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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

Model of Parkin activation by PINK1. Under basal conditions Parkin is kept in a closed inactive conformation by Ubl-mediated autoinhibition. Following mitochondrial depolarization, PINK1 phosphorylates Parkin at Ser65 thereby relieving Parkin autoinhibition and enabling Parkin to become active to ubiquitylate target substrates.
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RSOB120080F9: Model of Parkin activation by PINK1. Under basal conditions Parkin is kept in a closed inactive conformation by Ubl-mediated autoinhibition. Following mitochondrial depolarization, PINK1 phosphorylates Parkin at Ser65 thereby relieving Parkin autoinhibition and enabling Parkin to become active to ubiquitylate target substrates.

Mentions: Our data provide fundamental insights into the regulation of Parkin and elaborate a signalling pathway that may be central to neuronal loss in PD (figure 9). Our data would suggest that loss-of-function mutations in PINK1 would lead to suppression of Parkin E3 ligase activity and result in reduced ubiquitylation of Parkin's targets. This may explain why over-expression of Parkin in dPINK1 Drosophila restores ubiquitylation of targets and rescues the phenotype [4,5]. It is possible that the key Parkin targets are located at the mitochondria and indeed several candidate mitochondrial substrates for Parkin have been proposed, including Mitofusin1 [35], VDAC1 [12] and more recently PARIS [36] and Miro [37]. In future work, it would be vital to test whether phosphorylation of Parkin at Ser65 influences its ability to ubiquitylate these or other targets and define how this links to PD. Our data also suggest that small molecules that bind to and disrupt the Ubl domain-C-terminus auto-inhibitory interface may activate Parkin in a similar manner to Ser65 phosphorylation. If Parkin inactivation occurs in sporadic PD patients in addition to those harbouring PINK1 mutations, then such small molecule activators could hold significant therapeutic promise in combating disease progression in PD.Figure 9.


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)

Model of Parkin activation by PINK1. Under basal conditions Parkin is kept in a closed inactive conformation by Ubl-mediated autoinhibition. Following mitochondrial depolarization, PINK1 phosphorylates Parkin at Ser65 thereby relieving Parkin autoinhibition and enabling Parkin to become active to ubiquitylate target substrates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120080F9: Model of Parkin activation by PINK1. Under basal conditions Parkin is kept in a closed inactive conformation by Ubl-mediated autoinhibition. Following mitochondrial depolarization, PINK1 phosphorylates Parkin at Ser65 thereby relieving Parkin autoinhibition and enabling Parkin to become active to ubiquitylate target substrates.
Mentions: Our data provide fundamental insights into the regulation of Parkin and elaborate a signalling pathway that may be central to neuronal loss in PD (figure 9). Our data would suggest that loss-of-function mutations in PINK1 would lead to suppression of Parkin E3 ligase activity and result in reduced ubiquitylation of Parkin's targets. This may explain why over-expression of Parkin in dPINK1 Drosophila restores ubiquitylation of targets and rescues the phenotype [4,5]. It is possible that the key Parkin targets are located at the mitochondria and indeed several candidate mitochondrial substrates for Parkin have been proposed, including Mitofusin1 [35], VDAC1 [12] and more recently PARIS [36] and Miro [37]. In future work, it would be vital to test whether phosphorylation of Parkin at Ser65 influences its ability to ubiquitylate these or other targets and define how this links to PD. Our data also suggest that small molecules that bind to and disrupt the Ubl domain-C-terminus auto-inhibitory interface may activate Parkin in a similar manner to Ser65 phosphorylation. If Parkin inactivation occurs in sporadic PD patients in addition to those harbouring PINK1 mutations, then such small molecule activators could hold significant therapeutic promise in combating disease progression in PD.Figure 9.

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