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Radboud Universiteit Nijmegen, 24 januari 2014, Promotor : Zoelen, E.J.J. van Co-promotor : Leeuwen, J.E.M. van, Contains fulltext : 122889.pdf (publisher's version ) (Open Access)

Contains fulltext : 103221.pdf (publisher's version ) (Closed access)

Contains fulltext : 83789.pdf (publisher's version ) (Closed access)

RU Radboud Universiteit Nijmegen, 05 juli 2010, Promotor : Zoelen, E.J.J. van Co-promotor : Leeuwen, J.E.M. van, Contains fulltext : 77567.pdf (publisher's version ) (Open Access)

Contains fulltext : 75956.pdf (publisher's version ) (Closed access)

Contains fulltext : 75950.pdf (publisher's version ) (Closed access), The epidermal growth factor (EGF)-like growth factors bind their ErbB receptors in a highly selective manner. Recently, we have shown that the sequence YYDLL in the C-terminal linear region is compatible with binding to all ligand-binding ErbB receptors. In the present study, we show that introduction of the YYDLL sequence into the ErbB1 specific ligands EGF and transforming growth factor-alpha (TGFalpha) broadened their receptor specificity towards ErbB4. Upon introduction of the YYDLL sequence into epiregulin, which by itself binds ErbB1 and ErbB4 but not ErbB3, its binding specificity was broadened to ErbB3, concomitant with enhanced affinity for ErbB4. Introduction of the YYDLL sequence into NRG1beta resulted in a 10-fold increase in affinity for ErbB3, without affecting its receptor specificity. Remarkably, the strongly enhanced affinity for ErbB3 negatively influenced their mitogenic activity towards cells coexpressing ErbB2 and ErbB3. These observations are discussed in terms of the optimised ErbB affinity, selectivity and mitogenic potential that have taken place during evolution.

Contains fulltext : 36647.pdf (Publisher’s version ) (Open Access), 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.

Contains fulltext : 34663.pdf (publisher's version ) (Closed access)

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Contains fulltext : 129301.pdf (Publisher’s version ) (Open Access)

Contains fulltext : 79479.pdf (Publisher’s version ) (Open Access)