Characterization of PINK1 (PTEN-induced Putative Kinase 1) Mutations Associated with Parkinson Disease in Mammalian Cells and Drosophila*

Background: Mutations in PINK1 cause recessive Parkinson disease. Results: PINK1 mutations in the kinase domain hamper Parkin translocation to mitochondria, and their analogous mutations in Drosophila cannot rescue PINK1-null phenotypes. Conclusion: PINK1 kinase activity is essential for its function and for regulating Parkin functions in mitochondria. Significance: Understanding the roles of PINK1 mutations will be helpful for deciphering the pathogenic mechanism of PINK1-linked Parkinson disease. Mutations in PINK1 (PTEN-induced putative kinase 1) are tightly linked to autosomal recessive Parkinson disease (PD). Although more than 50 mutations in PINK1 have been discovered, the role of these mutations in PD pathogenesis remains poorly understood. Here, we characterized 17 representative PINK1 pathogenic mutations in both mammalian cells and Drosophila. These mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin. Furthermore, PINK1-null flies expressing the transgenic G426D mutant displayed defective phenotypes with increasing age, whereas L464P mutant-expressing flies exhibited the phenotypes at an earlier age. Collectively, these results strongly support the hypothesis that the kinase activity of PINK1 is essential for its function and for regulating downstream Parkin functions in mitochondria. We believe that this study provides the basis for understanding the molecular and physiological functions of various PINK1 mutations and provides insights into the pathogenic mechanisms of PINK1-linked PD.

Previously, we and others showed that PINK1 functions in a common pathway with Parkin to maintain mitochondrial integrity and function. Remarkably, PINK1 and Parkin mutant flies exhibit similar phenotypes, including mitochondrial defects, muscle degeneration, locomotor defects, disrupted spermatogenesis, and a reduced number of DA neurons (18 -21). Moreover, in cultured cells, Parkin overexpression compensates for mitochondrial dysfunction induced by PINK1 deficiency, indicating that Parkin acts downstream of PINK1 and that this linear pathway is well conserved in both mammals and Drosophila (22)(23)(24)(25).
Evidence linking mitochondria and PD has steadily grown (26 -29). Mitochondria are highly dynamic organelles that can change their shape, size, and subcellular localization depending on the cellular environment. These dynamic processes are regulated by fusion, fission, and transport, all of which are also linked to the maintenance of proper mitochondrial functions. There has been controversy over whether the PINK1/ Parkin pathway modulates mitochondrial fusion or fission. Cells derived from PINK1-defective patients have small fragmented mitochondria (30,31), whereas PINK1 and Parkin overexpression promote mitochondrial aggregation in mammalian cells (32). In Drosophila, PINK1 loss of function results in swelling or enlargement of mitochondria, and these defective phenotypes of the dPINK1-null flies are strongly suppressed by the overexpression of Drp1 (dynamin-related protein 1), which promotes mitochondrial fission, or the down-regulation of Opa1 (optic atrophy 1) or Marf (mitochondrial assembly regulatory factor), which promote fusion of the inner mitochondrial or the outer mitochondrial membrane, respectively (33)(34)(35)(36).
Recent studies have also shown that the PINK1/Parkin pathway is critical for mitophagy. Parkin is translocated to depolarized mitochondria upon treatment with CCCP, and Parkin-labeled mitochondria are subsequently eliminated by autophagy (mitophagy) (16,17,(37)(38)(39). Parkin ubiquitinates mitochondrial proteins at the outer membrane and facilitates the recruitment of adaptor proteins, such as p62, which links ubiquitinated cargo to the autophagic machinery (40 -43).
Despite this recent progress, the mechanism of PINK1linked PD pathogenesis still remains elusive. In this study, to investigate the consequences of PINK1 mutations, we selected 17 representative missense mutations and analyzed their effects on proteolytic processing and subcellular distribution of PINK1 and on Parkin mitochondrial translocation. Unexpectedly, PINK1 mutants exhibited normal protein stability and expression patterns and did not exhibit altered subcellular distributions under normal and damaged mitochondria conditions. However, Parkin was not localized to mitochondria in cells harboring PINK1 mutations within the kinase domain. These results were further confirmed in the Drosophila system. Taken together, the results of this study, in which we systemically analyzed the effect of PINK1 mutations in both in vitro and in vivo model systems, improve our understanding of how PINK1 mutations contribute to the pathogenesis of PD.
Generation of hPINK1 Mutants-For site-directed mutagenesis, a QuikChange TM kit (Stratagene) was used. For generation of mutations, a pcDNA3.1 zeo (ϩ) hPINK1 WT 3ϫMyc construct was used as a template. Information about the primers used in the experiments is available upon request.
Mammalian Cell Culture and Transfection-HeLa, HEK293T, and PINK1 KO mouse embryonic fibroblasts (MEFs) were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) at 37°C in a humidified atmosphere of 5% CO 2 . The transfection of expression plasmids was performed using Lipofectamine Plus reagent (Invitrogen) or PEI (Sigma) according to the manufacturer's instructions.
Immunocytochemistry-For immunocytochemistry, HeLa and PINK1 KO MEF cells were subcultured on 12-well culture plate coated with poly-L-lysine (Sigma). Appropriately treated cells were washed one time with PBS and were fixed in 2% paraformaldehyde for 15 min, followed by permeabilization with 0.5% Triton X-100 in PBS for 5 min. Then the cells were washed with 0.1% Triton X-100 in PBS (PBS-T) and incubated in blocking solution (4% BSA and 1% normal goat serum in PBS-T) for 1 h. Primary antibodies were added to the blocking solution, and the cells were incubated for overnight at 4°C. After washing with PBS-T three times, the cells were incubated with appropriate secondary antibodies in blocking solution for 45 min at room temperature. The antibody-labeled cells were washed with PBS-T six times and were mounted with mounting solution (100 mg/ml 1,4-diazabicyclo[2.2.2]octane in 90% glycerol). The slides were observed with a LSM710 laser scanning confocal microscope (Carl Zeiss). All of the immunostaining experiments with HeLa cells were conducted at least three times (n ϭ 300).
Fly Stocks-We have previously generated dPINK1 B9 , UAS-dPINK1 WT , and UAS-dPINK1 3KD line (18,33). UAS-dParkin line was previously described (44). The following fly stocks were obtained from the Bloomington Stock Center: hs-gal4 and gmr-gal4. All flies were grown on standard cornmeal-yeast-agar medium at 25°C. Details of the genotypes used in the experiments are available upon request.
Generation of Drosophila PINK1 Transgenic Mutants-For generation of G426D and L464P dPINK1 mutant transgenic lines, a QuikChange TM kit (Stratagene) was used. dPINK1 GD and LP DNAs were subcloned into HA-tagged pUAST vector and injected into w 1118 embryos.
Eye Phenotypes and SEM Analysis-Flies were crossed and maintained at 25°C. SEM images were analyzed by SUPRA 55VP (Carl Zeiss) in a variable pressure secondary electron mode.
Muscle Section and Mitochondria Staining-The samples embedded in Spurr's resin were trimmed and sectioned from the lateral side of the thorax (at a thickness of 4 mm and between 200 and 350 mm in depth), and the serial sections were then stained with toluidine blue dye. For streptavidin staining, fly thoraces after fixation were cut in half by dissecting vertically along the bristles in the middle of the thorax. Approximately 10 thoraces of 3-or 45-day-old flies were observed in light microscopy (Leica) for each genotype.
Quantification of Wing Phenotypes of Flies-For quantification, the percentages of male flies with normal wings at 3 or 45 days were measured (n Ͼ 100).
ATP Assay-ATP assays were carried out as previously described (33). The quantitative levels of ATP were measured (n Ͼ 5).
TH Immunostaining and Quantification of DA Neurons-45-day-old adult fly brains were fixed with 4% paraformaldehyde and stained with anti-TH antibody as described previously (18). The samples were observed and imaged by LSM 710 confocal microscope (Carl Zeiss). TH-positive neurons were counted under blinded conditions. For quantification of DA neurons, the first dorsolateral regions from 15 brains of each genotype were observed in a blind fashion to eliminate bias (n ϭ 30).
Statistical Analysis-The statistical analyses were performed using Student's t test for related samples. The p values calculated were pooled from three independent experiments.

RESULTS
Selection of 17 PINK1 Mutations-To understand how PD-linked mutations affect the function of PINK1 protein, we selected 17 missense mutations based on the high frequency of disease onset in patients carrying these mutations, the conservation of these amino acids in PINK1 across species, and the importance of these conserved residues in maintaining protein structure and PINK1 catalytic activity (Table 1) (6,(45)(46)(47)(48)(49)(50)(51)(52).
A majority of PINK1 mutations are observed in the Ser/Thr kinase domain, suggesting that loss of the kinase activity plays a crucial part in the pathogenesis of PINK1-linked PD ( Fig. 1A) (53)(54)(55). The A168P, P196L, G309D, T313M, L347P, G386A, G409V, and A427E mutations were selected to represent the mutations within the kinase domain in this study. In particular, G309D and L347P have been the most highly studied to have reduced kinase activity and protein stability, respectively (54). The Thr-313 residue was previously reported to be a phosphorylation site for microtubule affinity-regulating kinase 2 (MARK2/Par-1), and the T313M mutant exhibited defects in mitochondrial transport (56). The A168P, P196L, G386A, G409V, and A427E mutations were also reported to have effects on the kinase structure (57). An artificial kinase dead mutant (3KD; K219A/D362A/D384A) was used as a control in this study (54).
Mutations outside of the kinase domain were also selected. The P52L and R68P mutations within the MTS region were included in this study to determine the function of the MTS. Also, TM region mutants, C92F, C125G, Q126P and R147H, were selected to delineate the role of the TM region in PINK1 function. The C terminus of PINK1 has no homology to any known proteins; however, there are several reports that mutants with a truncated C terminus are associated with reduced PINK1 kinase activity (55, 58 -60).

Analysis of PD-causing PINK1 Mutations
Therefore, all of the known missense mutations in the Cterminal domain, N521T, D525N, and C575R, were also included in this study.
The cDNA for human PINK1 (hPINK1) containing 3ϫ Myc tag at the C terminus was inserted into the pcDNA 3.1 zeo (ϩ) vector, and all the mutations were generated in this construct using site-directed point mutagenesis method. It is well known that the C terminus-tagged PINK1 protein is detected as three protein bands in SDS-PAGE gels: the full-length protein (FL) and two processed proteins (P1 and P2) (61). Because of the triple Myc tag, FL, P1, and P2 of the WT hPINK1 can be detected as ϳ72, 63, and 54 kDa-sized proteins, respectively, in HeLa ( Fig. 1B) and HEK293T cell lines (data not shown).
Expression and Subcellular Localization Patterns of PINK1 Mutants under Normal Conditions-First, we examined the proteolytic processing patterns and protein stability of PINK1 mutants (Fig. 1B). Each mutant construct was transfected into HeLa cells, and whole cell lysates were subjected to immunoblot analysis using anti-human PINK1 or -Myc antibodies. None of the mutations altered the typical triplet pattern of PINK1 protein bands. The A168P mutant was not detected on using the anti-human PINK1 antibody because the mutation disrupts its antigenic structure of amino acids 140 -170 (Fig.  1B, lane 9). The presence of the typical cleavage pattern and stability in the A168P mutant was confirmed by immunoblotting using an anti-Myc antibody. Only the 3KD mutant showed a fast mobility shift, as previously shown (54). It is worth noting that the L347P mutation was reported to be unstable, because it does not bind to the Hsp90-Cdc37 chaperone complex (54,62,63). However, the L347P hPINK1 protein was as stable as the other mutants in our experimental conditions, even though the exogenously expressed L347P mutant interacted with the Hsp90-Cdc37 complex weakly (data not shown).
Given that PINK1 is targeted to mitochondria through its N-terminal MTS domain (64), we next assessed the subcellular distributions of hPINK1 mutants (Fig. 1C). Mitochondria were double labeled using MitoTracker, which accumulates in the mitochondria that have an intact ⌬ m , and the MTC02 antibody, which recognizes a nonglycosylated protein component of mitochondria found in human cells. The WT hPINK1 protein was localized in the cytoplasm, and it showed small punctate patterns that co-localized with mitochondria. All the mutants showed a localization pattern similar to that of WT hPINK1. Thus, the PINK1 patient mutations selected in this study had no significant effects on the proteolytic processing, stability, and subcellular localization of PINK1.
PINK1 Mutants Are Targeted to Mitochondria and Stabilized by CCCP Treatment-Recent studies suggested that the reduction of ⌬ m by the mitochondrial uncoupler CCCP leads to the accumulation of endogenous PINK1 (FL form) on mitochondria, and eventually results in Parkin-mediated mitophagy (16,17,(37)(38)(39)65). Therefore, we assessed the effects of PINK1 mutations on these processes in damaged mitochondria. Each PINK1 expression construct was expressed in HeLa cells, which were then treated with CCCP (20 M for 2 h), and subjected to immunocytochemical analysis. When cells were treated with CCCP, fragmented mitochondria significantly increased, and MitoTracker signals decreased because of the reduction in ⌬ m . The WT hPINK1 protein was mostly localized to punctate structures in CCCP-treated cells, compared with that in DMSO-treated cells (Fig. 2A). These punctate were co-local- Dividing lines indicate that samples were resolved on separate gels. Representative blots from three independent experiments are shown. C, confocal images of the subcellular localizations of hPINK1 mutants in HeLa cells. After 24 h of transfection, the cells were fixed and subjected to immunocytochemistry as described under "Experimental Procedures." Myc-tagged hPINK1 proteins were immunolabeled with Myc antibody (green), and the mitochondria were double-labeled using MitoTrackerா Red CMXRos (red) and MTC02 antibody (blue). Original magnification, ϫ800. C-term, C-terminal. FEBRUARY 22, 2013 • VOLUME 288 • NUMBER 8 ized with mitochondria, which were stained with an anti-MTC02 antibody. Interestingly, hPINK1 mutants showed similar distribution patterns under the low ⌬ m condition in HeLa (Fig. 2B) and PINK1-null MEFs cell lines (data not shown). In addition, as previously reported, CCCP induced the accumulation of FL-hPINK1 protein on mitochondria (17,(37)(38)(39), and all the missense mutants and 3KD mutants showed the same pattern as that of FL-PINK1 after CCCP treatment (Fig. 2C). In conclusion, we could not detect any significant differences between the subcellular localizations of the WT and mutant hPINK1 proteins under both normal and damaged mitochondria conditions.

Analysis of PD-causing PINK1 Mutations
Parkin Translocation to Mitochondria Is Impeded in Cells Expressing PINK1 Kinase Mutants-To investigate the effect of mutations on PINK1 activity, we used a specific downstream target of PINK1, Parkin, a product of the PARK2 gene. Exogenously expressed Parkin was distributed evenly throughout the cytoplasm and nucleus in HeLa cells. However, co-expression of WT hPINK1 and Parkin dramatically induced Parkin translocation to mitochondria and generated highly aggregated mitochondria around the perinuclear region ( Fig. 3A) (32). However, Parkin remained in the cytoplasm when co-expressed with the 3KD hPINK1 (Fig. 3A).
To examine the effect of the PINK1 mutants on Parkin translocation, we co-expressed each hPINK1 mutant with Parkin and then conducted immunocytochemical analyses (Fig. 3, B  and C). Interestingly, co-expression of hPINK1 proteins carrying most of the mutations within the PINK1 kinase domain was unable to completely promote the mitochondrial localization of Parkin. In addition, the C125G and Q126P mutants within the TM region failed to recruit Parkin to mitochondria. However, when PINK1 mutants within the MTS and C terminus region were co-expressed with Parkin, they successfully induced both Parkin localization in mitochondria and mitochondrial aggregation. These experiments were also reproduced in different cell lines, including COS-1 cells and PINK1-null MEFs (data not shown). Our results suggest that PINK1 acts upstream of Parkin in regulating Parkin mitochondrial localization and consequent mitochondrial aggregation, in a process dependent on its kinase activity.
Generation of Drosophila PINK1 Kinase Mutants Analogous to PD-linked Human Mutations-We subsequently investigated whether these PD-linked PINK1 mutations have similar effects on Parkin in vivo, utilizing the Drosophila model system. hPINK1 and Drosophila PINK1 (dPINK1) protein structures are well conserved (Fig. 4A). Among the kinase mutants, transgenic flies that express G426D (analogous to human G309D) and L464P (analogous to human L347P) were generated because of the high pathogenic relevance of the corresponding mutations in humans, and 3KD (K337R/D479R/D501A) kinase dead mutant flies were included as controls (Fig. 4B). There Is No Significant Genetic Interaction between dParkin and the dPINK1 Kinase Mutants-To investigate the genetic interaction between dParkin and dPINK1 mutants, we ex-pressed constructs encoding dParkin and dPINK1 mutants in adult fly eye tissue using the eye-specific gmr-gal4 driver. Rough eye phenotypes and disarrayed ommatidia were  FEBRUARY 22, 2013 • VOLUME 288 • NUMBER 8 observed in WT dPINK1-expressing flies (gmrϾdPINK1 WT ) upon analysis using light microscopy and a SEM (Fig. 4C). The 3KD (gmrϾdPINK1 3KD ), G426D (gmrϾdPINK1 GD ), and L464P mutant-expressing flies (gmrϾdPINK1 LP ) showed rough eye phenotypes similar to the WT dPINK1 expressing flies, whereas dParkin-expressing flies (gmrϾdParkin) had normal eyes. Next, the genetic interaction between dParkin and dPINK1 mutant flies were examined. As reported before, coexpression of WT dPINK1 and dParkin (gmrϾdParkin/ dPINK1 WT ) led to lethality (Fig. 4D) (14), indicating that PINK1 and Parkin have a strong genetic interaction in vivo. However, co-expression of dParkin and dPINK1 kinase mutants, including the 3KD (gmrϾdParkin/dPINK1 3KD ), G426D (gmrϾdParkin/dPINK1 GD ), and L464P mutants (gmrϾ dParkin/dPINK1 LP ), did not yield lethal phenotypes or additive eye defects. These results were further confirmed by SEM analysis. In conclusion, two PINK1 kinase mutations, G426D and L464P, which were defective in translocation of Parkin to mitochondria in mammalian cells, did not interact with Parkin in vivo.

Analysis of PD-causing PINK1 Mutations
The Phenotypes of dPINK1-null Mutant Are Not Rescued by the LP Mutant, but Are Rescued by the GD 3 Days after Eclosion-In a previous study, we found that PINK1-null mutant flies (dPINK1 B9 ) exhibit multiple PD-related phenotypes, including severe muscle degeneration, locomotor defects, and DA neuronal loss (18 -21). Transgenic flies expressing exogenous PINK1 kinase mutants in the dPINK1 B9 background were examined 3 days after eclosion. The most representative features of the dPINK1 B9 flies were the crushed thoraces and downturned wing postures, and these abnormalities were rescued by the WT dPINK1 expression (B9,hsϾdPINK1 WT ) but not by the 3KD (B9,hsϾdPINK1 3KD ) (Fig. 5, A and C). When we expressed PINK1 kinase mutants in the dPINK1 B9 background, the defects of the dPINK1 B9 flies were rescued by the G426D dPINK1 expression (B9,hsϾdPINK1 GD ) but not by the L464P dPINK1 expression (B9,hsϾdPINK1 LP ). To further observe the effects of the PINK1 mutants on mitochondrial structure and integrity, the thoracic muscles were dissected and then stained with toluidine blue dye and streptavidin antibodies (Fig. 5B). The dPINK1 B9 flies had swollen or enlarged mitochondria between disorganized muscle fibers. These structural defects were completely complemented by WT, but not by 3KD. The expression of L464P dPINK1 also failed to rescue the impaired muscle structures and mitochondrial defects in dPINK1 B9 . However, the expression of G426D dPINK1 rescued the defective muscles and mitochondria. In addition, the level of ATP was markedly reduced in the muscles in dPINK1 B9 flies compared with control flies (hs-gal4), and this reduced ATP level was rescued by WT or G426D expression, but not by 3KD or L464P dPINK1 (Fig. 5D). Because of the structural and functional defects, dPINK1 B9 flies could not fly (Fig. 5E). Again, the expression of WT or G426D dPINK1 rescued the flight ability of dPINK1 B9 flies, but the expression of 3KD or L464P dPINK1 did not. Collectively, these data showed that the expression of L464P and 3KD dPINK1 did not rescue the defective phenotypes of dPINK1-null mutant flies, but the expression of G426D dPINK1 rescued many of the defective phenotypes at 3 days after eclosion.
dPINK1-null Flies Expressing Exogenous GD Mutant Display Defective Phenotypes with Aging-The ability of G426D dPINK1 to rescue phenotype defects in the dPINK1 B9 flies was an unexpected result; therefore, we decided to observe this rescue further over a period of time. Notably, at 45 days of age, the G426D dPINK1-expressing flies (B9,hsϾdPINK1 GD ) showed downturned wing postures in contrast to the WT dPINK1-expressing dPINK1 B9 flies (B9,hsϾdPINK1 WT ) (Fig. 6, A and C). Moreover, abnormally swollen mitochondria between sparse muscle fibers were observed (Fig. 6B). Other age-dependent defects were also observed in the G426D dPINK1-expressing flies, such as reduced ATP levels (Fig. 6D) and flight abilities (Fig. 6E), at 45 days. Overall, we found that G426D dPINK1 expression, which rescued the defective phenotypes of young dPINK1-null mutants (Fig. 5), failed to sustain the rescue effects as the flies got older.
DA Neuronal Loss of dPINK1-null Mutant Is Not Rescued by Kinase Mutants-Because the loss of DA neurons is a hallmark of PD pathology, we examined DA neurons in the brains of adult dPINK1 B9 flies using anti-TH staining. Consistent with our previous reports (18), dPINK1 B9 flies displayed weak TH staining signals and reduced numbers of DA neurons in dorsolateral region 1 at 45 days (Fig. 7A). This loss of DA neurons was almost fully rescued by expression of WT (B9,hsϾdPINK1 WT ), but not 3KD (B9,hsϾdPINK1 3KD ), G426D (B9,hsϾdPINK1 GD ), or L464P dPINK1 mutant (B9,hsϾdPINK1 LP ) expression (Fig.  7). These results strongly suggest that the kinase activity is critical for the protective roles of PINK1 in DA neurons.

DISCUSSION
Because mutations in PINK1 are inherited primarily in a recessive manner, the loss of its function is thought to cause early onset PD. In this study, we dissected the underlying mechanism by which PINK1 missense mutations lead to PD pathogenesis using mammalian cells and an in vivo Drosophila model system.

Expression and Subcellular Localization Patterns of PINK1 Are Not Altered by Missense Mutations under Both Normal and
Damaged Mitochondria Conditions-We first examined the proteolytic patterns of hPINK1 mutants in mammalian cells. All of the mutants selected in the study generated typical triple bands in SDS-PAGE, similar to the WT hPINK1 protein (Fig.  1). In addition, they showed similar patterns of subcellular localization, both in the cytoplasm and in mitochondria. Also, their expression did not change mitochondrial morphology nor the ⌬ m .
We also investigated the role of hPINK1 mutations in response to damaged mitochondria. When treated with CCCP, cells expressing any of the 17 PINK1 mutants accumulated on mitochondria and the mutant proteins were stabilized just like the WT hPINK1 protein (Fig. 2). These results strongly suggest that the proteolysis, stability, and subcellular localization of PINK1 are not critical for the mechanism of PD pathogenesis associated with these missense mutations.
The Kinase Activity of PINK1 Is Indispensable for Its Function-The PINK1/Parkin pathway regulates mitochondrial integrity and function by modifying mitochondrial morphology and dynamics (32). In this study, Parkin was translocated to mitochondria when PINK1 was co-expressed with Parkin, and the mitochondria were highly aggregated. Surprisingly, in the cells expressing the hPINK1 with patient mutations in the kinase domain, Parkin translocation to mitochondria or mitochondrial aggregation did not occur (Fig. 3). Meanwhile, the subcellular localizations of PINK1 protein were not dramatically changed by Parkin co-expression. We anticipate that the catalytic activity of the overexpressed PINK1 protein localized in mitochondria, although its concentration may be much lower than the total PINK1 protein in the cell, is sufficient for mobilizing Parkin to mitochondria. The two PINK1 kinase mutants, human G309D (Drosophila G426D) and L347P (Drosophila L464P), which failed to mobilize Parkin to mitochondria in mammalian cells, also failed to genetically interact with Parkin in adult fly eye tissue (Fig. 4). These data consistently support the hypothesis that the kinase activity of PINK1 is essential for its function.
Other groups have studied the kinase activity of PINK1 in vivo or in vitro (32, 58 -60). PD-associated mutations in the kinase domain, including G309D, L347P, G386A, and G409V mutation, markedly inhibit PINK1 kinase activity (59,60). Previous biochemical studies confirmed that the PINK1 kinase domain mutants used in the present study have reduced kinase activity.
Notably, the C125G mutation within TM region also moderately suppresses PINK1 kinase activity, by 40% (59). Interestingly, co-expression of the C125G mutant or of PINK1 harboring the neighboring mutation, Q126P, failed to localize Parkin to mitochondria in our study, indicating that the TM region affects PINK1 kinase activity and subsequently regulates Parkin mobilization to mitochondria (Fig. 3). These results again emphasize the importance of PINK1 kinase activity in PD and also further substantiate that the role of PINK1 in regulating Parkin in a kinase activity-dependent manner.
Next, we investigated the mechanism by which PINK1 regulates Parkin. A growing body of evidence suggests that Parkin might be a direct substrate of PINK1 (32,58,66). A recent study clearly showed that Parkin is phosphorylated at Ser-65 in the ubiquitin-like domain by PINK1, when PINK1 is activated by CCCP (58). However, it is interesting to note that in our study, the Parkin S65A mutant was still recruited to mitochondria and induced mitochondrial aggregation, when PINK1 was co-expressed. Moreover, phosphomimetic Ser-65 mutants, S65D and S65E, were not translocated to mitochondria, nor did they induce mitochondrial aggregation (data not shown), suggesting that Ser-65 phosphorylation by PINK1 may not be critical for the functional regulation of Parkin. Previous work from our group also showed that PINK1 can directly phosphorylate Parkin at Thr-175 in the linker region (32). Mutational analysis confirmed that mitochondrial translocation of the Parkin T175A mutant was markedly reduced even when PINK1 was co-expressed. Currently we are conducting mapping analyses for Parkin phosphorylation in the presence and absence of PINK1 co-expression and CCCP treatment to delineate the regulation of their biochemical interactions more thoroughly.
Differential in Vivo Effects Were Shown Depending on the Levels of PINK1 Kinase Activity with Aging-Previous studies showed that hPINK1 L347P mutation completely abolished its kinase activity, whereas the G309D mutant still retained a modest level of kinase activity (54,59,60). Our Drosophila studies also showed differences in the activities of G426D (human G309D) and L464P dPINK1 (human L347P); although the L464P mutant, as well as the 3KD mutant, exhibited defective phenotypes at a young age, the G426D mutant exhibited defects only at older ages (Figs. 5-7).
As the prevalence of PD increases with aging, it is interesting that the G426D mutant flies showed age-dependent defective phenotypes in accordance with the level of kinase activity. Because both Parkin E3 ligase activity (60) and its localization to mitochondria are dependent on PINK1 kinase activity (Fig. 3), the mitochondrial substrate(s) of Parkin would be accumulated or deposited in the cell when the kinase activity of PINK1 is disrupted. For example, if the kinase activity of PINK1 is not sufficient to properly phosphorylate and regulate Parkin (e.g., the G426D mutant), its substrate(s) would be gradually accumulated in mitochondria and eventually cause progressive deterioration in mitochondrial and cellular functions. This may explain the mechanism underlying the age-dependent defects seen in our fly models and possibly the different disease onset ages of PD patients.
PINK1 May Regulate Various Cellular Responses through Different Effector Molecules-Meanwhile, we may need to consider that there are other substrates or effector molecules of PINK1 that are involved in regulating cellular responses, because not all of the PINK1 mutants were defective in Parkin translocation to mitochondria or in mitochondrial morphology (Figs. 1-3). Excitingly, recent proteomic analyses and genetic approaches have identified novel interaction partners for PINK1 (67)(68)(69)(70)(71)(72)(73), including TRAP1 (tumor necrosis factor receptor-associated protein 1), Omi/HtrA2 (a product of the PARK13 gene), Miro (an atypical GTPase that regulates mitochondrial trafficking), and FOXO (Forkhead box O transcription factor). Moreover, recent data suggested that PINK1 functions are not limited to mitochondria alone. For example, there was a study that addressed the role of PINK1 in the cytoplasm, showing that PINK1 exerts a cytoprotective function by activating Akt (71). Based on these data, we are currently investigating the possible connections between the PD-linked PINK1 mutations and the downstream targets of PINK1 in addition to Parkin.
In conclusion, we confirm that the kinase activity of PINK1 is critical for its function and the early onset of PD. We validated these data in the Drosophila model, proving the pathophysiological relevance of PD. Further studies addressing the modulation of PINK1 kinase activity or identifying novel targets of PINK1 are necessary to enhance our understanding of PINK1 mutation-associated PD pathogenesis.