polarity complex proteins.

MIB1 belongs to the RING domain containing family of E3 ubiquitin ligases. In vertebrates, MIB1 plays an essential role in activation of Notch signaling during development, through the ubiquitination and endocytosis of Notch ligands. More recently, Notch independent functions for MIB1 have been described in centriole homeostasis, dendritic spine outgrowth and directional cell migration. Here we use proximity-dependent biotin identification (BioID) to define the MIB1 interactome that included 163 high confidence interactions with polypeptides linked to centrosomes and cilia, endosomal trafficking, RNA and DNA processing, the ubiquitin system, and cell adhesion. Biochemical analysis identified several proteins within these groups including CCDC14 and EPS15 that were ubiquitinated but not degraded when co-expressed with MIB1. The MIB1 interactome included the epithelial cell polarity protein, EPB41L5. MIB1 binds to and ubiquitinates EPB41L5 resulting in its degradation. Furthermore, MIB1 ubiquitinates the EPB41L5-associated polarity protein CRB1, an important determinant of the apical membrane. In polarized cells, MIB1 localized to the lateral membrane with EPB41L5 and to the tight junction with CRB1, CRB3 and ZO1. Furthermore, over expression of MIB1 resulted in altered epithelial cell morphology and apical membrane expansion. These results support a role for MIB1 in regulation of polarized epithelial cell morphology.

The cell fate determinant numb interacts with EHD/Rme-1 family proteins and has a role in endocytic recycling.

The adaptor protein Numb is necessary for the cell fate specification of progenitor cells in the Drosophila nervous system. Numb is evolutionarily conserved and previous studies have provided evidence for a similar functional role during mammalian development. The Numb protein has multiple protein-protein interaction regions including a phosphotyrosine binding (PTB) domain and a carboxy-terminal domain that contains conserved interaction motifs including an EH (Eps15 Homology) domain binding motif and alpha-adaptin binding site. In this study we identify the EHD/Rme-1/Pincher family of endocytic proteins as Numb interacting partners in mammals and Drosophila. The EHD/Rme-1 proteins function in recycling of plasma membrane receptors internalized by both clathrin-mediated endocytosis and a clathrin-independent pathway regulated by ADP ribosylation factor 6 (Arf6). Here we report that Numb colocalizes with endogenous EHD4/Pincher and Arf6 and that Arf6 mutants alter Numb subcellular localization. In addition, we present evidence that Numb has a novel function in endosomal recycling and intracellular trafficking of receptors.

aPKC-mediated phosphorylation regulates asymmetric membrane localization of the cell fate determinant Numb.

In Drosophila, the partition defective (Par) complex containing Par3, Par6 and atypical protein kinase C (aPKC) directs the polarized distribution and unequal segregation of the cell fate determinant Numb during asymmetric cell divisions. Unequal segregation of mammalian Numb has also been observed, but the factors involved are unknown. Here, we identify in vivo phosphorylation sites of mammalian Numb and show that both mammalian and Drosophila Numb interact with, and are substrates for aPKC in vitro. A form of mammalian Numb lacking two protein kinase C (PKC) phosphorylation sites (Numb2A) accumulates at the cell membrane and is refractory to PKC activation. In epithelial cells, mammalian Numb localizes to the basolateral membrane and is excluded from the apical domain, which accumulates aPKC. In contrast, Numb2A is distributed uniformly around the cell cortex. Mutational analysis of conserved aPKC phosphorylation sites in Drosophila Numb suggests that phosphorylation contributes to asymmetric localization of Numb, opposite to aPKC in dividing sensory organ precursor cells. These results suggest a model in which phosphorylation of Numb by aPKC regulates its polarized distribution in epithelial cells as well as during asymmetric cell divisions.

LNX functions as a RING type E3 ubiquitin ligase that targets the cell fate determinant Numb for ubiquitin-dependent degradation.

LNX is a RING finger and PDZ domain containing protein that interacts with the cell fate determinant Numb. To investigate the function of LNX, we tested its RING finger domain for ubiquitin ligase activity. The isolated RING finger domain was able to function as an E2-dependent, E3 ubiquitin ligase in vitro and mutation of a conserved cysteine residue within the RING domain abolished its activity, indicating that LNX is the first described PDZ domain-containing member of the E3 ubiquitin ligase family. We have identified Numb as a substrate of LNX E3 activity in vitro and in vivo. In addition to the RING finger, a region of LNX, including the Numb PTB domain-binding site and the first PDZ domain, is required for Numb ubiquitylation. Expression of wild-type but not mutant LNX causes proteasome-dependent degradation of Numb and can enhance Notch signalling. These results suggest that the levels of mammalian Numb protein and therefore, by extension, the processes of asymmetric cell division and cell fate determination may be regulated by ubiquitin-dependent proteolysis.

Transgenic expression of numb inhibits notch signaling in immature thymocytes but does not alter T cell fate specification.

The conserved adaptor protein Numb is an intrinsic cell fate determinant that functions by antagonizing Notch-mediated signal transduction. The Notch family of membrane receptors controls cell survival and cell fate determination in a variety of organ systems and species. Recent studies have identified a role for mammalian Notch-1 signals at multiple stages of T lymphocyte development. We have examined the role of mammalian Numb (mNumb) as a Notch regulator and cell fate determinant during T cell development. Transgenic overexpression of mNumb under the control of the Lck proximal promoter reduced expression of several Notch-1 target genes, indicating that mNumb antagonizes Notch-1 signaling in vivo. However, thymocyte development, cell cycle, and survival were unperturbed by mNumb overexpression, even though transgenic Numb was expressed at an early stage in thymocyte development (CD4(-)CD8(-)CD3(-) cells that were CD44(+)CD25(+) or CD44(-)CD25(+); double-negative 2/3). Moreover, bone marrow from mNumb transgenic mice showed no defects in thymopoiesis in competitive repopulation experiments. Our results suggest that mNumb functions as a Notch-1 antagonist in immature thymocytes, but that suppression of Notch-1 signaling at this stage does not alter gammadelta/alphabeta or CD4/CD8 T cell fate specification.