title: Deubiquitinase FAM/USP9X interacts with the E3 ubiquitin ligase SMURF1 protein and protects it from ligase activity-dependent self-degradation.
abstract: Ubiquitination is an essential post-translational modification that mediates diverse cellular functions. SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) belongs to the Nedd4 family of HECT ubiquitin ligases that directly catalyzes ubiquitin conjugation onto diverse substrates. As a result, SMURF1 regulates a great variety of cellular physiologies including bone morphogenetic protein (BMP) signaling, cell migration, and planar cell polarity. Structurally, SMURF1 consists of a C2 domain, two WW domain repeats, and a catalytic HECT domain essential for its E3 ubiquitin ligase activity. This modular architecture allows for interactions with other proteins, which are either substrates or adaptors of SMURF1. Despite the increasing number of SMURF1 substrates identified, current knowledge regarding regulatory proteins and their modes of action on controlling SMURF1 activity is still limited. In this study, we employed quantitative mass spectrometry to analyze SMURF1-associated cellular complexes, and identified the deubiquitinase FAM/USP9X as a novel interacting protein for SMURF1. Through domain mapping study, we found the second WW domain of SMURF1 and the carboxyl terminus of USP9X critical for this interaction. SMURF1 is autoubiquitinated through its intrinsic HECT E3 ligase activity, and is degraded by the proteasome. USP9X association antagonizes this activity, resulting in deubiquitination and stabilization of SMURF1. In MDA-MB-231 breast cancer cells, SMURF1 expression is elevated and is required for cellular motility. USP9X stabilizes endogenous SMURF1 in MDA-MB-231 cells. Depletion of USP9X led to down-regulation of SMURF1 and significantly impaired cellular migration. Taken together, our data reveal USP9X as an important regulatory protein of SMURF1 and suggest that the association between deubiquitinase and E3 ligase may serve as a common strategy to control the cellular protein dynamics through modulating E3 ligase stability.
authors.affiliation: Developmental and Molecular Pathways, Novartis Institutes for BioMedical Research, Cambridge, Massachusetts 02139, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xie
authors.forename: Monika
authors.initials: M
authors.lastname: Avello
authors.forename: Markus
authors.initials: M
authors.lastname: Schirle
authors.forename: Elizabeth
authors.initials: E
authors.lastname: McWhinnie
authors.forename: Yan
authors.initials: Y
authors.lastname: Feng
authors.forename: Eva
authors.initials: E
authors.lastname: Bric-Furlong
authors.forename: Christopher
authors.initials: C
authors.lastname: Wilson
authors.forename: Robin
authors.initials: R
authors.lastname: Nathans
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Marc W
authors.initials: MW
authors.lastname: Kirschner
authors.forename: Shih-Min A
authors.initials: SM
authors.lastname: Huang
authors.forename: Feng
authors.initials: F
authors.lastname: Cong
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D015536:Down-Regulation; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D043222:Ubiquitin Thiolesterase / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000235:genetics
chemical_list: D012333:RNA, Messenger; C506234:USP9X protein, human; C498342:SMURF1 protein, human; D044767:Ubiquitin-Protein Ligases; D043222:Ubiquitin Thiolesterase; D046988:Proteasome Endopeptidase Complex