title: The Usp8 deubiquitination enzyme is post-translationally modified by tyrosine and serine phosphorylation.
abstract: The ERBB1-ERBB4 receptors belong to a family of receptor tyrosine kinases that trigger a network of signaling pathways after ligand binding, thereby regulating cellular growth, differentiation and development. Ligand-induced signaling through ERBB1, also known as EGFR, is attenuated by the clathrin-dependent receptor-mediated endocytosis and RING E3-ligase Cbl-mediated receptor ubiquitination, which is followed by incorporation into multi-vesicular bodies (MVBs) and subsequent degradation in lysosomes. Before incorporation into MVBs, the EGFR is deubiquitinated by Usp8. We previously demonstrated that Usp8 is tyrosine phosphorylated in an EGFR- and SRC-kinase dependent manner. In the present study we show that overexpression of constitutively active SRC enhances constitutive and ligand-induced Usp8 tyrosine phosphorylation. We also show that enhanced endosomal recycling of the EGFR induced by TGFα stimulation is associated with decreased Usp8 tyrosine phosphorylation. We therefore hypothesize that tyrosine phosphorylation of Usp8 could regulate the function of Usp8. To identify Usp8 tyrosine phosphorylation site(s), we used Usp8 deletion constructs, site-directed mutagenesis of nine individual Usp8 tyrosine residues and mass spectrometry (MS) analysis. Our results demonstrate that the MIT-domain is necessary for ligand-induced tyrosine phosphorylation of Usp8 1-504. However, mutation of three MIT domain tyrosine residues did not abolish Usp8 tyrosine phosphorylation. Similar results were obtained upon mutation of six exposed tyrosine residues in the Rhod domain and linker region. Repeated MS analysis of both Usp8 WT and C748A mutants readily detected serine phosphorylation, including the S680 14-3-3 binding site, but did not reveal any phospho-tyrosine residues. Notably, mutation of the tyrosine residue in the Usp8 14-3-3 binding motif (Y679) did not abolish phosphoserine-dependent binding of 14-3-3 to Usp8. Our findings are most consistent with the model that MIT domain-dependent recruitment of Usp8 to endosomal membranes is important for low stoichiometry SRC-mediated tyrosine phosphorylation of multiple Usp8 tyrosines. Our findings demonstrate that Usp8 is a target for the post-translational serine and tyrosine phosphorylation, most likely characterized by low abundant tyrosine phosphorylation on multiple residues, and high abundant serine phosphorylation on several residues.
authors: Department of Cell & Applied Biology, Faculty of Science, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen, Nijmegen, The Netherlands.
authors: Inez M J
authors: IM
authors: Meijer
authors: JoAnn
authors: J
authors: Kerperien
authors: Ana M
authors: AM
authors: Sotoca
authors: Everardus J J
authors: EJ
authors: van Zoelen
authors: Jeroen E M
authors: JE
authors: van Leeuwen
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016296:Mutagenesis; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism*; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000737:chemistry / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D016211:Transforming Growth Factor alpha; D014443:Tyrosine; D012694:Serine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D019061:src-Family Kinases; D010450:Endopeptidases; C541534:USP8 protein, human; D043222:Ubiquitin Thiolesterase