PINK1Mediated Bnip3Phosphorylation And Its Impacts On Mitophagy

Parkinson’s Disease (PD) is one of the most common neurodegenerative movement disorders, which affects1.7%of the China’s population over the age of65. Parkinson’s Disease (PD) is characterized by selective loss of dopaminergic neurons in substantia nigra region, the presence of abnormal protein aggregates and lewy bodies. So far,16pathogenic genes have been detected in families with Parkinson’s disease, and most of those genes are related to metabolic regulation of mitochondria. For example, Mutations in PINK1gene have been implicated in autosomal recessive early onset Parkinson Disease.And PINK1encodes a Ser/Thr kinase protein which locates on mitochondria with its N-terminal and participate in mitochondial quality control. Additionlly, recent studies indicate that the protective action of PINK1is mainly depends on its kinase activity. However only a few of cellular substrates for PINK1kinase were reported.Through mass spectrometric analysis, we previously found that Bnip3is a possible substrate for PINK1kinase. Bnip3is a member of the BH3-only protein subfamily, mainly located on the outer mitochondrial membrane and it will be upregulated in hypoxic environment. Overexpression of Bnip3induces dramatic mitophagy. On the contrary, knocking down Bnip3expression inbibits mitophagy and reduces cellular resistance to cytotoxicity.So the study of regulatory relationship of PINK1and Bnip3could show us a new pathway which is controlled by phosphorylation regulation of PINK1as well.Objective:To analyze of Bnip3phosphorylation mediated by PINK1and its impacts on mitophagy.Methods:1. In order to investigate whether PINK1can interact with Bnip3, we did the co-immunoprecipitation experiment. Further more, we tested if two kinase dead mutants PINK1G309D or PINK1D384N could interrupt the interaction between PINK1and Bnip3。2. Through testing endogenous Bnip3protein level after overex-pressing PINK1WT and its kinase dead mutants in HEK293, we can study that if the increase of PINK1kinase activity can change the protein level of Bnip3, or in the presence of CCCP treatment.3. In order to investigate whether phosphorylating serine95site of Bnip3affects mitophagy or not, we constructed the expression vectors of Bnip3S95D and Bnip3S95A. For expanding biological effects of S95phosphorylation, we also constructed expression vectors of Bnip3S92/93/95A and Bnip3S92/93/95D to simulate dephosphorylation and phosphorylation situation respectively. By overexpressing Bnip3wild-type and mutants in cells, we observed the effect of phosphorylation state of S95region in Bnip3on mitophagy using immunofluorescence staining and western blot.Results:PINK1can interacts with Bnip3, But phosphorylation state of S95region in Bnip3does not influence mitophagy activity of Bnip3.Conclusion:In general, we conclude that:PINK1interacts with Bnip3and may mediate phosphorylation of Bnip3at S95site, but neither increased nor decreased phosphorylation level of this site affects mitophagic function of Bnip3.