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

Kondapalli C, Kazlauskaite A, Zhang N, Woodroof HI, Campbell DG, Gourlay R, Burchell L, Walden H, Macartney TJ, Deak M, Knebel A, Alessi DR, Muqit MM - Open Biol (2012)

Bottom Line: 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.These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin.Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

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.

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PINK1 phosphorylation of Parkin at Ser65 mediates activation of Parkin E3 ligase activity. Wild-type (a) but not kinase-inactive (b) PINK1 activates wild-type Parkin, but does not affect the activity of Ser65Ala (S65A) mutant Parkin (c). Two micrograms of wild-type or S65A Parkin were incubated with indicated amounts of wild-type or kinase-inactive (D359A) MBP-TcPINK in a kinase reaction (50 mM Tris-HCl (pH 7.5), 0.1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM MgCl2, 1% 2-mercaptoethanol and 0.1 mM [γ-32P] ATP (approx. 500 cpm pmol−1) (in parallel to confirm the phosphorylation) for 60 min. The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris-HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl2, 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli, 0.05 mM Flag-Ubiquitin (MW approx. 9.5 kDa) (Boston Biochem) and 2 mM ATP). Reactions were terminated after 60 min by addition of SDS-PAGE loading buffer and resolved by SDS-PAGE. Ubiquitin, Parkin and PINK1 were detected using anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Incorporation of [γ-32P] ATP was detected by autoradiography (lower panel). Ubiquitin attached to the E1 (Ub-Ube1) and ubiquitin dimer (Ub2) formation occurred in the assay in all conditions (a–c). Ubiquitylation of PINK1 (Ub-PINK1) is indicated (a). Formation of polyubiquitin chains (poly-Ub) upon Parkin activation (a) is indicated. As mentioned in §4, further work is required to establish the nature of these chains and whether they are linked to UbcH7. Representative of five independent experiments.
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RSOB120080F2: PINK1 phosphorylation of Parkin at Ser65 mediates activation of Parkin E3 ligase activity. Wild-type (a) but not kinase-inactive (b) PINK1 activates wild-type Parkin, but does not affect the activity of Ser65Ala (S65A) mutant Parkin (c). Two micrograms of wild-type or S65A Parkin were incubated with indicated amounts of wild-type or kinase-inactive (D359A) MBP-TcPINK in a kinase reaction (50 mM Tris-HCl (pH 7.5), 0.1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM MgCl2, 1% 2-mercaptoethanol and 0.1 mM [γ-32P] ATP (approx. 500 cpm pmol−1) (in parallel to confirm the phosphorylation) for 60 min. The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris-HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl2, 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli, 0.05 mM Flag-Ubiquitin (MW approx. 9.5 kDa) (Boston Biochem) and 2 mM ATP). Reactions were terminated after 60 min by addition of SDS-PAGE loading buffer and resolved by SDS-PAGE. Ubiquitin, Parkin and PINK1 were detected using anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Incorporation of [γ-32P] ATP was detected by autoradiography (lower panel). Ubiquitin attached to the E1 (Ub-Ube1) and ubiquitin dimer (Ub2) formation occurred in the assay in all conditions (a–c). Ubiquitylation of PINK1 (Ub-PINK1) is indicated (a). Formation of polyubiquitin chains (poly-Ub) upon Parkin activation (a) is indicated. As mentioned in §4, further work is required to establish the nature of these chains and whether they are linked to UbcH7. Representative of five independent experiments.

Mentions: A recent study provided strong evidence that the Ubl domain of Parkin acts as an auto-inhibitory domain by binding to a region within the C-terminus thereby suppressing catalytic activity [24]. Given that Ser65 lies within the core of the Ubl domain, we hypothesized that phosphorylation of Ser65 might relieve the autoinhibition thereby activating the E3 ligase activity of Parkin. To investigate this, we set up an E3 ligase auto-ubiquitylation assay to assess Parkin catalytic activity using an approach that has been described previously employing highly purified full length recombinant Parkin expressed in E. coli with no epitope tags that can interfere with the autoinhibitory effect of the Ubl domain [24]. Prior to undertaking the E3 ligase activity assay, we phosphorylated Parkin with increasing levels of TcPINK1 in the presence of 32P-adenosine triphosphate (ATP) so that we could verify PINK1 was phosphorylating Parkin (middle panels in figure 2). To assess Parkin E3 ligase activity, aliquots of these reactions were added to a reaction containing E1 ubiquitin-activating ligase, UbcH7 conjugating E2 ligase, ubiquitin and Mg-ATP. After 60 min the reactions were terminated with SDS sample buffer in the presence of dithiothreitol (DTT) and reactions analysed by immunoblot analysis with antibodies that detect ubiquitin, Parkin and TcPINK1. In the absence of PINK1 phosphorylation we confirmed previous findings and found that Parkin displayed no significant E3 ligase activity and no evidence of formation of polyubiquitin chains were observed (lane 1 on figure 2a). Excitingly, when increasing levels of TcPINK1 were added to the reaction at concentrations in which phosphorylation of Parkin was detected (middle panel of figure 2a), we observed the marked dose-dependent appearance of non-DTT-reducible low molecular weight polyubiquitylated species migrating between approximately 30 and 50 kDa (top panel of figure 2a). Consistent with this being mediated by phosphorylation of Parkin at Ser65 by PINK1, the appearance of polyubiquitin chains was inhibited by introducing a point mutation in PINK1 that ablates catalytic activity (figure 2b) or by mutating Ser65 in Parkin to a non-phosphorylatable Ala residue (figure 2c).Figure 2.


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

Kondapalli C, Kazlauskaite A, Zhang N, Woodroof HI, Campbell DG, Gourlay R, Burchell L, Walden H, Macartney TJ, Deak M, Knebel A, Alessi DR, Muqit MM - Open Biol (2012)

PINK1 phosphorylation of Parkin at Ser65 mediates activation of Parkin E3 ligase activity. Wild-type (a) but not kinase-inactive (b) PINK1 activates wild-type Parkin, but does not affect the activity of Ser65Ala (S65A) mutant Parkin (c). Two micrograms of wild-type or S65A Parkin were incubated with indicated amounts of wild-type or kinase-inactive (D359A) MBP-TcPINK in a kinase reaction (50 mM Tris-HCl (pH 7.5), 0.1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM MgCl2, 1% 2-mercaptoethanol and 0.1 mM [γ-32P] ATP (approx. 500 cpm pmol−1) (in parallel to confirm the phosphorylation) for 60 min. The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris-HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl2, 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli, 0.05 mM Flag-Ubiquitin (MW approx. 9.5 kDa) (Boston Biochem) and 2 mM ATP). Reactions were terminated after 60 min by addition of SDS-PAGE loading buffer and resolved by SDS-PAGE. Ubiquitin, Parkin and PINK1 were detected using anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Incorporation of [γ-32P] ATP was detected by autoradiography (lower panel). Ubiquitin attached to the E1 (Ub-Ube1) and ubiquitin dimer (Ub2) formation occurred in the assay in all conditions (a–c). Ubiquitylation of PINK1 (Ub-PINK1) is indicated (a). Formation of polyubiquitin chains (poly-Ub) upon Parkin activation (a) is indicated. As mentioned in §4, further work is required to establish the nature of these chains and whether they are linked to UbcH7. Representative of five independent experiments.
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RSOB120080F2: PINK1 phosphorylation of Parkin at Ser65 mediates activation of Parkin E3 ligase activity. Wild-type (a) but not kinase-inactive (b) PINK1 activates wild-type Parkin, but does not affect the activity of Ser65Ala (S65A) mutant Parkin (c). Two micrograms of wild-type or S65A Parkin were incubated with indicated amounts of wild-type or kinase-inactive (D359A) MBP-TcPINK in a kinase reaction (50 mM Tris-HCl (pH 7.5), 0.1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM MgCl2, 1% 2-mercaptoethanol and 0.1 mM [γ-32P] ATP (approx. 500 cpm pmol−1) (in parallel to confirm the phosphorylation) for 60 min. The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris-HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl2, 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli, 0.05 mM Flag-Ubiquitin (MW approx. 9.5 kDa) (Boston Biochem) and 2 mM ATP). Reactions were terminated after 60 min by addition of SDS-PAGE loading buffer and resolved by SDS-PAGE. Ubiquitin, Parkin and PINK1 were detected using anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Incorporation of [γ-32P] ATP was detected by autoradiography (lower panel). Ubiquitin attached to the E1 (Ub-Ube1) and ubiquitin dimer (Ub2) formation occurred in the assay in all conditions (a–c). Ubiquitylation of PINK1 (Ub-PINK1) is indicated (a). Formation of polyubiquitin chains (poly-Ub) upon Parkin activation (a) is indicated. As mentioned in §4, further work is required to establish the nature of these chains and whether they are linked to UbcH7. Representative of five independent experiments.
Mentions: A recent study provided strong evidence that the Ubl domain of Parkin acts as an auto-inhibitory domain by binding to a region within the C-terminus thereby suppressing catalytic activity [24]. Given that Ser65 lies within the core of the Ubl domain, we hypothesized that phosphorylation of Ser65 might relieve the autoinhibition thereby activating the E3 ligase activity of Parkin. To investigate this, we set up an E3 ligase auto-ubiquitylation assay to assess Parkin catalytic activity using an approach that has been described previously employing highly purified full length recombinant Parkin expressed in E. coli with no epitope tags that can interfere with the autoinhibitory effect of the Ubl domain [24]. Prior to undertaking the E3 ligase activity assay, we phosphorylated Parkin with increasing levels of TcPINK1 in the presence of 32P-adenosine triphosphate (ATP) so that we could verify PINK1 was phosphorylating Parkin (middle panels in figure 2). To assess Parkin E3 ligase activity, aliquots of these reactions were added to a reaction containing E1 ubiquitin-activating ligase, UbcH7 conjugating E2 ligase, ubiquitin and Mg-ATP. After 60 min the reactions were terminated with SDS sample buffer in the presence of dithiothreitol (DTT) and reactions analysed by immunoblot analysis with antibodies that detect ubiquitin, Parkin and TcPINK1. In the absence of PINK1 phosphorylation we confirmed previous findings and found that Parkin displayed no significant E3 ligase activity and no evidence of formation of polyubiquitin chains were observed (lane 1 on figure 2a). Excitingly, when increasing levels of TcPINK1 were added to the reaction at concentrations in which phosphorylation of Parkin was detected (middle panel of figure 2a), we observed the marked dose-dependent appearance of non-DTT-reducible low molecular weight polyubiquitylated species migrating between approximately 30 and 50 kDa (top panel of figure 2a). Consistent with this being mediated by phosphorylation of Parkin at Ser65 by PINK1, the appearance of polyubiquitin chains was inhibited by introducing a point mutation in PINK1 that ablates catalytic activity (figure 2b) or by mutating Ser65 in Parkin to a non-phosphorylatable Ala residue (figure 2c).Figure 2.

Bottom Line: 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.These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin.Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.

View Article: PubMed Central - PubMed

Affiliation: MRC Protein Phosphorylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.

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.

Show MeSH
Related in: MedlinePlus