title: Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes.
abstract: Protein ubiquitination is mediated sequentially by ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3. Uba1 was thought to be the only E1 until the recent identification of Uba6. To differentiate the biological functions of Uba1 and Uba6, we applied an orthogonal ubiquitin transfer (OUT) technology to profile their ubiquitination targets in mammalian cells. By expressing pairs of an engineered ubiquitin and engineered Uba1 or Uba6 that were generated for exclusive interactions, we identified 697 potential Uba6 targets and 527 potential Uba1 targets with 258 overlaps. Bioinformatics analysis reveals substantial differences in pathways involving Uba1- and Uba6-specific targets. We demonstrate that polyubiquitination and proteasomal degradation of ezrin and CUGBP1 require Uba6, but not Uba1, and that Uba6 is involved in the control of ezrin localization and epithelial morphogenesis. These data suggest that distinctive substrate pools exist for Uba1 and Uba6 that reflect non-redundant biological roles for Uba6.
authors.affiliation: Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Xianpeng
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA.|School of Pharmacy, Shanghai Jiao Tong University, Shanghai 20040, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Zhao
authors.affiliation: Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Limin
authors.initials: L
authors.lastname: Sun
authors.affiliation: Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA.|Department of Chemistry, Center for Diagnostics &Therapeutics, Georgia State University, Atlanta, Georgia 30303, USA.
authors.forename: Karan
authors.initials: K
authors.lastname: Bhuripanyo
authors.affiliation: Department of Chemistry, Center for Diagnostics &Therapeutics, Georgia State University, Atlanta, Georgia 30303, USA.
authors.forename: Yiyang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Preventive Medicine, Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Yingtao
authors.initials: Y
authors.lastname: Bi
authors.affiliation: Department of Preventive Medicine, Northwestern University, Chicago, Illinois 60611, USA.|Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Ramana V
authors.initials: RV
authors.lastname: Davuluri
authors.affiliation: Integrated Proteomics Core, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Duc M
authors.initials: DM
authors.lastname: Duong
authors.affiliation: Chemistry of Life Processes Institute, Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Dhaval
authors.initials: D
authors.lastname: Nanavati
authors.affiliation: Department of Chemistry, University of Chicago, Chicago, Illinois 60637, USA.|Department of Chemistry, Center for Diagnostics &Therapeutics, Georgia State University, Atlanta, Georgia 30303, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Yin
authors.affiliation: Department of Pharmacology, Northwestern University, Chicago, Illinois 60611, USA.|Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, Illinois 60611, USA.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Kiyokawa
mesh_terms: D000067879:CELF1 Protein / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D045744:Cell Line, Tumor; D019295:Computational Biology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004848:Epithelium / Q000254:growth & development; D057809:HEK293 Cells; D006801:Humans; D009024:Morphogenesis / Q000502:physiology; D016254:Mutagenesis, Insertional; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D059748:Proteolysis; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013379:Substrate Specificity / Q000502:physiology; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044764:Ubiquitin-Activating Enzymes / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000502:physiology*; D000099149:Ezrin
chemical_list: D000067879:CELF1 Protein; D003598:Cytoskeletal Proteins; D046988:Proteasome Endopeptidase Complex; D034741:RNA, Small Interfering; D025801:Ubiquitin; D044764:Ubiquitin-Activating Enzymes; D000099149:Ezrin; C102843:CELF1 protein, human; C471538:UBA1 protein, human; C521757:UBA6 protein, human; C491802:NAE protein, human