Abstract
Downregulation of receptor tyrosine kinases and growth factor signaling is imperative for the prevention of malignancy formation. Eps15 is a ubiquitin-binding endocytic adaptor protein essential for clathrin-dependent endocytosis and this process. Although it has been well-studied at the plasma membrane, Eps15 has also been found at low levels on early endosomes. However, the basis of this localization is currently unknown. Thus, determining why Eps15 is recruited to different membranes is important for understanding its involvement in the attenuation of downstream signaling. Using geldanamycin (GA), we induced the internalization and ubiquitination of ErbB2, a member of the EGF family of growth receptors. We found that in geldanamycin-treated SK-BR-3 breast cancer cells, FLAG-Eps15 colocalized with ErbB2-containing vesicles remarkably well as compared to steady-state cells. This recruitment suggested Eps15 may localize to endosomes in response to ubiquitinated cargo. To further test the existence of ubiquitin-dependent binding, we used constructs containing ubiquitin fused in-frame targeted to the plasma membrane (PM-GFP-Ub) or early endosomes (GFP-FYVE(Hrs)-Ub∆GG). We found that FLAG-Eps15 also colocalized with these constructs at EEA1-positive endosomes. This implied the recruitment of Eps15 to endosomes is ubiquitin-dependent. Colocalization analysis of Eps15 mutant constructs also revealed that the UIM domains (ubiquitin interacting motifs) but not the EH domains were required for endosomal recruitment. Hrs, a highly conserved ubiquitin sorting protein localizes to early endosomes and helps traffic ubiquitinated cargo to lysosomes for degradation. One possible method of Eps15 recruitment may be via its known interaction with this key endosomal sorting protein. However, silencing of the endosomal Eps15 binding partner Hrs did not affect recruitment of Eps15 to ubiquitin-enriched endosomes, suggesting an alternate mechanism. Taken together, our findings suggest that Eps15 can be recruited to endosomes by directly binding ubiquitinated cargo. Recruitment of Eps15 to ubiquitin-rich endosomes may act as a rheostat, sequestering the protein away from the plasma membrane, slowing endocytosis to allow time for clearance of ubiquitinated cargo from endosomes. This provides evidence for the first time that ubiquitin is sufficient in recruiting Eps15 differentially to membranes, independent of clathrin-mediated endocytosis.
