Cases of inherited Parkinson disease, while rare, have been eagerly studied for clues to causes of the far more prevalent sporadic form of the disease. So far, three genes—parkin, DJ1, and PINK1—have been identified that cause a recessive form of early onset PD. PINK1, the newest member of the trio (see ARF related news story) bears all the sequence hallmarks of a mitochondrial targeted protein kinase. Like parkin (a ubiquitin ligase), and DJ1 (a mitochondrial protein of unknown function; see ARF related news story), PINK1 plays a role in protecting cells from stress, but the details of its role in normal cells and in PD are not yet understood. Now, Mark Cookson and his colleagues at the National Institute on Aging have characterized the activity and cellular localization of wild-type PINK1 and two PD-causing mutants. The authors' work, which appeared in PNAS online on April 11, confirms that the PD mutations compromise PINK1 kinase activity and raises some interesting questions about the protein stability and cellular localization of the protein.

Natural substrates for PINK1 have not yet been identified, but first author Alexandra Beilina and colleagues were able to purify a fragment of the protein that retains autophosphorylation activity. While perhaps not physiologically relevant, the authors found that this activity was halved by the G309D mutation found in a Spanish family, while the L347P mutation found in the Philippines practically abolished kinase activity. But the authors also found that the L347P mutation dramatically reduces protein stability, in either bacteria or mammalian COS cells, suggesting that loss of PINK1 can be causal for PD. The link between the G309D mutant and PD is less obvious, however, given that the protein is relatively stable and retains substantial activity, though this mutation has been shown to leave cells susceptible to mitochondria-induced apoptosis (see ARF related news story).

Of course, evidence linking mitochondria and Parkinson disease has steadily grown, and PINK1 does have a mitochondrial localization signal, which prompted Beilina and colleagues to test the protein's cellular distribution. They found that when overexpressed in mammalian cells, PINK1 is localized mainly to the mitochondria, but that the protein is proteolytically cleaved and a small amount of the C-terminal ends up in the cytosol. The significance of the cytosolic location of the protein is unknown. Though the authors used two different C-terminal tags, both giving similar localization patterns, the cytosolic protein could be an artifact of overexpression. They also found that localization was not affected by PD mutations, suggesting changes in cell localization do not underlie the pathogenic effect of the mutations.

“The outstanding problem now is that we don’t know what the kinase targets are,” said Cookson, who is now fishing for PINK1-associated proteins that could be cellular substrates.—Pat McCaffrey.

Reference:
Beilina A, Van Der Brug M, Ahmad R, Kesavapany S, Miller DW, Petsko GA, Cookson MR. Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability. Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5703-8. Epub 2005 Apr 11. Abstract

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  1. This paper by Mark Cookson’s group is very exciting: While it starts answering some of the numerous issues regarding PINK1 function, at the same time it poses new, intriguing questions. There are at least two important messages in this paper. First, the authors showed that at least one of the mutations—L347P— dramatically decreased the stability of PINK1 protein and its kinase activity. Kinase activity is also reduced for the stable G309D mutant, although in a less significant way. This demonstrates for the first time that PINK1 mutations act with a loss-of-function mechanism and that kinase activity of PINK1 is crucial to its function. The recent identification of another PD gene encoding a protein—dardarin—with likely kinase activity makes these findings even more interesting. Secondly, by expressing the protein with a variety of C-terminus tags in addition to those at the N-terminus, Beilina and co-workers clearly showed that PINK1 is processed in the mitochondria to a mature form about 8-10 kD smaller than the native protein, and that this mature form is enriched in the mitochondrial fraction after subcellular fractionation. These data show the cleavage of the predicted mitochondrial leading peptide at the N-terminus and indeed confirm the mitochondrial targeting of PINK1. But that’s not all, and some of the data presented in this work raise new, interesting queries. For instance, a consistent proportion of the mature PINK1 protein seems to localize in the cytosol. Although this could simply represent an artifact of protein overexpression, it is worth noting that cytoplasmic localization is not observed for N-terminus tagged PINK1, which represents the preprotein prior to mitochondrial cleavage. The authors suggest that, after processing of the leader peptide, a proportion of mature PINK1 could be exported back into the cytoplasm. This challenging hypothesis implies that mitochondria might not be the only scenario of action for PINK1.
    The cloning of PINK1, encoding the first mitochondrial protein directly linked to Parkinson disease, seemed to crown a large amount of evidence that pointed at mitochondrial dysfunction as the key pathogenetic event of PD, such as the mitochondrial effects observed in parkin knockout animal and cellular models, and the demonstration of mitochondrial relocalization of DJ-1 after oxidative stress. Now, should the cytoplasmic localization of a pool of mature PINK1 be confirmed for the endogenous protein, we will face a more complex scenario where the pathogenetic pathways of distinct PD genes can variably intersect at different cellular levels. At present, the understanding of this intricate interplay is one of the most thrilling challenges of PD research.

  2. Might we expect that the anti-inflammatory PPAR-γ agonists which upregulate PTEN may be especially beneficial for those with PINK1-PTEN-induced putative kinase 1 mutations?

    References:

    . PPAR-gamma modulates allergic inflammation through up-regulation of PTEN. FASEB J. 2005 Jun;19(8):1033-5. PubMed.

    . Tumor suppressor and anti-inflammatory actions of PPARgamma agonists are mediated via upregulation of PTEN. Curr Biol. 2001 May 15;11(10):764-8. PubMed.

  3. Of further interest is the finding by Kim et al. that DJ-1 is a regulator of PTEN.

    References:

    . DJ-1, a novel regulator of the tumor suppressor PTEN. Cancer Cell. 2005 Mar;7(3):263-73. PubMed.

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References

News Citations

  1. Pink Mutations Link Parkinson’s Disease to Mitochondria
  2. Protection Against Parkinson’s—How the DJ Changes Station

Paper Citations

  1. . Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability. Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5703-8. PubMed.

Further Reading

Papers

  1. . Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability. Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5703-8. PubMed.

News

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  2. Loss of Parkin in Mammals Takes Steam Out of Mitochondria

Primary Papers

  1. . Mutations in PTEN-induced putative kinase 1 associated with recessive parkinsonism have differential effects on protein stability. Proc Natl Acad Sci U S A. 2005 Apr 19;102(16):5703-8. PubMed.