ERBB2 is a target for USP8-mediated deubiquitination.
Overexpression and poor downregulation of ErbB receptor tyrosine kinases are associated with enhanced signaling and tumorigenesis. Attenuation of EGF-receptor (EGFR) signaling is mediated by endocytosis and ubiquitination by the E3-ligase Cbl. En route to lysosomes, but before incorporation of the EGFR into internal vesicles of MVBs, the EGFR undergoes Usp8-mediated deubiquitination. ErbB2 displays enhanced recycling back to the cell surface, and therefore we hypothesized that Usp8 is not part of the ErbB2 trafficking pathway. Here, we demonstrate, in the context of a chimeric EGFR-ErbB2 receptor, that (i) EGF induces pY1091 Cbl binding site-dependent K63-polyubiquitination of EGFR-ErbB2, (ii) Cbl is tyrosine phosphorylated upon stimulation of EGFR-ErbB2 wt and Y1091F mutant receptor, (iii) EGF-induced activation of EGFR-ErbB2 induces Usp8 tyrosine phosphorylation, and (iv) ubiquitination of the EGFR-ErbB2 wt and Y1091F mutant is enhanced upon coexpression of catalytically inactive Usp8-C748A in the presence and absence of EGF. We further show that Usp8 tyrosine phosphorylation upon stimulation of EGFR-ErbB2 is (a) independent of Y1091, (b) dependent on Src- and EGFR-ErbB2-kinase activity, (c) enhanced upon coexpression of Usp8-C748A, and (d) partly dependent on the Microtubule Interacting and Transport (MIT) domain of Usp8. Our findings demonstrate that Usp8 is part of the ErbB2 endosomal trafficking pathway.
Department of Cell & Applied Biology, Faculty of Science, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
Inez M J
IM
Meijer
Jeroen E M
JE
van Leeuwen
D016475:3T3 Cells; D000818:Animals; D010450:Endopeptidases / Q000502:physiology*; D056827:Endosomal Sorting Complexes Required for Transport / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D010766:Phosphorylation; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000502:physiology*; D054875:Ubiquitination; D019061:src-Family Kinases / Q000378:metabolism
D010450:Endopeptidases; D056827:Endosomal Sorting Complexes Required for Transport; D050721:Proto-Oncogene Proteins c-cbl; D018719:Erb-b2 Receptor Tyrosine Kinases; D043222:Ubiquitin Thiolesterase; D019061:src-Family Kinases; C541534:USP8 protein, human