PMID- 27503909 OWN - NLM STAT- MEDLINE DCOM- 20170612 LR - 20220129 IS - 1470-8728 (Electronic) IS - 0264-6021 (Print) IS - 0264-6021 (Linking) VI - 473 IP - 20 DP - 2016 Oct 15 TI - Gsk3beta and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease. PG - 3563-3580 AB - Fbxo7 is a clinically relevant F-box protein, associated with both cancer and Parkinson's disease (PD). Additionally, SNPs within FBXO7 are correlated with alterations in red blood cell parameters. Point mutations within FBXO7 map within specific functional domains, including near its F-box domain and its substrate recruiting domains, suggesting that deficiencies in SCF(Fbxo7/PARK15) ubiquitin ligase activity are mechanistically linked to early-onset PD. To date, relatively few substrates of the ligase have been identified. These include HURP (hepatoma up-regulated protein), whose ubiquitination results in proteasome-mediated degradation, and c-IAP1 (inhibitor of apoptosis protein 1), TNF receptor-associated factor 2 (TRAF2), and NRAGE, which are not destabilized as a result of ubiquitination. None of these substrates have been linked directly to PD, nor has it been determined whether they would directly engage neuronal cell death pathways. To discover ubiquitinated substrates of SCF(Fbxo7) implicated more directly in PD aetiology, we conducted a high-throughput screen using protein arrays to identify new candidates. A total of 338 new targets were identified and from these we validated glycogen synthase kinase 3beta (Gsk3beta), which can phosphorylate alpha-synuclein, and translocase of outer mitochondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCF(Fbxo7) substrates both in vitro and in vivo Ubiquitin chain restriction analyses revealed that Fbxo7 modified Gsk3beta using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3beta activity, rather than its levels or localization. In addition, Fbxo7 ubiquitinated Tomm20, and its levels correlated with Fbxo7 expression, indicating a stabilizing effect. None of the PD-associated mutations in Fbxo7 impaired Tomm20 ubiquitination. Our findings demonstrate that SCF(Fbxo7) has an impact directly on two proteins implicated in pathological processes leading to PD. CI - (c) 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society. FAU - Teixeira, Felipe Roberti AU - Teixeira FR AD - Department of Genetics and Evolution, Federal University of Sao Carlos, Sao Carlos, Brazil Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K. Department of Biochemistry and Immunology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil. FAU - Randle, Suzanne J AU - Randle SJ AD - Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K. FAU - Patel, Shachi P AU - Patel SP AD - Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K. FAU - Mevissen, Tycho E T AU - Mevissen TE AD - MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K. FAU - Zenkeviciute, Grasilda AU - Zenkeviciute G AD - Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K. FAU - Koide, Tie AU - Koide T AD - Department of Biochemistry and Immunology, Ribeirao Preto Medical School, University of Sao Paulo, Ribeirao Preto, Brazil. FAU - Komander, David AU - Komander D AD - MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K. FAU - Laman, Heike AU - Laman H AD - Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K. LA - eng GR - 309756/ERC_/European Research Council/International GR - BB/J007846/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom GR - MC_U105192732/MRC_/Medical Research Council/United Kingdom PT - Journal Article DEP - 20160808 PL - England TA - Biochem J JT - The Biochemical journal JID - 2984726R RN - 0 (Antigens, Neoplasm) RN - 0 (Cell Cycle Proteins) RN - 0 (DLGAP5 protein, human) RN - 0 (F-Box Proteins) RN - 0 (FBXO7 protein, human) RN - 0 (Inhibitor of Apoptosis Proteins) RN - 0 (MAGED1 protein, human) RN - 0 (Membrane Transport Proteins) RN - 0 (Mitochondrial Precursor Protein Import Complex Proteins) RN - 0 (Neoplasm Proteins) RN - 0 (Receptors, Cell Surface) RN - 0 (TNF Receptor-Associated Factor 2) RN - 0 (TOMM20 protein, human) RN - EC 2.3.2.27 (SKP Cullin F-Box Protein Ligases) RN - EC 2.7.11.1 (Glycogen Synthase Kinase 3 beta) SB - IM MH - Antigens, Neoplasm/genetics/metabolism MH - Cell Cycle Proteins/genetics/metabolism MH - Cell Line, Tumor MH - F-Box Proteins/genetics/*metabolism MH - Fluorescent Antibody Technique MH - Glycogen Synthase Kinase 3 beta MH - HEK293 Cells MH - Humans MH - Immunoprecipitation MH - Inhibitor of Apoptosis Proteins/genetics/metabolism MH - Membrane Transport Proteins/genetics/*metabolism MH - Mitochondrial Precursor Protein Import Complex Proteins MH - Neoplasm Proteins/genetics/metabolism MH - Parkinson Disease/*enzymology/genetics/*metabolism MH - Point Mutation/genetics MH - Polymorphism, Single Nucleotide/genetics MH - Receptors, Cell Surface/genetics/*metabolism MH - SKP Cullin F-Box Protein Ligases/genetics/metabolism MH - TNF Receptor-Associated Factor 2/genetics/metabolism MH - Ubiquitination/genetics/physiology PMC - PMC5260939 MID - EMS71054 OTO - NOTNLM OT - Fbxo7/PARK15 OT - Tomm20 OT - glycogen synthase kinase OT - mitophagy OT - protein array OT - ubiquitin ligases COIS- The Authors declare that there are no competing interests associated with the manuscript. EDAT- 2016/08/10 06:00 MHDA- 2017/06/13 06:00 PMCR- 2016/10/11 CRDT- 2016/08/10 06:00 PHST- 2016/04/27 00:00 [received] PHST- 2016/08/08 00:00 [accepted] PHST- 2016/08/10 06:00 [pubmed] PHST- 2017/06/13 06:00 [medline] PHST- 2016/08/10 06:00 [entrez] PHST- 2016/10/11 00:00 [pmc-release] AID - BCJ20160387 [pii] AID - BCJ-2016-0387 [pii] AID - 10.1042/BCJ20160387 [doi] PST - ppublish SO - Biochem J. 2016 Oct 15;473(20):3563-3580. doi: 10.1042/BCJ20160387. Epub 2016 Aug 8.