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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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Knock-down of endogenous PINK1 abrogates Parkin Ser65 phosphorylation. (a) Timecourse of endogenous PINK1 stabilization by CCCP treatment. HEK293 cells were stimulated at the indicated time points with 10 μM of CCCP. One milligram of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) or pre-immune IgG covalently coupled to protein G Sepharose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody (Novus). Representative of three independent experiments. (b) Knock-down of endogenous PINK1 abrogates Parkin Ser65 phosphorylation. HEK293 cells were co-transfected with PINK1 siRNA (#1 or #2) or scrambled siRNA (scrambled) and untagged wild-type (WT) or Ser65Ala (S65A) mutant Parkin as indicated using TransFectin reagent (Bio-Rad). Forty-eight hours post-transfection, cells were treated with or without 10 μM CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with GST-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser65 antibody in the presence of dephosphorylated peptide. Five per cent of the IP was immunoblotted with total anti-Parkin antibody. 0.25 mg of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) and immunoblotted with anti-PINK1 antibody (Novus). Representative of three independent experiments.
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RSOB120080F4: Knock-down of endogenous PINK1 abrogates Parkin Ser65 phosphorylation. (a) Timecourse of endogenous PINK1 stabilization by CCCP treatment. HEK293 cells were stimulated at the indicated time points with 10 μM of CCCP. One milligram of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) or pre-immune IgG covalently coupled to protein G Sepharose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody (Novus). Representative of three independent experiments. (b) Knock-down of endogenous PINK1 abrogates Parkin Ser65 phosphorylation. HEK293 cells were co-transfected with PINK1 siRNA (#1 or #2) or scrambled siRNA (scrambled) and untagged wild-type (WT) or Ser65Ala (S65A) mutant Parkin as indicated using TransFectin reagent (Bio-Rad). Forty-eight hours post-transfection, cells were treated with or without 10 μM CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with GST-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser65 antibody in the presence of dephosphorylated peptide. Five per cent of the IP was immunoblotted with total anti-Parkin antibody. 0.25 mg of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) and immunoblotted with anti-PINK1 antibody (Novus). Representative of three independent experiments.

Mentions: To test whether we could detect endogenous PINK1 we subjected HEK293 cell lysates to immunoprecipitation employing a PINK1 antibody that we raised against a recombinant fragment of human PINK1 (residues 175–250) and immunoblotted the immunoprecipitates with a commercial PINK1 antibody (raised against residues 175–250; Novus Biologicals). This revealed the presence of a band that migrated at the size predicted for endogenous PINK1 and that was strikingly stabilized by CCCP in a time-dependent manner (up to 9 h; upper band figure 4a). This protein was not observed in control immunoprecipitates undertaken with pre-immune Immunoglobulin G (IgG; lower band figure 4a). Consistent with this band representing endogenous PINK1 it was significantly reduced when cells were transfected for 48 h with two independent PINK1 siRNA oligos but not scrambled siRNA (figure 4b). Importantly, siRNA-mediated knockdown using two different siRNA probes targeting PINK1 severely abrogated phosphorylation of Parkin Ser65, suggesting that the endogenous PINK1 protein does indeed regulate Parkin phosphorylation at Ser65 (figure 4b).Figure 4.


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)

Knock-down of endogenous PINK1 abrogates Parkin Ser65 phosphorylation. (a) Timecourse of endogenous PINK1 stabilization by CCCP treatment. HEK293 cells were stimulated at the indicated time points with 10 μM of CCCP. One milligram of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) or pre-immune IgG covalently coupled to protein G Sepharose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody (Novus). Representative of three independent experiments. (b) Knock-down of endogenous PINK1 abrogates Parkin Ser65 phosphorylation. HEK293 cells were co-transfected with PINK1 siRNA (#1 or #2) or scrambled siRNA (scrambled) and untagged wild-type (WT) or Ser65Ala (S65A) mutant Parkin as indicated using TransFectin reagent (Bio-Rad). Forty-eight hours post-transfection, cells were treated with or without 10 μM CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with GST-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser65 antibody in the presence of dephosphorylated peptide. Five per cent of the IP was immunoblotted with total anti-Parkin antibody. 0.25 mg of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) and immunoblotted with anti-PINK1 antibody (Novus). Representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376738&req=5

RSOB120080F4: Knock-down of endogenous PINK1 abrogates Parkin Ser65 phosphorylation. (a) Timecourse of endogenous PINK1 stabilization by CCCP treatment. HEK293 cells were stimulated at the indicated time points with 10 μM of CCCP. One milligram of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) or pre-immune IgG covalently coupled to protein G Sepharose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody (Novus). Representative of three independent experiments. (b) Knock-down of endogenous PINK1 abrogates Parkin Ser65 phosphorylation. HEK293 cells were co-transfected with PINK1 siRNA (#1 or #2) or scrambled siRNA (scrambled) and untagged wild-type (WT) or Ser65Ala (S65A) mutant Parkin as indicated using TransFectin reagent (Bio-Rad). Forty-eight hours post-transfection, cells were treated with or without 10 μM CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with GST-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser65 antibody in the presence of dephosphorylated peptide. Five per cent of the IP was immunoblotted with total anti-Parkin antibody. 0.25 mg of whole-cell lysates were immunoprecipitated with anti-PINK1 antibody (S085D) and immunoblotted with anti-PINK1 antibody (Novus). Representative of three independent experiments.
Mentions: To test whether we could detect endogenous PINK1 we subjected HEK293 cell lysates to immunoprecipitation employing a PINK1 antibody that we raised against a recombinant fragment of human PINK1 (residues 175–250) and immunoblotted the immunoprecipitates with a commercial PINK1 antibody (raised against residues 175–250; Novus Biologicals). This revealed the presence of a band that migrated at the size predicted for endogenous PINK1 and that was strikingly stabilized by CCCP in a time-dependent manner (up to 9 h; upper band figure 4a). This protein was not observed in control immunoprecipitates undertaken with pre-immune Immunoglobulin G (IgG; lower band figure 4a). Consistent with this band representing endogenous PINK1 it was significantly reduced when cells were transfected for 48 h with two independent PINK1 siRNA oligos but not scrambled siRNA (figure 4b). Importantly, siRNA-mediated knockdown using two different siRNA probes targeting PINK1 severely abrogated phosphorylation of Parkin Ser65, suggesting that the endogenous PINK1 protein does indeed regulate Parkin phosphorylation at Ser65 (figure 4b).Figure 4.

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