PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

Open Biol. 2012 May;2(5):120080. doi: 10.1098/rsob.120080.

Abstract

Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.

Keywords: PINK1; Parkin; Parkinson's disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone / pharmacology
  • Enzyme Activation / drug effects
  • HEK293 Cells
  • Humans
  • Insect Proteins / genetics
  • Insect Proteins / physiology
  • Membrane Potential, Mitochondrial / physiology*
  • Parkinson Disease / metabolism
  • Phosphorylation / drug effects
  • Phosphoserine / metabolism
  • Phosphothreonine / metabolism
  • Protein Kinases / genetics
  • Protein Kinases / physiology*
  • Protein Processing, Post-Translational / drug effects*
  • Protein Stability / drug effects
  • Protein Structure, Tertiary
  • RNA Interference
  • RNA, Small Interfering / pharmacology
  • Receptor-Like Protein Tyrosine Phosphatases, Class 2 / pharmacology
  • Recombinant Fusion Proteins / physiology
  • Tribolium / enzymology
  • Tribolium / genetics
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • Insect Proteins
  • RNA, Small Interfering
  • Recombinant Fusion Proteins
  • Phosphothreonine
  • Phosphoserine
  • Carbonyl Cyanide m-Chlorophenyl Hydrazone
  • Ubiquitin-Protein Ligases
  • parkin protein
  • Protein Kinases
  • PTEN-induced putative kinase
  • Receptor-Like Protein Tyrosine Phosphatases, Class 2