DNA damage induces nuclear translocation of parkin

Abstract

Parkinson's disease (PD) is the second most common form of human degenerative disorder. Mutation of parkin is one of the most prevalent causes of autosomal recessive PD. Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here I show that parkin translocates into nucleus upon DNA damage. Nuclear translocation of parkin appears to be required to promote DNA repair. These findings suggest that DNA damage induces nuclear translocation of parkin leading to the PCNA interaction and possibly other nuclear proteins involved in DNA repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in parkinsonism.

Figure 1

DNA damage and nuclear translocation of parkin. (A) DNA induces nuclear translocation of parkin. SH-SY5Y cells stably transfected with parkin were UV-irradiated (60 J/m²), or treated with H₂O₂ (300 μM) or etoposide (50 μM) to induce DNA damage, followed by the isolation of nuclear and cytoplasmic fractions and resolution of parkin by immunoblotting. Note that each DNA damaging agent induced an increase in the absolute and relative levels of parkin in the nucleus. Absence of cross-contamination of the fractions was confirmed by immunoblotting for lamin B, a nuclear marker, and β-actin, a cytoplasmic marker. Quantitation of the relative nuclear ratio of parkin is shown, and represents the mean ± S.D., n = 3. *P < 0.05 relative to control by ANOVA with post-hoc Student Neumann-Kiels tests. (B) Nuclear translocation of parkin in SH-SY5Y cells was confirmed by immunofluorescence assays. SH-SY5Y cells stably over-expressing parkin were UV-irradiated (60 J/m²) or mock-treated. Two hours later the cellular localization of parkin was determined by immunofluorescent microscopy. Green: parkin. Blue: Hoechst staining of nucleus.

Figure 2

Parkin interacts with damaged DNA. (A) Parkin was co-transfected in HeLa cells with a calmodulin 1 promoter construct under control conditions (-UV) or after the calmodulin construct was damaged by UV irradiation in vitro (+UV)(100 J/M²). To determine whether parkin interacts with damaged DNA, parkin was immunoprecipitated followed by PCR amplification of the calmodulin promoter construct. Note the markedly increased co-precipitation of parkin with DNA damaged by UV irradiation. Input DNA, non-specific IgG controls, and parkin, β-actin immunoblots are shown. (B) Parkin binds to chromatin in the aging human brain. ChIP assays were performed on postmortem human cortical samples with anti-parkin or anti-8-oxoguanine followed by PCR amplification of a calmodulin-1 promoter sequence that is damaged in human cortical samples from aged and young adult individuals. Note increased binding of parkin to chromatin from some aged cortical samples (≥ 73 years old) relative to young adult samples (<40 years old), which correlates with increased 8-oxoguanine content. Input and non-specific IgG ChIP controls are shown.

Figure 3

Parkin reduces DNA damage induced by UV irradiation. Stably SH-SY5Y cell lines expressing wild-type parkin, the Del3–4 parkin mutant, or the pcDNA control vector were UV-irradiated (100 J/M²) and then assessed for DNA damage by immunocytochemical staining for cyclopyrimidine dimers (CPD) (red). Nuclei were stained with Hoechst dye. All three cell lines show similar robust CPD staining 1 hr after UV irradiation. Decreased DNA damage in SH-SY5Y cells expressing parkin, but not the pcDNA control and Del3–4 parkin mutant was detected. Quantitation of CPD positive cells in three cell lines after UV irradiation is shown, and represents the mean ± S.D., n = 5. *P < 0.05, **P > 0.05 relative to control by ANOVA with post-hoc Student Neumann-Kiels tests.

Figure 4

Nuclear localized parkin promotes DNA excision repair. (A) HCR assays showed Parkin facilitates DNA repair of UV induced DNA lesions in SH-SY5Y cells (pTK-Renilla irradiated by UV-C at 250 J/m²). Values are expressed relative to the transfected undamaged reporter, and represent the mean ± s.d.; n = 3. Asterisks indicate P < 0.05 relative to control by ANOVA with post-hoc Student Neumann-Kiels tests. Western blot analysis showed the expression of wild type parkin and parkin Del3–4 mutant. (B) HCR assays showed the ability of wild type Parkin, Parkin-NLS, and Parkin-NES to regulate DNA repair activities. n = 3. * P < 0.05, ** P > 0.05 relative to control by ANOVA with post-hoc Student Neumann-Kiels tests. Western blot analysis showed the expression of wild type parkin, parkin-NLS, and parkin-NES.