TY - JOUR AU - Alwan, H.A.J. AU - Leeuwen, J.E.M. van PY - 2007 UR - https://hdl.handle.net/2066/36647 AB - 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. TI - UBPY-mediated epidermal growth factor receptor (EGFR) de-ubiquitination promotes EGFR degradation EP - 1669 SN - 1083-351X IS - iss. 3 SP - 1658 JF - Journal of Biological Chemistry VL - vol. 282 DO - https://doi.org/10.1074/jbc.M604711200 ER - TY - JOUR AU - Alwan, H.A.J. AU - Leeuwen, J.E.M. van PY - 2006 UR - https://hdl.handle.net/2066/34663 TI - UBPY-mediated EGFR deubiquitination promotes EGFR degradation EP - 1669 SN - 0021-9258 IS - iss. 3 SP - 1658 JF - Journal of Biological Chemistry ER - TY - JOUR AU - Woning, S.P. van der AU - Rotterdam, W. van AU - Nabuurs, S.B. AU - Venselaar, H. AU - Jacobs-Oomen, S. AU - Wingens, M. AU - Vriend, G. AU - Stortelers, C. AU - Zoelen, E.J.J. van PY - 2006 UR - https://hdl.handle.net/2066/35633 AB - Epidermal growth factor (EGF)-like growth factors bind their ErbB receptors in a highly selective manner, but the molecular basis for this specificity is poorly understood. We have previously shown that certain residues in human EGF (Ser(2)-Asp(3)) and TGFalpha (Glu(26)) are not essential for their binding to ErbB1 but prevent binding to ErbB3 and ErbB4. In the present study, we have used a phage display approach to affinity-optimize the C-terminal linear region of EGF-like growth factors for binding to each ErbB receptor and thereby shown that Arg(45) in EGF impairs binding to both ErbB3 and ErbB4. By omitting all these so-called negative constraints from EGF, we designed a ligand designated panerbin that binds ErbB1, ErbB3, and ErbB4 with similarly high affinity as their wild-type ligands. Homology models, based on the known crystal structure of TGFalpha-bound ErbB1, showed that panerbin is able to bind ErbB1, ErbB3, and ErbB4 in a highly similar manner with respect to position and number of interaction sites. Upon in silico introduction of the experimentally known negative constraints into panerbin, we found that Arg(45) induced local charge repulsion and Glu(26) induced steric hindrance in a receptor-specific manner, whereas Ser(2)-Asp(3) impaired binding due to a disordered conformation. Furthermore, radiolabeled panerbin was used to quantify the level of all three receptors on human breast cancer cells in a single radioreceptor assay. It is concluded that the ErbB specificity of EGF-like growth factors primarily results from the presence of a limited number of residues that impair the unintended interaction with other ErbB receptors. TI - Negative constraints underlie the ErbB specificity of epidermal growth factor-like ligands EP - 40040 SN - 1083-351X IS - iss. 52 SP - 40033 JF - Journal of Biological Chemistry VL - vol. 281 DO - https://doi.org/10.1074/jbc.M603168200 ER - TY - JOUR AU - Alwan, H.A.J. AU - Zoelen, E.J.J. van AU - Leeuwen, J.E.M. van PY - 2003 UR - https://hdl.handle.net/2066/129301 TI - Ligand-induced lysosomal epidermal growth factor receptor (EGFR) degradation is preceded by proteasome-dependent EGFR de-ubiquitination EP - 35790 SN - 1083-351X SP - 35781 JF - Journal of Biological Chemistry VL - vol. 278 DO - https://doi.org/10.1074/jbc.M301326200 ER - TY - JOUR AU - Stortelers, C. AU - Woning, S.P. van der AU - Jacobs-Oomen, S. AU - Wingens, M. AU - Zoelen, E.J.J. van PY - 2003 UR - https://hdl.handle.net/2066/129304 TI - Selective formation of ErbB-2/ErbB-3 heterodimers depends on the ErbB-3 affinity of epidermal growth factor-like ligands EP - 12063 SN - 1083-351X SP - 12055 JF - Journal of Biological Chemistry VL - vol. 278 DO - https://doi.org/10.1074/jbc.M211948200 ER - TY - JOUR AU - Wingens, M. AU - Walma, T. AU - Ingen, H. van AU - Stortelers, C. AU - Leeuwen, J.E.M. van AU - Zoelen, E.J.J. van AU - Vuister, G.W. PY - 2003 UR - https://hdl.handle.net/2066/79479 TI - Structural analysis of an epidermal growth factor/transforming growth factor-alpha chimera with unique ErbB binding specificity EP - 39123 SN - 1083-351X IS - iss. 40 SP - 39114 JF - Journal of Biological Chemistry VL - vol. 278 DO - https://doi.org/10.1074/jbc.M305603200 ER - TY - JOUR AU - Lenferink, A.E.G. AU - Zoelen, E.J.J. van AU - Vugt, M. van AU - Grothe, S. AU - Rotterdam, W. van AU - Poll, M.L.M. van de AU - O'Connor-McCourt, M.D. PY - 2000 UR - https://hdl.handle.net/2066/129332 TI - Superagonistic activation of ErbB-1 by EGF-related growth factors with enhanced association and dissociation rate constants EP - 26753 SN - 1083-351X SP - 26748 JF - Journal of Biological Chemistry VL - vol. 275 ER - TY - JOUR AU - Poll, M.L.M. van de AU - Lenferink, A.E.G. AU - Vugt, A.H.M. van AU - Jacobs, J.J.L. AU - Janssen, J.W.H. AU - Joldersma, M. AU - Zoelen, E.J.J. van PY - 1995 UR - https://hdl.handle.net/2066/216426 TI - A single amino acid exchange, Arg 45 to Ala, generates an epidermal growth facor (EGF) mutant with high affinity for the chicken EGF receptor EP - 22343 SN - 1083-351X SP - 22337 JF - Journal of Biological Chemistry VL - vol. 270 DO - https://doi.org/10.1074/jbc.270.38.22337 L1 - https://repository.ubn.ru.nl/bitstream/handle/2066/216426/216426.pdf?sequence=1 ER -