SPATIAL REGULATION OF SIGNALING BY THE ENDOCYTIC PROTEIN NUMB

NUMB was originally identified as a cell fate determinant that controls signaling outputs during asymmetric cell division. NUMB is a tumor suppressor protein that regulates NOTCH and p53 signaling pathways. Loss of NUMB occurs in 50% of all breast tumors and in 30% of lung cancers, and correlates with an increased aggressiveness and poor prognosis. NUMB has been also characterized for its role in trafficking, acting as an adaptor protein and regulating signaling emanating from various plasma membrane receptors, including NOTCH, Receptor Tyrosine Kinases (RTKs), such as EGFR and c-MET, and integrins. Consistent with this latter notion, NUMB interacts with key players of the Clathrin-mediated endocytosis, regulating the internalization of various receptors. However, its action cannot be restricted to the initial steps of internalization at the level of the cell surface, as it also localizes, in C. Elegans and D. Melanogaster, in recycling endosomes and regulates the delivery of cargos back to the plasma membrane. Similarly, in mammalian cells, NUMB and EH (Eps15- Homology)-domain containing proteins co-localize in ARF6-associated recycling vesicles, and NUMB regulates post-endocytic sorting of NOTCH1. The combination of endocytic/recycling trafficking and actin related dynamics is one of the major mechanisms through which spatial restriction of signaling is achieved. Indeed, the correct recycling of active RAC via ARF6 to the plasma membrane is required for the formation of migratory protrusions induced upon RTK stimulation, such as Circular Dorsal Ruffles (CDRs). To identify novel endocytic/trafficking proteins involved in actin dynamics and directional cell migration, we performed an RNAi screening using Peripheral Ruffles (PRs) and CDRs as readout markers. We found that the endocytic adaptor NUMB is as a negative regulator of CDR formation without affecting PR formation. In this thesis, we have characterized NUMB as a negative regulator of CDR formation downstream of c-MET and PDGFR. This function of NUMB is accompanied by an increased mesenchymal mode of motility and cell invasion. In agreement with the notion that spatial recycling of active RAC to the plasma membrane mediated by ARF6 is required for the formation of CDRs, we found that NUMB is enriched in ARF6 recycling compartments and inhibits MHC I and RAC recycling. These evidences suggest that NUMB might act as a negative regulator of ARF6-dependent recycling. Since GEFs are the primary regulatory targets of small GTP-ase, we focused our attention on ARF6 GEFs, which are reported to control actin remodeling, in particular on the EFA6A-D and Cytohesin family members. Among these GEFs, only EFA6B contains an NPxF motif in its N-terminal domain and directly binds the NUMB PTB domain. Functionally, the removal of EFA6B by itself does not alter CDR formation, but it inhibits the increase of CDR formation that is brought about by NUMB down-regulation. Whether NUMB affects EFA6B GEF activity and, as a consequence, ARF6 activation, is currently under investigation. Our data suggest that NUMB may regulate the ARF6-dependent recycling pathway, which is required for migration and invasion of tumor cells, by interacting with and possibly modulating its GEF EFA6B. Of note, up-regulation of this pathway in the absence of NUMB may even account for the aggressiveness of NUMB-negative tumors.