Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation
Hui Xiong, … , Xiaoxi Zhuang, Zhuohua Zhang
Hui Xiong, … , Xiaoxi Zhuang, Zhuohua Zhang
Published February 23, 2009
Citation Information: J Clin Invest. 2009;119(3):650-660. https://doi.org/10.1172/JCI37617.
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Research Article

Parkin, PINK1, and DJ-1 form a ubiquitin E3 ligase complex promoting unfolded protein degradation

Abstract

Mutations in PARKIN, pten-induced putative kinase 1 (PINK1), and DJ-1 are individually linked to autosomal recessive early-onset familial forms of Parkinson disease (PD). Although mutations in these genes lead to the same disease state, the functional relationships between them and how their respective disease-associated mutations cause PD are largely unknown. Here, we show that Parkin, PINK1, and DJ-1 formed a complex (termed PPD complex) to promote ubiquitination and degradation of Parkin substrates, including Parkin itself and Synphilin-1 in neuroblastoma cells and human brain lysates. Genetic ablation of either Pink1 or Dj-1 resulted in reduced ubiquitination of endogenous Parkin as well as decreased degradation and increased accumulation of aberrantly expressed Parkin substrates. Expression of PINK1 enhanced Parkin-mediated degradation of heat shock–induced misfolded protein. In contrast, PD-pathogenic Parkin and PINK1 mutations showed reduced ability to promote degradation of Parkin substrates. This study identified a functional ubiquitin E3 ligase complex consisting of PD-associated Parkin, PINK1, and DJ-1 to promote degradation of un-/misfolded proteins and suggests that their PD-pathogenic mutations impair E3 ligase activity of the complex, which may constitute a mechanism underlying PD pathogenesis.

Authors

Hui Xiong, Danling Wang, Linan Chen, Yeun Su Choo, Hong Ma, Chengyuan Tang, Kun Xia, Wei Jiang, Ze’ev Ronai, Xiaoxi Zhuang, Zhuohua Zhang

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Figure 9

PD-pathogenic Parkin or PINK1 mutants impair Parkin degradation.

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PD-pathogenic Parkin or PINK1 mutants impair Parkin degradation. (A) Int...
(A) Interaction of PINK1WT with PD-pathogenic ParkinΔE4. Cells expressing PINK1WT alone (Control), PINK1WT and ParkinΔE4 (ΔE4), or PINK1WT and ParkinWT (Parkin) were immunoprecipitated with an anti-flag antibody (to precipitate PINK1), followed by immunoblotting with an anti-myc antibody (to detect Parkin variants). (B) Interaction of ParkinWT with PINK1G309D. Cells expressing ParkinWT alone (control), ParkinWT and PINK1G309D (G309D), or ParkinWT and PINK1WT (PINK1) were immunoprecipitated with anti-myc antibody (to precipitate Parkin), followed by immunoblotting with an anti-flag antibody (to detect PINK1 variants). (C) PINK1 promoted degradation of PD-pathogenic Parkin mutants. ParkinWT (WT) and PD-pathogenic ParkinR42P, -T240W, and -ΔE4 were cotransfected with or without PINK1. Steady-state levels of Parkin, PINK1, and tubulin were analyzed by immunoblotting. PINK1 promoted significant degradation of ParkinWT but not ParkinR42P, -T240W, or -ΔE4. (D) PD-pathogenic PINK1 mutants were impaired in promoting Parkin degradation. PINK1WT (WT) and PD-pathogenic PINK1G309D, -T313M, and -P399L were cotransfected with or without Parkin. Steady-state levels of Parkin, PINK1, and tubulin were detected by immunoblotting. PD-pathogenic mutants showed little or reduced ability to promote Parkin degradation. (E and F) Quantitation of Parkin degradation affected by pathogenic Parkin (E) or PINK1 (F) mutations. The data were from 3 independent experiments. Relative Parkin levels were normalized to either the level of Parkin variants without PINK1 expression (E, black bars) or the level of ParkinWT without PINK1 expression (Ctrl; F) in the same experiment.