title: PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.
abstract: PINK1 kinase activates the E3 ubiquitin ligase Parkin to induce selective autophagy of damaged mitochondria. However, it has been unclear how PINK1 activates and recruits Parkin to mitochondria. Although PINK1 phosphorylates Parkin, other PINK1 substrates appear to activate Parkin, as the mutation of all serine and threonine residues conserved between Drosophila and human, including Parkin S65, did not wholly impair Parkin translocation to mitochondria. Using mass spectrometry, we discovered that endogenous PINK1 phosphorylated ubiquitin at serine 65, homologous to the site phosphorylated by PINK1 in Parkin's ubiquitin-like domain. Recombinant TcPINK1 directly phosphorylated ubiquitin and phospho-ubiquitin activated Parkin E3 ubiquitin ligase activity in cell-free assays. In cells, the phosphomimetic ubiquitin mutant S65D bound and activated Parkin. Furthermore, expression of ubiquitin S65A, a mutant that cannot be phosphorylated by PINK1, inhibited Parkin translocation to damaged mitochondria. These results explain a feed-forward mechanism of PINK1-mediated initiation of Parkin E3 ligase activity.
authors.affiliation: Biochemistry Section, Surgical Neurology Branch, and 2 Protein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20824.
authors.forename: Lesley A
authors.initials: LA
authors.lastname: Kane
authors.forename: Michael
authors.initials: M
authors.lastname: Lazarou
authors.forename: Adam I
authors.initials: AI
authors.lastname: Fogel
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Koji
authors.initials: K
authors.lastname: Yamano
authors.forename: Shireen A
authors.initials: SA
authors.lastname: Sarraf
authors.forename: Soojay
authors.initials: S
authors.lastname: Banerjee
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Youle
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D004331:Drosophila melanogaster; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D020125:Mutation, Missense; D010766:Phosphorylation / Q000502:physiology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D000099202:PTEN-Induced Putative Kinase
chemical_list: D011494:Protein Kinases; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000099202:PTEN-Induced Putative Kinase; C111567:parkin protein
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