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Human Molecular Genetics, 2003, Vol. 12, No. 13 1535-1542
DOI: 10.1093/hmg/ddg168
© 2003 Oxford University Press

Dentatorubral-pallidoluysian atrophy protein is phosphorylated by c-Jun NH2-terminal kinase

Yuko Okamura-Oho1, Toshiyuki Miyashita1, Kazuaki Nagao1, Seigo Shima1, Yukie Ogata1, Toshiaki Katada2, Hiroshi Nishina2 and Masao Yamada1,*

1Department of Genetics, National Research Institute for Child Health and Development, 3-35-31 Taishido, Setagaya-ku, Tokyo, 154-8567, Japan and 2Department of Physiological Chemistry, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

Received February 25, 2003; Accepted April 30, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominant-inherited neurodegenerative disease characterized by selective cell loss in particular neuronal pathways. This is caused by expansion of CAG repeats in the coding region of the DRPLA gene, and the extended polyglutamine tract (polyQ) confers a toxic activity. It is valuable to characterize disease gene products for elucidation of the mechanism underlying neuron death at specific anatomical areas of the brain. Here, we define the DRPLA protein as a phosphoprotein, and c-Jun NH2-terminal kinase (JNK) is one of the major factors involved in its phosphorylation. Endogenous DRPLA protein was serine-phosphorylated. Phosphorylation was demonstrated in a recombinant JNK activation system in vitro and also in overexpressing cells by transfection after the JNK activation with osmotic pressure. One of the phospho-acceptor sites for JNK appearing in the DRPLA sequence was indeed phosphorylated, which was confirmed by a specific antibody raised against the phosphopeptide. Kinetic studies in the JNK recombinant system showed that expanded polyQ slightly reduced the affinity of JNK to the protein. Thus, the abnormal DRPLA protein seems to be slowly phosphorylated in a certain condition of JNK activation in patients. It may delay a process that is essential in keeping neurons alive.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominantly inherited neurodegenerative disease characterized by selective neuron loss in the cerebellar and pallidal outflow pathways (1). The disease is caused by expansion of CAG repeats in the coding region of the DRPLA gene, and the extended polyglutamine tract (polyQ) confers a toxic activity to a subset of neurons (2–4). Several other neurodegenerative diseases including Huntington disease are also known to be caused by expansion of CAG repeats in the coding region of the respective genes (5). Thus, these diseases are collectively called polyglutamine diseases. It has been demonstrated that over expression of extended polyQ induces apoptosis in a variety of cells (6,7), but the precise mechanism leading to apoptosis has not yet been settled. Previous studies have implied impairment of transcription, retardation in cleaning of misfolding proteins, sequestering of essential factors or formation of pores with extended polyQ (8–20). Although several studies have excluded the involvement of aggregates visible under a microscope in these processes (21–23), all the proposed mechanisms seem to imply the cohesive force of polyQ.

We have been studying the normal functions of the DRPLA product (24–27), also known as ‘atrophin-1’. As a specific subset of neurons are degenerated in different polyglutamine diseases, polyQ alone is not enough to elucidate the pathogenesis of patients. We previously reported binding partners with DRPLA protein (25). Among them, IRSp53 and DVL1 potentially activate signaling cascades of c-Jun NH2-terminal kinase (JNK), a family member of mitogen activated protein kinases (MAPKs), which phosphorylates serine/threonine (S/T) residues followed by proline (S/TP) (28–34). Seven other binding partners use phosphorylated S/T as a target (25,35). As there are many S/TP sequences in the DRPLA protein (4) (Fig. 1), we are interested on the phosphorylation state of the product. Recently, a DRPLA-like protein in Drosophila, Atro (or Grunge), was reported to have multiple functions in transcriptional regulation (36,37). It functions as a transcriptional co-repressor in the earliest stage of embryogenesis, and further participates in body patterning in multiple developmental stages as transcriptional regulators. Expression of the abnormal DRPLA protein with extended polyQ in Drosophila embryos causes deregulation of transcription. Atro/Grunge has evolved into two descendants, DRPLA and RERE, in mammals (4,26). Although human DRPLA protein lacks a putative DNA-binding domain in the N-terminal portion of the Drosophila homolog, it may be still be involved in transcriptional regulation as it gains additional motifs in the flanking region of polyQ. In light of these observations, we have characterized the phosphorylated state of the DRPLA protein and detected phosphorylation by JNK.



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  		Figure 1. Putative phosphorylation sites in the DRPLA protein. (A) The amino acid sequence of the human DRPLA protein containing polyglutamine (Qn), serine (s) or threonine (t) followed by a proline residue (S/TP), fragments of the DRPLA protein (DR-a–i), and an antigen fragment used for generating the anti-human DRPLA antibody (anti-DRa) are indicated. (B) Conserved amino acid residues in the DR-i fragment of the human DRPLA protein and in the homologous portion of other species. Accession numbers of the genes are AF068719, NM_079249, XM_132846, NM_017228 and D31840. Conserved residues at least in four species are bold. Serine 734 in the DRPLA protein and serine 98 in human BCL-2 are double-underlined. Amino acid residues for generating an anti-DRPLA phospho-serine 734 antibody (anti-DRs) and an anti-DRPLA peptide antibody (anti-DRb) are underlined.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Endogenous DRPLA protein was phosphorylated
We first examined a phosphorylated state of the DRPLA protein in cultured cells (Fig. 2). The major form of the DRPLA protein in human neuroblastoma cells moved to the position of a 160 kDa protein through SDS–PAGE and was visualized with anti-human DRPLA antibody (anti-DRa), as shown previously (24). When the cell lysate was treated with alkaline phosphatase, the major form moved faster through SDS–PAGE and migrated to the same position as the in vitro translated product without post-translational modification (Fig. 2A). The major form precipitated with the anti-DRa antibody from the cell lysate was immuno-reactive to an anti-phosphoserine antibody (anti-pS), and it almost lost the reactivity when treated with the alkaline phosphatase (Fig. 2B and C). These results indicated that the major form of DRPLA protein in cultured cells was serine-phosphorylated.



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  		Figure 2. Phosphorylation of the DRPLA protein in cultured cells. (A) Treatment with alkaline phosphatase increases the mobility of the DRPLA protein through SDS–PAGE. Aliquots of neuroblastoma (SH-SY5Y) cell lysate were incubated with or without alkaline phosphatase (Alp + and -), applied to SDS–PAGE and blotted with an anti-recombinant DRPLA antibody (anti-DRa). The in vitro translation product of the DRPLA protein was in the parallel run. (B) and (C) DRPLA protein is serine-phosphorylated. Aliquots of cell lysate were immunoprecipitated with the anti-DRa antibody or a pre-immune serum (anti- DRa or cont), and analyzed by western blotting with an anti-phosphoserine antibody ( pS). The molecular size of the DRPLA protein is indicated in theparallel run of cell lysate blotted with the anti-DRa antibody. The immunoprecipitated protein was treated with or without the alkaline phosphatase (Alp + and - in panel C). Bands indicated by asterisks are the heavy chains of immunoglobulin.

 
DRPLA protein was phosphorylated by JNK in vitro
In an attempt to establish a biochemical basis for the phosphorylation, we adopted an in vitro activation system for JNK in which purified forms of recombinant protein kinases were mixed together. In this mixture, JNK3 is synergistically activated by two forms of MAPK kinase (MKK), constitutive active MKK7 and native MKK4 that is activated by constitutive active MEK kinase 1 (MEKK1), as described previously (38–40).

In vitro translation products of DRPLA with normal or expanded repeats were subjected to the activation system and analyzed by SDS–PAGE. When incubated in the JNK mixture, the translation products migrated more slowly than the untreated counterparts (Fig. 3A, upper panel). As the mobility shift was not observed by omission of any member of the four kinases from the reaction, the shift must be due to synergistically activated JNK as previously demonstrated. The products translated in unlabeled conditions, were radiolabeled after incubation in the mixture containing [{gamma}-32P]ATP (Fig. 3A, lower panel). When JNK-reacted products were further treated with alkaline phosphatase, the products moved faster than the untreated counterparts (Fig. 3B). Thus, we concluded that the normal and abnormal forms of the DRPLA protein were phosphorylated by JNK in vitro.



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  		Figure 3. Phosphorylation of the full-length DRPLA protein in the recombinant activation system for JNK. In vitro translation products of the full-length DRPLA protein containing normal (Q19) and abnormal (Q71) polyQ were incubated in the recombinant JNK mixture. (A) The DRPLA protein labeled with [35S]methionine (35S-Met) was treated with or without the JNK reaction mixture (+ or -) for 1 h and applied on SDS–PAGE (upper panel). Non-labeled DRPLA protein was incubated in the mixtures containing [{gamma}-32P]ATP for 1 h and the phosphorylation was detected by autoradiography (lower panel). (B) The DRPLA protein labeled with [35S]methionine was treated with or without alkaline phosphatase (Alp + or -), and resolved through SDS–PAGE with parallel run of products incubated in an incomplete JNK reaction mixture (JNK-). The indicated lanes were the results after omission of recombinant JNK, omission of any member of four kinases from the reaction abolished the mobility shift as well as incorporation of [{gamma}-32P]ATP. (C–E) Kinetic analysis of phosphorylation of the DRPLA protein by JNK. In vitro translation products (diamond, 1 U; square, 2 U; and triangle, 4 U) of the normal (Q19, solid symbols) and abnormal (Q71, open symbols) DRPLA proteins were incubated in 10 µl of the JNK mixture containing [{gamma}-32P]ATP for the indicated periods, and subjected to SDS–PAGE. A representative result of the autoradiographs in duplicates of three independent experiments is shown in (C). The extent of phosphorylation was quantified with radioactivity in the target proteins and plotted along incubation periods (D). The initial velocity (v) was calculated in the substrate content (s), and the double reciprocal plot (1/v versus 1/s) was used for evaluation of Km (E).

 
A time course study of phosphorylation showed that both the normal and abnormal DRPLA proteins were fully phosphorylated within 120 min (Fig. 3C and D). However, a kinetic analysis showed that the affinity of JNK for the abnormal protein was lower than that of the normal protein (Km=0.59 U/µl for the normal protein and 1.11 U/µl for the abnormal protein in Fig. 3E). Thus, the expanded polyQ reduced the affinity of JNK for the DRPLA protein.

To further investigate the phospho-acceptor sites, we produced seven non-overlapping fragments of the DRPLA protein (DR-a–i, shown in Fig. 1A). When subjected to the recombinant system, one of the fragments, DR-f, was strongly phosphorylated in the mixture (Fig. 4). In the absence of recombinant JNK3, the phosphorylation was completely eliminated (lanes f and f * in Fig. 4; note that the band for JNK3 also disappeared in lane f *). This clearly showed that the DR-f fragment was directly phosphorylated by JNK3. When the fragment was separated into two portions (DR-h and -i in Fig. 1A), both were phosphorylated (Fig. 4, lanes h and i). Thus, DR-f was thought to contain at least two phospho-acceptor sites, one of which was located in DR-h and the other in DR-i. Although the details were not examined, two other fragments, DR-a and -g, seem to have additional minor phospho-acceptor sites (Fig. 4, lanes a and g).



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  		Figure 4. Phosphorylation of fragments of the DRPLA protein in the recombinant activation system for JNK. Equivalent amounts of the fragments of the DRPLA protein (DR-a–i, illustrated in Fig. 1A) were produced as GST-fusion proteins and visualized in western blotting with an anti-GST antibody (lower panels). They were incubated in the mixture containing JNK3 (closed triangle in upper panels), MKK4 (open triangle), constitutive active forms of MKK7 and MEKK1, and [{gamma}-32P]ATP for 30 min. The phosphorylation is detected with the radioactivity of the fragments (upper panels). Recombinant JNK3 was omitted in the last lane (f *). GST and c-Jun were used as positive and negative control substrates. Experiments were repeated three times and representative results are shown.

 
DRPLA protein over-expressed in cells was phosphorylated by JNK
Endogenous DRPLA protein was phosphorylated as shown in Figure 2. Then, we tested the phosphorylated state of over expressed forms of the normal and abnormal protein, tagged with HA at the N-terminal end, in neuroblastoma cells (Fig. 5). We chose a high osmotic pressure by adding sorbitol to the cultured medium for transient activation of JNK. As demonstrated in Figure 5A, active JNK was increased to be detectable by western blotting with an anti-active JNK antibody. Along with the stimulation, amounts of the overexpressed forms of the DRPLA protein decreased afterward. It seemed that the N-terminal portion was cleaved off in the process, although the smaller fragments were not detected with the anti-HA antibody.



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  		Figure 5. The overexpressed DRPLA protein is phosphorylated by JNK. Cells expressing HA-tagged normal and abnormal forms of the DRPLA protein (HA-DRQ19 and Q71) were incubated under a high osmotic condition with sorbitol for the indicated period (otherwise 30 min). The over-expressed protein was detected by western blotting with an anti-HA antibody (HA). The cell lysates were treated with alkaline phosphatase (Alp + in the panel C). The cells were incubated with the addition of JNK inhibitor, SP600125 (SP + in the panel D). The activity of JNK was monitored by western blotting with anti-active JNK antibody and by immune-complex kinase assay with c-Jun (A and D). The amount of total JNK and an unrelated protein (acid ß-galactosidase) were monitored by western blotting with anti-JNK and ß-galactosidase antibodies (D).

 
We examined the mobility shift of the normal DRPLA protein expressed in cells after osmotic stress. The mobility shift of the normal DRPLA was clear after 30 min of the stimulation (Fig. 5B), although the extent of the mobility shift shown here was less than that shown in the recombinant system (Q19 in Fig. 3A). The mobility shift of the abnormal protein was imperceptible. After the treatment of cell lysates with alkaline phosphatase, the normal protein with the osmotic stress migrated to the basal position without stress (Fig. 5C). Thus, the mobility shift was due to the phosphorylation state. When the cells were treated with a selective inhibitor of JNK, SP600125 (41) in the osmotic stress condition, the JNK kinase activity was inhibited as expected, and the normal protein migrated to the same position as the cells without osmotic stress (Fig. 5D). These results showed that DRPLA protein overexpressed in cultured cells was phosphorylated by JNK.

Serine 734 of the DRPLA protein is a phospho-acceptor site by JNK
We focused on phosphorylation of serine 734 (S734) in the DR-i fragment of DRPLA protein (double-underlined in Fig. 1B), because the flanking sequence, PESP, exactly matched the consensus sequence of phosphorylation by MAPKs (33). Moreover, the following sequence to S734 (SPVPP) is also found in BCL-2 (Fig. 1B) and the serine residue in the BCL-2 sequence is phosphorylated by JNK (42). To determine if the S734 residue in the DRPLA protein is essential for phosphorylation with JNK, we constructed a mutant fragment, DR-i734A, in which S734 was substituted to alanine. In contrast to the normal counterpart, the mutant fragment was not phosphorylated in the recombinant activation system and the immune complex kinase assay for JNK (Fig. 6A and B).



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  		Figure 6. Serine 734 in the DRPLA protein is phosphorylated by JNK in vitro. (A) The fragment of DRPLA protein (DR-i), and its mutant form (DR-i734A, in which serine 734 was substituted to alanine) were subjected to the phosphorylation study with the recombinant activation system for JNK. (B) The indicated fragments of the DRPLA protein and c-Jun (with arrows) were used as a substrate for the immune-complex kinase assay of JNK. The neuroblastoma cells were treated with sorbitol for the indicated periods. Immunoprecipitated JNK was incubated with substrates and [{gamma}-32P]ATP. The phosphorylation of substrates was visualized by autoradiography. The amounts of precipitated JNK were monitored by western blotting (the last panel). (C) Specificity of two anti-peptide antibodies was tested by ELISA. Synthesized phosphorylated peptides (solid symbols), used for generating antibodies (DRs and DRb), and the non-phosphorylated counterparts (open symbols) were the antigen. Solid lines are for antibodies, and dotted lines for non-immune sera. (D) Anti-DRs antibody reacts with the fragment of DRPLA protein after phosphorylation by JNK. The fragment DR-i, treated with or without the recombinant JNK (+ or -), was blotted with anti-DRs, DRb and GST antibodies.

 
To show immunological evidence for phosphorylation of S734, we produced two antibodies (epitopes of which are shown in Fig. 1B) and assessed their specific reactivity in ELISA assay (Fig. 6C). The raised anti-phosphoserine 734 antibody (anti-DRs) reacted with the phosphorylated DR-i fragment in the recombinant JNK system, but not with non-phosphorylated counterparts in western blotting. The anti-DRPLA peptide antibody (anti-DRb) reacted with both (Fig. 6D). These results were consistent with the recombinant JNK system described above, and the S734 residue was the main target by JNK.

Serine 734 is phosphorylated in the rat brain
The rat DRPLA protein has 93% homology to its human counterpart and is smaller by two amino acid residues (43). Since the peptide sequences used for raising the anti-phosphopeptide antibodies are exactly the same in both species (Fig. 1B), we analyzed the phosphorylated state of the rat DRPLA protein by western blotting with the raised antibodies (Fig. 7). The expression level of the DRPLA protein detected with the anti-DRb antibody was consistent with our previous results on the level of mRNA in northern blotting (4); where it was high in the brain and low in the pancreas and testis (Fig. 7A). The major form in the brain (150 kDa protein) was also reactive with the anti-DRs antibody, which indicated that S734 in the DRPLA protein in the rat brain tissues was phosphorylated (Fig. 7B). This form was also detected with the anti-DRa antibody (data not shown). When the brain samples were treated with alkaline phosphatase, the major form migrated faster and almost lost the immunoreactivity to the anti-DRs antibody (Fig. 7C). These data were consistent with phosphorylation at S734.



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  		Figure 7. Serine 734 in the DRPLA protein in rat tissues is phosphorylated. (A–C) Western blotting of the DRPLA protein in rat tissues. Samples (30 µg of protein) obtained from the cerebrum (cer), cerebellum (cbl), pancreas (pancr) and testis were blotted with the anti-DRb, anti-DRs and anti-ß actin antibodies. The brain samples in the parallel run were blotted with the anti-DRs in B. Samples were treated with or without alkaline phosphatase (Alp + or -) in (C).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We report evidence for phosphorylation of the DRPLA protein, the product of the gene responsible for the polyglutamine disease, dentatorubral-pallidoluysian atrophy. The endogenous form was phosphorylated and the phosphorylation was mainly mediated by JNK.

JNK is activated in the critical process of embryonic morphogenesis, as well as in response to environmental stress such as radiation and high osmotic pressure (44). The activation of JNK requires its phosphorylation mediated by MKK 4 and 7 (38,39). These two MKKs are activated by MKK kinases such as MEKK1. Activated JNK phosphorylates subsets of proteins including c-Jun, and up-regulates transcriptions of stress-responsive genes in various cells (45,46). In neurons, JNK3 is highly and consistently phosphorylated because of the synergistic activation of MKK4 and 7. Thus, DRPLA, one of the substrates of JNK3 as demonstrated in this report, may have a function in the brain coupled with activated JNK (47–49).

We identified the S734 residue of the DRPLA protein as a phospho-acceptor site in the recombinant system as well as in the brain tissues. However, phosphorylation of this residue was not detectable with the endogenous form of DRPLA protein in cultured cells with the same technique (data not shown). This may be accounted for by a weaker activity of JNK in cultured cells, while consistent activation of JNK is known in the brain at a similar level of the recombinant JNK system (49). The phosphorylation may be coupled to the activation of a protease. The molecular size of DRPLA protein detected in the rat brain with the specific phosphopeptide antibody was 150 kDa, which was slightly smaller than that expected from the sequence and the results with the human protein. The phosphorylated forms of HA-tagged human DRPLA gradually disappeared after osmotic treatment, as demonstrated in Figure  5. Although we did not directly detect degraded forms, caspase-3 may be one of the candidates involved. We previously reported the DRPLA protein is a substrate of caspase-3 and about 10 kDa from the N-terminus was cleaved off (24).

Both the normal and abnormal forms of DRPLA protein were phosphorylated by the JNK recombinant system. However, precise kinetic analyses showed a reduced affinity of JNK for the abnormal protein. Furthermore, the abnormal protein expressed in cultured cells showed a subtle mobility shift in the JNK activation condition with osmotic pressure. It may be possible that a slight reduction of the affinity in the in vitro system causes considerable effects in a physiological condition, i.e. delay in a certain cascade reaction. Two other polyglutamine disease products, Huntingtin and androgen receptor, have recently been reported to be phosphorylated by an S/T kinase, Akt, which mediates the survival signal of IGF-I (50–52). As the DRPLA protein is already known to be situated in the insulin/IGF-I signaling cascade (25), three polyglutamine diseases products seem to be connected by the signal transduction of IGF-I. As IGF-I is one of the main neurotrophic factors, neurodegeneration in polyglutamine diseases may be solved by a function of IGF-I.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid constructions
Original cDNA clones for DRPLA were previously described (2,4). The DNA fragment including the entire coding region of the DRPLA gene was subcloned into pBluescript SK(-) phagemid (Stratagene) for in vitro translation experiments, and a cpDNA-3xHA vector (kindly provided by Dr John C. Reed) for expression of HA-tagged proteins. cDNA fragments (shown in Fig. 1A) amplified by PCR methods were subcloned into a pGEX-3X vector (Pharmacia) to generate GST fusion proteins. Plasmid harboring a point mutation was obtained by PCR-mediated mutagenesis (53).

Antibodies
Epitopes for the following three antibodies are indicated in Figure 1. A rabbit polyclonal antibody which specifically reacted to a phosphorylated serine 734 residue in the DRPLA protein (anti-DRs) was raised against phosphopeptide ETPEpSPVPP (MBL Nagoya Japan). The serum was affinity-purified with the antigen peptide, and the reactivity to the non-phosphorylated peptide was eliminated with the affinity column. An anti-DRPLA peptide antibody (anti-DRb) was raised against phosphopeptide RSPpSPPPK, but it unexpectedly reacted to the non-phosphorylated counterpart as well as the antigen peptide. A rabbit polyclonal antibody raised against the recombinant human DRPLA protein (anti-DRa) was described previously (24). The following antibodies are also used: polyclonal anti-JNK1 antibody C-17 (Santa Cruz), monoclonal anti-JNK antibody (Pharmingen), anti-activated JNK antibody (Promega), monoclonal anti-phosphoserine antibody PSR-45 (Sigma), anti-HA antibody (Roche), anti-ß actin antibody (Santa Crutz) and anti-human acid ß-galactosidase antibody (54).

Preparation of recombinant proteins
Fragments of the DRPLA protein fused with GST were produced and purified as described previously (25). Translation products of the full-length DRPLA genes were produced with the TNT coupled reticulocyte lysate mixture in vitro (Promega). The radio-labeled and non-labeled products were made in the presence and absence of [35S]methionine, respectively. To quantify the translation products, the labeled mixture was resolved by SDS–PAGE, and the radioactivity of the DRPLA protein was measured as Photo-Stimulated Luminescence (PSL) with a Fuji BAS 2000 PhosphorImager (Fuji Film, Japan). One unit (U) was defined as an amount of the mixture which gave 1 PSL unit/min of 35S. The non-labeled mixtures, equivalent to 1, 2 and 4 U of the labeled counterparts, were subjected to the in vitro phosphorylation study.

In vitro phosphorylation assay in a recombinant activation system for JNK
In the recombinant system, JNK3 was synergistically activated by two forms of MKK, constitutive active MKK7 and native MKK4 that is activated by constitutive active MEKK1, as described previously (38–40). The mixture contains 100 ng of four GST-fusion proteins, JNK3, MKK4, MKK7 and MEKK1, in kinase buffer (50 mM Tris–Cl pH 7.5, 10 mM MgCl2, 125 µM ATP, 1 mM DTT, 2 mM EDTA-Na and 500 µM NaVO4). The fragments of the DRPLA protein fused with GST (100 ng equivalent) and the in vitro translation products of the DRPLA protein (1–4 U) were incubated in 10 µl of the mixture, with 5 µCi of [{gamma}-32P]ATP if needed, at 30°C for the indicated periods. Samples were resolved by SDS–PAGE and visualized with the PhosphorImager.

Cell culture
A human neuroblastoma cell line, SH-SY5Y, was maintained in RPMI1640 with 10% fetal bovine serum and transfected with plasmids using Lipofectamine PLUS (GIBCOBRL). For the activation of JNK with high osmotic pressure, cells were pre-incubated in the medium with 500 µM of Na3VO4 for 15 min. Then D(-)-sorbitol was added at a final concentration of 1 M and the incubation was continued for the indicated periods. For inhibition of the JNK kinase activity, a reversible ATP-competitive inhibitor, SP600125 (25 µM, BIOMOL), was added to the media 15 min prior to the sorbitol stimulation (41).

Immune complex protein kinase assay
SH-SY5Y cells were lysed in a lysis buffer (20 mM Tris–Cl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerolphosphate, 1 mM Na3VO4, 1 µg/ml leupeptin and 1 mM PMSF). The lysates were sonicated and centrifuged. An aliquot of cleared lysates (100 µg of protein) was immunoprecipitated with the anti-JNK antibody C-17 for 2 h. The immune complex was used for a kinase assay with 5 µCi of [{gamma}-32P]ATP and 2 µg of the GST-fusion protein in a kinase buffer (20 mM Hepes pH 7.4, 10 mM MgCl2 and 200 µM Na3VO4). After incubation at 30°C for 30 min, the samples were resolved by SDS–PAGE. One-tenth of the immune complex was analyzed by western blotting with an anti-monoclonal anti-JNK antibody.

Phosphatase treatment
SH-SY5Y cells and tissues from adult rats (male, Wistar rats) were homogenized in the Tris/MgCl2 buffer (50 mM Tris-Cl pH 7.5 and 1 mM MgCl2), sonicated for 30 s and centrifuged. Cleared lysates were incubated with calf intestine alkaline phosphatase (2 U for 10 µg protein, Sigma) at 30°C for 15 min, and the reaction was terminated with phosphatase inhibitors (Sigma).

Western blotting, immunoprecipitation and ELISA assay
The details of procedures for immunoprecipitation and western blotting were described previously (25). Briefly, an aliquot containing 100 ng of GST-fusion proteins, and 30 µg of protein extracts was subjected to western blotting with 1 : 500, 1 : 500, 1 : 200, 1 : 200 and 1 : 1000 diluted anti-GST, DRa, DRb, DRs and HA antibodies, respectively. To confirm equal loading of protein samples, the blotted membranes were re-probed with the 1 : 400 and 1 : 250 diluted anti-ß actin and anti-ß galactosidase antibodies, respectively. The endogenous DRPLA protein was immunoprecipitated with the anti-DRa antibody from 500 µg protein of cell lysates and blotted with the 1:100 diluted anti-phosphoserine antibody. The ELISA assay using synthetic peptides as antigens and titration assays with anti-DRb and anti-DRs antibodies were performed according to the standard methods.


   ACKNOWLEDGEMENTS
 
We wish to acknowledge Professor Yoshiyuki Suzuki (International University of Health and Welfare) for important suggestions and critical reading of the manuscript. We also thank A. Asaka and Y. Ohtsuka for technical assistance and K. Saito for preparing the manuscript. This study was supported in part by Grants for Human Genome, Brain Science and Pediatric Research from the Ministry of Health, Labor and Welfare, and a Grant for Organized Research Combination System from the Ministry of Education, Culture, Sports, Science and Technology, Japan.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +81 334160181; Fax: +81 334122259; Email: myamada{at}nch.go.jp Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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