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.