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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

Identification and characterization of a novel autophosphorylation site of PINK1 induced by the mitochondrial uncoupling agent CCCP. (a) CCCP induces a band-shift in wild-type but not kinase-inactive PINK1. Flp-In T-Rex HEK293 cell lines stably expressing FLAG alone, wild-type or kinase-inactive PINK1-FLAG were induced to express protein by addition of 0.1 μg ml−1 of doxycycline in the culture medium for 24 h. Cells were then treated with 10 μM of CCCP for 3 h and lysates subjected to sub-cellular fractionation. Twenty-five micrograms of cytoplasmic or mitochondrial lysate were resolved by 8% SDS-PAGE. Relative purity of the fractions was confirmed using cytoplasmic and mitochondrial markers, namely GAPDH and HSP60, respectively. Whole-cell extracts from the same cells were also made in parallel using 1% Triton lysis as described in the methods. In mitochondrial and whole-cell extracts, both wild-type and kinase-inactive PINK1 became stabilized by CCCP but a band-shift was noted for wild-type PINK1 which was revealed to be a doublet on lower exposure. The upper band was absent from kinase-inactive PINK1 treated with CCCP. (b) Identification of Thr257 phosphorylation site on PINK1. Flp-In T-Rex HEK293 cell lines stably expressing FLAG alone, or wild-type PINK1-FLAG were treated with DMSO or 10 μM of CCCP for 3 h. Recombinant PINK1 was immunoprecipitated from 10 mg of mitochondrial extract for each condition using anti-FLAG-agarose and subjected to 4–12% gradient SDS-PAGE and stained with colloidal Coomassie blue. The Coomassie-stained bands migrating with the expected molecular mass of PINK1-FLAG were excised from the gel, digested with trypsin, and subjected to precursor-ion scanning mass spectroscopy. The major phosphopeptide that is indicated ‘Thr257’ was seen from cells expressing wild-type PINK1-FLAG treated with CCCP and this was not seen in bands from the other two conditions. The figure shows the signal intensity (cps, counts of ions per second) of the HPO3− ion (−79 Da) seen in negative precursor ion scanning mode versus the ion distribution (m/z) for the Thr257 phosphopeptide. The observed values of 722.4 and 788.4 are for the VALAGEYGAVTYR and VALAGEYGAVTYRK variants, respectively, of the Thr257 peptide as [M-2H]2− ions. Other phosphopeptides marked with an asterisk were observed but we were unable to assign phosphorylation site(s). (c) Evidence that CCCP induces PINK1 auto-phosphorylation using a phospho-specific Thr257 antibody. 0.5 mg of mitochondrial extracts (treated with DMSO or 10 μM of CCCP for 3 h) of Flp-In T-Rex stable cell lines expressing FLAG empty, wild-type PINK1-FLAG, kinase-inactive PINK1-FLAG (D384A) and phospho-mutant Thr257Ala (T257A) were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Blots were probed with anti-phospho-Thr257 PINK1 antibody and anti-PINK1 antibody. (d) Mutation of Thr257Ala PINK1 does not affect Parkin Ser65 phosphorylation. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, kinase-inactive PINK1-FLAG and T257A PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser65Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser65 Parkin antibody in the presence of dephosphorylated peptide. Ten per cent of the IP was immune-blotted with total anti-Parkin antibody. One milligram of 1% Triton whole-cell lysate were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Blots were probed with anti-phospho-Thr257 PINK1 and anti-PINK1 antibody. (e) PINK1 dephosphorylation by lambda phosphatase inhibits PINK1 activity. C-terminal-FLAG tagged wild-type or kinase-inactive (D384A) PINK1 were immunoprecipitated from 5 mg of mitochondrial enriched extracts using anti-FLAG agarose beads. Wild-type PINK1 was incubated with or without 1000 U of lambda phosphatase or treated with lambda phosphatase along with 50 mM EDTA. Kinase-inactive PINK1 was incubated in buffer alone without lambda phosphatase. The beads were washed thrice in 50 mM Tris pH 7.5, 0.1 mM EGTA and then used in an in vitro kinase assay with GST-Parkin Ubl (1–108) as the substrate. Samples were analysed as described in legend to figure 1.
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RSOB120080F6: Identification and characterization of a novel autophosphorylation site of PINK1 induced by the mitochondrial uncoupling agent CCCP. (a) CCCP induces a band-shift in wild-type but not kinase-inactive PINK1. Flp-In T-Rex HEK293 cell lines stably expressing FLAG alone, wild-type or kinase-inactive PINK1-FLAG were induced to express protein by addition of 0.1 μg ml−1 of doxycycline in the culture medium for 24 h. Cells were then treated with 10 μM of CCCP for 3 h and lysates subjected to sub-cellular fractionation. Twenty-five micrograms of cytoplasmic or mitochondrial lysate were resolved by 8% SDS-PAGE. Relative purity of the fractions was confirmed using cytoplasmic and mitochondrial markers, namely GAPDH and HSP60, respectively. Whole-cell extracts from the same cells were also made in parallel using 1% Triton lysis as described in the methods. In mitochondrial and whole-cell extracts, both wild-type and kinase-inactive PINK1 became stabilized by CCCP but a band-shift was noted for wild-type PINK1 which was revealed to be a doublet on lower exposure. The upper band was absent from kinase-inactive PINK1 treated with CCCP. (b) Identification of Thr257 phosphorylation site on PINK1. Flp-In T-Rex HEK293 cell lines stably expressing FLAG alone, or wild-type PINK1-FLAG were treated with DMSO or 10 μM of CCCP for 3 h. Recombinant PINK1 was immunoprecipitated from 10 mg of mitochondrial extract for each condition using anti-FLAG-agarose and subjected to 4–12% gradient SDS-PAGE and stained with colloidal Coomassie blue. The Coomassie-stained bands migrating with the expected molecular mass of PINK1-FLAG were excised from the gel, digested with trypsin, and subjected to precursor-ion scanning mass spectroscopy. The major phosphopeptide that is indicated ‘Thr257’ was seen from cells expressing wild-type PINK1-FLAG treated with CCCP and this was not seen in bands from the other two conditions. The figure shows the signal intensity (cps, counts of ions per second) of the HPO3− ion (−79 Da) seen in negative precursor ion scanning mode versus the ion distribution (m/z) for the Thr257 phosphopeptide. The observed values of 722.4 and 788.4 are for the VALAGEYGAVTYR and VALAGEYGAVTYRK variants, respectively, of the Thr257 peptide as [M-2H]2− ions. Other phosphopeptides marked with an asterisk were observed but we were unable to assign phosphorylation site(s). (c) Evidence that CCCP induces PINK1 auto-phosphorylation using a phospho-specific Thr257 antibody. 0.5 mg of mitochondrial extracts (treated with DMSO or 10 μM of CCCP for 3 h) of Flp-In T-Rex stable cell lines expressing FLAG empty, wild-type PINK1-FLAG, kinase-inactive PINK1-FLAG (D384A) and phospho-mutant Thr257Ala (T257A) were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Blots were probed with anti-phospho-Thr257 PINK1 antibody and anti-PINK1 antibody. (d) Mutation of Thr257Ala PINK1 does not affect Parkin Ser65 phosphorylation. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, kinase-inactive PINK1-FLAG and T257A PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser65Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser65 Parkin antibody in the presence of dephosphorylated peptide. Ten per cent of the IP was immune-blotted with total anti-Parkin antibody. One milligram of 1% Triton whole-cell lysate were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Blots were probed with anti-phospho-Thr257 PINK1 and anti-PINK1 antibody. (e) PINK1 dephosphorylation by lambda phosphatase inhibits PINK1 activity. C-terminal-FLAG tagged wild-type or kinase-inactive (D384A) PINK1 were immunoprecipitated from 5 mg of mitochondrial enriched extracts using anti-FLAG agarose beads. Wild-type PINK1 was incubated with or without 1000 U of lambda phosphatase or treated with lambda phosphatase along with 50 mM EDTA. Kinase-inactive PINK1 was incubated in buffer alone without lambda phosphatase. The beads were washed thrice in 50 mM Tris pH 7.5, 0.1 mM EGTA and then used in an in vitro kinase assay with GST-Parkin Ubl (1–108) as the substrate. Samples were analysed as described in legend to figure 1.

Mentions: To investigate whether human PINK1 has the potential to phosphorylate Parkin, we over-expressed full-length human Parkin in human HEK293 Flp-In TRex cells stably expressing wild-type human PINK1, or kinase-inactive human PINK1 (D384A) (figure 3a). Cells were treated with or without the mitochondrial uncoupling agent, CCCP, for 3 h—conditions that induce stabilization and activation of PINK1 at the mitochondria (see §2 and also figure 6). Parkin was immunoprecipitated and phosphorylation site analysis undertaken by mass spectrometry. This strikingly revealed that Parkin was phosphorylated at Ser65, but only in cells expressing wild-type human PINK1 that had been stimulated with CCCP (figure 3a). No detectable phosphorylation of Ser65 was observed in the absence of CCCP treatment or in cells expressing kinase-inactive PINK1 (figure 3a). This result suggests that CCCP treatment might activate PINK1 enabling it to phosphorylate Parkin (this is explored below). We also detected phosphorylation of a previously reported phosphorylation site (Ser131). In contrast to Ser65, phosphorylation of Ser131 was constitutive and not modulated by CCCP or PINK1 (figure 3a). We failed to detect phosphorylation of Parkin at another previously reported site (Thr175) [25].Figure 3.


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)

Identification and characterization of a novel autophosphorylation site of PINK1 induced by the mitochondrial uncoupling agent CCCP. (a) CCCP induces a band-shift in wild-type but not kinase-inactive PINK1. Flp-In T-Rex HEK293 cell lines stably expressing FLAG alone, wild-type or kinase-inactive PINK1-FLAG were induced to express protein by addition of 0.1 μg ml−1 of doxycycline in the culture medium for 24 h. Cells were then treated with 10 μM of CCCP for 3 h and lysates subjected to sub-cellular fractionation. Twenty-five micrograms of cytoplasmic or mitochondrial lysate were resolved by 8% SDS-PAGE. Relative purity of the fractions was confirmed using cytoplasmic and mitochondrial markers, namely GAPDH and HSP60, respectively. Whole-cell extracts from the same cells were also made in parallel using 1% Triton lysis as described in the methods. In mitochondrial and whole-cell extracts, both wild-type and kinase-inactive PINK1 became stabilized by CCCP but a band-shift was noted for wild-type PINK1 which was revealed to be a doublet on lower exposure. The upper band was absent from kinase-inactive PINK1 treated with CCCP. (b) Identification of Thr257 phosphorylation site on PINK1. Flp-In T-Rex HEK293 cell lines stably expressing FLAG alone, or wild-type PINK1-FLAG were treated with DMSO or 10 μM of CCCP for 3 h. Recombinant PINK1 was immunoprecipitated from 10 mg of mitochondrial extract for each condition using anti-FLAG-agarose and subjected to 4–12% gradient SDS-PAGE and stained with colloidal Coomassie blue. The Coomassie-stained bands migrating with the expected molecular mass of PINK1-FLAG were excised from the gel, digested with trypsin, and subjected to precursor-ion scanning mass spectroscopy. The major phosphopeptide that is indicated ‘Thr257’ was seen from cells expressing wild-type PINK1-FLAG treated with CCCP and this was not seen in bands from the other two conditions. The figure shows the signal intensity (cps, counts of ions per second) of the HPO3− ion (−79 Da) seen in negative precursor ion scanning mode versus the ion distribution (m/z) for the Thr257 phosphopeptide. The observed values of 722.4 and 788.4 are for the VALAGEYGAVTYR and VALAGEYGAVTYRK variants, respectively, of the Thr257 peptide as [M-2H]2− ions. Other phosphopeptides marked with an asterisk were observed but we were unable to assign phosphorylation site(s). (c) Evidence that CCCP induces PINK1 auto-phosphorylation using a phospho-specific Thr257 antibody. 0.5 mg of mitochondrial extracts (treated with DMSO or 10 μM of CCCP for 3 h) of Flp-In T-Rex stable cell lines expressing FLAG empty, wild-type PINK1-FLAG, kinase-inactive PINK1-FLAG (D384A) and phospho-mutant Thr257Ala (T257A) were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Blots were probed with anti-phospho-Thr257 PINK1 antibody and anti-PINK1 antibody. (d) Mutation of Thr257Ala PINK1 does not affect Parkin Ser65 phosphorylation. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, kinase-inactive PINK1-FLAG and T257A PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser65Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser65 Parkin antibody in the presence of dephosphorylated peptide. Ten per cent of the IP was immune-blotted with total anti-Parkin antibody. One milligram of 1% Triton whole-cell lysate were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Blots were probed with anti-phospho-Thr257 PINK1 and anti-PINK1 antibody. (e) PINK1 dephosphorylation by lambda phosphatase inhibits PINK1 activity. C-terminal-FLAG tagged wild-type or kinase-inactive (D384A) PINK1 were immunoprecipitated from 5 mg of mitochondrial enriched extracts using anti-FLAG agarose beads. Wild-type PINK1 was incubated with or without 1000 U of lambda phosphatase or treated with lambda phosphatase along with 50 mM EDTA. Kinase-inactive PINK1 was incubated in buffer alone without lambda phosphatase. The beads were washed thrice in 50 mM Tris pH 7.5, 0.1 mM EGTA and then used in an in vitro kinase assay with GST-Parkin Ubl (1–108) as the substrate. Samples were analysed as described in legend to figure 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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RSOB120080F6: Identification and characterization of a novel autophosphorylation site of PINK1 induced by the mitochondrial uncoupling agent CCCP. (a) CCCP induces a band-shift in wild-type but not kinase-inactive PINK1. Flp-In T-Rex HEK293 cell lines stably expressing FLAG alone, wild-type or kinase-inactive PINK1-FLAG were induced to express protein by addition of 0.1 μg ml−1 of doxycycline in the culture medium for 24 h. Cells were then treated with 10 μM of CCCP for 3 h and lysates subjected to sub-cellular fractionation. Twenty-five micrograms of cytoplasmic or mitochondrial lysate were resolved by 8% SDS-PAGE. Relative purity of the fractions was confirmed using cytoplasmic and mitochondrial markers, namely GAPDH and HSP60, respectively. Whole-cell extracts from the same cells were also made in parallel using 1% Triton lysis as described in the methods. In mitochondrial and whole-cell extracts, both wild-type and kinase-inactive PINK1 became stabilized by CCCP but a band-shift was noted for wild-type PINK1 which was revealed to be a doublet on lower exposure. The upper band was absent from kinase-inactive PINK1 treated with CCCP. (b) Identification of Thr257 phosphorylation site on PINK1. Flp-In T-Rex HEK293 cell lines stably expressing FLAG alone, or wild-type PINK1-FLAG were treated with DMSO or 10 μM of CCCP for 3 h. Recombinant PINK1 was immunoprecipitated from 10 mg of mitochondrial extract for each condition using anti-FLAG-agarose and subjected to 4–12% gradient SDS-PAGE and stained with colloidal Coomassie blue. The Coomassie-stained bands migrating with the expected molecular mass of PINK1-FLAG were excised from the gel, digested with trypsin, and subjected to precursor-ion scanning mass spectroscopy. The major phosphopeptide that is indicated ‘Thr257’ was seen from cells expressing wild-type PINK1-FLAG treated with CCCP and this was not seen in bands from the other two conditions. The figure shows the signal intensity (cps, counts of ions per second) of the HPO3− ion (−79 Da) seen in negative precursor ion scanning mode versus the ion distribution (m/z) for the Thr257 phosphopeptide. The observed values of 722.4 and 788.4 are for the VALAGEYGAVTYR and VALAGEYGAVTYRK variants, respectively, of the Thr257 peptide as [M-2H]2− ions. Other phosphopeptides marked with an asterisk were observed but we were unable to assign phosphorylation site(s). (c) Evidence that CCCP induces PINK1 auto-phosphorylation using a phospho-specific Thr257 antibody. 0.5 mg of mitochondrial extracts (treated with DMSO or 10 μM of CCCP for 3 h) of Flp-In T-Rex stable cell lines expressing FLAG empty, wild-type PINK1-FLAG, kinase-inactive PINK1-FLAG (D384A) and phospho-mutant Thr257Ala (T257A) were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Blots were probed with anti-phospho-Thr257 PINK1 antibody and anti-PINK1 antibody. (d) Mutation of Thr257Ala PINK1 does not affect Parkin Ser65 phosphorylation. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, kinase-inactive PINK1-FLAG and T257A PINK1-FLAG were co-transfected with untagged wild-type (WT) or Ser65Ala (S65A) mutant Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. 0.25 mg of 1% Triton whole-cell lysate were subjected to immunoprecipitation with anti-Parkin antibody (S966C) covalently coupled to protein G Sepharose and then immunoblotted with anti-phospho-Ser65 Parkin antibody in the presence of dephosphorylated peptide. Ten per cent of the IP was immune-blotted with total anti-Parkin antibody. One milligram of 1% Triton whole-cell lysate were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Blots were probed with anti-phospho-Thr257 PINK1 and anti-PINK1 antibody. (e) PINK1 dephosphorylation by lambda phosphatase inhibits PINK1 activity. C-terminal-FLAG tagged wild-type or kinase-inactive (D384A) PINK1 were immunoprecipitated from 5 mg of mitochondrial enriched extracts using anti-FLAG agarose beads. Wild-type PINK1 was incubated with or without 1000 U of lambda phosphatase or treated with lambda phosphatase along with 50 mM EDTA. Kinase-inactive PINK1 was incubated in buffer alone without lambda phosphatase. The beads were washed thrice in 50 mM Tris pH 7.5, 0.1 mM EGTA and then used in an in vitro kinase assay with GST-Parkin Ubl (1–108) as the substrate. Samples were analysed as described in legend to figure 1.
Mentions: To investigate whether human PINK1 has the potential to phosphorylate Parkin, we over-expressed full-length human Parkin in human HEK293 Flp-In TRex cells stably expressing wild-type human PINK1, or kinase-inactive human PINK1 (D384A) (figure 3a). Cells were treated with or without the mitochondrial uncoupling agent, CCCP, for 3 h—conditions that induce stabilization and activation of PINK1 at the mitochondria (see §2 and also figure 6). Parkin was immunoprecipitated and phosphorylation site analysis undertaken by mass spectrometry. This strikingly revealed that Parkin was phosphorylated at Ser65, but only in cells expressing wild-type human PINK1 that had been stimulated with CCCP (figure 3a). No detectable phosphorylation of Ser65 was observed in the absence of CCCP treatment or in cells expressing kinase-inactive PINK1 (figure 3a). This result suggests that CCCP treatment might activate PINK1 enabling it to phosphorylate Parkin (this is explored below). We also detected phosphorylation of a previously reported phosphorylation site (Ser131). In contrast to Ser65, phosphorylation of Ser131 was constitutive and not modulated by CCCP or PINK1 (figure 3a). We failed to detect phosphorylation of Parkin at another previously reported site (Thr175) [25].Figure 3.

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