Functional and biochemical study of human PTEN-induced kinase (PINK1), a protein associated with early onset parkinsonism.

摘要

Mutations in the PTEN-induced kinase (PINK1) gene are associated with recessive parkinsonism. The encoded protein is predicted to be a Ser/Thr protein kinase targeted to mitochondria. First, we examine localization and processing of PINK1 in transfected mammalian cells using constructs that were tagged with myc or GFP at either end of the protein. Our results show that full length PINK1 is processed at the N terminus, resulting in the formation of the mature kinase lacking approximately 80--100 amino acids. Additionally, we verified the mitochondrial localization of PINK1 and showed that the mature protein is also present in the cytosol. We were able to confirm kinase activity in vitro with the recombinant kinase domain of PINK1 and produce artificial kinase impaired mutants that lack this activity. We have also investigated the effects of PINK1 mutations on autophosphorylation activity, expression levels, processing, and localization in mammalian cells. We examined nine point mutations in PINK1 described in patients with early onset parkinsonism. We show that C92F, L347P, G386S, and E417G mutations destabilize the protein and reduce autophosphorylation activity. The G309D mutation was stable, but demonstrated a decrease in autophosphorylation activity. To investigate the role of PINK1 in neurodegeneration, we have generated cell lines stably expressing either wild-type, kinase impaired or G309D mutations. We show a protective effect of wild type PINK1 on cell death mediated by the mitochondrial complex I inhibitors. In contrast, kinase-inactive mutants abrogated the protective effect of PINK1. To understand the protective function of PINK1 against cell death and the mechanism of protection we searched for potential PINK1 substrates using protein-based microarray technology. We showed that PINK1 phosphorylates several members of the MST family kinases involved in cell survival and apoptosis. The role of these phosphorylations has yet to be elucidated. Importantly, further analysis has demonstrated decreased MST kinases phosphorylation by most PINK1 mutants studied herein.