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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

Human Parkin Ser65 is a substrate of human PINK1 upon CCCP stimulation. (a) Confirmation by mass spectrometry that Ser65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser131 and Ser65 phosphopeptide (3+ R.NDWTVQNCDLDQQSIVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y-axis and retention time is plotted on the x-axis. The m/z value corresponding to the Ser131 phosphopeptide was detected in all conditions whilst that of the Ser65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. (b) Characterization of Parkin phospho-Ser65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive 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 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.
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RSOB120080F3: Human Parkin Ser65 is a substrate of human PINK1 upon CCCP stimulation. (a) Confirmation by mass spectrometry that Ser65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser131 and Ser65 phosphopeptide (3+ R.NDWTVQNCDLDQQSIVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y-axis and retention time is plotted on the x-axis. The m/z value corresponding to the Ser131 phosphopeptide was detected in all conditions whilst that of the Ser65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. (b) Characterization of Parkin phospho-Ser65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive 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 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.

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)

Human Parkin Ser65 is a substrate of human PINK1 upon CCCP stimulation. (a) Confirmation by mass spectrometry that Ser65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser131 and Ser65 phosphopeptide (3+ R.NDWTVQNCDLDQQSIVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y-axis and retention time is plotted on the x-axis. The m/z value corresponding to the Ser131 phosphopeptide was detected in all conditions whilst that of the Ser65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. (b) Characterization of Parkin phospho-Ser65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive 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 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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RSOB120080F3: Human Parkin Ser65 is a substrate of human PINK1 upon CCCP stimulation. (a) Confirmation by mass spectrometry that Ser65 of human Parkin is phosphorylated by CCCP-induced activation of human wild-type PINK1-FLAG. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive PINK1-FLAG (D384A) were co-transfected with HA-Parkin, induced with doxycycline and stimulated with 10 μM of CCCP for 3 h. Whole-cell extracts were obtained following lysis with 1% Triton and approximately 30 mg of whole-cell extract were subjected to immunoprecipitation with anti-HA-agarose and run on 10% SDS-PAGE and stained with colloidal Coomassie blue. Coomassie-stained bands migrating with the expected molecular mass of HA-Parkin were excised from the gel, digested with trypsin, and subjected to high performance liquid chromatography with tandem mass spectrometry (LC-MS-MS) on an LTQ-Orbitrap mass spectrometer. Extracted ion chromatogram analysis of Ser131 and Ser65 phosphopeptide (3+ R.NDWTVQNCDLDQQSIVHIVQRPWR.K+P). The total signal intensity of the phosphopeptide is plotted on the y-axis and retention time is plotted on the x-axis. The m/z value corresponding to the Ser131 phosphopeptide was detected in all conditions whilst that of the Ser65 phosphopeptide was only detected in samples from wild-type PINK1-FLAG-expressing cells following CCCP treatment. (b) Characterization of Parkin phospho-Ser65 antibody. Flp-In T-Rex HEK293 cells expressing FLAG-empty, wild-type PINK1-FLAG, and kinase-inactive 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 antibody in the presence of dephosphorylated peptide. Ten per cent of the immunoprecipitate (IP) was immunoblotted with total anti-Parkin antibody. Twenty five micrograms of whole cell lysate was immunoblotted with total PINK1 antibody.
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