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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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Human PINK1 directly phosphorylates Parkin Ser65 upon CCCP stimulation in vitro. Flp-In T-Rex HEK293 cells expressing wild-type PINK1-FLAG, and 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. Five milligrams of mitochondrial lysate was subjected to immunoprecipitation with anti-FLAG agarose and used in an in vitro radioactive kinase assay with [γ-32P]-Mg2+ATP and E. coli expressed recombinant GST-Parkin Ubl domain (residues 1–108) (Ubl) and mutant GST-Parkin (residues 1–108) Ser65Ala (Ubl S65A), purified from E. coli. One half of the assay reaction was run on a 10% SDS-PAGE and was subjected to autoradiography. Colloidal Coomassie stained gel shows equal loading of recombinant substrate. The other half of the reaction was immunoblotted with anti-phospho-Thr257 PINK1 and total PINK1 antibodies following 8% SDS-PAGE.
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RSOB120080F5: Human PINK1 directly phosphorylates Parkin Ser65 upon CCCP stimulation in vitro. Flp-In T-Rex HEK293 cells expressing wild-type PINK1-FLAG, and 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. Five milligrams of mitochondrial lysate was subjected to immunoprecipitation with anti-FLAG agarose and used in an in vitro radioactive kinase assay with [γ-32P]-Mg2+ATP and E. coli expressed recombinant GST-Parkin Ubl domain (residues 1–108) (Ubl) and mutant GST-Parkin (residues 1–108) Ser65Ala (Ubl S65A), purified from E. coli. One half of the assay reaction was run on a 10% SDS-PAGE and was subjected to autoradiography. Colloidal Coomassie stained gel shows equal loading of recombinant substrate. The other half of the reaction was immunoblotted with anti-phospho-Thr257 PINK1 and total PINK1 antibodies following 8% SDS-PAGE.

Mentions: Our results suggest that human PINK1 is activated by CCCP treatment and thereby rendered capable of phosphorylating Parkin at Ser65. To test this, we first immunoprecipitated wild-type or kinase-inactive PINK1 from the mitochondrial fraction of cells treated with CCCP and tested to see whether it could phosphorylate the Ubl domain of Parkin in vitro (figure 5). This revealed that wild-type PINK1 isolated from CCCP-treated cells but not from non-treated cells could indeed phosphorylate the Ubl domain of Parkin (figure 5). Importantly, kinase-inactive PINK1 isolated from CCCP-stimulated cells failed to phosphorylate the Ubl domain of Parkin. Mutation of Ser65 to Ala also prevented wild-type PINK1 isolated from CCCP-stimulated cells from phosphorylating the Ubl domain of Parkin (figure 5). These observations indicate that CCCP treatment is indeed leading to the activation of human PINK1 enabling it to directly phosphorylate Parkin at Ser65.Figure 5.


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 PINK1 directly phosphorylates Parkin Ser65 upon CCCP stimulation in vitro. Flp-In T-Rex HEK293 cells expressing wild-type PINK1-FLAG, and 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. Five milligrams of mitochondrial lysate was subjected to immunoprecipitation with anti-FLAG agarose and used in an in vitro radioactive kinase assay with [γ-32P]-Mg2+ATP and E. coli expressed recombinant GST-Parkin Ubl domain (residues 1–108) (Ubl) and mutant GST-Parkin (residues 1–108) Ser65Ala (Ubl S65A), purified from E. coli. One half of the assay reaction was run on a 10% SDS-PAGE and was subjected to autoradiography. Colloidal Coomassie stained gel shows equal loading of recombinant substrate. The other half of the reaction was immunoblotted with anti-phospho-Thr257 PINK1 and total PINK1 antibodies following 8% SDS-PAGE.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120080F5: Human PINK1 directly phosphorylates Parkin Ser65 upon CCCP stimulation in vitro. Flp-In T-Rex HEK293 cells expressing wild-type PINK1-FLAG, and 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. Five milligrams of mitochondrial lysate was subjected to immunoprecipitation with anti-FLAG agarose and used in an in vitro radioactive kinase assay with [γ-32P]-Mg2+ATP and E. coli expressed recombinant GST-Parkin Ubl domain (residues 1–108) (Ubl) and mutant GST-Parkin (residues 1–108) Ser65Ala (Ubl S65A), purified from E. coli. One half of the assay reaction was run on a 10% SDS-PAGE and was subjected to autoradiography. Colloidal Coomassie stained gel shows equal loading of recombinant substrate. The other half of the reaction was immunoblotted with anti-phospho-Thr257 PINK1 and total PINK1 antibodies following 8% SDS-PAGE.
Mentions: Our results suggest that human PINK1 is activated by CCCP treatment and thereby rendered capable of phosphorylating Parkin at Ser65. To test this, we first immunoprecipitated wild-type or kinase-inactive PINK1 from the mitochondrial fraction of cells treated with CCCP and tested to see whether it could phosphorylate the Ubl domain of Parkin in vitro (figure 5). This revealed that wild-type PINK1 isolated from CCCP-treated cells but not from non-treated cells could indeed phosphorylate the Ubl domain of Parkin (figure 5). Importantly, kinase-inactive PINK1 isolated from CCCP-stimulated cells failed to phosphorylate the Ubl domain of Parkin. Mutation of Ser65 to Ala also prevented wild-type PINK1 isolated from CCCP-stimulated cells from phosphorylating the Ubl domain of Parkin (figure 5). These observations indicate that CCCP treatment is indeed leading to the activation of human PINK1 enabling it to directly phosphorylate Parkin at Ser65.Figure 5.

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