Alwan HA, van Leeuwen JE.. UBPY-mediated epidermal growth factor receptor (EGFR) de-ubiquitination promotes EGFR degradation. J Biol Chem 282: 1658-1669
Department of Cell Biology, Institute for Neuroscience, Faculty of Natural Sciences, Mathematics and Informatics, Radboud University Nijmegen, 6525 ED Nijmegen, The Netherlands.Journal of Biological Chemistry (Impact Factor: 4.57). 02/2007; 282(3):1658-69. DOI: 10.1074/jbc.M604711200
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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- "MVB sorting of ubiquitylated EGFR involves several ubiquitin-binding protein complexes, termed endosomal sorting complex required for transport (ESCRT)-0, -I and -II (Bishop et al., 2002; Doyotte et al., 2005; Garrus et al., 2001; Henne et al., 2011; Hurley and Stenmark, 2011; Komada and Soriano, 1999; Malerød et al., 2007; Raiborg and Stenmark, 2009; Raiborg et al., 2002; Razi and Futter, 2006; Ren and Hurley, 2010; Slagsvold et al., 2005; Wollert and Hurley, 2010). Although the function of ESCRT-II is less well characterised, ESCRT-0 and ESCRT-I combine with the scaffold protein, his domain protein tyrosine phosphatase (HD-PTP, also known as PTPN23) (Ali et al., 2013; Doyotte et al., 2008; Stefani et al., 2011) and the deubiquitylating enzyme, UBPY/USP8 (Alwan and van Leeuwen, 2007; Bowers et al., 2006; Pareja et al., 2012; Row et al., 2006), to facilitate the transfer of EGFR to a further complex, ESCRT-III. ESCRT-III drives the formation of the intralumenal vesicles (ILVs) that capture the receptor within the MVB (Henne et al., 2012; Wollert and Hurley, 2010). "
ABSTRACT: ESCRT-I is essential for the multivesicular body (MVB) sorting of ubiquitinated cargo such as epidermal growth factor receptor, as well as for divergent cellular functions such as cell division and retroviral budding. ESCRT-I has four subunits; TSG101, VPS28, VPS37 and MVB12. There are several members of VPS37 and MVB12 families in mammalian cells, and their differential incorporation into ESCRT-I could provide function-specific variants of the complex. However, it remains unclear whether these different forms of VPS37 and MVB12 combine randomly or generate selective pairings within ESCRT-I, and what the mechanistic basis for such pairing would be. Here we show that the incorporation into ESCRT-I of two MVB12 members, UBAP1 and MVB12A, is highly selective with respect to their VPS37 partners. We map the selective assembly of UBAP1/VPS37A to the core ESCRT-I binding domain of VPS37A. In contrast, selective integration of UBAP1 requires both the minimal ESCRT-I binding region and a neighbouring predicted helix. The biochemical specificity in ESCRT-I assembly is matched by functional specialisation, since siRNA-mediated depletion of UBAP1, but not MVB12A or MVB12B, disrupts ubiquitin-dependent sorting at the MVB.Journal of Cell Science 11/2013; 127(3). DOI:10.1242/jcs.140673 · 5.43 Impact Factor
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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 · 3.53 Impact Factor
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- "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. "
ABSTRACT: Wnt/b-catenin signalling is initiated by binding of secreted Wnt ligands to Frizzled and LRP5/6/Arrow co-receptors. A new study in this issue of The EMBO Journal provides compelling evidence that the level of cell surface Frizzled is controlled by a cycle of mono-ubiquitylation-deubiquitylation, the latter being mediated by the deubiquitylating enzyme UBPY/USP8. The amount of Frizzled on the plasma membrane appears to be a major rate-limiting factor in determining a cell's Wnt responsiveness. © 2010 European Molecular Biology Organization | All Rights Reserved.The EMBO Journal 07/2010; 29(13):2099-100. DOI:10.1038/emboj.2010.132 · 10.43 Impact Factor
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