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PINK1-dependent phosphorylation of PINK1 and Parkin is essential for mitochondrial quality control.

ABSTRACT: Mitochondrial dysfunction has been linked to the pathogenesis of a large number of inherited diseases in humans, including Parkinson's disease, the second most common neurodegenerative disorder. The Parkinson's disease genes pink1 and parkin, which encode a mitochondrially targeted protein kinase, and an E3 ubiquitin ligase, respectively, participate in a key mitochondrial quality-control pathway that eliminates damaged mitochondria. In the current study, we established an in vivo PINK1/Parkin-induced photoreceptor neuron degeneration model in Drosophila with the aim of dissecting the PINK1/Parkin pathway in detail. Using LC-MS/MS analysis, we identified Serine 346 as the sole autophosphorylation site of Drosophila PINK1 and found that substitution of Serine 346 to Alanine completely abolished the PINK1 autophosphorylation. Disruption of either PINK1 or Parkin phosphorylation impaired the PINK1/Parkin pathway, and the degeneration phenotype of photoreceptor neurons was obviously alleviated. Phosphorylation of PINK1 is not only required for the PINK1-mediated mitochondrial recruitment of Parkin but also induces its kinase activity toward Parkin. In contrast, phosphorylation of Parkin by PINK1 is dispensable for its translocation but required for its activation. Moreover, substitution with autophosphorylation-deficient PINK1 failed to rescue pink1 null mutant phenotypes. Taken together, our findings suggest that autophosphorylation of PINK1 is essential for the mitochondrial translocation of Parkin and for subsequent phosphorylation and activation of Parkin.

SUBMITTER: Zhuang N

PROVIDER: S-EPMC5261015 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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PINK1-dependent phosphorylation of PINK1 and Parkin is essential for mitochondrial quality control.

Zhuang Na N Li Lin L Chen She S Wang Tao T

Cell death & disease 20161201 12

Mitochondrial dysfunction has been linked to the pathogenesis of a large number of inherited diseases in humans, including Parkinson's disease, the second most common neurodegenerative disorder. The Parkinson's disease genes pink1 and parkin, which encode a mitochondrially targeted protein kinase, and an E3 ubiquitin ligase, respectively, participate in a key mitochondrial quality-control pathway that eliminates damaged mitochondria. In the current study, we established an in vivo PINK1/Parkin-i ...[more]

PMID: 27906179

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Project description:We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (?Ubl-Parkin) is robustly activated by ubiquitinPhospho-Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho-Ser65) can also activate ?Ubl-Parkin similarly to ubiquitinPhospho-Ser65. Thirdly, we establish that ubiquitinPhospho-Ser65, but not non-phosphorylated ubiquitin or UblPhospho-Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho-Ser65, suggesting that small molecules that mimic ubiquitinPhospho-Ser65 could hold promise as novel therapies for Parkinson's disease.

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PINK1-dependent phosphorylation of PINK1 and Parkin is essential for mitochondrial quality control.

Publications

PINK1-dependent phosphorylation of PINK1 and Parkin is essential for mitochondrial quality control.

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