title: Gsk3β and Tomm20 are substrates of the SCFFbxo7/PARK15 ubiquitin ligase associated with Parkinson's disease.
abstract: 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 SCFFbxo7/PARK15ubiquitin 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 SCFFbxo7implicated 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 3β (Gsk3β), which can phosphorylate α-synuclein, and translocase of outer mitochondrial membrane 20 (Tomm20), a mitochondrial translocase that, when ubiquitinated, promotes mitophagy, as SCFFbxo7substrates both in vitro and in vivo Ubiquitin chain restriction analyses revealed that Fbxo7 modified Gsk3β using K63 linkages. Our results indicate that Fbxo7 negatively regulates Gsk3β 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 SCFFbxo7has an impact directly on two proteins implicated in pathological processes leading to PD.
authors: Department of Genetics and Evolution, Federal University of São Carlos, São Carlos, Brazil Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K. Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.
authors: Felipe Roberti
authors: FR
authors: Teixeira
authors: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.
authors: Suzanne J
authors: SJ
authors: Randle
authors: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.
authors: Shachi P
authors: SP
authors: Patel
authors: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K.
authors: Tycho E T
authors: TE
authors: Mevissen
authors: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.
authors: Grasilda
authors: G
authors: Zenkeviciute
authors: Department of Biochemistry and Immunology, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil.
authors: Tie
authors: T
authors: Koide
authors: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH, U.K.
authors: David
authors: D
authors: Komander
authors: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, U.K.
authors: Heike
authors: H
authors: Laman
mesh_terms: D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D000071679:Glycogen Synthase Kinase 3 beta; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010300:Parkinson Disease / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000235:genetics / Q000502:physiology
chemical_list: D000951:Antigens, Neoplasm; D018797:Cell Cycle Proteins; C465041:DLGAP5 protein, human; D044783:F-Box Proteins; C489149:FBXO7 protein, human; D051034:Inhibitor of Apoptosis Proteins; C120897:MAGED1 protein, human; D026901:Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009363:Neoplasm Proteins; D011956:Receptors, Cell Surface; D047992:TNF Receptor-Associated Factor 2; C084008:TOMM20 protein, human; D044843:SKP Cullin F-Box Protein Ligases; D000071679:Glycogen Synthase Kinase 3 beta
keywords: Fbxo7/PARK15; Tomm20; glycogen synthase kinase; mitophagy; protein array; ubiquitin ligases