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The Journal of Neuroscience, August 31, 2005, 25(35):7968-7978; doi:10.1523/JNEUROSCI.2172-05.2005
Previous Article | Next Article
Neurobiology of Disease
Accumulation of the Authentic Parkin Substrate Aminoacyl-tRNA Synthetase Cofactor, p38/JTV-1, Leads to Catecholaminergic Cell Death
Han Seok Ko,1,2 Rainer von Coelln,1,2 Sathya R. Sriram,1,2,6 Seong Who Kim,1,2 Kenny K. K. Chung,1,2 Olga Pletnikova,5 Juan Troncoso,2,5 Brett Johnson,1,2 Roya Saffary,1,2 Eyleen L. Goh,1,2 Hongjun Song,1,2,3,6 Bum-Joon Park,7 Min Jung Kim,7 Sunghoon Kim,7 Valina L. Dawson,1,2,3,4,6 andTed M. Dawson1,2,3,6 1Institute for Cell Engineering, Departments of 2Neurology, 3Neuroscience, 4Physiology, and 5Pathology, and 6Graduate Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and 7National Creative Research Initiatives Center for Human Aminoacyl-tRNA Synthetases Network, College of Pharmacy, Seoul National University, Seoul 151-742, Korea
Abstract
Top
Abstract
Introduction
Autosomal-recessive juvenile parkinsonism (AR-JP) is caused by loss-of-function mutations of the parkin gene. Parkin, a RING-type E3 ubiquitin ligase, is responsible for the ubiquitination and degradation of substrate proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). Accordingly, the abnormal accumulation of neurotoxic parkin substrates attributable to loss of parkin function may be the cause of neurodegeneration in parkin-related parkinsonism. We evaluated the known parkin substrates identified to date in parkin null mice to determine whether the absence of parkin results in accumulation of these substrates. Here we show that only the aminoacyl-tRNA synthetase cofactor p38 is upregulated in the ventral midbrain/hindbrain of both young and old parkin null mice. Consistent with upregulation in parkin knock-out mice, brains of AR-JP and idiopathic PD and diffuse Lewy body disease also exhibit increased level of p38. In addition, p38 interacts with parkin and parkin ubiquitinates and targets p38 for degradation. Furthermore, overexpression of p38 induces cell death that increases with tumor necrosis factor-treatment and parkin blocks the pro-cell death effect of p38, whereas the R42P, familial-linked mutant of parkin, fails to rescue cell death. We further show that adenovirus-mediated overexpression of p38 in the substantia nigra in mice leads to loss of dopaminergic neurons. Together, our study represents a major advance in our understanding of parkin function, because it clearly identifies p38 as an important authentic pathophysiologic substrate of parkin. Moreover, these results have important implications for understanding the molecular mechanisms of neurodegeneration in PD.
Key words: Parkinson's disease; parkin; ubiquitination; proteasome degradation; p38/JTV1; dopaminergic neuronal cell death
Introduction
Parkinson's disease (PD) is one of the most common neurodegenerative disorders characterized by the progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and the presence of intracellular inclusions, named Lewy bodies (Dawson and Dawson, 2003). Deficiency of these neurons causes progressive motor impairments, including tremor, rigidity, and bradykinesia. Although the majority of PD is sporadic, the identification of familial PD-linked mutations in genes encoding
-tubulin, cyclin E, synaptotamin XI (SytXI), parkin-associated endothelin-like receptor (Pael-R), and p38/JTV-1 subunit of the multi-tRNA synthetase complex (Zhang et al., 2000
Materials and Methods
cDNA, cell culture, and antibodies. Full-length human p38 cDNA was cloned into the mammalian expression vector pCMV-2A-FLAG (Stratagene, La Jolla, CA). Expression plasmids for FLAG-tagged human parkin and V5-tagged human heat shock protein 70 (Hsp70) were kindly provided by R. Takahashi (RIKEN Brain Science Institute, Tokyo, Japan), and hemagglutinin (HA)-tagged mouse C-terminus of hsc70-interacting protein (CHIP) was kindly provided by S. Hatakeyama (Kyushu University, Fukuoka-shi, Japan). Human full-length parkin and its deletion mutants and ubiquitin cDNAs were cloned into pRK5-HA vector as described previously (Chung et al., 2001Human neuroblastoma SK-N-MC and SH-SY5Y cells were purchased from American Type Culture Collection (Manassas, VA). They were maintained in modified Eagle's medium and DMEM supplemented with 10% (v/v) heat-inactivated fetal calf serum at 37°C in a humidified 5% CO2/95% air atmosphere, respectively. Human neuroblastoma SK-N-MC and SH-SY5Y cells were transiently transfected with the target vector by the Lipofectamine method according to the instructions of the manufacturers (Invitrogen, Carlsbad, CA).
Adult neural stem cells were isolated from the brains of adult parkin null mice and their littermates and cultured as described previously (Zhao et al., 2003
To prepare the stable transformed cells, SH-SY5Y cells were transfected with pcDNA3-HA-p38. Selections were started 2 d later using a medium containing 700 µg/ml geneticin (Invitrogen). Individual clones were isolated, and their characterization was examined by Western blotting with an antibody against anti-HA.
To generate a peptide antigen of p38, a peptide containing the last 18 amino acids of the C terminal of p38 was synthesized and cross-linked to keyhole limpet hemocyanin. The conjugated peptide was then used to immunize a New Zealand white rabbit (JH745–748) (Cocalico Biologicals, Reamstown, PA). Antisera were purified by affinity chromatography using the same peptide immobilized on SulfoLink gel matrix (Pierce, Rockford, IL) according to the protocol of the manufacturer. Antibody specificity was confirmed by the ability to preabsorb the immunostaining with excess purified p38 protein and with p38 knock-out mice. p38 monoclonal antibody was kindly provided by S. Kim (University of Seoul, Seoul, Korea), and Pael-R monoclonal antibody was kindly provided by R. Takahashi (RIKEN Brain Science Institute, Tokyo, Japan).
In vitro pull-down assay and immunoprecipitation. The p38 cDNA was cloned into the pGEX-KT vector (Amersham Biosciences, Piscataway, NJ) to produce the fusion protein glutathione S-transferase (GST)-p38. Expression and purification of GST-p38 and GST alone (as a control) using glutathione-Sepharose 4B (Amersham Biosciences) was performed as recommended by the manufacturer. In vitro translation of parkin wild-type (WT) and 77-465 were performed with rabbit reticulocyte lysate (Promega, Madison, WI) and 35S-methionine according to instructions of the manufacturer. The glutathione-Sepharose beads with bound GST-fusion proteins and in vitro-translated parkin WT and 77-465 were incubated in bead-binding buffer [50 mM K-phosphate, pH 7.5, 100 mM KCl, and 10% glycerol (v/v)/0.1% Triton X-100] at 4°C for 2 h. The beads were washed four times with bead-binding buffer without glycerol and Triton X-100. Beads were then heated for 5 min at 95°C in SDS-sample buffer and analyzed by SDS-PAGE. Bands were visualized by autoradiography.
For coimmunoprecipitation from cell cultures, SH-SY5Y cells were transfected with 2 µg of each plasmid. After 48 h, cells were washed with cold PBS and harvested in immunoprecipitation buffer (1% Triton X-100, 2 µg/ml aprotinin, and 100 µg/ml PMSF in PBS). The lysate was then rotated at 4°C for 1 h, followed by centrifugation at 14,000 rpm for 20 min. The supernatants were then combined with 50 µl of protein G Sepharose (Amersham Biosciences) preincubated with antibodies against HA or myc (Roche, Indianapolis, IN), followed by rotating at 4°C for 2 h. The protein G Sepharose was pelleted and washed three times using immunoprecipitation buffer or buffer with additional 500 mM NaCl, followed by three washes with PBS. The precipitates were resolved on SDS-PAGE gel and subjected to Western blot analysis. Immunoblot signals were visualized with enhanced chemiluminescence (Amersham Biosciences).
For coimmunoprecipitation of the endogenous proteins from human brain, frontal cortex gray matter was homogenized in 4 vol of ice-cold PBS containing 320 mM sucrose and 0.1% Triton X-100 with protease inhibitor cocktail (Sigma, St. Louis, MO). The tissue homogenate was centrifuged at 37,000 x g at 4°C for 20 min. The supernatant was used for immunoprecipitation with one of the following antibodies: anti-HA (Roche), anti-myc (Roche), anti-p38, or anti-parkin. Immunoprecipitates were separated by SDS-PAGE and subjected to Western blot analysis with an anti-p38 monoclonal antibody. Immunoblot signals were visualized with enhanced chemiluminescence (Amersham Biosciences). For mapping of the binding region between parkin and p38, the myc-tagged parkin deletion constructs were generated as described previously (Chung et al., 2001
In vivo ubiquitination assay. For in vivo ubiquitination assay from cell cultures, SH-SY5Y cells were transiently transfected with 2 µg of pRK5-myc-tagged parkin or myc-tagged parkin mutants, pCMV-FLAG-p38, and 2 µg of pMT123-HA-ubiquitin plasmids for 24 h and then treated with the proteasome inhibitor MG132 (5 µM) (Sigma) for 18 h. Total cell lysates were prepared by harvesting the cells after washing with PBS, followed by solubilizing the pellets in 200 µl of 2% SDS, followed by sonication. The lysates were then rotated at 4°C for 1 h, diluted to 1 ml with TBS, and then boiled and sonicated. A total of 50 µl of the samples were used as input and for immunoprecipitation. Immunoprecipitation was performed with an antibody against FLAG. The precipitates were subjected to Western blotting with anti-HA or anti-FLAG antibodies.
Assessment of cell viability. To examine the effect of p38 on cell death, we cotransfected SK-N-MC cells with either control plasmid and HA tagged-p38 or HA tagged-p38 together with FLAG-parkin and HA tagged-p38 together with Arg42Pro parkin mutant and incubated for 24 h, followed by treatment with tumor necrosis factor-
Preparation of human and mouse brain tissue. Frontal cortical tissue from five control brains and eight PD and diffuse Lewy body (DLB) brains with high Lewy body burden that were age matched with similar postmortem intervals as described previously (Chung et al., 2004
Detergent-soluble and -insoluble fractions were prepared from human brain tissue and mouse brain tissue by homogenization of samples in lysis buffer [10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 10 mM Na-
Western blotting was performed with an anti-parkin (PRK8 mouse monoclonal), anti-p38, anti-CDCrel-1 (Zhang et al., 2000
Animals and stereotaxic injection of p38. All procedures were performed in compliance with the guidelines set forth by the Laboratory Animal Manual of the National Institute of Health Guide to the Care and Use of Animals and were approved by the Johns Hopkins Medical Institute Animal Care Committee. Female BC57 mice were bred and maintained at the animal facility of the Johns Hopkins School of Medicine. They were housed in groups of three per cage in a temperature- and humidity-controlled room with a 12 h light/dark cycle. Food and water were available ad libitum.
Animals weighing 30 g were anesthetized with phentobarbital (60 mg/kg, i.p.). An injection cannula (26.5 gauge) was placed stereotaxically into the SN (anteroposterior, –3.1 mm from bregma; mediolateral, 1.3 mm; dorsoventral, 4.3 mm) according to the atlas of Paxinos et al. (1985
Immunohistochemistry. Animals were deeply anesthetized (80 mg/kg phentobarbital, i.p.) and transcardially perfused with PBS, followed by ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. Brains were promptly removed and postfixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h at room temperature. Each brain was cryoprotected in 30% sucrose until equilibrated and then stored at –80°C until processed.
Brain tissue was cut into 40 µm sections on a HM440E microtome (Microm, Walldorf, Germany), and the sections were collected in the phosphate buffer and incubated in 0.1 M PBS, pH 7.4, containing 1% BSA and 0.2% Triton X-100. After washing in the rinsing buffer containing the PBS and 0.5% BSA, the sections were incubated with antisera against tyrosine hydroxylase (TH) (1:2000, polyclonal; Novus Biologicals, Littleton, CO) overnight at 4°C with shaking. After washes in the rinsing buffer, the sections were incubated for 1 h in the appropriate biotinylated secondary antisera, washed with the rinsing buffer, and then further incubated for 1 h in the avidin–biotin complex. Antigens were visualized by reaction with 0.05% 3,3'-diaminobenzidine in the presence of 0.003% hydrogen peroxide. After two washes in the phosphate buffer, the sections were mounted on gelatin-coated slides, dehydrated through graded ethyl alcohols, cleared in xylene, and coverslipped with Permount. To verify whether a reduction in TH cell count necessarily implies cell death, some TH sections were counterstained for Nissl (0.2% cresyl violet for 2 min, followed by dehydration with serial concentration of EtOH), and, for each TH section, an adjacent section was stained with cresyl violet.
For immunostaining, human brain tissue fixed with Formalin and then paraffin embedded sections (10 µm) were deparaffinized, treated with H2O2, blocked with 3% normal goat serum in TBS, and incubated with anti-p38 polyclonal antibody (1:50) overnight. Subsequently, the tissue was incubated with biotinylated secondary antibodies (anti-rabbit IgG for 1 h), and immunoreactions were visualized with the ABC complex (Vector Laboratories, Burlingame, CA) and DAB (Vector Laboratories).
For double immunofluorescence labeling with
Cell counting of SNpc. TH-immunopositive cells in the SNpc were counted as described previously (Yuan et al., 2005
Statistical analysis. Densitometric analysis of protein bands was analyzed on an AlphaImager 2000 densitometer (Alpha Inotech, Wohlen, Switzerland). Data are expressed as mean ± SEM. The results were statistically evaluated for significance by applying the unpaired two-tailed test. Differences were considered significant when p < 0.05.
Results
The steady-state level of aminoacyl-tRNA synthetase cofactor p38/JTV1 is upregulated in ventral midbrain/hindbrain of Parkin null mice
A number of putative substrates have been identified for parkin, but it is not clear which one, if any, are authentic parkin substrates (von Coelln et al., 2004a
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Figure 1. Increased steady-state levels of p38 but not other substrates in parkin null mice. Ventral midbrain/hindbrains of 18-month-old wild-type (n = 5/n = 4 animals) and parkin null (n = 6/n = 5 animals) mice were homogenized. Soluble (A) and insoluble (C) fractions were analyzed by Western blotting with antibodies to p38, CDCrel-1,
The specificity of p38 rabbit polyclonal antibody was assessed by preparing extracts from wild-type, heterozygote, and p38 null brain extracts. The p38 antibody only recognizes a single band on Western blot analysis in wild-type brain, in heterozygote mice, there is reduced immunoreactivity, and in p38 null brain extracts, there is no immunoreactivity, thus confirming the specificity of our polyclonal p38 antibody (Fig. 2A). To examine whether in vitro knock-out of parkin leads to the accumulation of p38, we isolated adult neuronal stem cells from parkin null mice. We observe a trend toward increased levels of p38 in parkin null neuronal stem cells (Fig. 2B). To determine whether the accumulation of p38 is age dependent, we examined the relative level of p38 in 2-month-old ventral midbrain/hindbrain extracts compared with age-matched wild-type control ventral midbrain/hindbrain extracts (Fig. 2C). We observed a statistically significant accumulation (relative level of p38 is 15%) of p38 in ventral midbrain/hindbrain at 2 months of age (Fig. 2C). Next, to determine whether p38 accumulates in other brain regions, we examined the level of p38 in cortex of 2 and 18 months of age (Fig. 2D,E) and cerebellum, brainstem, and striatum at 18 months of age (Fig. 2F). We failed to observe a significant increase in p38 in cortex, cerebellum, brain stem, and striatum of parkin null mice versus age-matched wild-type controls (Fig. 2D–F and data not shown). Together, these results suggest that p38 accumulates primarily in the midbrain/hindbrain of parkin knock-out mice.p38 accumulates in AR-JP, PD, and DLB disease
To ascertain whether the upregulation of p38 in parkin knock-out mice has potential pathophysiologic relevance, Western blot analysis was performed from AR-JP cortex, and the level of p38 was compared in age-matched controls. Increased levels of p38 in AR-JP brains is observed compared with controls (Fig. 3A). To determine whether the other putative parkin substrates accumulate in AR-JP brains, we assessed the level of CDCrel-1, synphilin-1,
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Figure 2. Significant accumulation of p38 in ventral midbrain/hindbrain of 2-month-old parkin null mice. A, Specificity of polyclonal antibodies to p38 was confirmed on a Western blot (WB) against brain homogenates from p38 null (–/–), wild-type (+/+), and heterozygous (+/–) mice (top). The blot was stripped and reprobed with an anti-actin antibody to confirm equivalent loading in all lanes (bottom). B, Cell extracts (40 µg) of adult neuronal stem cells isolated from parkin null and wild-type mice were subjected to SDS-PAGE and probed on a Western blot with anti-p38 (top), anti-parkin (middle), and anti-actin (bottom). C, Soluble fraction of brain lysates from ventral midbrain/hindbrain of 2-month-old wild-type and parkin null mice were subjected to SDS-PAGE and probed on a Western blot with anti-p38 (top) and anti-actin (bottom) antibodies. Densitometric analyses of band intensities normalized to actin levels are presented as mean ± SEM. **p < 0.005, Student's t test. D, Lysates (40 µg) prepared from cortex of 2-month-old wild-type and parkin null mice were analyzed for immunoreactivity to anti-p38 and anti-actin antibodies. E, F, Lysates (40 µg) prepared from cortex (E), cerebellum, brainstem, and striatum (F) of 18-month-old wild-type and parkin null mice were analyzed by Western blotting with antibodies to p38 and actin. Densitometric analysis is presented for cortex as mean ± SEM (E).
Parkin interacts with aminoacyl-tRNA synthetase p38
The accumulation of p38 in parkin knock-out midbrain/hindbrain, AR-JP brains, and PD/DLBD brains with S-nitrosylative stress suggests that p38 interacts with parkin. To further characterize the interaction of p38 with parkin, a GST pull-down assay was performed (Fig. 4A). The fusion protein GST-p38 was expressed, purified, and incubated with in vitro-translated 35S-Met-labeled parkin and parkin 77-465. After extensive washing, the proteins bound to glutathione-Sepharose beads were separated by SDS-PAGE and detected by autoradiography. GST-p38 (lane 2), but not GST alone (lane 1), is able to efficiently retain parkin and parkin 77-465 (Fig. 4A).To investigate the physical interaction in vivo, immunoprecipitation was performed using the human neuroblastoma cell line SH-SY5Y (Fig. 4B) and human brain homogenates (Fig. 4C). Cotransfection experiments with myc-tagged p38 and HA-tagged parkin were performed, followed by coimmunoprecipitation using the anti-myc antibody. p38 coimmunoprecipitates with parkin after cotransfection in SH-SY5Y cells (Fig. 4B). To determine whether p38 interacts with parkin in human brain, coimmunoprecipitation using an antibody against parkin from human cortex was performed, followed by Western blot analysis with a monoclonal antibody against p38. p38 coimmunoprecipitates with parkin from human cortex, whereas the immunoprecipitation with anti-myc or anti-HA antibodies fails to immunoprecipitate p38 (Fig. 4C). We also observed that p38 strongly coimmunoprecipitates with parkin from human midbrain and striatum (data not shown). We next monitored whether the interaction of parkin and p38 is disrupted or altered by familial-PD-associated mutations in parkin. Myc-tagged wild-type and mutant parkin, R42P and Q311stop, were transiently transfected into SH-SY5Y cells along with HA-tagged p38 to examine the relative binding abilities of p38 to wild-type versus mutant parkin. The R42P mutation in parkin reduces its ability to interact with p38, whereas the Q311stop mutant binds more avidly to p38 then wild-type parkin (Fig. 4D). Parkin exists in a macromolecular protein complex with CHIP and Hsp70, in which this complex participates in the ubiquitination and degradation of parkin substrates (Imai et al., 2002
p38 interacts with parkin through its RING1 domain, and parkin associates with p38 through its N-terminal domain
To ascertain which domain of parkin binds to p38, various deletion constructs of myc-tagged parkin were constructed (Fig. 5A). Each truncated construct was coexpressed with HA-tagged p38 in SH-SY5Y cells. After 48 h of transfection, protein extracts were immunoprecipitated with anti myc-conjugated agarose, followed by Western blot analysis with anti-HA antibody (Fig. 5A). WT parkin and mutants containing the RING1 (R1) domain interact with p38, whereas mutants lacking R1 domain fail to interact with p38 (Fig. 5A). These data suggest that amino acid residues 220–318, which contain the RING1 domain, are important for the interaction of parkin with p38. Next, we constructed a series of myc-tagged truncated p38 constructs to determine the domain of p38 that interacts with parkin (Fig. 5B). The series of truncation of myc-tagged p38 were coexpressed with HA-tagged parkin and followed by coimmunoprecipitation using anti-myc (Fig. 5B). Truncated p38 containing the whole or part of the 82–162 domain (WT, F1, F3, and F4) interact with parkin. However, truncated p38 lacking amino acid residues 82–162 fails to interact with the full-length parkin. This mapping study indicates that parkin mainly interacts with the 82–162 amino acid domain of p38 (Fig. 5B).
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Figure 3. p38 accumulates in AR-JP, PD, and DLBD brains. A, Homogenates of human frontal cortex from control and AR-JP brains were immunoblotted with anti-p38 antibody (top). The blot was stripped and reprobed with anti-actin antibody to confirm equivalent loading in all lanes (bottom). Densitometric analyses of band intensities normalized to actin levels are presented as mean ± SEM. *p < 0.05, Student's t test. B, Homogenates of human frontal cortex from control (Con) and AR-JP cases were analyzed by Western blotting (WB) for steady-state levels of
Parkin ubiquitinates p38, and familial-linked parkin mutations interfere with p38 proteasomal degradation
To determine whether parkin ubiquitinates p38, in vivo ubiquitination experiments were examined. SH-SY5Y cells were cotransfected with FLAG-tagged p38, myc-tagged parkin, and HA-tagged ubiquitin, and FLAG-p38 was immunoprecipitated with an anti-FLAG antibody from the total cell extract. p38 is ubiquitinated by parkin, as shown by the significant anti-HA immunoreactivity (Fig. 6, top panel, lane 4) and anti-FLAG (Fig. 6, middle panel, lane 4) in the form of smear, which is characteristic of polyubiquitinated proteins, suggesting that parkin ubiquitinates p38. To examine whether familial-linked parkin mutations affect the parkin-mediated ubiquitination of p38 (Fig. 6, lanes 5, 6), we cotransfected SH-SY5Y cells with FLAG-tagged p38, myc-tagged Q311Stop, R42P, and HA-tagged ubiquitin, and immunoprecipitated FLAG-p38 with an anti-FLAG antibody. We find that Q311Stop mutant impairs the ability of parkin to ubiquitinate p38, whereas the R42P mutant enhances p38 ubiquitination, suggesting that the two mutants affected parkin function by distinct mechanisms.Multi-ubiquitinated substrates are degraded by the 20S proteolytic subunits of 26S proteasome. To evaluate whether parkinubiquitinated p38 might be a target for degradation by 26S proteasome, we stably transfected SH-SY5Ycells with HA-tagged p38 and determined whether parkin promotes p38 degradation of HA-tagged p38 by analyzing the steady-state level of p38. There is a dose-dependent decrease in p38 in the presence of parkin (Fig. 7A), and the proteasome inhibitors MG132 (5 µM) and
p38 overexpression leads to neuronal toxicity in vitro and in vivo
To address the functional significance of the interaction of p38 with parkin, we examined the role of p38 in cell death. Overexpression of myc-tagged p38 in native SK-N-MC cells causes an almost threefold increase in cell death compared with mock-transfected cells, and coexpression of FLAG-parkin partially protects from p38-induced toxicity but R42P mutant parkin fails to the rescue (Fig. 8A). Moreover, overexpression of p38 enhances the sensitivity of human neuroblastoma SK-N-SH to TNF
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Figure 4. Parkin interacts with aminoacyl-tRNA synthetase cofactor p38/JTV-1. A, GST-pull down of parkin by p38. GST-p38 was incubated with in vitro-translated 35S-Met-labeled wild-type and UBL-deleted (77-465) parkin. Proteins bound to glutathione-Sepharose beads were washed extensively, subjected to SDS-PAGE, and detected by autoradiography. Both wild-type and 77-465 parkin were efficiently retained by GST-p38 (lane 2) but not GST alone (lane 1). B, Parkin and p38 interact in SH-SY5Y cells. Lysates prepared from SH-SY5Y cells transfected with myc-tagged p38 and HA-tagged parkin were subjected to immunoprecipitation (IP) with anti-myc, followed by anti-HA immunoblotting (middle). The blot was stripped and reprobed with anti-myc antibody (bottom) to show an equivalent amount of immunoprecipitated p38. C, Coimmunoprecipitation of p38 and parkin from human brain extract. Human frontal cortex homogenate was subjected to immunoprecipitation with anti-myc, anti-HA, anti-p38, or anti-parkin, followed by immunoblotting with a monoclonal antibody to p38. D, Familial-associated mutations in parkin alter the interaction with p38. Lysates prepared from SH-SY5Y cells transfected with HA-tagged p38 and myc-tagged wild-type, R42P, or Q311Stop parkin constructs were subjected to immunoprecipitation with anti-myc, followed by anti-HA immunoblotting (middle). The blot was stripped and reprobed with anti-myc to show levels of immunoprecipitated parkin (bottom). E, p38 interacts with CHIP. Lysates from SH-SY5Y cells transfected with FLAG-tagged p38, HA-tagged CHIP, or both were subjected to immunoprecipitation with anti-FLAG, followed by anti-HA immunoblotting (middle). The blot was stripped and reprobed with anti-FLAG to show equivalent amounts of immunoprecipitated p38 (bottom). F, p38 interacts with Hsp70. Lysates from SH-SY5Y cells transfected with FLAG-tagged p38, V5-tagged Hsp70, or both were immunoprecipitated with anti-FLAG, followed by immunoblotting with anti-V5 (middle) and anti-FLAG (bottom). G, Treatment with proteasome inhibitor promotes the interaction between parkin and p38. SH-SY5Y cells were transfected with HA-tagged p38 and FLAG-tagged parkin, followed by treatment with proteasome inhibitor MG132 (5 µM) for 18 h. Lysates were subjected to immunoprecipitation with anti-FLAG antibody, followed by Western blotting with anti-HA (middle) and anti-FLAG (bottom).
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Figure 5. p38 interacts with the R1 ring finger domain of parkin, and parkin associates with the N-terminal domain of p38. A, Lysates from SH-SY5Y cells transfected with HA-p38 and variousmyc-tagged parkin domain constructs were subjected to immunoprecipitation (IP) with anti-myc, followed by anti-HA immunoblotting (middle). The blot was stripped and reprobed with anti-myc to illustrate the levels of parkin constructs that were expressed (bottom). Putative functional domains of parkin used in the mapping experiments are shown at the bottom of the figure. B, Lysates prepared from SH-SY5Y cells transfected with HA-tagged parkin and various myc-tagged fragments of p38 were subjected to immunoprecipitation with anti-myc antibodies, followed by anti-HA immunoblotting (middle). The blot was stripped and reprobed with anti-myc to show the relative levels of immunoprecipitated p38 fragments (bottom). A schematic representation of the different p38 fragments used in the mapping experiments is shown.
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Figure 6. Parkin ubiquitinates p38, and familial-associated mutations in parkin alter the ubiquitination of p38. Lysates prepared from SH-SY5Y cells transfected with myc-tagged wild-type, Q311Stop or R42P parkin, HA-tagged ubiquitin, and FLAG-tagged p38 were subjected to immunoprecipitation (IP) with anti-FLAG, followed by immunoblotting with anti-HA (top) and anti-FLAG (middle). Lysates were also probed with an anti-myc antibody to show parkin expression (bottom). Arrow indicates immunoprecipitated p38, and brackets indicate ubiquitinated p38.
In addition, a significant reduction in cell number is also observed after Nissl staining of p38-injected SNpc compared with the respective contralateral side (30.92 ± 3.63% reduction; p < 0.01) (Fig. 8Ce,Cf). Together, these results show that p38 overexpression in dopamine neurons leads to cell death.
Discussion
The major findings of the current study are that the aminoacyl-tRNA synthetase (ARS) cofactor p38/JTV-1 is an authentic parkin substrate and that overexpression of p38 in dopaminergic neurons leads to cell death. p38 accumulates in the ventral midbrain/hindbrain of parkin knock-out mice, AR-JP brains, and PD/DLBD brains with evidence of nitrosative stress. Moreover, we confirm and extend previous observations that parkin interacts with p38, leading to its ubiquitination and subsequent proteasomal degradation (Corti et al., 2003
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Figure 7. p38 is degraded by the proteasome. A, SH-SY5Y cells stably expressing HA-tagged p38 were transiently transfected with increasing concentrations of FLAG-parkin. Lysates were immunoblotted with anti-HA antibody to show the steady-state levels of p38 (top). The blot was stripped and reprobed with anti-FLAG (middle) to show relative levels of parkin and with anti-actin to confirm equivalent loading (bottom). Densitometric analyses of relative band intensities of p38 normalized to actin are presented as the mean ± SEM of three independent experiments. *p < 0.05, Student's t test. B, SH-SY5Y cells stably expressing HA-tagged p38 were transiently transfected with FLAG-parkin and treated for 18 h with DMSO vehicle (lane 2), 5 µM MG132 (lane 3), or 10 µM lactacystin (lane 4) before harvesting. Lysates (20 µg) were analyzed on a Western blot (WB) by immunoblotting with anti-HA (top). The blot was also stripped and reprobed with anti-FLAG (middle) to show relative expression of parkin and with anti-actin (bottom) to confirm equivalent loading in all lanes. Densitometric analyses of relative p38 band intensities normalized to actin are presented as the mean ± SEM of three independent experiments. *p < 0.05, Student's t test. C, SH-SY5Y cells stably expressing HA-tagged p38 were transiently transfected with mock vector or myc-tagged wild-type, R42P, and Q311Stop parkin. Cell lysates were analyzed by immunoblotting with anti-HA antibody (top). The blot was stripped and reprobed with anti-myc to show the expression of parkin (middle) and with anti-actin to confirm equivalent loading (bottom). Densitometric analyses of relative p38 band intensities normalized to actin are presented as the mean ± SEM of three independent experiments. *p < 0.05, Student's t test.
Parkin interacts with p38 through the RING finger 1 domain. The RING-IBR-RING domain binds to specific coenzymes, such as UbcH7, UbcH8, Hsp70, CHIP (Zhang et al., 2000
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Figure 8. p38 overexpression leads to neurotoxicity in vitro and in vivo. A, Human SK-N-MC neuroblastoma cells were transiently transfected with mock vector, HA-tagged p38 only, or myc-tagged parkin and R42P together with HA-tagged p38. Cell death was assessed by trypan blue staining. Data are shown as the mean ± SEM for three independent experiments. *p < 0.05, Student's t test. B, Human SK-N-SH neuroblastoma cells were transiently transfected with mock vector, HA-tagged p38 only, or myc-tagged wild-type and R42P parkin together with HA-tagged p38 and treated with TNF
The wide variety of parkin mutations discovered in AR-JP patients is hypothesized to result in a loss of the ubiquitin ligase activity of parkin. Because wild-type parkin binds and ubiquitinates p38, targeting it for degradation to the proteasome, we studied the effect of two mutations in parkin, a missense point mutation (R42P) and a nonsense point mutation (Q311Stop), on their relative ability to bind, ubiquitinate, and degrade p38. Coimmunoprecipitation studies showed that both mutants are defective in binding p38; whereas the R42P mutant decreases the ability of parkin to bind p38, the Q311Stop mutant shows enhanced binding. In addition, the ubiquitination profile of p38 in the presence of these parkin mutants is also disrupted. The Q311Stop mutation abolishes the ability of parkin to ubiquitinate p38, whereas the R42P mutation increases the ubiquitin modification on p38 compared with wild-type parkin. Although these familial-associated mutations are observed to have different effects on the function of parkin, both mutants failed to enhance the degradation of p38. The increased accumulation of p38 with the Q311Stop mutant may be explained by its diminished ability to ubiquitinate p38. Furthermore, the observed enhanced interaction between p38 and the Q311Stop mutant suggests a role for the C-terminal portion of parkin in substrate binding and release. The R42P point mutation lies in the ubiquitin-like domain of parkin, which is predicted to interact with the proteasome (Sakata et al., 2003Mutations in parkin are thought to be the most common cause of inherited PD, with the frequency of the mutations estimated at 50% in families with AR-JP (Lucking et al., 2000
Recently, we reported that S-nitrosylation-mediated inhibition of parkin activity could be a potential link between the genetic and sporadic forms of PD (Chung et al., 2004
There is growing evidence that overexpression of a variety of parkin substrates may exert cytotoxicity and that overexpression of parkin protects against substrate toxicity (Imai et al., 2001
The molecular mechanism by which p38 leads to cell death is not known. p38 is an important component of the multi-tRNA synthetase complex present in mammalian systems (Quevillon et al., 1999
Footnotes
Received May 28, 2005; revised July 12, 2005; accepted July 14, 2005.This work was supported by National Institutes of Health–National Institute of Neurological Disorders and Stroke Grants NS 38377 and NS 48206, the Lee Martin Trust, the Sylvia Nachlas Trust, a grant from the National Creative Research Initiatives of the Ministry of Science and Technology, Korea, and Korea Research Foundation Grant KRF-2004-037-E00002. T.M.D. is the Leonard and Madlyn Abramson Professor in Neurodegenerative Diseases. We thank Yoshikuni Mizuno for the gift of AR-JP and control brains and Simone Engelender for the gift of the synphilin-1 antibody.
Correspondence should be addressed to Dr. Ted M. Dawson, Institute for Cell Engineering, Johns Hopkins University School of Medicine, 733 North Broadway Street, Suite 731, Baltimore, MD 21205. E-mail: tdawson{at}jhmi.edu.
M. J. Kim's present address: Department of Genetics and Development, Columbia University Medical Center, New York, NY 10032.
DOI:10.1523/JNEUROSCI.2172-05.2005
Copyright © 2005 Society for Neuroscience 0270-6474/05/257968-11$15.00/0
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