Pinilla-Macua, I. | Search bioRxiv

bioRxiv2026

Traffickome infers directed, compartment-resolved membrane trafficking from proteomic data

Membrane trafficking determines the subcellular localization and compartment-residence time of receptors, channels, transporters and other transmembrane proteins and thereby influences where in the cell and how long these proteins function. Functional-enrichment computational approaches may predict the membrane-trafficking regulations but do not resolve the directionality of the transport between cellular compartments. We present Traffickome, a deterministic method organizing 754 trafficking proteins into 11 directed transitions across seven compartments and 1,837 signaling proteins across 22 pathways. Implemented as an interactive browser and open-source Python package, Traffickome infers compartment-to-compartment transport, cargo fate and signaling-pathway activity and enables in silico perturbation from a single proteomic comparison. Across 29 proteomic comparisons spanning 14 studies encompassing over 66,000 gene-level observations across over 10,000 genes, Traffickome ranked the expected trafficking transition first in 23 cases (79%), outperforming the best conventional enrichment methods (17 of 29, 59%). Mean accuracy fell to 18% after randomizing the protein-to-transition assignments. To illustrate the power of Traffickome, we performed time-resolved analysis of the phosphoproteome in EGF-stimulated cells with the goal to distinguish signaling by internalized versus plasma-membrane EGFR. In the resulting datasets, comprising over 18,000 phosphosite-level measurements, Traffickome revealed localization-dependent differences in EGF-induced phosphorylation and prioritized trafficking regulators for testing their effects on signaling processes.

cell biology↗

bioRxiv2026

Small-molecule CBLB inhibitor abolishes EGFR ubiquitination, reduces receptor endocytosis and diminishes cell motility signaling

Endocytosis of the epidermal growth factor receptor (EGFR) is considered a key regulator of the receptor signaling activity. However, the molecular mechanisms underlying EGFR endocytosis are incompletely understood. Although ligand-induced ubiquitination of EGFR is known to promote its endocytic trafficking, the importance of EGFR ubiquitination in clathrin-mediated endocytosis, the primary physiological route of EGFR internalization, remains debated, and the relative contributions of ubiquitination-dependent and - independent mechanisms are not defined. Hence, we used NX-1013, a novel small-molecule inhibitor of the CBLB E3 ubiquitin ligase, to dissect the role of EGFR ubiquitination in its endocytic trafficking and signaling. Strikingly, brief treatment with NX-1013 completely abolished EGF-induced EGFR ubiquitination, demonstrating that this process is exclusively mediated by the closely related CBLB and CBL ligases. NX-1013 inhibited clathrin-mediated internalization of activated EGFR by 60-70%. The remaining, ubiquitination-independent internalization required EGFR kinase activity, was highly clathrin-dependent, and was significantly impaired by depletion of the AP-2 clathrin adaptor complex. Interestingly, inhibition of CBLs and EGFR endocytosis by NX-1013 did not affect major downstream signaling pathways in human oral squamous cell carcinoma cells, with the exception of Rac1 activation and EGFR-dependent cell migration, both of which were suppressed. Significance StatementCBL E3 ubiquitin ligases mediate ubiquitin conjugation of EGFR but their functional contributions to EGFR endocytic trafficking and signaling remain poorly defined. Here, we describe a newly developed small-molecule inhibitor of CBL proteins that potently blocks EGFR ubiquitination. This tool allowed us to dissect ubiquitination-dependent versus - independent components of the clathrin-mediated endocytosis and ligand-induced downregulation of EGFR. Strikingly, while inhibition of CBLs suppressed EGFR-driven cell motility signaling, it spared other major downstream pathways in EGFR-dependent human oral squamous cell carcinoma cells. These findings establish acute inhibition of CBLs as a powerful approach to interrogate ubiquitin-mediated receptor regulation and highlight its potential for therapeutic targeting of cancer cell migration.

cell biology↗