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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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Timecourse of CCCP-induced activation of PINK1. (a) Timecourse of PINK1 autophosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing PINK1-FLAG wild-type and kinase-inactive (D384A) were stimulated at the indicated time points with 10 μM of CCCP. 0.5 mg of mitochondrial extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with anti-phospho-Thr275 antibody or total PINK1 antibody. (b) No time-dependent activation of cytoplasmic PINK1 in vivo. As in (a) cytoplasmic extracts were obtained at the indicated time-points and immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with PINK1 anti-phospho-Thr275 antibody or total PINK1 antibody. (c) Timecourse of Parkin Ser65 phosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing wild-type 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 at the indicated time points. 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. One per cent of the IP was immunoblotted with total anti-Parkin antibody. 1.5 mg of whole-cell extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody.
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RSOB120080F7: Timecourse of CCCP-induced activation of PINK1. (a) Timecourse of PINK1 autophosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing PINK1-FLAG wild-type and kinase-inactive (D384A) were stimulated at the indicated time points with 10 μM of CCCP. 0.5 mg of mitochondrial extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with anti-phospho-Thr275 antibody or total PINK1 antibody. (b) No time-dependent activation of cytoplasmic PINK1 in vivo. As in (a) cytoplasmic extracts were obtained at the indicated time-points and immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with PINK1 anti-phospho-Thr275 antibody or total PINK1 antibody. (c) Timecourse of Parkin Ser65 phosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing wild-type 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 at the indicated time points. 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. One per cent of the IP was immunoblotted with total anti-Parkin antibody. 1.5 mg of whole-cell extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody.

Mentions: We next investigated the timecourse of the PINK1 stabilization, band-shift, autophosphorylation of Thr257 and ability of PINK1 to phosphorylate Parkin following CCCP treatment. This revealed that the stabilization of full-length PINK1 at the mitochondria is rapid with significant stabilization seen within 5 min of CCCP treatment and is maximal by 40 min and then sustained for up to 3 h (figure 7a). Loss of the cleaved form of PINK1 observed in the cytosol is particularly rapid and almost disappears within 5 min of CCCP treatment (figure 7b). However, the appearance of the band-shift and autophosphorylation of Thr257 occurred more slowly and was observed only after 40 min of CCCP treatment and was sustained for up to 3 h (figure 7a). There was no phosphorylation of Thr257 or band-shift of cytoplasmic-associated PINK1 indicating that mitochondrial association is required for this (figure 7b). In contrast, monitoring Parkin Ser65 phosphorylation using the phospho-specific antibody against phospho-Ser65 indicated that Parkin Ser65 phosphorylation occurs at 5 min (figure 7c) and becomes maximal and sustained from 40 min onwards. This suggests that the kinetics of PINK1 activation against its substrate are significantly faster than the kinetics of PINK1 autophosphorylation.Figure 7.


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)

Timecourse of CCCP-induced activation of PINK1. (a) Timecourse of PINK1 autophosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing PINK1-FLAG wild-type and kinase-inactive (D384A) were stimulated at the indicated time points with 10 μM of CCCP. 0.5 mg of mitochondrial extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with anti-phospho-Thr275 antibody or total PINK1 antibody. (b) No time-dependent activation of cytoplasmic PINK1 in vivo. As in (a) cytoplasmic extracts were obtained at the indicated time-points and immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with PINK1 anti-phospho-Thr275 antibody or total PINK1 antibody. (c) Timecourse of Parkin Ser65 phosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing wild-type 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 at the indicated time points. 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. One per cent of the IP was immunoblotted with total anti-Parkin antibody. 1.5 mg of whole-cell extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376738&req=5

RSOB120080F7: Timecourse of CCCP-induced activation of PINK1. (a) Timecourse of PINK1 autophosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing PINK1-FLAG wild-type and kinase-inactive (D384A) were stimulated at the indicated time points with 10 μM of CCCP. 0.5 mg of mitochondrial extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with anti-phospho-Thr275 antibody or total PINK1 antibody. (b) No time-dependent activation of cytoplasmic PINK1 in vivo. As in (a) cytoplasmic extracts were obtained at the indicated time-points and immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with PINK1 anti-phospho-Thr275 antibody or total PINK1 antibody. (c) Timecourse of Parkin Ser65 phosphorylation in vivo. Flp-In T-Rex HEK293 cells stably expressing wild-type 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 at the indicated time points. 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. One per cent of the IP was immunoblotted with total anti-Parkin antibody. 1.5 mg of whole-cell extracts were immunoprecipitated with anti-FLAG agarose and resolved by 8% SDS-PAGE. Immunoblotting was performed with total PINK1 antibody.
Mentions: We next investigated the timecourse of the PINK1 stabilization, band-shift, autophosphorylation of Thr257 and ability of PINK1 to phosphorylate Parkin following CCCP treatment. This revealed that the stabilization of full-length PINK1 at the mitochondria is rapid with significant stabilization seen within 5 min of CCCP treatment and is maximal by 40 min and then sustained for up to 3 h (figure 7a). Loss of the cleaved form of PINK1 observed in the cytosol is particularly rapid and almost disappears within 5 min of CCCP treatment (figure 7b). However, the appearance of the band-shift and autophosphorylation of Thr257 occurred more slowly and was observed only after 40 min of CCCP treatment and was sustained for up to 3 h (figure 7a). There was no phosphorylation of Thr257 or band-shift of cytoplasmic-associated PINK1 indicating that mitochondrial association is required for this (figure 7b). In contrast, monitoring Parkin Ser65 phosphorylation using the phospho-specific antibody against phospho-Ser65 indicated that Parkin Ser65 phosphorylation occurs at 5 min (figure 7c) and becomes maximal and sustained from 40 min onwards. This suggests that the kinetics of PINK1 activation against its substrate are significantly faster than the kinetics of PINK1 autophosphorylation.Figure 7.

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