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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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PINK1 is specifically activated by inducers of mitochondrial membrane depolarization. (a) Table of agonists tested. (b) PINK1 phosphorylation of Parkin at Ser65 is specific to mitochondrial uncouplers. Flp-In T-Rex HEK293 cells expressing wild-type PINK1-FLAG were co-transfected with untagged wild-type Parkin, induced with 0.1 μg ml−1 doxycycline for 24 h and stimulated with the indicated agonists for 3 h except for Deferiprone (24 h treatment). 0.25 mg of 1% Triton whole-cell lysate was 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 IP was immunoblotted with total anti-Parkin antibody. Twenty-five micrograms of whole-cell lysate was immunoblotted with total PINK1 antibody (Novus), phospho-JNK (CST), total-JNK (CST), phospho-ERK1/2, total-ERK1/2 (CST), phospho-ACC (CST), total ACC (CST), phospho-AMPK (CST) and total AMPK (CST). Representative of two independent experiments.
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RSOB120080F8: PINK1 is specifically activated by inducers of mitochondrial membrane depolarization. (a) Table of agonists tested. (b) PINK1 phosphorylation of Parkin at Ser65 is specific to mitochondrial uncouplers. Flp-In T-Rex HEK293 cells expressing wild-type PINK1-FLAG were co-transfected with untagged wild-type Parkin, induced with 0.1 μg ml−1 doxycycline for 24 h and stimulated with the indicated agonists for 3 h except for Deferiprone (24 h treatment). 0.25 mg of 1% Triton whole-cell lysate was 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 IP was immunoblotted with total anti-Parkin antibody. Twenty-five micrograms of whole-cell lysate was immunoblotted with total PINK1 antibody (Novus), phospho-JNK (CST), total-JNK (CST), phospho-ERK1/2, total-ERK1/2 (CST), phospho-ACC (CST), total ACC (CST), phospho-AMPK (CST) and total AMPK (CST). Representative of two independent experiments.

Mentions: Within the inner mitochondrial membrane (IMM), the electron transport chain transfers electrons through a series of oxidation–reduction reactions coupled to the transfer of protons across the IMM and this efflux creates a proton electrochemical gradient known as the protomotive force. The protomotive force drives the re-entry of protons through the proton channel of the F1F0-ATP synthase crucial for ATP production and comprises mainly of an electrical component—the mitochondrial membrane potential (Δψm)—and a transmembrane pH gradient [26]. CCCP dissipates both Δψm and the pH gradient leading to impaired mitochondrial ATP synthesis. To determine the mechanism of activation of PINK1, we tested a panel of agonists that have previously been reported to disrupt mitochondria by diverse modes of action (figure 8a). Under the conditions tested only the proton ionophores CCCP, FCCP and the potassium uniporter valinomycin were able to induce activation of PINK1 leading to Parkin Ser65 phosphorylation (figure 8b). In contrast to CCCP and FCCP, valinomycin depolarizes the Δψm but does not affect the pH gradient suggesting that PINK1 is specifically activated by loss of the Δψm. We did not observe any effect of an inhibitor of ATP synthase (oligomycin) or various inhibitors of the electron transport chain complexes that have previously been implicated in neurodegeneration models (MPP+, rotenone, 3-nitropropionic acid).Figure 8.


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)

PINK1 is specifically activated by inducers of mitochondrial membrane depolarization. (a) Table of agonists tested. (b) PINK1 phosphorylation of Parkin at Ser65 is specific to mitochondrial uncouplers. Flp-In T-Rex HEK293 cells expressing wild-type PINK1-FLAG were co-transfected with untagged wild-type Parkin, induced with 0.1 μg ml−1 doxycycline for 24 h and stimulated with the indicated agonists for 3 h except for Deferiprone (24 h treatment). 0.25 mg of 1% Triton whole-cell lysate was 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 IP was immunoblotted with total anti-Parkin antibody. Twenty-five micrograms of whole-cell lysate was immunoblotted with total PINK1 antibody (Novus), phospho-JNK (CST), total-JNK (CST), phospho-ERK1/2, total-ERK1/2 (CST), phospho-ACC (CST), total ACC (CST), phospho-AMPK (CST) and total AMPK (CST). Representative of two independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120080F8: PINK1 is specifically activated by inducers of mitochondrial membrane depolarization. (a) Table of agonists tested. (b) PINK1 phosphorylation of Parkin at Ser65 is specific to mitochondrial uncouplers. Flp-In T-Rex HEK293 cells expressing wild-type PINK1-FLAG were co-transfected with untagged wild-type Parkin, induced with 0.1 μg ml−1 doxycycline for 24 h and stimulated with the indicated agonists for 3 h except for Deferiprone (24 h treatment). 0.25 mg of 1% Triton whole-cell lysate was 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 IP was immunoblotted with total anti-Parkin antibody. Twenty-five micrograms of whole-cell lysate was immunoblotted with total PINK1 antibody (Novus), phospho-JNK (CST), total-JNK (CST), phospho-ERK1/2, total-ERK1/2 (CST), phospho-ACC (CST), total ACC (CST), phospho-AMPK (CST) and total AMPK (CST). Representative of two independent experiments.
Mentions: Within the inner mitochondrial membrane (IMM), the electron transport chain transfers electrons through a series of oxidation–reduction reactions coupled to the transfer of protons across the IMM and this efflux creates a proton electrochemical gradient known as the protomotive force. The protomotive force drives the re-entry of protons through the proton channel of the F1F0-ATP synthase crucial for ATP production and comprises mainly of an electrical component—the mitochondrial membrane potential (Δψm)—and a transmembrane pH gradient [26]. CCCP dissipates both Δψm and the pH gradient leading to impaired mitochondrial ATP synthesis. To determine the mechanism of activation of PINK1, we tested a panel of agonists that have previously been reported to disrupt mitochondria by diverse modes of action (figure 8a). Under the conditions tested only the proton ionophores CCCP, FCCP and the potassium uniporter valinomycin were able to induce activation of PINK1 leading to Parkin Ser65 phosphorylation (figure 8b). In contrast to CCCP and FCCP, valinomycin depolarizes the Δψm but does not affect the pH gradient suggesting that PINK1 is specifically activated by loss of the Δψm. We did not observe any effect of an inhibitor of ATP synthase (oligomycin) or various inhibitors of the electron transport chain complexes that have previously been implicated in neurodegeneration models (MPP+, rotenone, 3-nitropropionic acid).Figure 8.

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