Alwan HA, van Leeuwen JE.. UBPY-mediated epidermal growth factor receptor (EGFR) de-ubiquitination promotes EGFR degradation. J Biol Chem 282: 1658-1669
ABSTRACT Whereas poly-ubiquitination targets protein substrates for proteasomal degradation, mono-ubiquitination is known to regulate protein trafficking in the endosomal system and to target cargo proteins for lysosomal degradation. The role of the de-ubiquitinating enzymes AMSH and UBPY in endosomal trafficking of cargo proteins such as the epidermal growth factor receptor (EGFR) has only very recently been the subject of study and is already a matter of debate. Although one report (Mizuno, E., Iura, T., Mukai, A., Yoshimori, T., Kitamura, N., and Komada, M. (2005) Mol. Biol. Cell 16, 5163-5174) concludes that UBPY negatively regulates EGFR degradation by de-ubiquitinating the EGFR on endosomes, another report (Row, P. E., Prior, I. A., McCullough, J., Clague, M. J., and Urbe, S. (2006) J. Biol. Chem. 281, 12618-12624) concludes that UBPY-mediated EGFR de-ubiquitination is essential for EGFR degradation. Here, we demonstrate that Usp8/UBPY, the mammalian ortholog of budding yeast Ubp4/Doa4, constitutively co-precipitates in a bivalent manner with the EGFR. Moreover, UBPY is a substrate for Src-family tyrosine kinases that are activated after ligand-induced EGFR activation. Using overexpression of three different recombinant dominant negative UBPY mutants (UBPY C748A mutant, UBPY 1-505, and UBPY 640-1080) in NIH3T3 and HEK293 cells, we demonstrate that UBPY affects both constitutive and ligand-induced (i) EGFR ubiquitination, (ii) EGFR expression levels, and (iii) the appearance of intermediate EGFR degradation products as well as (iv) downstream mitogen-activated protein kinase signal transduction. Our findings provide further evidence in favor of the model that UBPY-mediated EGFR de-ubiquitination promotes EGFR degradation.
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- "Finally, Devor and co-workers (Balut et al., 2011) chose to pursue USP8 because that DUB had been reported in deubiquitylating proteins targeted to the lysosome for degradation (Alwan and van Leeuwen, 2007). After which, they used USP8 overexpression and knockdown experiments to unequivocally demonstrate that USP8 was imperative for proper deubiquitylation of KCa3.1 and delivery of KCa3.1 to the lysosome for degradation (Balut et al., 2011). "
ABSTRACT: The field of ubiquitylation and deubiquitylation of proteins in molecular physiology is growing at a rapid rate. Our understanding of molecular physiology of these processes may become limited by the advancement of technologies that scientists can employ. Therefore, it is important to approach physiological questions of ubiquitylation and deubiquitylation of proteins from a multiple methodological direction. Indeed, the role of ubiquitylation and deubiquitylation of proteins in cellular function has been implicated in the pathophysiology of human diseases including cancer, viral diseases, and neurodegenerative disorders. There are many modulators (activators and inhibitors) of ubiquitylation and deubiquitylation. Therefore, the link is being able to rapidly assess potential modulators of ubiquitylation and deubiquitylation and determine which specific modulators play a role(s) within a particular physiological setting. After the specific modulators have been identified, further experimentation is required to assess the downstream use as potential clinical targets for a particular disease. The first step is to identify the specific modulators. This perspective highlights a multi-prong technologies approach that uses three novel technologies (BLAP-tagged proteins, TUBES, and DUB-Chips) that can rapidly identify a number of potential candidates that modulate ubiquitylation and deubiquitylation of cellular proteins.Frontiers in Physiology 05/2012; 3:137. DOI:10.3389/fphys.2012.00137 · 3.50 Impact Factor
The EMBO Journal 07/2010; 29(13):2099-100. DOI:10.1038/emboj.2010.132 · 10.75 Impact Factor
- "Mukai et al found no evidence that Fz ubiquitylation– deubiquitylation is influenced by Wnt ligand. This is different from the situation with EGFR, wherein mono-ubiquitylation is ligand dependent (Mizuno et al, 2005; Alwan and van Leeuwen, 2007; Niendorf et al, 2007; Row et al, 2007). Rather than acting in response to Wnt, Fz recycling is important for setting the level of responsiveness of the receiving cells to the Wnt ligand. "
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- "Interestingly, these enzymes appear to exert opposing effects on lysosomal degradation of activated RTKs, though their precise involvement remains unclear (Alwan and van Leeuwen, 2007; McCullough et al., 2006). "
ABSTRACT: Genetic screens performed in worms identified major regulators of the epidermal growth factor receptor (EGFR) pathway, including the ubiquitin ligase Cbl/SLI-1. Here we focus on the less-characterized Lst2 protein and confirm suppression of MAPK signals. Unexpectedly, human Lst2, a monoubiquitinylated phosphoprotein, does not localize to endosomes, despite an intrinsic phosphoinositol-binding FYVE domain. By constructing an ubiquitinylation-defective mutant and an ubiquitin fusion, we conclude that endosomal localization of Lst2, along with an ability to divert incoming EGFR molecules to degradation in lysosomes, is regulated by ubiquitinylation/deubiquitinylation cycles. Consistent with bifurcating roles, Lst2 physically binds Trim3/BERP, which interacts with Hrs and a complex that biases cargo recycling. These results establish an ubiquitin-based endosomal switch of receptor sorting, functionally equivalent to the mechanism inactivating Hrs via monoubiquitinylation.Developmental Cell 06/2009; 16(5):687-98. DOI:10.1016/j.devcel.2009.03.015 · 10.37 Impact Factor