Human Molecular Genetics

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Human Molecular Genetics, 2003, Vol. 12, No. 12 1427-1437
DOI: 10.1093/hmg/ddg159
© 2003 Oxford University Press

The p38 subunit of the aminoacyl-tRNA synthetase complex is a Parkin substrate: linking protein biosynthesis and neurodegeneration

Olga Corti^1,, Cornelia Hampe^1,, Hana Koutnikova¹, Frédéric Darios¹, Sandrine Jacquier¹, Annick Prigent¹, Jean-Charles Robinson², Laurent Pradier³, Merle Ruberg¹, Marc Mirande², Etienne Hirsch¹, Thomas Rooney³, Alain Fournier³ and Alexis Brice^1,*

¹INSERM U289, Hôpital de la Salpêtrière, Bâtiment Pharmacie, 47 boulevard de l'Hôpital, 75651 Paris Cedex 13, France, ²Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, 91190 Gif-sur-Yvette, France and ³CNS Department and Yeast Genomics, Aventis-Pharma, 94400 Vitry, France

Received January 22, 2003; Revised April 4, 2003; Accepted April 22, 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Parkinson's disease (PD) is a severe neurological disorder, characterized by the progressive degeneration of the dopaminergic nigrostriatal pathway and the presence of Lewy bodies (LBs). The discovery of genes responsible for familial forms of the disease has provided insights into its pathogenesis. Mutations in the parkin gene, which encodes an E3 ubiquitin–protein ligase involved in the ubiquitylation and proteasomal degradation of specific protein substrates, have been found in nearly 50% of patients with autosomal-recessive early-onset parkinsonism. The abnormal accumulation of substrates due to loss of Parkin function may be the cause of neurodegeneration in parkin-related parkinsonism. Here, we demonstrate that Parkin interacts with, ubiquitylates and promotes the degradation of p38, a key structural component of the mammalian aminoacyl-tRNA synthetase complex. We found that the ubiquitylation of p38 is abrogated by truncated variants of Parkin lacking essential functional domains, but not by the pathogenic Lys161Asn point mutant. Expression of p38 in COS7 cells resulted in the formation of aggresome-like inclusions in which Parkin was systematically sequestered. In the human dopaminergic neuroblastoma-derived SH-SY5Y cell line, Parkin promoted the formation of ubiquitylated p38-positive inclusions. Moreover, the overexpression of p38 in SH-SY5Y cells caused significant cell death against which Parkin provided protection. Analysis of p38 expression in the human adult midbrain revealed strong immunoreactivity in normal dopaminergic neurons and the labeling of LBs in idiopathic PD. This suggests that p38 plays a role in the pathogenesis of PD, opening the way for a detailed examination of its potential non-canonical role in neurodegeneration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Parkinson's disease (PD) is a common neurodegenerative disorder clinically characterized by resting tremor, rigidity and bradykinesia. These severe neurological symptoms are caused by the progressive and selective degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). Lewy bodies (LBs), which are ubiquitylated neuronal cytoplasmic inclusions, are a pathological hallmark of PD (1).

Insights into the molecular mechanisms of the pathogenesis of PD were provided by the discovery of genes implicated in rare monogenic forms (2). Missense mutations in the -synuclein gene (A30P, A53T), encoding a presynaptic protein of unknown function, have been described in a few families with autosomal dominant PD (3,4). Alpha-synuclein is a major component of LBs, suggesting that this protein plays a more general role in the pathogenesis of sporadic PD (5,6). A point mutation (I93M) has been found in the gene encoding the de-ubiquitylating enzyme, UCHL-1, another component of LBs, in two members of a family with autosomal-dominant PD (7). More recently, a deletion and a point mutation (L166P) in DJ-1, which encodes a protein thought to be involved in the oxidative stress response, were shown to be linked to autosomal-recessive early-onset parkinsonism in two European families (8).

In 1998, the parkin gene was shown to be responsible for a distinct clinical and genetic entity in Japan, defined as autosomal-recessive juvenile parkinsonism (9). It was subsequently demonstrated that almost 50% of patients with familial autosomal-recessive early-onset parkinsonism from various populations carry a series of parkin exon rearrangements and point mutations (10–12). These mutations are associated with a wide range of ages at onset and a broad phenotypic spectrum, including cases that are clinically indistinguishable from idiopathic PD (12,13). The general lack of LBs in patients with parkin-related PD suggests that Parkin plays a role in the biogenesis of these inclusions (14–16). This hypothesis is supported by the presence of Parkin immunoreactivity in LBs from patients with sporadic and familial PD (17).

Parkin is a 465 amino acid protein homologous to ubiquitin at its N-terminus (ubiquitin-like domain) and with a C-terminal cysteine-rich RING-IBR-RING motif common to proteins and protein complexes with ubiquitin–protein ligase activity (9,18). Parkin has E3 ubiquitin–protein ligase activity (19–21), which enables it to interact with the cytoplasmic ubiquitin-conjugating E2 enzymes, UbcH7 and UbcH8, and with the endoplasmic reticulum resident E2 enzymes, UBC6 and UBC7, thus promoting the ubiquitylation and proteasomal degradation of specific protein substrates (19–22). Loss of Parkin function is thought to result in the gradual accumulation of non-ubiquitylated, potentially toxic, substrates, leading to neurodegeneration.

Parkin ubiquitylates five cellular proteins with distinct putative functions: the synaptic vesicle associated protein, CDCrel-1; the -synuclein interacting protein, synphilin; the putative G-protein-coupled transmembrane protein, Pael R; an O-glycosylated isoform of -synuclein; and the pro-apoptotic cyclin E (21–25). The specific pathological roles of these substrates remain to be established, but it is likely that the alteration of the degradation rates of several Parkin substrates act synergistically to compromise dopaminergic cell survival. We performed a yeast two-hybrid screen to isolate further Parkin substrates and identified p38, which is a key structural component of the macromolecular aminoacyl-tRNA synthetase (ARS) complex involved in protein biosynthesis (26,27).

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
The scaffold p38 subunit of the mammalian ARS complex is a Parkin substrate
To identify novel Parkin substrates, we performed a yeast two-hybrid screen of a HeLa cDNA library using a human Parkin sequence corresponding to amino acids 135–290 as bait (Fig. 1A). This led to the isolation of a 1141 bp cDNA clone encoding the 320 amino acids of p38, a central scaffold component of the multiARS complex (26–28). The interaction between Parkin and p38 was specific, as p38 failed to interact with the unrelated protein Ras or with the cytoplasmic domain of the amyloid precursor protein (data not shown). To confirm these results, we carried out co-immunoprecipitation experiments in COS7 cells. Myc epitope-tagged p38 (myc-p38) was overexpressed alone or with an HA epitope-tagged variant of Parkin devoid of its N-terminal ubiquitin-like motif (Fig. 1A; HA-Parkin^77-465). Myc-p38 specifically co-immunoprecipitated with HA-Parkin^77-465 using anti-HA antibodies (Fig. 1B).

View larger version (30K): [in this window] [in a new window]   Figure 1. Parkin interacts with and ubiquitylates p38. (A) Schematic representation of human Parkin, showing its functional domains (Ubl, ubiquitin-like). (B) Co-immunoprecipitation of myc-p38 with HA-Parkin77-465 in COS7 cells. Immunoprecipitates obtained with anti-HA antibodies (anti-HA IP) or aliquots of cell lysates were analyzed by western blot (WB) using anti-HA or anti-myc antibodies. (C) Analysis of the ubiquitylation of myc-p38, HA-CDCrel-1 and EGFP in SH-SY5Y and SH-SY5Y-HA-Parkin1-465 cells transfected with expression vectors encoding the indicated proteins. His-ubi, his-tagged ubiquitin. Purified his-ubi proteins or cell lysates were analyzed by WB using anti-myc, anti-HA, anti-GFP or anti-Parkin (54) antibodies. (D, E) Analysis of p38 ubiquitylation in SH-SY5Y cells transiently transfected with vectors encoding myc-p38, his-ubi and HA-Parkin1-86, HA-Parkin77-465, HA-ParkinLys161Asn or HA-Parkin1-465. Results of representative experiments are shown. The asterisks indicate the non-specific binding of non-ubiquitylated myc-p38 to the Probond affinity resin.

 
We then determined whether Parkin is involved in the ubiquitylation of p38 in a human dopaminergic neuroblastoma-derived cell line stably expressing full-length HA-Parkin (SH-SY5Y-HA-Parkin^1-465). Myc-p38, an HA-tagged Parkin substrate CDCrel-1 (positive control) and EGFP (enhanced green fluorescent protein; negative control) were overexpressed alone or with his-tagged ubiquitin in SH-SY5Y or SH-SY5Y-HA-Parkin^1-465 cells. His-ubiquitylated proteins were purified by affinity chromatography and the covalent modification of p38, CDCrel-1 and EGFP by ubiquitin was examined by western blotting using anti-myc, anti-HA and anti-EGFP antibodies, respectively. We observed high molecular weight proteins corresponding to ubiquitylated p38 and CDCrel-1 in cells overexpressing Parkin, but not in native SH-SY5Y cells, consistent with the Parkin-mediated ubiquitylation of these proteins (Fig. 1C). In contrast, EGFP was not ubiquitylated in native SH-SY5Y or in SH-SY5Y-HA-Parkin^1-465 cells.

We next evaluated whether the deletion of the functional domains involved in the E3 ubiquitin–protein ligase activity of Parkin affects the Parkin-mediated ubiquitylation of p38 in SH-SY5Y cells (19,20). Myc-p38 and his-ubiquitin were transiently overexpressed in native SH-SY5Y cells, alone or together with HA-Parkin^1-465 or truncated isoforms lacking a large portion of the linker region and the entire RING-IBR-RING domain (HA-Parkin^1-86) or the ubiquitin-like motif (HA-Parkin^77-465) (Fig. 1A). Consistent with the results obtained in SH-SY5Y-HA-Parkin^1-465 cells, full-length Parkin promoted the ubiquitylation of p38 (Fig. 1D). In contrast, p38 was not ubiquitylated when it was overexpressed with Parkin^1-86 or Parkin^77-465. In a similar experiment, we evaluated the ability of the pathogenic Parkin mutant Lys161Asn (HA-Parkin^Lys161Asn) to ubiquitylate p38 in SH-SY5Y cells (Fig. 1E). This mutant affects the central domain of Parkin used as bait for the yeast two hybrid screen, which directly interacts with p38. Surprisingly, Parkin^Lys161Asn ubiquitylated p38 to the same extent as normal Parkin.

To investigate whether the Parkin-mediated ubiquitylation of p38 accelerates its degradation, we carried out pulse-chase experiments to examine the turnover of myc-p38 in native SH-SY5Y and SH-SY5Y-HA-Parkin^1-465 cells. Parkin significantly promoted myc-p38 degradation in SH-SY5Y-HA-Parkin^1-465 cells (Fig. 2). Accordingly, about 50% of de novo synthesized myc-p38 was degraded after a 0.5 h chase in SH-SY5Y-HA-Parkin^1-465 cells, whereas a chase of 1.75 h was required for the degradation of a similar amount of myc-p38 in SH-SY5Y cells.

View larger version (29K): [in this window] [in a new window]   Figure 2. Parkin promotes the degradation of p38. Pulse-chase analysis of the turnover of myc-p38 in SH-SY5Y and SH-SY5Y-HA-Parkin1-465 cells. Data from three independent experiments are expressed as means±SEM. Asterisks indicate statistical significance; P0.003 versus SH-SY5Y-HA-Parkin1-465.

 
Parkin is recruited into p38-positive aggresome-like inclusions in COS7 cells
We further analyzed the functional interaction between Parkin and p38 by examining their intracellular distribution and potential co-localization in COS7 cells (Fig. 3). When full-length Parkin was overexpressed alone, Parkin immunoreactivity displayed a homogeneous punctuate distribution throughout the cell body and processes and was particularly intense in the perinuclear region; frequently, immunoreactivity was also present in the nucleus (Fig. 3). A similar pattern of immunoreactivity was observed for endogenous Parkin (data not shown). Importantly, there was a striking overlap in the distribution of Parkin and that of endogenous p38, in particular in the perinuclear region and the cell processes, and less frequently in the nucleus of COS7 cells. Occasionally, endogenous p38 immunostaining was also associated with discrete cytoplasmic granules (Fig. 3A). The overexpression of p38 alone led to formation of large p38-immunoreactive perinuclear inclusions in 30% of the transfected cells in basal conditions, and in most cells when proteasome activity was inhibited by epoxomicin (data not shown) (29). When overexpressed with p38, full-length Parkin was systematically sequestered in large p38-positive inclusions (Fig. 3B). Such a redistribution was also observed for Parkin^77-465 and Parkin^Lys161Asn (Fig. 3B), but not when EGFP was overexpressed with p38 (data not shown).

View larger version (58K): [in this window] [in a new window]   Figure 3. p38 co-localizes with Parkin in COS7 cells. (A) Analysis of the localization of endogenous p38 and overexpressed HA-Parkin1-465 revealed with anti-HA and anti-p38 antibodies. (B) HA-Parkin1-465, HA-Parkin77-465 and HA-ParkinLys161Asn are recruited in aggregates formed by overexpressed myc-p38. Immunocytochemistry using anti-Parkin (54) and anti-myc antibodies. Cells were analyzed by laser-scanning confocal microscopy.

 
We next characterized the p38 immunoreactive inclusions to determine whether they were related to aggresomes, which are pericentriolar structures formed by cells in response to the presence of misfolded proteins (30,31). Double immunofluorescence for the centrosome marker -tubulin and p38 revealed that, similarly to aggresomes, the inclusions assembled at the microtubule organizing center (Fig. 4A, upper panel). This process was dependent on intact microtubules, since treatment of the transfected cells with the microtubule depolymerizing agent nocodazole caused the generation of smaller p38 aggregates that were dispersed throughout the cell body (Fig. 4A, upper panel). In contrast, cytochalasin D, a compound that destabilizes actin filaments, had no effect on the formation of the inclusions (data not shown). The assembly of the inclusions caused the reorganization of the intermediate filament vimentin such that it formed a ring-like structure at the periphery of aggregates, as reported for aggresomes (Fig. 4A, lower panel). Most inclusions were ubiquitin-positive and co-localized with the proteasomal 20S subunit and the cytoplasmic chaperones, Hsp70 and Hdj-2 (Fig. 4B).

View larger version (48K): [in this window] [in a new window]   Figure 4. p38 positive inclusions in COS7 cells overexpressing myc-p38 are related to aggresomes. Laser confocal scanned images illustrating (A, upper panel) the co-localization of p38-positive inclusions and the centrosome marker -tubulin; the effect of nocodazole treatment on the appearance of p38-positive aggregates; (lower panel) the redistribution of vimentin at the periphery of inclusions. (B) Double immunofluorescence for p38 and endogenous ubiquitin, the proteasomal 20S complex, the cytoplasmic chaperone, Hsp70 and the co-factor, Hdj-2.

 
Parkin promotes the formation of ubiquitylated p38-positive inclusion bodies and protects from p38-induced toxicity in the dopaminergic SH-SY5Y cell line
To characterize the functional relationship between Parkin and p38 further, we examined the intracellular distribution of myc-p38 in SH-SY5Y and SH-SY5Y-HA-Parkin^1-465 cells. In SH-SY5Y cells, p38 was homogeneously distributed throughout the cytoplasm and the processes (data not shown). p38-positive ubiquitylated cytoplasmic inclusions were also observed in about 2% of the transfected cells (Fig. 5A and B). The percentage of cells displaying these inclusions was significantly higher when p38 was overexpressed in SH-SY5Y-HA-Parkin^1-465 cells (about 9%; Fig. 5B). The aggregation of p38 was considerably enhanced in the presence of epoxomicin: p38-positive inclusions were observed in 6 and 14% of the transfected SH-SY5Y and SH-SY5Y-HA-Parkin^1-465 cells, respectively (Fig. 5B). Similar results were obtained with an EGFP-p38 fusion protein. In contrast, the control protein EGFP did not form visible aggregates in SH-SY5Y or SH-SY5Y-HA-Parkin^1-465 cells (data not shown).

View larger version (57K): [in this window] [in a new window]   Figure 5. Parkin promotes the aggregation of p38 and protects from p38-induced cell death in SH-SY5Y cells. (A) Laser confocal scanned image illustrating a p38-positive inclusion in SH-SY5Y cells overexpressing myc-p38 and double labeled for ubiquitin and p38. (B) Quantification of the percentage of transfected SH-SY5Y and SH-SY5Y-HA-Parkin1-465 cells displaying p38-positive inclusions, after treatment or not with the proteasome inhibitor epoxomicin. aP0.002; bP<0.02. (C) Quantification of the percentage of dead EGFP-positive cells in SH-SY5Y or SH-SY5Y-HA-Parkin1-465 cells overexpressing EGFP or a EGFP-p38 fusion protein. Asterisks, P<0.001. Results of one representative experiment (n=3) are shown.

 
To determine whether the accumulation of p38 could be toxic, EGFP or EGFP-p38 were overexpressed in native SH-SY5Y or SH-SY5Y-HA-Parkin^1-465 cells, then the viability of EGFP-positive cells was estimated (Fig. 5C). Levels of cell death were similar in SH-SY5Y and SH-SY5Y-HA-Parkin^1-465 cells producing EGFP (about 5%). Cell death increased 6-fold when EGFP-p38 was overepressed in native SH-SY5Y cells. The overexpression of Parkin in SH-SY5Y cells partially but significantly protected from p38-induced toxicity, leading to a 2-fold decrease in cell death.

p38 is present in LBs in patients with idiopathic PD
We next examined the distribution of p38 in the human midbrain, using specific antibodies (Fig. 6). In lysates from SH-SY5Y cells overexpressing myc-p38, these antibodies detected both native endogenous p38 and exogenous myc-p38. In homogenates of human cortex (Cx) and caudate putamen (C/P) two p38 immunoreactive bands were visible. Their apparent molecular mass was slightly higher than that of endogenous p38 in SH-SY5Y cells (Fig. 6, lanes ‘anti-p38’). Therefore, these bands are likely to correspond to post-translationally modified isoforms of p38 and/or to products of alternatively spliced mRNAs. None of these bands were detected when the blots were hybridized with anti-p38 antibodies pre-absorbed with a large excess of recombinant p38 or with pre-immune serum (Fig. 6, lanes ‘anti-p38 pre-absorbed’ and ‘pre-immune serum’). Analysis of p38 immunoreactivity in the midbrains of two control subjects and three patients with PD post-mortem, revealed strong labeling in the human SN, predominantly in cells with a neuronal phenotype and in nerve fibers (Fig. 7A and B). This immunoreactivity was abolished when the antibodies were pre-absorbed with an excess of recombinant p38 (Fig. 7C and E). Intense staining was observed in perikarya and processes of melanized dopaminergic neurons and of non-dopaminergic neurons, in the SNc (Fig. 7D). The p38 immunostaining was heterogeneously distributed and appeared to be associated with discrete granules in the cytoplasm and the perinuclear region (Fig. 7D). No major differences in the type or intensity of p38 immunoreactivity were observed between control subjects and patients. However, the number of p38-positive melanized neurons was markedly lower in the SNc of patients with PD, consistent with the neuronal loss associated with the disease (data not shown). To determine whether p38 immunoreactivity could be associated with nigral pathology and in particular with LBs, we double-labeled midbrain sections from PD subjects for ubiquitin and p38. In the three PD patients examined, p38 immunostaining was frequently observed in the cores of classical LBs in pigmented neurons (Fig. 7F), as well as in the cores of non-somatic LBs (Fig. 7G) and occasionally in ubiquitin-positive neurites (Fig. 7H).

View larger version (15K): [in this window] [in a new window]   Figure 6. Specificity of novel polyclonal anti-p38 antibodies. Western blot analysis of cleared homogenates of (T) SH-SY5Y cells overexpressing myc-p38, (NT) native SH-SY5Y, (Cx) human cortex, (C/P) human caudate-putamen hybridized with anti-myc antibodies, anti-p38 antibodies, anti-p38 antibodies pre-absorbed with recombinant p38, or pre-immune serum.

 

View larger version (74K): [in this window] [in a new window]   Figure 7. Immunohistochemical analysis of the distribution of p38 in the human SN adult post-mortem and its localization in LBs. Sections of the SN analyzed after immunolabeling with (A, B) specific anti-p38 antibodies, or (C, E) the same antibodies pre-absorbed with an excess of recombinant p38. Note that in (C) and (E) only endogenous pigmentation of neuromelanin is visible. (D) Granular p38 immunoreactivity in melanized dopaminergic neurons of the SNc. Laser confocal scanned images illustrating anti-p38 immunoreactivity in (F) classical intracellular LBs, (G) extracellular LBs, and (H) ubiquitin-positive neurites in the SN of subjects with idiopathic PD, after double labeling for ubiquitin and p38. SNc, SN pars compacta; SNr, SN pars reticulata. Scale bar=0.5 mm in (A); 0.1 mm in (B) and (C); and 20 µm in (D) and (E).

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
We showed that the p38 subunit of the mammalian multiARS complex is a Parkin substrate. p38 was isolated in a yeast two-hybrid screen, using a Parkin peptide excluding both the ubiquitin-like and most of the RING-IBR-RING domain as bait. This bait was chosen to ensure that we did not isolate components of the ubiquitin-proteasome pathway that interact with these motifs (32,33). Physical interaction was confirmed in mammalian cells by co-immunoprecipitating p38 with a Parkin variant containing the bait peptide. Co-localization studies in COS7 cells showed a marked overlap in the intracellular distribution of Parkin and endogenous p38, supporting a physiological interaction between the two proteins. In the human dopaminergic neuroblastoma-derived SH-SY5Y cell line, full-length Parkin promoted the ubiquitylation and proteasomal degradation of p38. Importantly, the deletion of either the ubiquitin-like domain or the RING-IBR-RING motif, which are essential for the E3 ubiquitin ligase activity of Parkin (19,20), abrogated the Parkin-mediated ubiquitylation of p38. In contrast, in our experimental conditions the pathogenic point mutant Lys161Asn ubiquitylated p38 to the same extent as normal Parkin. Although apparently surprising, this finding is in agreement with other studies reporting at least partial ubiquitin– protein ligase activity for several pathogenic Parkin mutants. Ubiquitylation of the Parkin substrate synphilin was observed with Parkin mutants Arg275Trp, Gln311Stop and Thr415Asn, whereas this process was compromised with mutants Thr240Arg, Arg256Cys and Trp453Stop (23). Similarly, Imai and colleagues observed ubiquitylation of Parkin substrate PaelR in the presence of Parkin mutant Thr240Arg, but not of an exon 4 deleted mutant (22). In another study, Hyun and collaborators (34) reported full ubiquitin–protein ligase activity for an exon 3–5 deleted mutant of Parkin. Finally, in a pure in vitro ubiquitylation assay developed by Rankin and colleagues (35), E3 ubiquitin–protein ligase activity was measured with normal Parkin, but also with a truncated variant devoid of the N-terminal 35 amino acid residues, and with the point mutants Cys238Ala and Cys332Ala. It appears therefore that the pathogenic mutations identified so far do not systematically abrogate the enzymatic activity of Parkin, raising the possibility that various molecular mechanisms underlie neuronal degeneration in parkin-related Parkinson's disease (e.g. loss of interaction with essential partners and/or specific substrates). This hypothesis is also highlighted by the recent finding that Parkin functions in a multiprotein ubiquitin ligase complex that includes the F-box protein h-Sel10 and Cullin 1 (25).

Several lines of evidence suggest that the novel Parkin substrate p38 contributes to neurodegeneration in PD. The overexpression of p38 resulted in its accumulation in aggresome-like inclusions. These inclusions systematically recruited Parkin, but also the ubiquitin-like deleted Parkin mutant and the pathogenic mutant Lys161Asn, suggesting that these variants maintain the ability to interact with p38. In addition, the p38 immunoreactive aggresomes sequestered proteasomal 20S subunit, as well as the molecular chaperones Hsp70 and Hdj-2. A propensity to form aggregates upon overexpression has already been reported for two other Parkin substrates: the -synuclein-interacting protein, synphilin and the unfolded putative transmembrane protein, Pael-R (22,23). Pael-R, in particular, forms multiprotein complexes with Parkin, the E4-like protein CHIP, Hsp70 and Hdj-2 in cells, and these proteins appear to regulate its folding and degradation (36). Similarly, a finely tuned balance between protein synthesis, folding and degradation may be required to maintain appropriate p38 levels in the cell. In the simian kidney-derived COS7 cell line, numerous ubiquitylated p38-positive inclusions formed in the absence of exogenous Parkin. In contrast, in neural SH-SY5Y cells, the overexpression of Parkin promoted their formation. Therefore, in SH-SY5Y cells, the Parkin-mediated ubiquitylation of p38 appears to be required both for its proteasomal degradation and for its aggregation. As suggested by the significant increase in the number of p38-positive inclusions observed following inhibition of proteasome activity, the aggregation of p38 may reflect the overwhelming of the cellular protein degradation machinery.

Remarkably, p38 immunoreactivity was observed in LBs in patients with idiopathic PD, demonstrating that p38 aggregates in humans and suggesting that this process contributes to dopaminergic neurodegeneration. p38 was frequently observed in the central cores of LBs, a localization that is also typical of Parkin and the Parkin substrate, synphilin (17,37). This localization is indicative of a primary accumulation in inclusions rather than a deposition on pre-existing LBs, and suggests that the Parkin-mediated ubiquitylation of p38 is an early event in LB formation. Whether LBs are actively involved in the degenerative process, represent a mere epiphenomenon, or play a neuroprotective role in PD is still unclear. The general absence of LBs in parkin-related parkinsonism (14–16) suggests that these inclusions are not the cause of dopaminergic cell death in PD. This hypothesis is also supported by the absence of LBs in most dopaminergic neurons undergoing apoptosis in PD, and the lack of correlation between the presence of LBs and the occurrence of apoptotic features in these neurons (38). Interestingly, the overexpression of p38 in native SH-SY5Y cells induced significant cell death, providing evidence that the accumulation of non-ubiquitylated, non-aggregated p38 could be deleterious. Notably, exogenous Parkin promoted the accumulation of p38 in ubiquitin-positive inclusions and also partially prevented p38-induced cell death, suggesting that, in this model, the aggregation of p38 has a protective effect.

Our results provide a link between a protein involved in the degeneration of dopaminergic nigral neurons through a loss of function mechanism, and a key structural component of ARS complexes that is essential for protein biosynthesis. ARSs belong to a family of ubiquitous enzymes that catalyze the esterification of amino acids with their cognate tRNA. In mammals, at least 10 of these enzymes assemble into two distinct high molecular mass complexes (39,40). The multiARS complex integrates nine ARS activities and the three auxiliary proteins, p18, p38 and p43. p38 is a key scaffold subunit that plays a central role in the in vivo assembly of this supramolecular structure (26,27). This essential function is illustrated by the dramatic consequences of the deficiency of p38 in homozygous mutant mice, leading to the complete disintegration of the ARS complex, instability of the component enzymes and auxiliary proteins, and early post-natal lethality (41).

Considerable evidence has accumulated suggesting that components of the ARS complex have non-canonical functions and that they are involved in cell death pathways (42). Human glutaminyl-RS has been proposed to be a negative regulator of the apoptosis signal-regulating kinase 1, a protein kinase that plays a crucial role in Fas-activated apoptosis (43). In the presence of apoptotic stimuli, mammalian tyrosyl-RS is secreted and split into two fragments with distinct potent pro-apoptotic cytokine activities (44,45). Similarly, p43, a tRNA-binding protein that functions as a co-factor of aminoacylation is the precursor of the inflammatory cytokine EMAPII (endothelial monocyte-activating peptide II) (46–50). EMAPII is a substrate of caspase 7, which is cleaved off p43 and released from the multisynthetase complex in apoptotic conditions, before being secreted into the extracellular matrix where it activates the cell death signaling pathway (49,50). EMAP-II immunoreactivity was recently detected in activated microglial cells after the neurotoxic lesion of the rat hippocampus, suggesting that it plays a role in neurodegenerative processes (51).

Our observations suggest that the loss of Parkin function in parkin-related parkinsonism results in the accumulation of non-ubiquitylated p38 above a toxicity threshold that could be deleterious via an as yet unidentified molecular mechanism. This threshold may be reached more rapidly in neurons, a cell type in which p38 immunoreactivity was particularly intense. By contributing to the detrimental effects of other Parkin substrates, p38 may induce dopaminergic neuronal degeneration. Alternatively, the uncontrolled intracellular accumulation of p38 may alter multiARS complex assembly and/or spatially dislocate components of this complex from the subcellular compartments of the protein synthesis machinery (52,53), thereby directly impairing the cellular translational potential. Conversely, the cleavage and subsequent release of components of the multiARS complex under apoptotic conditions (49,50) may destabilize the complex and favor the aggregation of p38. This aggregation may also be promoted by a decrease in proteasomal enzymatic activities, which is observed in the SNc of patients with sporadic PD (54,55). As is the case for several pathological proteins that form inclusions in neurodegenerative diseases (56), and consistent with our observations in cultured cells, the sequestration of key cellular components (i.e. proteasomal subunits, ubiquitin, Parkin, chaperones) in p38-positive aggregates may perturb the cellular metabolism and amplify cellular stress.

In conclusion, we have provided evidence for a functional interaction between Parkin and the p38 component of the multiARS complex and suggest that p38 contributes to dopaminergic cell death in PD. Further studies will be required to determine whether and by which mechanisms p38 plays a dual function in protein biosynthesis and in the pathogenesis of PD.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Yeast two-hybrid screening
The bait plasmid pLex-Parkin^135-290 was generated by subcloning the human Parkin cDNA sequence corresponding to protein residues 135–290 into pLex9/pBTM11. The yeast strain L40 was transformed with pBTM116-lexA-Parkin^135-290, then the HeLa cDNA library in pGAD-GH (Clontech) was introduced into it. We screened 3.5x10⁶ yeast transformants for their ability to grow on Trp^- Leu^- His^- selection medium. The specificity of the interaction was confirmed by co-transforming L40 yeast with each of the selected plasmids and with the control vectors pLex9-cAPP (A. Fournier) or pLex9-RasVal12 (a generous gift from A. Vojtek).

Plasmids, cell culture and transfections
The Parkin cDNAs were subcloned into the EcoRI and XbaI sites of pcDNA3-HA (A. Fournier). The p38 cDNA was inserted into the BamHI and NotI sites of pcDNA3-myc (L. Pradier), and into the BglII and ApaI sites of pEGFP-C1 (Clontech). The integrity of the constructs was confirmed by sequencing. pEGFP-C1 was purchased from Clontech and p6his-ubiquitin (pMT107) provided by G. Bossis.

Cells were grown in DMEM (Invitrogen) with 10% fetal calf serum (FCS) in a 5% CO₂ atmosphere. Transfections were performed with DMRIE-C (Invitrogen) according to the manufacturer's instructions. Stable clones were obtained by cotransfecting SH-SY5Y cells with pcDNA3-HA-Parkin^1-465 and with pTK-Hyg (Clontech), at a 1 : 10 molar ratio. Cells were plated out at a low density (2–8x10⁵/10 cm Petri dish) in a medium containing hygromycin (90 µg/ml, Invitrogen). The expression of the transgene in the selected hygromycin-resistant clones was verified by western blot using anti-HA (clone 16B12, BabCO, 1 : 1000) and anti-Parkin antibodies (1 : 400) (57,58).

Co-immunoprecipitation and ubiquitylation assays
Forty-eight hours after transfection, cells were lysed in co-IP buffer [50 mM Tris–HCl pH 8, 150 mM NaCl, 5% glycerol, 0.5% NP40, 0.2 mM Na₃VO₄, 4 mg/ml NaF, complete protease inhibitors (Roche)]. Lysates were centrifuged at 4°C for 30 min at 13 000g. Supernatants were pre-cleared with protein G Sepharose (Amersham) for 1 h at 4°C, combined with 2 µg monoclonal protein G Sepharose-coupled anti-HA antibodies (clone 16B12, BAbCO), and gently rocked for 2 h at 4°C. Immunoprecipitated proteins were washed four times in co-IP buffer and once in PBS, and analyzed by western blot with monoclonal anti-myc antibodies (clone 9E10, Santa-Cruz, 1 : 400) or monoclonal anti-HA antibodies (clone 16B12, BabCO, 1 : 1000).

For ubiquitylation assays, cells were treated with 1 µM epoxomicin (Affiniti) for 8 h, 36 h after transfection, and lysed in denaturing lysis buffer (6 M guanidium–HCl, 0.1 M Na₂HPO₄/NaH₂PO₄, 0.01 M Tris–HCl, pH 8). Six-his ubiquitylated proteins were affinity purifed in denaturing conditions (ProBond resin, Invitrogen), according to the manufacturer's instructions, then eluted from beads with 200 mM imidazole in 5% SDS, 0.15 M Tris–HCl pH 6.7, 30% glycerol, 0.72 M ß-mercaptoethanol. Purified proteins and aliquots from cell lysates were analyzed by western blot for myc-p38, HA-CDCrel-1, HA-Parkin or EGFP (monoclonal anti-GFP, Roche, 0.4 µg/ml). Proteins were visualized using enhanced chemioluminescence (Pierce).

Pulse-chase analysis of p38 turnover
Pulse-chase experiments were performed 36 h after the transfection of cells with pcDNA3-myc-p38. Cells were starved in methionine/cysteine-free DMEM–2% FCS (1 h), pulsed with 200 µCi/ml of [³⁵S]-methionine/cysteine (Perkin Elmer, 30 min), rinsed and chased for the indicated periods of time in DMEM–10% FCS. Cell lysates obtained in co-IP buffer were immunoprecipitated with anti-myc antibodies (clone 9E10, Santa Cruz; 4 µg). Immunoprecipitates were resolved by SDS–PAGE, visualized by phosphoimaging and quantified with Aida analysis software.

Quantification of p38-positive inclusions and viability assays
Cells were plated on collagen-coated coverslips in 24-well culture clusters. To determine the number of cells containing p38-positive aggregates, 48 h after transfection, cells were treated or not with 0.5 µM epoxomicin over-night, then processed for anti-myc immunocytochemistry. For each condition, at least 150 p38-positive cells were counted from each of four independent wells. For viability assays, cells from three wells were transfected with pEGFP-C1 and cells from 10 wells with pEGFP-C1-p38. Thirty-six hours after transfection, cells were labeled with ethidium homodimer 1 (EthD-1, Viability/Cytotoxicity Kit, Molecular Probes), then at least 200 EGFP-positive cells were counted blindly from coded coverslips. The percentage cell death was calculated as the ratio of double-labeled EthD-1/EGFP-positive cells to EGFP-positive cells.

Immunochemistry
Thirty-six hours after transfection, cells were treated or not with nocodazole (15 µg/ml, over-night) or cytochalasin D (200 nM, over-night), fixed in 4% paraformaldehyde and analyzed by standard immunocytochemical procedures. Primary antibodies were monoclonal anti-myc (clone 9E10, Santa Cruz, 1 : 400), monoclonal anti--tubulin (clone GTU-88, Sigma, 1 : 10 000), monoclonal anti-vimentin (clone V9, Dako, 1 : 100), polyclonal anti-p38 (1 : 20 000), polyclonal anti-20S proteasome (Affiniti, 1 : 2500), polyclonal anti-ubiquitin (Dako, 1 : 100), polyclonal anti-Hsp70 (Stressgen, 1 : 2500), and polyclonal anti-Parkin (1 : 400) (57,58). Secondary antibodies were Alexa Fluor 488-conjugated goat anti-mouse IgG (Interchim, 1 : 200) and CY3-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, 1 : 600). Polyclonal anti-p38 antibodies were raised in rabbit against hamster p38 expressed in E. coli and purified to homogeneity as previously described (27). Mono-specific antibodies were affinity-purified on p38 immobilized on CNBr-activated Sepharose 4B. The specificity of these antibodies was assessed by western blot analysis of cleared homogenates of human brain tissue. Homogenates, generated by disrupting the tissue in 50 mM Hepes, pH 7.5, 150 mM NaCl, 150 mM MgCl₂, 10% glycerol, 1% Triton-X100, 10 mM Na pyrophosphate, 100 mM NaF, 1 mM EDTA, complete protease inhibitors (Roche), were centrifuged at 15 000g, for 30 min at 4°C. Supernatants (30 µg) were immunoblotted using anti-p38 antibodies (1 : 100 000) or pre-immune serum (1 : 5000).

Human brain tissue was selected and prepared as previously described (59). Conventional immunohistochemistry was carried out with polyclonal anti-p38 antibodies (1 : 20 000) on free-floating human brain sections, as described previously (58). Immunoreactivity was revealed using the avidin–biotin peroxidase complex (Vectastain, Vector) with diaminobenzidine as the chromogenic substrate. For pre-absorptions, antibodies were incubated with a 2000-fold excess of recombinant his-p38 (26) prior to use. After antigen retrieval (microwave method), immunofluorescent labeling was performed with polyclonal anti-p38 (1 : 5000) and monoclonal anti-ubiquitin antibodies (clone ubi-1, Zymed, 1 : 50), as described previously (60). Secondary antibodies were as above.

Statistical analyses
ANOVA and the Fisher LSD test were used to determine whether differences between groups were significant. Data are expressed as means±SEM. Statistical significance was defined as P<0.05.

	ACKNOWLEDGEMENTS

 
We thank Y. Zhang for kindly providing pRK-HA-CDCrel-1, and G. Bossis for providing phis-Ubi and for excellent advice on ubiquitylation experiments in cultured cells. This work was supported by INSERM, Aventis-Pharma, the Association France Parkinson, the Fondation de France and the GIP Fonds de Recherche Aventis.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +33 142162183; Fax: +33 144243658; Email: brice{at}ccr.jussieu.fr

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

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				E. Avraham, R. Rott, E. Liani, R. Szargel, and S. Engelender Phosphorylation of Parkin by the Cyclin-dependent Kinase 5 at the Linker Region Modulates Its Ubiquitin-Ligase Activity and Aggregation J. Biol. Chem., April 27, 2007; 282(17): 12842 - 12850. [Abstract] [Full Text] [PDF]

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				C. Hampe, H. Ardila-Osorio, M. Fournier, A. Brice, and O. Corti Biochemical analysis of Parkinson's disease-causing variants of Parkin, an E3 ubiquitin-protein ligase with monoubiquitylation capacity Hum. Mol. Genet., July 1, 2006; 15(13): 2059 - 2075. [Abstract] [Full Text] [PDF]

				H. S. Ko, S. W. Kim, S. R. Sriram, V. L. Dawson, and T. M. Dawson Identification of Far Upstream Element-binding Protein-1 as an Authentic Parkin Substrate J. Biol. Chem., June 16, 2006; 281(24): 16193 - 16196. [Abstract] [Full Text] [PDF]

				J. W. Um, D. S. Min, H. Rhim, J. Kim, S. R. Paik, and K. C. Chung Parkin Ubiquitinates and Promotes the Degradation of RanBP2 J. Biol. Chem., February 10, 2006; 281(6): 3595 - 3603. [Abstract] [Full Text] [PDF]

				N. Matsuda, T. Kitami, T. Suzuki, Y. Mizuno, N. Hattori, and K. Tanaka Diverse Effects of Pathogenic Mutations of Parkin That Catalyze Multiple Monoubiquitylation in Vitro J. Biol. Chem., February 10, 2006; 281(6): 3204 - 3209. [Abstract] [Full Text] [PDF]

				W. Springer, T. Hoppe, E. Schmidt, and R. Baumeister A Caenorhabditis elegans Parkin mutant with altered solubility couples {alpha}-synuclein aggregation to proteotoxic stress Hum. Mol. Genet., November 15, 2005; 14(22): 3407 - 3423. [Abstract] [Full Text] [PDF]

				S. R. Sriram, X. Li, H. S. Ko, K. K.K. Chung, E. Wong, K. L. Lim, V. L. Dawson, and T. M. Dawson Familial-associated mutations differentially disrupt the solubility, localization, binding and ubiquitination properties of parkin Hum. Mol. Genet., September 1, 2005; 14(17): 2571 - 2586. [Abstract] [Full Text] [PDF]

				H. S. Ko, R. von Coelln, S. R. Sriram, S. W. Kim, K. K. K. Chung, O. Pletnikova, J. Troncoso, B. Johnson, R. Saffary, E. L. Goh, et al. Accumulation of the Authentic Parkin Substrate Aminoacyl-tRNA Synthetase Cofactor, p38/JTV-1, Leads to Catecholaminergic Cell Death J. Neurosci., August 31, 2005; 25(35): 7968 - 7978. [Abstract] [Full Text] [PDF]

				S. G. Park, H. J. Kim, Y. H. Min, E.-C. Choi, Y. K. Shin, B.-J. Park, S. W. Lee, and S. Kim From The Cover: Human lysyl-tRNA synthetase is secreted to trigger proinflammatory response PNAS, May 3, 2005; 102(18): 6356 - 6361. [Abstract] [Full Text] [PDF]

				L. Zhong, Y. Tan, A. Zhou, Q. Yu, and J. Zhou RING Finger Ubiquitin-Protein Isopeptide Ligase Nrdp1/FLRF Regulates Parkin Stability and Activity J. Biol. Chem., March 11, 2005; 280(10): 9425 - 9430. [Abstract] [Full Text] [PDF]

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				D. J. Moore, L. Zhang, J. Troncoso, M. K. Lee, N. Hattori, Y. Mizuno, T. M. Dawson, and V. L. Dawson Association of DJ-1 and parkin mediated by pathogenic DJ-1 mutations and oxidative stress Hum. Mol. Genet., January 1, 2005; 14(1): 71 - 84. [Abstract] [Full Text] [PDF]

				H. Jiang, Y. Ren, J. Zhao, and J. Feng Parkin protects human dopaminergic neuroblastoma cells against dopamine-induced apoptosis Hum. Mol. Genet., August 15, 2004; 13(16): 1745 - 1754. [Abstract] [Full Text] [PDF]

				R. von Coelln, B. Thomas, J. M. Savitt, K. L. Lim, M. Sasaki, E. J. Hess, V. L. Dawson, and T. M. Dawson Loss of locus coeruleus neurons and reduced startle in parkin null mice PNAS, July 20, 2004; 101(29): 10744 - 10749. [Abstract] [Full Text] [PDF]

				K. K. K. Chung, B. Thomas, X. Li, O. Pletnikova, J. C. Troncoso, L. Marsh, V. L. Dawson, and T. M. Dawson S-Nitrosylation of Parkin Regulates Ubiquitination and Compromises Parkin's Protective Function Science, May 28, 2004; 304(5675): 1328 - 1331. [Abstract] [Full Text] [PDF]

				I. Marin, J. I. Lucas, A.-C. Gradilla, and A. Ferrus Parkin and relatives: the RBR family of ubiquitin ligases Physiol Genomics, May 19, 2004; 17(3): 253 - 263. [Abstract] [Full Text] [PDF]

				J. J. Palacino, D. Sagi, M. S. Goldberg, S. Krauss, C. Motz, M. Wacker, J. Klose, and J. Shen Mitochondrial Dysfunction and Oxidative Damage in parkin-deficient Mice J. Biol. Chem., April 30, 2004; 279(18): 18614 - 18622. [Abstract] [Full Text] [PDF]

				I. F. Mata, P. J. Lockhart, and M. J. Farrer Parkin genetics: one model for Parkinson's disease Hum. Mol. Genet., April 1, 2004; 13(90001): R127 - 133. [Abstract] [Full Text] [PDF]

				M. M.K. Muqit, S. M. Davidson, M. D. Payne Smith, L. P. MacCormac, S. Kahns, P. H. Jensen, N. W. Wood, and D. S. Latchman Parkin is recruited into aggresomes in a stress-specific manner: over-expression of parkin reduces aggresome formation but can be dissociated from parkin's effect on neuronal survival Hum. Mol. Genet., January 1, 2004; 13(1): 117 - 135. [Abstract] [Full Text] [PDF]

				H. C. Ardley, G. B. Scott, S. A. Rose, N. G. S. Tan, A. F. Markham, and P. A. Robinson Inhibition of Proteasomal Activity Causes Inclusion Formation in Neuronal and Non-Neuronal Cells Overexpressing Parkin Mol. Biol. Cell, November 1, 2003; 14(11): 4541 - 4556. [Abstract] [Full Text] [PDF]

				M. S. Goldberg, S. M. Fleming, J. J. Palacino, C. Cepeda, H. A. Lam, A. Bhatnagar, E. G. Meloni, N. Wu, L. C. Ackerson, G. J. Klapstein, et al. Parkin-deficient Mice Exhibit Nigrostriatal Deficits but Not Loss of Dopaminergic Neurons J. Biol. Chem., October 31, 2003; 278(44): 43628 - 43635. [Abstract] [Full Text] [PDF]

				D. P. Huynh, D. R. Scoles, D. Nguyen, and S. M. Pulst The autosomal recessive juvenile Parkinson disease gene product, parkin, interacts with and ubiquitinates synaptotagmin XI Hum. Mol. Genet., October 16, 2003; 12(20): 2587 - 2597. [Abstract] [Full Text] [PDF]

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