CHARACTERIZATION OF THE UBIQUITIN LIGASE, UBE4B, IN ENDOCYTIC TRAFFICKING

Endocytosis is a process by which cells internalize membrane proteins to remove them from the plasma membrane, allowing cells to regulate the cell surface expression of transmembrane proteins. In this manner, cellular responses to extracellular cues may be tuned by limiting the number of proteins available at the cell surface. One particular class of proteins, receptor tyrosine kinases (RTK), is internalized upon binding to extracellular ligands during their residence at the cell surface. The epidermal growth factor receptor (EGFR) is an RTK whose trafficking through the endocytic pathway through the cell is well-documented. Stimulation of EGFR with its cognate ligand, EGF, prompts EGFR entry into the endocytic pathway and simultaneously activates downstream signal transduction pathways that regulate physiological responses, such as survival, proliferation, and differentiation. Activated EGFRs continue to signal as they traverse the endocytic pathway until the ligand-receptor complex is included into vesicles that bud into the lumen of the multivesicular body (MVB). Inclusion into internal MVB vesicles designates EGFR for lysosomal proteolysis and extinguishes their signaling activity. Ubiquitination is a post-translational modification that underlies some aspects of membrane protein trafficking. Ubiquitin modification allows EGFR recognition by endosomal protein complexes that mediate protein inclusion into MVB vesicles from those that remain on the MVB membrane for incorporation into other cellular structures (e.g. plasma membrane, Golgi). The endosomal sorting machinery consists of a core group of cytosolic proteins that are recruited to the endosomal membrane, called the endosomal sorting complexes required for transport (ESCRT) machinery. A subset of ESCRT proteins bind directly to ubiquitin, allowing the sorting machinery to engage and manipulate the movement of protein cargo into inwardly budded MVB vesicles. Lack of a ubiquitin tag precludes EGFR from inclusion into vesicles that bud into the lumen of the MVB. The precise nature of coordination between the cellular machineries that govern ubiquitination and endosomal sorting are not well understood. I have identified a protein interaction between UBE4B, an E3/E4 ubiquitin ligase, and the ESCRT-0 components, Hrs and STAM. ESCRT-0 is the first complex of the endosomal sorting machinery to bind to endosomes, and recognition by ESCRT-0 is required for EGFR sorting and degradation. Immunoprecipitation and ubiquitination assays revealed that UBE4B binds and can ubiquitinate the EGFR. Depletion of UBE4B resulted in impaired EGFR inclusion into inwardly-budded MVB vesicles. EGFR inclusion was rescued more efficiently with the addition of recombinant UBE4B versus the addition of a ubiquitination-defective mutant, UBE4B(P1140A). These data suggest that the ubiquitination of EGFR by UBE4B is required for efficient EGFR sorting. Altering UBE4B expression in neuroblastoma cells revealed a negative correlation between UBE4B expression and proliferation, as well as altered proliferative responses to EGR inhibitors. These findings suggest a mechanism by which UBE4B may regulate cell proliferation, and reveals UBE4B as a potential target in neuroblastoma therapeutic development