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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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Related in: MedlinePlus

(Overleaf.) TcPINK1 phosphorylates human Parkin at Ser65in vitro. (a) The indicated PD-linked proteins (1 μM) were incubated with either full-length MBP-fusion of wild-type TcPINK1 (1–570) or kinase-inactive (KI) TcPINK1 (D359A) (0.5 μg) and [γ-32P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel). Similar results were obtained in three independent experiments. Fine dividing lines indicate that reactions were resolved on separate gels. The substrate bands on the Coomassie gel are denoted with a small red asterisk. All substrates were of human sequence and expressed in E. coli unless otherwise indicated. Tags on the substrates used for this experiment were glutathione s-transferase (GST)-α-synuclein, Parkin (no tag as His-SUMO tag cleaved off), His-UCHL1, GST-DJ1, GST-LRRK2 KI (1326-end D2017A), MBP-ATP13A2, GST-Omi, MBP-PLA2G6, GST-FBX07, GST-GAK-kinase-inactive (D191A), VPS35 (no tag as GST-tag cleaved off). (b) As in (a) except that proteins reported to interact with PINK1 were tested as PINK1 substrates. Human DJ1, Omi, TRAP1, PARL, NCS1 and Miro2 were expressed in E. coli with an N-terminal GST tag. Similar results were obtained in three independent experiments. (c) Timecourse of phosphorylation of Parkin by wild-type TcPINK1. MBP-TcPINK1 (0.5 μg) was incubated in the presence of GST-Parkin (1 μg) and [γ-32P] ATP for the times indicated and assays terminated by addition of SDS loading buffer. Samples were subjected to SDS-PAGE and proteins detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel). Gel pieces were quantified by Cerenkov counting for calculation of the stoichiometry of Parkin phosphorylation. Similar results were obtained in two independent experiments. (d) Mapping of phosphopeptides on Parkin after phosphorylation by TcPINK1 in vitro. Full-length GST-Parkin (1 μg) was incubated with 2 μg of either wild-type TcPINK1 (1–570) or KI TcPINK1 (D359A) in the presence of Mg2+[γ-32P] ATP for 60 min. Assays were terminated by addition of LDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining and phosphorylated Parkin was digested with trypsin. The resultant peptides were separated by reverse phase HPLC on a Vydac C18 column (Vydac 218TP5215) equilibrated in 0.1% (v/v) trifluoroacetic acid and the column developed with an acetonitrile gradient (diagonal line). The flow rate was 0.2 ml min−1 and fractions (0.1 ml each) were collected and analysed for 32P radioactivity by Cerenkov counting. Two major 32P-labelled peaks (P1, P2) were identified following incubation with wild-type TcPINK1 (left). No peaks were identified following incubation with kinase-inactive TcPINK1 (right). (e) Schematic of domain organization of Parkin illustrating that Ser65 lies within the Ubl domain (upper panel) and sequence alignment of residues around Ser65 in human Parkin and a variety of lower organisms showing high degree of conservation. Abbreviations: Ubl, ubiquitin-like; IBR, in-between-RING; RING, really interesting new gene. (f) Mutation of Ser65Ala (S65A) abolishes Parkin phosphorylation by TcPINK1. Full-length wild-type TcPINK1 (1–570) and kinase inactive TcPINK1 (D359A) against wild-type or S65A mutants of full-length Parkin, or the isolated Ubl-domain-containing N-terminal fragment (residues 1–108). The indicated substrates (2 μM) were incubated in the presence of the indicated enzyme (1 μg) and [γ-32P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel).
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RSOB120080F1: (Overleaf.) TcPINK1 phosphorylates human Parkin at Ser65in vitro. (a) The indicated PD-linked proteins (1 μM) were incubated with either full-length MBP-fusion of wild-type TcPINK1 (1–570) or kinase-inactive (KI) TcPINK1 (D359A) (0.5 μg) and [γ-32P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel). Similar results were obtained in three independent experiments. Fine dividing lines indicate that reactions were resolved on separate gels. The substrate bands on the Coomassie gel are denoted with a small red asterisk. All substrates were of human sequence and expressed in E. coli unless otherwise indicated. Tags on the substrates used for this experiment were glutathione s-transferase (GST)-α-synuclein, Parkin (no tag as His-SUMO tag cleaved off), His-UCHL1, GST-DJ1, GST-LRRK2 KI (1326-end D2017A), MBP-ATP13A2, GST-Omi, MBP-PLA2G6, GST-FBX07, GST-GAK-kinase-inactive (D191A), VPS35 (no tag as GST-tag cleaved off). (b) As in (a) except that proteins reported to interact with PINK1 were tested as PINK1 substrates. Human DJ1, Omi, TRAP1, PARL, NCS1 and Miro2 were expressed in E. coli with an N-terminal GST tag. Similar results were obtained in three independent experiments. (c) Timecourse of phosphorylation of Parkin by wild-type TcPINK1. MBP-TcPINK1 (0.5 μg) was incubated in the presence of GST-Parkin (1 μg) and [γ-32P] ATP for the times indicated and assays terminated by addition of SDS loading buffer. Samples were subjected to SDS-PAGE and proteins detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel). Gel pieces were quantified by Cerenkov counting for calculation of the stoichiometry of Parkin phosphorylation. Similar results were obtained in two independent experiments. (d) Mapping of phosphopeptides on Parkin after phosphorylation by TcPINK1 in vitro. Full-length GST-Parkin (1 μg) was incubated with 2 μg of either wild-type TcPINK1 (1–570) or KI TcPINK1 (D359A) in the presence of Mg2+[γ-32P] ATP for 60 min. Assays were terminated by addition of LDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining and phosphorylated Parkin was digested with trypsin. The resultant peptides were separated by reverse phase HPLC on a Vydac C18 column (Vydac 218TP5215) equilibrated in 0.1% (v/v) trifluoroacetic acid and the column developed with an acetonitrile gradient (diagonal line). The flow rate was 0.2 ml min−1 and fractions (0.1 ml each) were collected and analysed for 32P radioactivity by Cerenkov counting. Two major 32P-labelled peaks (P1, P2) were identified following incubation with wild-type TcPINK1 (left). No peaks were identified following incubation with kinase-inactive TcPINK1 (right). (e) Schematic of domain organization of Parkin illustrating that Ser65 lies within the Ubl domain (upper panel) and sequence alignment of residues around Ser65 in human Parkin and a variety of lower organisms showing high degree of conservation. Abbreviations: Ubl, ubiquitin-like; IBR, in-between-RING; RING, really interesting new gene. (f) Mutation of Ser65Ala (S65A) abolishes Parkin phosphorylation by TcPINK1. Full-length wild-type TcPINK1 (1–570) and kinase inactive TcPINK1 (D359A) against wild-type or S65A mutants of full-length Parkin, or the isolated Ubl-domain-containing N-terminal fragment (residues 1–108). The indicated substrates (2 μM) were incubated in the presence of the indicated enzyme (1 μg) and [γ-32P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel).

Mentions: As some of the known PD-linked proteins may function in a signalling network [19], we tested whether catalytically active recombinant insect TcPINK1 could directly phosphorylate 11 different PD-linked proteins and seven putative PINK1 interacting proteins (figure 1a,b). Strikingly, this revealed that wild-type but not kinase-inactive TcPINK1 phosphorylated full-length Parkin, but not any of the other proteins tested, including Omi [20], TRAP1 [21] or Miro2 [22,23] (figure 1a,b).Figure 1.


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)

(Overleaf.) TcPINK1 phosphorylates human Parkin at Ser65in vitro. (a) The indicated PD-linked proteins (1 μM) were incubated with either full-length MBP-fusion of wild-type TcPINK1 (1–570) or kinase-inactive (KI) TcPINK1 (D359A) (0.5 μg) and [γ-32P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel). Similar results were obtained in three independent experiments. Fine dividing lines indicate that reactions were resolved on separate gels. The substrate bands on the Coomassie gel are denoted with a small red asterisk. All substrates were of human sequence and expressed in E. coli unless otherwise indicated. Tags on the substrates used for this experiment were glutathione s-transferase (GST)-α-synuclein, Parkin (no tag as His-SUMO tag cleaved off), His-UCHL1, GST-DJ1, GST-LRRK2 KI (1326-end D2017A), MBP-ATP13A2, GST-Omi, MBP-PLA2G6, GST-FBX07, GST-GAK-kinase-inactive (D191A), VPS35 (no tag as GST-tag cleaved off). (b) As in (a) except that proteins reported to interact with PINK1 were tested as PINK1 substrates. Human DJ1, Omi, TRAP1, PARL, NCS1 and Miro2 were expressed in E. coli with an N-terminal GST tag. Similar results were obtained in three independent experiments. (c) Timecourse of phosphorylation of Parkin by wild-type TcPINK1. MBP-TcPINK1 (0.5 μg) was incubated in the presence of GST-Parkin (1 μg) and [γ-32P] ATP for the times indicated and assays terminated by addition of SDS loading buffer. Samples were subjected to SDS-PAGE and proteins detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel). Gel pieces were quantified by Cerenkov counting for calculation of the stoichiometry of Parkin phosphorylation. Similar results were obtained in two independent experiments. (d) Mapping of phosphopeptides on Parkin after phosphorylation by TcPINK1 in vitro. Full-length GST-Parkin (1 μg) was incubated with 2 μg of either wild-type TcPINK1 (1–570) or KI TcPINK1 (D359A) in the presence of Mg2+[γ-32P] ATP for 60 min. Assays were terminated by addition of LDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining and phosphorylated Parkin was digested with trypsin. The resultant peptides were separated by reverse phase HPLC on a Vydac C18 column (Vydac 218TP5215) equilibrated in 0.1% (v/v) trifluoroacetic acid and the column developed with an acetonitrile gradient (diagonal line). The flow rate was 0.2 ml min−1 and fractions (0.1 ml each) were collected and analysed for 32P radioactivity by Cerenkov counting. Two major 32P-labelled peaks (P1, P2) were identified following incubation with wild-type TcPINK1 (left). No peaks were identified following incubation with kinase-inactive TcPINK1 (right). (e) Schematic of domain organization of Parkin illustrating that Ser65 lies within the Ubl domain (upper panel) and sequence alignment of residues around Ser65 in human Parkin and a variety of lower organisms showing high degree of conservation. Abbreviations: Ubl, ubiquitin-like; IBR, in-between-RING; RING, really interesting new gene. (f) Mutation of Ser65Ala (S65A) abolishes Parkin phosphorylation by TcPINK1. Full-length wild-type TcPINK1 (1–570) and kinase inactive TcPINK1 (D359A) against wild-type or S65A mutants of full-length Parkin, or the isolated Ubl-domain-containing N-terminal fragment (residues 1–108). The indicated substrates (2 μM) were incubated in the presence of the indicated enzyme (1 μg) and [γ-32P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel).
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RSOB120080F1: (Overleaf.) TcPINK1 phosphorylates human Parkin at Ser65in vitro. (a) The indicated PD-linked proteins (1 μM) were incubated with either full-length MBP-fusion of wild-type TcPINK1 (1–570) or kinase-inactive (KI) TcPINK1 (D359A) (0.5 μg) and [γ-32P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel). Similar results were obtained in three independent experiments. Fine dividing lines indicate that reactions were resolved on separate gels. The substrate bands on the Coomassie gel are denoted with a small red asterisk. All substrates were of human sequence and expressed in E. coli unless otherwise indicated. Tags on the substrates used for this experiment were glutathione s-transferase (GST)-α-synuclein, Parkin (no tag as His-SUMO tag cleaved off), His-UCHL1, GST-DJ1, GST-LRRK2 KI (1326-end D2017A), MBP-ATP13A2, GST-Omi, MBP-PLA2G6, GST-FBX07, GST-GAK-kinase-inactive (D191A), VPS35 (no tag as GST-tag cleaved off). (b) As in (a) except that proteins reported to interact with PINK1 were tested as PINK1 substrates. Human DJ1, Omi, TRAP1, PARL, NCS1 and Miro2 were expressed in E. coli with an N-terminal GST tag. Similar results were obtained in three independent experiments. (c) Timecourse of phosphorylation of Parkin by wild-type TcPINK1. MBP-TcPINK1 (0.5 μg) was incubated in the presence of GST-Parkin (1 μg) and [γ-32P] ATP for the times indicated and assays terminated by addition of SDS loading buffer. Samples were subjected to SDS-PAGE and proteins detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel). Gel pieces were quantified by Cerenkov counting for calculation of the stoichiometry of Parkin phosphorylation. Similar results were obtained in two independent experiments. (d) Mapping of phosphopeptides on Parkin after phosphorylation by TcPINK1 in vitro. Full-length GST-Parkin (1 μg) was incubated with 2 μg of either wild-type TcPINK1 (1–570) or KI TcPINK1 (D359A) in the presence of Mg2+[γ-32P] ATP for 60 min. Assays were terminated by addition of LDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining and phosphorylated Parkin was digested with trypsin. The resultant peptides were separated by reverse phase HPLC on a Vydac C18 column (Vydac 218TP5215) equilibrated in 0.1% (v/v) trifluoroacetic acid and the column developed with an acetonitrile gradient (diagonal line). The flow rate was 0.2 ml min−1 and fractions (0.1 ml each) were collected and analysed for 32P radioactivity by Cerenkov counting. Two major 32P-labelled peaks (P1, P2) were identified following incubation with wild-type TcPINK1 (left). No peaks were identified following incubation with kinase-inactive TcPINK1 (right). (e) Schematic of domain organization of Parkin illustrating that Ser65 lies within the Ubl domain (upper panel) and sequence alignment of residues around Ser65 in human Parkin and a variety of lower organisms showing high degree of conservation. Abbreviations: Ubl, ubiquitin-like; IBR, in-between-RING; RING, really interesting new gene. (f) Mutation of Ser65Ala (S65A) abolishes Parkin phosphorylation by TcPINK1. Full-length wild-type TcPINK1 (1–570) and kinase inactive TcPINK1 (D359A) against wild-type or S65A mutants of full-length Parkin, or the isolated Ubl-domain-containing N-terminal fragment (residues 1–108). The indicated substrates (2 μM) were incubated in the presence of the indicated enzyme (1 μg) and [γ-32P] ATP for 30 min. Assays were terminated by addition of SDS loading buffer and separated by SDS-PAGE. Proteins were detected by Colloidal Coomassie blue staining (lower panel) and incorporation of [γ-32P] ATP was detected by autoradiography (upper panel).
Mentions: As some of the known PD-linked proteins may function in a signalling network [19], we tested whether catalytically active recombinant insect TcPINK1 could directly phosphorylate 11 different PD-linked proteins and seven putative PINK1 interacting proteins (figure 1a,b). Strikingly, this revealed that wild-type but not kinase-inactive TcPINK1 phosphorylated full-length Parkin, but not any of the other proteins tested, including Omi [20], TRAP1 [21] or Miro2 [22,23] (figure 1a,b).Figure 1.

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