C-terminal truncation and Parkinson's disease-associated mutations down-regulate the protein serine/threonine kinase activity of PTEN-induced kinase-1

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia.
Human Molecular Genetics (Impact Factor: 6.39). 12/2006; 15(21):3251-62. DOI: 10.1093/hmg/ddl398
Source: PubMed


The Parkinson's disease (PD) causative PINK1 gene encodes a mitochondrial protein kinase called PTEN-induced kinase 1 (PINK1). The autosomal recessive pattern of inheritance of PINK1 mutations suggests that PINK1 is neuroprotective and therefore loss of PINK1 function causes PD. Indeed, overexpression of PINK1 protects neuroblastoma cells from undergoing neurotoxin-induced apoptosis. As a protein kinase, PINK1 presumably exerts its neuroprotective effect by phosphorylating specific mitochondrial proteins and in turn modulating their functions. Towards elucidation of the neuroprotective mechanism of PINK1, we employed the baculovirus-infected insect cell system to express the recombinant protein consisting of the PINK1 kinase domain either alone [PINK1(KD)] or with the PINK1 C-terminal tail [PINK1(KD+T)]. Both recombinant enzymes preferentially phosphorylate the artificial substrate histone H1 exclusively at serine and threonine residues, demonstrating that PINK1 is indeed a protein serine/threonine kinase. Introduction of the PD-associated mutations, G386A and G409V significantly reduces PINK1(KD) kinase activity. Since Gly-386 and Gly-409 reside in the conserved activation segment of the kinase domain, the results suggest that the activation segment is a regulatory switch governing PINK1 kinase activity. We also demonstrate that PINK1(KD+T) is approximately 6-fold more active than PINK1(KD). Thus, in addition to the activation segment, the C-terminal tail also contains regulatory motifs capable of governing PINK1 kinase activity. Finally, the availability of active recombinant PINK1 proteins permits future studies to search for mitochondrial proteins that are preferentially phosphorylated by PINK1. As these proteins are likely physiological substrates of PINK1, their identification will shed light on the mechanism of pathogenesis of PD.

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    • "Fig. S4), the conformation of which is important in many kinases for regulating their protein kinase activity. Variations in the corresponding glycine residue have been reported to alter the function of human PTEN-induced kinase (PINK1), Cyclindependent kinase (Cdk1) in D. melanogaster, and calcium and calmodulin-dependent serine/threonine-protein kinase (CCaMK) in the garden pea Pisum sativum [Clegg et al., 1993; Lévy et al., 2004; Sim et al., 2006]. The [G1179S]Roco4 kinase domain with the homologous Gly-1179 in the activation loop mutated to serine was generated by Gilsbach et al. (2012) to mimic the G2019S variation in LRRK2. "
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    ABSTRACT: Genetic variations of leucine-rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras-like GTPase (ROC) domain and C-terminal of Roc (COR) domain, a protein kinase domain and four repeat domains. PD-causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three-dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three-dimensional structures of the functional domains of LRRK2 have yet to be determined. Based upon the known three-dimensional structures of repeat domains of other proteins, the tandem Roc-COR domains of the C. tepidum Rab family protein, and the kinase domain of the D. discoideum Roco4 protein, we predicted (i) the motifs essential for protein-protein interactions in all domains, (ii) the motifs critical for catalysis and substrate recognition in the tandem Roc-COR and kinase domains, and (iii) the effects of some PD-associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2. This article is protected by copyright. All rights reserved.
    Human Mutation 04/2014; 35(4). DOI:10.1002/humu.22515 · 5.14 Impact Factor
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    • "Several mutations in PTEN-induced putative kinase 1 (PINK1) gene have been reported to be associated with recessive PD, which was considered as the second common virulence gene besides Parkin [3]–[6]. The encoded protein PINK1 is a 581 amino acid protein with a mitochondrial localization signal (MLS) and a functional serine/threonine kinase domain, which was identified to be degraded by the UPS [4], [7], [8]. Previous studies have demonstrated that degradation of PINK1 by ubiquitin proteasome system (UPS) is regulated by Parkin through a direct interaction between them [9]–[14]; and expression of wild-type DJ-1 increased steady-state levels of PINK1, whereas expression of DJ-1A39S reduced steady-state levels of PINK1 [15]. "
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    ABSTRACT: Mutations in PTEN-induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism, and loss of the stability of PINK1 may also contribute to sporadic Parkinson's disease (PD). Degradation of PINK1 occurs predominantly through the ubiquitin proteasome system (UPS), however, to date, few of the proteins have been found to regulate the degradation of PINK1. Using the yeast two-hybrid system and pull-down methods, we identified bcl-2-associated athanogene 5 (BAG5), a BAG family member, directly interacted with PINK1. We showed that BAG5 stabilized PINK1 by decreasing the ubiquitination of PINK1. Interestingly, BAG5 rescued MPP(+)- and rotenone-induced mitochondria dysfunction by up-regulating PINK1 in vitro. In PINK1-null mice and MPTP-treated mice, BAG5 significantly increased in the substantia nigra pars compacta (SNpc) although PINK1 was decreased. Our findings indicated that BAG5, as a key protein to stabilize PINK1, is a promising therapeutic tool for preventing mitochondrial dysfunction following oxidative stress.
    PLoS ONE 01/2014; 9(1):e86276. DOI:10.1371/journal.pone.0086276 · 3.23 Impact Factor
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    • "PINK1 is a 581 amino acid protein ubiquitously transcribed and encodes a serine/threonine kinase, showing high homology with the Ca 2+ /calmodulin kinase family. Also, PINK1 contains a N-terminal mitochondrial targeting sequence and a C-terminal autoregulatory domain (Beilina et al., 2005; Silvestri et al., 2005; Sim et al., 2006) is predominantly localized to mitochondria, but also is present in the cytosol (Haque et al., 2008; Valente et al., 2004; Weihofen et al., 2008; Zhou et al., 2008). Full-length PINK1 (FL-PINK1), is approximately 63 kDa, and is transcribed in the nucleus, translated in the cytoplasm and imported intact into mitochondria. "
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    ABSTRACT: Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment was due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24hours. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3hours, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection.
    Neurobiology of Disease 10/2013; 62(100). DOI:10.1016/j.nbd.2013.10.021 · 5.08 Impact Factor
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