In contrast to human PINK1, we report that insect orthologues of PINK1, including Drosophila melanogaster, T. castaneum (TcPINK1) and P. humanus corporis (PhcPINK1), exhibit significant catalytic kinase activity in vitro (). Using the most active orthologue, TcPINK1, we have been able to describe the first investigation of the substrate specificity of PINK1 and we have used this information to elaborate a novel peptide substrate, KKWIpYRRSPRRR (), which we have designated PINKtide. We have employed PINKtide to probe the effect that Parkinson's disease-associated mutations exert on PINK1 kinase activity (; electronic supplementary material, figure S10). A key question concerns why PINK1 from insect species is active, whereas human PINK1 is inactive when expressed in a similar manner (a; electronic supplementary material, figure S5). It is possible that mammalian PINK1 is not active as it is regulated in a more complex manner, and that additional interactors and/or covalent modifications not present in E. coli or insect hosts (used to express human PINK1 in our studies) are required to activate human PINK1.
Our results suggest that missense PINK1 mutations situated within the kinase domain exert their Parkinson's disease-causing effects by markedly suppressing kinase activity (a
). This emphasizes the importance of identifying the key physiological substrates of PINK1 in order to understand how the loss of kinase activity leads to neurodegeneration in Parkinson's disease. To date, no robust substrates of PINK1 have been discovered, partly owing to the unavailability of active recombinant PINK1. We have also evaluated some of the previously described PINK1 interactors (TRAP1 [14
] and Omi [15
]), and found that neither of them were significantly phosphorylated in vitro
by insect PINK1 (electronic supplementary material, figure S3), therefore suggesting that these are not directly phosphorylated by PINK1. It would also be interesting to establish whether sites of PINK1 phosphorylation identified in substrates in the future lie within Ser/Thr-Pro motifs that might be predicted from our initial analysis of PINK1 substrate specificity. We have also attempted to map the sites on myelin basic protein that were phosphorylated by TcPINK1 and found numerous sites were phosphorylated at a very low stoichiometry (data not shown). This confirms that myelin basic protein is not an optimal substrate for PINK1. We would predict that PINK1 would phosphorylate a genuine substrate at a distinct site(s) at significant stoichiometry.
The C-terminus of PINK1 has no homology to any known protein. We have found that removal of the C-terminus of TcPINK1 (486–570), equivalent to residues 513–581 in human PINK1, abolishes TcPINK1 kinase activity, and also that the three C-terminal truncating disease mutants W437X, Q456X and R492X ablate kinase activity (b
; electronic supplementary material, figure S10). Our data are also consistent with recent Drosophila
data showing that human wild-type PINK1 but not C-terminal-truncated human PINK1 (residues 1–509) could rescue the Drosophila
PINK1 null phenotype [13
]. It would be interesting to undertake further analysis to establish how the C-terminal domain might regulate kinase activity.
In the future, it will also be vital to solve the atomic structure of PINK1 to fully understand how the enzyme is regulated and to understand the molecular mechanism by which mutations inhibit kinase activity. The observation that the C125G mutation lying outside the kinase domain only modestly impaired kinase activity suggests that this mutation affects PINK1 function by a distinct mechanism. As the C125G mutation is close to the transmembrane mitochondrial-binding region, it could impact on recruitment of PINK1 to the mitochondrial membrane. It could also affect the interaction of PINK1 with an upstream activator or physiological substrate.
Many kinase inhibitors are being developed for the treatment of diseases involving aberrant protein phosphorylation. As it has not previously been possible to assay PINK1 kinase activity, it is not known whether the numerous kinase inhibitors currently in preclinical development or clinical use may also inhibit PINK1. It should also be noted that a recent study has advocated that PINK1 inhibitors might have utility in treating certain forms of colorectal cancer with mutations in mismatch repair genes MSH2, MLH1 and MSH6 [20
]. A key issue with this is whether administration of PINK1 inhibitors to human cancer patients could have the potential to induce Parkinson's disease. In the literature, the youngest age of the onset of Parkinson's disease in patients harbouring homozygous PINK1 mutations is approximately 10 (A217D mutation) [21
], suggesting that at least a decade of PINK1 inhibition is required before Parkinson's disease symptoms develop. It is thus likely that short periods of exposure to compounds that inhibit PINK1 for cancer treatments would carry a reduced risk of Parkinson's disease compared with chronic long-term treatment. Furthermore, chemical inhibition of kinases rarely achieves a null effect and more often confers a hypomorphic effect on kinase activity. Taking these factors into consideration, we feel that PINK1 inhibitors would be associated with a lower risk compared with disease-causing Parkinson's disease mutations that inactivate PINK1 (a
; electronic supplementary material, figure S10). We also suggest that insect PINK1 could be introduced into kinase panels used to profile kinase inhibitors being developed for the treatment of human disease, at least until it is worked out how to activate and assay human PINK1. Insect PINK1 could also be deployed to attempt to identify PINK1 inhibitors for the treatment of cancer, although further work would be needed to ascertain whether an inhibitor of insect PINK1 would also effectively suppress mammalian PINK1.
In conclusion, we report for the first time a novel method to express an active form of PINK1. This has enabled us to develop an assay to quantitatively assess PINK1 activity and investigate its substrate specificity. Our work suggests that, with the exception of the C125G mutation, all other Parkinson's disease mutations assessed are likely to exert their disease-causing effects by suppressing kinase catalytic activity. These observations emphasize the importance of PINK1 kinase activity in preventing the onset of Parkinson's disease, and that the key challenge in future will be to identify PINK1 substrates and study the relevance of these in Parkinson's disease. We hope that the results presented in this study will aid with assaying PINK1 catalytic activity and in the hunt for substrates of this enzyme.