Several mutations in PTEN-induced putative kinase 1 (PINK1) gene have been reported to be associated with recessive parkinsonism. The encoded protein is predicted to be a Ser͞Thr protein kinase targeted to mitochondria. In this study, we have investigated the effects of mutations on PINK1 kinase activity in vitro and on expression levels and localization in mammalian cells. We chose to examine two point mutations: G309D, which was originally reported to be stable and properly localized in cells and L347P, which is of interest because it is present at an appreciable carrier frequency in the Philippines. We were able to confirm kinase activity and produce artificial ''kinase-dead'' mutants that are stable but lack activity. The L347P mutation grossly destabilizes PINK1 and drastically reduces kinase activity, whereas G309D has much more modest effects on these parameters in vitro. This finding is in line with predictions based on homology modeling. We also examined the localization of PINK1 in transfected mammalian cells by using constructs that were tagged with myc or GFP at either end of the protein. These results show that PINK1 is processed at the N terminus in a manner consistent with mitochondrial import, but the mature protein also exists in the cytosol. The physiological relevance of this observation is not yet clear, but it implies that a portion of PINK1 may be exported after processing in the mitochondria. mitochondria ͉ PARK6 ͉ Parkinson's disease S everal genes have now been identified that are causally associated with recessive parkinsonism. Mutations in parkin (1) are a relatively frequent cause of parkinsonism with onset before the age of 50, having a varied but generally mild phenotype. DJ-1 mutations are less common than parkin mutations, but the phenotype is broadly similar (2). Given that recessive mutations in either of these two genes cause parkinsonism, it is likely that mutations in either parkin or DJ-1 lead to loss of dopaminergic neurons in the substantia nigra that project to the striatum. Positron-emission tomography demonstrates a loss of dopaminergic function in parkin (e.g., 3-6) and DJ-1 (7, 8) patients, supporting this idea. Therefore, although detailed pathology of these two genetic forms of parkinsonism is not available, there are clear phenotypic overlaps. This conclusion argues that there are relationships between parkin and DJ-1, but the normal functions of the two wild-type proteins are not obviously linked. Parkin is an E3 protein-ubiquitin ligase (9), whereas DJ-1 may have a number of roles (10) but can affect the ability of neurons to survive oxidative stress generated as a result of mitochondrial damage (11).Recently, mutations in the PTEN-induced putative kinase 1 (PINK1) gene have been described that are also associated with recessive parkinsonism. Initially, three pedigrees were described with identified mutations: a G309D point substitution in one family and a truncation mutation (W437X) in two additional families (12). Subsequently, several studies have described co...
The extracellular aggregation of amyloid b (Ab) peptides and the intracellular hyperphosphorylation of tau at specific epitopes are pathological hallmarks of neurodegenerative diseases such as Alzheimer's disease (AD). Cdk5 phosphorylates tau at AD-specific phospho-epitopes when it associates with p25. p25 is a truncated activator, which is produced from the physiological Cdk5 activator p35 upon exposure to Ab peptides. We show that neuronal infections with Cdk5 inhibitory peptide (CIP) selectively inhibit p25/ Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. Furthermore, Ab 1À42 -induced apoptosis of these cortical neurons was also reduced by coinfection with CIP. Of particular importance is our finding that CIP did not inhibit endogenous or transfected p35/ Cdk5 activity, nor did it inhibit the other cyclin-dependent kinases such as Cdc2, Cdk2, Cdk4 and Cdk6. These results, therefore, provide a strategy to address, and possibly ameliorate, the pathology of neurodegenerative diseases that may be a consequence of aberrant p25 activation of Cdk5, without affecting 'normal' Cdk5 activity.
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