PRAK Interacts with DJ-1 and Prevents Oxidative Stress-Induced Cell Death

As a core member of p38 MAPK signal transduction pathway, p38 regulated/activated kinase (PRAK) is activated by cellular stresses. However, the function of PRAK and its downstream interacting partner remain undefined. Using a yeast two-hybrid system, we identified DJ-1 as a potential PRAK interacting protein. We further verified that DJ-1 bound to PRAK in vitro and in vivo and colocalized with PRAK in the nuclei of NIH3T3 cells. Furthermore, following H2O2 stimulation the majority of endogenous DJ-1 in PRAK+/+ cells still remained in the nucleus, whereas most DJ-1 in PRAK−/− cells translocated from the nucleus into the cytoplasm, indicating that PRAK is essential for DJ-1 to localize in the nucleus. In addition, PRAK-associated phosphorylation of DJ-1 was observed in vitro and in vivo of H2O2-challenged PRAK+/+ cells. Cytoplasmic translocation of DJ-1 in H2O2-treated PRAK−/− cells lost its ability to sequester Daxx, a death protein, in the nucleus, and as a result, Daxx gained access to the cytoplasm and triggered cell death. These data highlight that DJ-1 is the downstream interacting target for PRAK, and in response to oxidative stress PRAK may exert a cytoprotective effect by facilitating DJ-1 to sequester Daxx in the nucleus, thus preventing cell death.

Endogenous PRAK is primarily located in the cytoplasm, whereas exogenous PRAK predominates in the nucleus [26]. A sequence analysis of PRAK revealed that PRAK contains a putative nuclear localization sequence (NLS) and a nuclear 2 Oxidative Medicine and Cellular Longevity export sequence (NES), and both of them are required for the shuttling of PRAK between nucleus and cytoplasm. Following stimulation with arsenite, the nuclear PRAK was markedly reduced due to a decrease in the nuclear import of PRAK and an increase in the nuclear export of PRAK [26]. Furthermore, the nuclear import of PRAK was independent of p38 activation, whereas the nuclear export required p38mediated phosphorylation of PRAK. However, the function of PRAK shuttling between nucleus and cytoplasm in response to different cellular stresses remains unclear.
Here, we report that DJ-1, originally found as a mitogendependent oncogene product [27], is a downstream interacting protein for PRAK. DJ-1 bound to PRAK both in vitro and in vivo and colocalized with PRAK in the nuclei of NIH3T3 cells. Functional studies revealed that PRAK can activate DJ-1 and help DJ-1 to localize in the nucleus. Phosphorylation of DJ-1 following H 2 O 2 treatment was observed in PRAK +/+ cells but not in PRAK −/− cells. Consistently, endogenous DJ-1 in PRAK +/+ cells was mainly located in the nucleus even after the cells were challenged with H 2 O 2 , whereas most DJ-1 in PRAK −/− cells translocated from the nucleus into the cytoplasm in response to oxidative stress. As a result, DJ-1 was unable to sequester its interacting partner, a death protein Daxx in the nuclei of PRAK −/− cells, thereby causing an increased cell death.

Yeast Two-Hybrid
Screening. The Matchmaker GAL4 Two-hybrid System 3 (Clontech, protocol PT3247-1) was used to screen for proteins that interact with PRAK. pGBKT7-PRAK was transformed into the yeast strain AH109 as the bait, and a human heart cDNA library previously transformed into the yeast strain Y187 (Clontech) was used as the prey. Approximately 1 × 10 6 transformants were screened. After being mated, the mixtures of the bait and the prey were plated onto SD/-Ade/-His/-Leu/-Trp/X--gal plates and allowed to grow at 30 ∘ C for 4-6 days. The yeast colonies were assayed for -galactosidase activity using a colony-lift filter and positive clones were subjected to sequencing.

Yeast Two-Hybrid Interaction
Assay. Plasmids pGBKT7-PRAK and pGADT7-DJ-1 were transformed into the yeast strains Y187 and AH109, respectively. Yeast clones of AH109 expressing AD-DJ-1 and Y187 expressing DBD-PRAK were mixed and mated at 30 ∘ C for 24 hrs. To select the diploid, mated mixtures were spread on SD/-Leu/-Trp plates and the yeast colonies were then transferred onto SD/-Ade/-His/ -Leu/-Trp/X--gal plates for assessing -galactosidase activity. Positive and negative controls were performed in parallel.

In Vitro Binding
Assay of PRAK and DJ-1. pGEX-KG-DJ-1 and pET-14b-PRAK were transformed into E. coli BL21 strain to produce GST-tagged DJ-1 fusion protein and His-tagged PRAK fusion protein, respectively. GST-DJ-1 was purified with GST-bind resin (Novagen) and eluted by reduced glutathione. His-PRAK was purified with Ni-NTA resin (Qiagen) and eluted by elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, and 250 mM imidazole, pH 8.0). After incubation with either GST-DJ-1 fusion protein or GST, His-PRAK fusion protein was pulled down with Ni-NTA beads, and the precipitate was separated by SDS-PAGE.

Immunoprecipitation and Immunoblotting.
After transfection, human HEK293 cells were lysed in lysis buffer containing 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mMglycerolphosphate, 1 mM Na 3 VO 4 , and a mixture of protease inhibitors and phosphatase inhibitors (Roche). Equal amounts of extracted protein were mixed with 10 L anti-Flag M2 beads (Sigma) and incubated on ice for 6 hrs. The samples were spun briefly and washed five times with lysis buffer containing 0.1% Tween-20. Loading buffer (20 L) was added to each sample and boiled for 5 min. The samples were then separated by SDS-PAGE, transferred onto nitrocellulose membranes, and probed with either anti-HA antibody (Cell Signaling Technology) or anti-Flag M2 antibody (Stratagene).
PRAK +/+ cells and PRAK −/− cells were treated with H 2 O 2 (300 M) for different time periods, and nonstimulated Hela cells were lysed as described above. Protein A/G beads (20 L) (sigma) were incubated with 2 g anti-DJ-1 antibody (Abcam), 2 g anti-PRAK antibody (BD Biosciences), or 2 g nonspecific IgG antibody (BD Biosciences) at 4 ∘ C for 6 hrs, spun briefly, and washed five times with lysis buffer containing 0.1% Tween-20. Equal amounts of extracted protein from PRAK +/+ cells, PRAK −/− cells, and Hela cells were incubated with 20 L protein A/G beads coupled with anti-DJ-1 antibody, anti-PRAK antibody, or IgG on ice for 6 hrs. The samples were spun briefly and washed five times with lysis buffer containing 0.1% Tween-20. Loading buffer (20 L) was added to each sample and boiled for 5 min. The samples were then subjected to immunoblot analysis with anti-Ser phosphorylation antibody (Calbiochem), anti-DJ-1 antibody (Abcam), or anti-PRAK antibody (BD Biosciences).
NIH3T3 cells were lysed after transfection as described above. The resultant lysates were centrifuged and supernatants containing the cytoplasmic proteins were collected. For nuclear protein extraction, the pellets were further lysed in nuclear extraction buffer containing 20 mM Hepes, pH7.9, 420 mM NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 1 mM DTT, 25% glycerol, and a mixture of protease inhibitors and phosphatase inhibitors (Roche). Equal amounts of protein extracted from either cytoplasm or nucleus were subjected to immunoblot analysis.

Immunofluorescence
Assay. NIH3T3 cells were cotransfected with pcDNA-HA-PRAK, pcDNA3-Flag-DJ-1, and PEGFP-DJ-1 for 24 hrs. PRAK +/+ cells and PRAK −/− cells were starved for 48 hrs and were further challenged with H 2 O 2 (300 M) for 6 hrs. Cells were fixed in 4% paraformaldehyde for 10 min, washed twice with PBS, and permeabilized with 0.1% sodium tetrahydroborate for 5 min. After being washed three times with PBST (PBS + 0.2% Triton X-100) and blocked with 3% BSA for 1 hr, cells were incubated with the indicated antibodies diluted in 3% BSA at room temperature for 1 hr and washed three times with PBST. Cells were further incubated with the indicated secondary antibodies diluted in 3% BSA at room temperature for 1 hr and washed three times with PBST. Cell nucleus was stained with 10 M DAPI. Fluorescent images were recorded and analyzed using a fluorescence microscope (DMRA2, Leica) equipped with FW4000 software. To correct fluorescence bleed through into the FRET channel, cells transfected with either pECFP-DJ-1 alone or pEYFP-PRAK alone were used to determine the donor or the acceptor correction factor. Images of CFP-DJ-1 and YFP-PRAK expression in cotransfected cells were sequentially acquired with the donor (CFP) channel, acceptor (YFP) channel, and FRET channel under identical conditions. The image obtained with the FRET channel was evaluated using Carl Zeiss AxioVision FRET 4.6 software and values of FRET were calculated as described previously [28].

Cell Viability
Assay. PRAK +/+ and PRAK −/− cells were plated in 96-well tissue culture plates (2.5 × 10 4 cells per well) and exposed to H 2 O 2 (300 M) for different time periods. Cell viability was quantified using a Cell Titer 96 Aqueous One solution cell proliferation assay kit (Promega) with a HTS7000 Bio-Assay Reader (Perkin Elmer).
2.11. Statistical Analysis. All data are expressed as mean ± SD. Statistical analysis was performed by the Student's -test and ANOVA. Differences were judged statistically significant when the value was less than 0.05.

Interaction of DJ-1 and PRAK in Yeast.
To screen the PRAK-binding proteins, we amplified a full-length human PRAK cDNA (1415 bp) from pcDNA3-HA-PRAK by PCR and subcloned it into the pGBKT7 vector. pGBKT7-PRAK was transformed into the yeast strain AH109 and placed on SD/ -Trp plates, which expresses Myc-DBD-PRAK fusion protein as confirmed by Western blot analysis (Figure 1(a)).
A Gal4-based yeast two-hybrid system was used to identify proteins that interact with PRAK. The pGBKT7-PRAK plasmid was transformed into the yeast strain AH109 as the bait and a human heart cDNA library was previously transformed into the yeast strain Y187 as the prey. Approximately 1 × 10 6 transformants were screened and a total of seven positive clones were obtained. NCBI blast results revealed that clone 4 was identical to the coding sequence (58-189 aa) of human DJ-1 (Figure 1(b)).
To further test the interaction between DJ-1 and PRAK, yeast strains AH109 expressing active domain-(AD-) fused DJ-1 and Y187 expressing DNA binding domain-(DBD-) fused PRAK were mixed, mated for 24 hrs, and plated on SD/-Leu/-Trp plates. After the yeast colonies were transferred onto SD/-Ade/-His/-Leu/-Trp/X--gal plates,galactosidase activity was measured to assess the interaction between DJ-1 and PRAK. Yeasts transformed with both DBD-PRAK and AD-DJ-1 expressed the LacZ phenotype (Figure 1(c)), whereas yeasts transformed with DBD and AD-DJ-1 failed to show any LacZ activity (Figure 1(d)), confirming that DJ-1 interacts with PRAK, but not DBD, in yeast. To assess the interaction of PRAK with DJ-1 in vivo, human HEK293 cells were transfected with pcDNA3-HA-PRAK plasmid with or without pcDNA3-Flag-DJ-1 plasmid. Twenty-four hours after transfection, cell extracts were prepared and subjected to immunoprecipitation with an anti-Flag antibody. The precipitates were then blotted with an anti-HA antibody. HA-PRAK was coprecipitated with Flag-DJ-1 in cells cotransfected with Flag-DJ-1 and HA-PRAK, but not in cells transfected with HA-PRAK alone (Figure 2(c)), indicating that PRAK specifically binds to DJ-1 in HEK293 cells.

In Vitro and In Vivo
To examine whether PRAK binds to DJ-1 under physiological conditions, cell extracts prepared from Hela cells were immunoprecipitated with an anti-PRAK antibody or a nonspecific IgG, and the precipitates were further immunoblotted against an anti-DJ-1 antibody. The anti-PRAK antibody did precipitate PRAK, and furthermore, DJ-1 was detected in the precipitates with the anti-PRAK antibody but not with a control IgG (Figure 2(d)). These data clearly indicate that there is a constitutive binding of PRAK with DJ-1 in nonstimulated cells.
To further confirm the above finding in live cells, we constructed a pair of plasmids encoding either CFP-DJ-1 or YFP-PRAK to perform a fluorescence resonance energy transfer (FRET) assay. Hela cells were cotransfected with pECFP-DJ-1 and pEYFP-PRAK plasmids, and the coexpression of CFP and YFP was evaluated by FRET. As shown in Figure 2(e), cells cotransfected with both CFP-DJ-1 and YFP-PRAK depicted a distinct color-coded FRET region, with the efficiency ranging from 15% to 17%, which illustrates an interaction between PRAK and DJ-1. In contrast, cells cotransfected with either CFP and YFP-PRAK or YFP and CFP-DJ-1 failed to display any significant FRET (Figure 2(e)).

Colocalization between PRAK and DJ-1.
To examine the intracellular localization of PRAK and DJ-1, NIH3T3 cells were cotransfected with pcDNA3-HA-PRAK and pEGFP-DJ-1 and further stained with an anti-HA antibody and visualized with a Texas red-conjugate secondary antibody. We observed that exogenously introduced HA-PRAK colocalized with GFP-DJ-1 in the nuclei of the NIH3T3 cells (Figure 3(a)).
It has been reported that, in the normal circumstance, endogenous PRAK is mainly located in the cytoplasm of the cells [26], whereas the location of endogenous DJ-1 is cell cycle related and present in both cytoplasm and nucleus [27]. We stained PRAK +/+ cells with antibodies against PRAK and DJ-1 and FITC-and Texas red-conjugated secondary antibodies, to assess whether endogenous PRAK colocalized with endogenous DJ-1; however, there was no obvious colocalization between PRAK and DJ-1 observed in the nucleus (Figure 3(b)). In contrast, when PRAK +/+ cells were synchronized by serum starvation for 48 hrs and then treated with 300 M of H 2 O 2 for 6 hrs, we did observe the colocalization of endogenous PRAK with DJ-1 in the nucleus (Figure 3(b)), indicating that, in response to oxidative stress, endogenous PRAK moves into the nucleus and colocalizes with DJ-1.

The Effect of PRAK on Subcellular Localization and
Phosphorylation of DJ-1. NIH3T3 cells were transfected with pcDNA3-Flag-DJ-1, in combination with either pcDNA3-HA-PRAK or pcDNA-HA. Western blot analysis of cytoplasmic and nuclear extracts revealed that Flag-DJ-1 was mainly located in the cytoplasm when it was transfected alone; however, Flag-DJ-1 distributed in both cytoplasm and nucleus when it was cotransfected with HA-PRAK (Figures  4(a) and 4(b)), suggesting that overexpression of PRAK leads to a shift of DJ-1 from the cytoplasm to the nucleus. This finding was further supported by the results from fluorescent micrographs. GFP-DJ-1 was observed in the cytoplasm and nucleus when cells were transfected with pcDNA3-EGFP-DJ-1 alone (Figure 4(c)); however, more GFP-DJ-1 aggregated in the nucleus when cells were cotransfected with both pcDNA3-EGFP-DJ-1 and pcDNA3-HA-PRAK (Figure 4(c)).
Next, we examined the localization of endogenous DJ-1 in PRAK +/+ and PRAK −/− cells after the cells were synchronized by serum starvation for 48 hrs and treated with 300 M of H 2 O 2 for 6 hrs. In PRAK +/+ cells, endogenous DJ-1 mainly located in the nucleus even after the cells were treated with H 2 O 2 for 6 hrs (Figures 5(a) and 5(c)). However, in nonstimulated PRAK −/− cells, more endogenous DJ-1 appeared in the cytoplasm when compared with nonstimulated PRAK +/+ cells (Figures 5(b) and 5(d)). Furthermore, most endogenous DJ-1 in PRAK −/− cells translocated into the cytoplasm from the nucleus after the cells being treated with H 2 O 2 for 6 hrs (Figures 5(b) and 5(d)).
To assess whether PRAK can directly phosphorylate DJ-1, GST-tagged DJ-1 was incubated with His-tagged PRAK or p38 fusion proteins. Coincubation of His-PRAK but not His-p38 with GST-DJ-1 induced phosphorylation of DJ-1 (

PRAK Facilitates DJ-1 to Sequester Daxx in the Nucleus and Prevent Cell Death.
Previous studies reported that Daxx interacts with apoptosis signal-regulating kinase 1 (ASK1) and causes activation of this kinase, which subsequently triggers cell death [29], whereas DJ-1 can hamper the interaction between Daxx and ASK1 by recruiting Daxx in the nucleus, thereby inhibiting ASK1 activation and cell death [30]. We found that endogenous DJ-1 normally located in the nuclei of PRAK +/+ cells; however, in PRAK −/− cells, DJ-1 translocated from the nucleus into the cytoplasm following H 2 O 2 treatment ( Figure 5). Based on these findings, we hypothesized that, under oxidative stress, DJ-1, in the absence of PRAK, is unable to sequester Daxx in the nucleus, and more Daxx translocate into the cytoplasm, thereby causing ASK1 activation and cell death. To confirm this, we assessed DJ-1 and Daxx localization in both PRAK +/+ and PRAK −/− cells following H 2 O 2 treatment. In PRAK +/+ cells, DJ-1 and Daxx colocalized in the nucleus (Figures 7(a) and 7(c)). After the cells were treated with 300 M H 2 O 2 for 6 hrs, DJ-1 still remained in the nucleus and the majority of Daxx was kept in the nucleus despite a small amount of Daxx which translocated into the cytoplasm (Figures 7(a) and 7(c)). In contrast, most DJ-1 in PRAK −/− cells translocated into the cytoplasm in response to the H 2 O 2 challenge and failed to sequester Daxx in the nucleus (Figures 7(b) and 7(d)). As a result, Daxx translocated from the nucleus into the cytoplasm (Figures 7(b) and 7(d)). To

Discussion
DJ-1, first identified by Nagakubo et al. [27] as a mitogendependent oncogene product, is ubiquitously expressed in almost all human tissues as homodimers and participates in many physiological and pathological processes including tumorigenesis [31][32][33], fertilization [34,35], regulation of the androgen receptor [36][37][38][39][40], posttranslational modification of protein SUMO-1, a ubiquitin-like modifier [41], oxidative stress [42][43][44], and the development of Parkinson's disease [45][46][47][48][49]. However, it is undefined whether DJ-1 is a downstream interacting target for PRAK. In the present study, using a yeast two-hybrid system, we identified that DJ-1 is a potential PRAK interacting partner. A pull-down assay demonstrated that His-PRAK exclusively bound to GST-DI-1. Immunoprecipitation and immunoblotting data from human HEK293 cells revealed that PRAK was coprecipitated with DJ-1 in cells cotransfected with pCDNA3-HA-PRAK and pCDNA3-Flag-DJ-1 plasmids but not in cells transfected with pCDNA3-HA-PRAK alone. In addition, a constitutive binding of endogenous PRAK with DJ-1 was observed in nonstimulated Hela cells as confirmed by immunoprecipitation with anti-PRAK antibody and immunoblotting with anti-DJ-1 antibody. Using a FRET-based technique, we further illustrated an interaction between PRAK and DJ-1 in Hela cells. These results clearly demonstrate that PRAK binds to and interacts with DJ-1 both in vitro and in vivo.
It has been shown that endogenous PRAK is mainly located in the cytoplasm, whereas exogenous PRAK predominates in the nucleus [26]. On the other hand, endogenous DJ-1 is present in both cytoplasm and nucleus [27]. However, it is unclear whether PRAK preferentially colocalizes with DJ-1, thus affecting the intracellular distribution of DJ-1. We first examined the intracellular colocalization of either exogenously introduced or endogenous PRAK and DJ-1. We cotransfected NIH3T3 cells with pCDNA3-HA-PRAK and pEGFP-DJ-1 plasmids and observed colocalization of exogenously introduced PRAK with DJ-1 in the nucleus. Although there was no apparent colocalization of endogenous PRAK translocated from the cytoplasm into the nucleus. Similarly, endogenous DJ-1 in PRAK +/+ cells was mainly located in the nucleus even after the cells were treated with H 2 O 2 for 6 hrs; in contrast, most endogenous DJ-1 in PRAK −/− cells translocated from the nucleus into the cytoplasm in response to H 2 O 2 challenge. These results demonstrate that PRAK preferentially colocalizes with DJ-1 and helps DJ-1 to localize in the nucleus in response to oxidative stress. On the other hand, it has been reported that DJ-1 can shuttle between cytoplasm and nucleus [27], but it contains no NLS [50], indicating that there must be some other protein(s) which interact with DJ-1 and decide the subcellular localization of DJ-1. Our data support the notion that PRAK is one of such candidates that interacts with DJ-1 and assists its shuttling between nucleus and cytoplasm. It is important to clarify whether interaction of PRAK with DJ-1, in addition to facilitating the intracellular localization of DJ-1, also leads to DJ-1 phosphorylation. Using an in vitro assay system, we found that phosphorylation of DJ-1 was achieved only when GST-DJ-1 was coincubated with His-PRAK fusion protein. Furthermore, a substantially increased phosphorylation of endogenous DJ-1 in response to H 2 O 2 -induced oxidative stress was observed in PRAK +/+ cells but not in PRAK −/− cells. These data clearly demonstrate a PRAK-dependent phosphorylation of DJ-1.
Next, we attempted to clarify the biological significance of sequestering DJ-1 in the nucleus by PRAK in response to oxidative stress. Recent studies have revealed that DJ-1 functions as a new type of H 2 O 2 scavenger [51]; however, DJ-1 protects against oxidative stress-induced cell death via its sequestration of Daxx, a death protein in the nucleus, thus preventing subsequent activation of ASK1-mediated cell death pathway, rather than its direct effect of scavenging H 2 O 2 [30]. Based on these findings, we hypothesized that PRAK facilitates DJ-1 to sequester Daxx in the nucleus, thus protecting against oxidative stress-induced cell death. To test this, we treated cells with H 2 O 2 and observed that, in PRAK +/+ cells, the majority of DJ-1 and Daxx were still colocalized in the nucleus, whereas most DJ-1 and Daxx in PRAK −/− cells translocated from the nucleus into the cytoplasm, demonstrating that, without PRAK, DJ-1 fails to sequester Daxx in the nucleus in response to oxidative stress; as a result, more Daxx translocate into the cytoplasm where it triggers ASK1-associated cell death pathway. Consistent with this, we observed a substantially increased cell death in H 2 O 2treated PRAK −/− cells compared to H 2 O 2 -treated PRAK +/+ cells. In supporting our finding, a recent study by Han and colleagues [52] has reported that PRAK plays a key role in rasinduced senescence and tumor suppression by directly phosphorylating and activating the tumor-suppressor protein p53, indicating that PRAK possesses a diverse range of biological functions dependent on its downstream interacting partners.
Taken together, we identified DJ-1 as a novel interacting protein for PRAK. PRAK preferentially colocalizes with DJ-1 and leads to DJ-1 activation, which in turn facilitates DJ-1 to sequester Daxx in the nucleus, preventing oxidative stressinduced cell death. Further elucidation of molecular mechanisms underlying the interaction of PRAK, DJ-1, and Daxx may unravel a novel cytoprotective function of PRAK in response to oxidative stress.