Recycling of EGFR and ErbB2 is associated with impaired Hrs tyrosine phosphorylation and decreased deubiquitination by AMSH.
ErbB receptors play an important role in normal cellular growth, differentiation and development, but overexpression or poor downregulation can result in enhanced signaling and cancerous growth. ErbB signaling is terminated by clathrin-dependent receptor-mediated endocytosis, followed by incorporation in multi-vesicular bodies and subsequent degradation in lysosomes. In contrast to EGFR, ErbB2 displays poor ligand-induced downregulation and enhanced recycling, but the molecular mechanisms underlying this difference are poorly understood. Given our previous observation that both EGFR and an EGFR-ErbB2 chimera undergo Cbl-mediated K63-polyubiquitination, we investigated in the present study whether activation of the EGFR and the EGFR-ErbB2 chimera is associated with tyrosine phosphorylation of the ESCRT-0 complex subunit Hrs and AMSH-mediated deubiquitination. EGF stimulation of the EGFR resulted in efficient Hrs tyrosine phosphorylation and deubiquitination by the K63-polyubiquitin chain-specific deubiquitinating enzyme AMSH. In contrast, EGF activation of EGFR-ErbB2 showed significantly decreased Hrs tyrosine phosphorylation and deubiquitination by AMSH. To test whether this phenotype is the result of endosomal recycling, we induced recycling of the EGFR by stimulation with TGFα. Indeed, even though TGFα-stimulation of EGFR is associated with efficient ligand-stimulated K63-polyubiquitination, we observed that Hrs tyrosine phosphorylation as well as AMSH-mediated deubiquitination is significantly reduced under these conditions. Using various EGFR-ErbB2 chimeras, we demonstrate that enhanced recycling, decreased Hrs tyrosine phosphorylation and decreased AMSH mediated deubiquitination of EGFR-ErbB2 chimeras is primarily due to the presence of ErbB2 sequences or the absence of EGFR sequences C-terminal to the Cbl binding site. We conclude that endosomal recycling of the EGFR and ErbB2 receptors is associated with significantly impaired tyrosine phosphorylation of the ESCRT-0 subunit Hrs as well as decreased deubiquitination by AMSH, which is consistent with the finding that recycling receptors are not efficiently incorporated in the MVB 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
Walter
W
van Rotterdam
Everardus J J
EJ
van Zoelen
Jeroen E M
JE
van Leeuwen
D000818:Animals; D015536:Down-Regulation / Q000187:drug effects; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D016211:Transforming Growth Factor alpha / Q000494:pharmacology; D043222:Ubiquitin Thiolesterase / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
D056827:Endosomal Sorting Complexes Required for Transport; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D010750:Phosphoproteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; D043222:Ubiquitin Thiolesterase; C486696:STAMBP protein, human; C096164:hepatocyte growth factor-regulated tyrosine kinase substrate