MOLECULAR CHARACTERIZATION OF THE ROLE OF CBL PROTEINS IN EGFR ENDOCYTOSIS

c-Cbl is the major E3 ligase involved in ubiquitination of Epidermal Growth Factor Receptor (EGFR). Ubiquitination by c-Cbl plays a critical role in EGFR endocytosis by targeting receptors to lysosomal degradation. Its involvement at early internalization steps is still debated, also due to the fact that multiple internalization pathways were described. Indeed, EGFR ubiquitination is required for non-clathrin mediated endocytosis (NCE), while it is not essential for clathrin endocytosis (CME). However, c-Cbl might still play a crucial function also in CME since, in addition to its role as an E3 ligase, it works also as an adaptor, by recruiting several proteins involved in the early phases of this process. Importantly, c-Cbl has been found mutated in different disorders, from myeloproliferative disease to Noonan syndrome and non-small cell lung cancer (NSCLC). Most of these mutations are located within the Ring finger domain and in the regulatory linker region, and are therefore predicted to affect E3 ligase activity. However, some mutations map outside this region, suggesting that they might impinge on the adaptor function without altering E3 ligase activity. None of these mutations was characterized in detail at the mechanistic level. In order to draw a more precise molecular picture of c-Cbl activity in EGFR ubiquitination and endocytosis, we investigated the effects of different set of cancer-relevant mutations, combining two distinct approaches: 1) RNA interference-based functional assays and 2) in vitro ubiquitination assays. 1) First, we characterized the effect of the knockdown (KD) of c-Cbl (and its family members) on EGFR ubiquitination and endocytosis in two different cell systems, murine fibroblast and HeLa cells. From our data, we confirmed that c-Cbl is essential for NCE, by ubiquitinating the EGFR; however, it also plays a role in CME. Importantly, reconstitution experiments with RING finger mutants demonstrated that c-Cbl E3 ligase activity is also required for CME. Since EGFR ubiquitination is not essential for CME, we hypothesize that this activity is exerted not directly on the receptor but on endocytic adaptors. In agreement, Eps15 monoubiquitination is impaired upon c-Cbl KD. 2) We were able to reconstitute the EGFR ubiquitination reaction in vitro, and now we can use this tool to study the molecular details of c-Cbl catalysis. Moreover, in order to dissect c-Cbl adaptor function vs E3 ligase activity, we plan to investigate the phenotype of c-Cbl mutations that map outside the E3 ligase domain in EGFR internalization and ubiquitination, exploiting both in vivo analyses (through reconstitution experiments in cell lines) and in vitro ubiquitination assay

RILP regulates vacuolar ATPase through interaction with the V1G1 subunit

Rab-interacting lysosomal protein (RILP) is a downstream effector of the Rab7 GTPase. GTP-bound Rab7 recruits RILP to endosomal membranes and, together, they control late endocytic traffic, phagosome and autophagosome maturation and are responsible for signaling receptor degradation. We have identified, using different approaches, the V1G1 (officially known as ATP6V1G1) subunit of the vacuolar ATPase (V-ATPase) as a RILP-interacting protein. V1G1 is a component of the peripheral stalk and is fundamental for correct V-ATPase assembly. We show here that RILP regulates the recruitment of V1G1 to late endosomal and lysosomal membranes but also controls V1G1 stability. Indeed, we demonstrate that V1G1 can be ubiquitylated and that RILP is responsible for proteasomal degradation of V1G1. Furthermore, we demonstrate that alterations in V1G1 expression levels impair V-ATPase activity. Thus, our data demonstrate for the first time that RILP regulates the activity of the V-ATPase through its interaction with V1G1. Given the importance of V-ATPase in several cellular processes and human diseases, these data suggest that modulation of RILP activity could be used to control V-ATPase function

The Rab-interacting lysosomal protein (RILP) regulates vacuolar ATPase acting on the V1G1 subunit

RILP is a downstream effector of the Rab7 GTPase. GTP-bound Rab7 recruits RILP on endosomal membranes and, together, they control late endocytic traffic, phagosome and autophagosome maturation and are responsible for signaling receptor degradation. We have identified, using different approaches, the V1G1 subunit of the vacuolar ATPase (V-ATPase) as a RILP interacting protein. V1G1 is a component of the peripheral stalk and it is fundamental for correct V-ATPase assembly. We established that RILP regulates the recruitment of V1G1 subunit to late endosomal/lysosomal membranes but also controls V1G1 stability. Indeed, we demonstrated that V1G1 is ubiquitinated and that RILP is responsible for proteasomal degradation of V1G1. Furthermore, we demonstrated that alterations of V1G1 expression levels impair V-ATPase activity. Thus, our data demonstrate for the first time that RILP regulates the activity of the V-ATPase through the interaction with V1G1. Given the importance of V-ATPase in several cellular processes and human diseases, these data suggest that modulation of RILP activity could be used to control V-ATPase function

RILP regulates vacuolar ATPase through interaction with the V1G1 subunit

Erratum for RILP regulates vacuolar ATPase through interaction with the V1G1 subunit. [J Cell Sci. 2014

Threshold-controlled ubiquitination of the EGFR directs receptor fate

How the cell converts graded signals into threshold-activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)-activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose-response curve of EGFR ubiquitination correlate precisely with the non-clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR-NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re-engineered on a phosphorylation/ubiquitination-incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation

Chromopynones are pseudo natural product glucose uptake inhibitors targeting glucose transporters GLUT-1 and -3

The principles guiding the design and synthesis of bioactive compounds based on natural product (NP) structure, such as biology-oriented synthesis (BIOS), are limited by their partial coverage of the NP-like chemical space of existing NPs and retainment of bioactivity in the corresponding compound collections. Here we propose and validate a concept to overcome these limitations by de novo combination of NP-derived fragments to structurally unprecedented ‘pseudo natural products’. Pseudo NPs inherit characteristic elements of NP structure yet enable the efficient exploration of areas of chemical space not covered by NP-derived chemotypes, and may possess novel bioactivities. We provide a proof of principle by designing, synthesizing and investigating the biological properties of chromopynone pseudo NPs that combine biosynthetically unrelated chromane- and tetrahydropyrimidinone NP fragments. We show that chromopynones define a glucose uptake inhibitor chemotype that selectively targets glucose transporters GLUT-1 and -3, inhibits cancer cell growth and promises to inspire new drug discovery programmes aimed at tumour metabolism