Abstract
BackgroundPINK1 (PTEN-induced putative kinase 1) gene is the causal gene for recessive familial type 6 of Parkinson’s disease (PARK6), which is an early-onset autosomal recessive inherited neurodegenerative disease. PINK1 has been reported to exert both autophosphorylation and phosphorylation activity, affecting cell damage under stress and other physiological responses. However, there has been no report on the identification of PINK1 autophosphorylation sites and their physiological functions.Methods(1) We adopted mass spectrometry assay to identify the autophosphorylation site of PINK1, and autoradiography assay was further conducted to confirm this result. (2) Kinase activity assay was used to compare the kinase activity of both Ser465 mutant PINK1 and disease-causing mutant PINK1. (3) We use Pulse-chase analysis to measure whether Ser465 may affect PINK1 degradation. (4) Immunocytochemistry staining was used to study the PINK1 subcellular localization and Parkin transition in subcellular level.ResultIn our study, we identified the 465th serine residue (Ser465) as one of the autophosphorylation sites in PINK1 protein. The inactivation of Ser465 can decrease the kinase activity of PINK1. Either dissipated or excessive Ser465 site phosphorylation of PINK1 can slow down its degradation. PINK1 autophosphorylation contributes to the transit of Parkin to mitochondria, and has no effect on its subcellular localization. PARK6 causal mutations, T313 M and R492X, display the same characteristics as Ser465A mutation PINK1 protein, such as decreasing PINK1 kinase activity and affecting its interaction with Parkin.ConclusionSer465 was identified as one of the autophosphorylation sites of PINK1, which affected PINK1 kinase activity. In addition, Ser465 is involved in the degradation of PINK1 and the transit of Parkin to mitochondria. T313 M and R492X, two novel PARK6 mutations on Thr313 and Arg492, were similar to Ser465 mutation, including decreasing PINK1 phosphorylation activity and Parkin subcellular localization.
Highlights
PINK1 (PTEN-induced putative kinase 1) gene is the causal gene for recessive familial type 6 of Parkinson’s disease (PARK6), which is an early-onset autosomal recessive inherited neurodegenerative disease
Ser465 is involved in the degradation of PINK1 and the transit of Parkin to mitochondria
Plasmid construction 293A human embryonic kidney cells, pGEX-5X-1 vector, pKH3-PINK1 and EGFP-Parkin plasmid were provided by the school of Life Science of University of Science and Technology in China. pKH3-PINK1T313 M, pKH3-PINK1-R492X and pGEX-5X-1-PINK1 plasmids were constructed in our previous work [3, 22]. pGEX-5X-1-PINK1-T313 M, pGEX-5X-1-PINK1-R492X, pGEX-5X-1-PINK1-S465A, pGEX-5X-1-PINK1-S465D, pKH3-PINK1-S465A and pKH3-PINK1- S465D plasmids were all constructed by this study
Summary
PINK1 (PTEN-induced putative kinase 1) gene is the causal gene for recessive familial type 6 of Parkinson’s disease (PARK6), which is an early-onset autosomal recessive inherited neurodegenerative disease. PINK1 has been reported to exert both autophosphorylation and phosphorylation activity, affecting cell damage under stress and other physiological responses. PINK1 (PTEN-induced putative kinase 1) gene is the causative gene for PARK6, which was cloned in 2004 by Valente et al [20]. Recent studies showed that PINK1 may directly phosphorylate other proteins, exerting anti-oxidative stress and anti-apoptosis effects. TRAP1 protein is found to be the substrate of PINK1 [14], through which, PINK1 can reduce the release of mitochondrial cytochrome c and fill the gap caused by cell damage and even cell death under stress. In 2008, Guo et al Translational Neurodegeneration (2017) 6:34
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