Exp Neurobiol 2011; 20(1): 35-44
Published online March 31, 2011
© The Korean Society for Brain and Neural Sciences
Hyoung Kyoung Choi and Kwang Chul Chung*
Department of Biology, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-2-2123-2653, FAX: 82-2-312-5657
Dual-specificity tyrosine (Y)-phosphorylation-regulated protein kinase 1A (Dyrk1A) is the mammalian homologue of
Keywords: ASK1, cell death, Dyrk1A, JNK, signal transduction
Dual-specificity tyrosine (Y)-phosphorylation-regulated protein kinase 1A (Dyrk1A) is firstly identified as Minibrain in
Diverse types of proteins have been identified as the substrates of Dyrk1A, including transcription factors, including NF-AT and Forkhead (Woods et al., 2001; Arron et al., 2006; Gwack et al., 2006), several endocytosis and synaptic vesicle recycling proteins, such as dynamin 1 and amphiphysin 1 (Chen-Hwang et al., 2002; Murakami et al., 2006), and cytosolic proteins, such as APP and tau (Ryoo et al., 2007; Ryoo et al., 2008), implying that Dyrk1A participates in various biological responses. Regarding to the functional role of Dyrk1A, active Dyrk1A phosphorylates the transcription factor cAMP response element (CRE)-binding protein (CREB), which subsequently leads to the stimulation of CRE-mediated gene transcription during neuronal differentiation (Yang et al., 2001). Yak1p, the
Recently, Dyrk1A appears to be involved in the pathogenesis of several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease, and Huntington's disease (HD). For example, Dyrk1A phosphorylates α-synuclein and regulates its inclusion formation, and potentially affecting neuronal cell viability (Kim et al., 2006). In addition, up-regulation of Dyrk1A in immortalized hippocampal progenitor H19-7 cells causes AD-like pathogenesis through the formation of tau inclusion and the generation of β-amyloid fragment (Park et al., 2007). However, the exact role of Dyrk1A and its signal transduction leading to cell death is not clearly elucidated yet.
Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, is composed of an inhibitory N-terminal domain, a kinase domain, and a C-terminal regulatory domain (Ichijo et al., 1997). ASK1 can promote apoptosis in response to common pro-apoptotic stresses, such as oxidative stress (Song et al., 2002), death receptor ligands (Nishitoh et al., 1998), and endoplasmic reticulum stress (Nishitoh et al., 2002). ASK1 also phosphorylates and activates both p38 and JNK pathways (Ichijo et al., 1997). The mechanism of ASK1 activation is positively regulated by its binding proteins such as TNF receptor-ssociated factors 2/6 (Noguchi et al., 2005) and Daxx (Chang et al., 1998). On the other hand, several cellular proteins, including thioredoxin (Saitoh et al., 1998), Hsp90 (Zhang et al., 2005), and 14-3-3 (Zhang et al., 1999), have been reported to interact with ASK1 and inhibit ASK1 activity.
In the present study, we investigated whether and how Dyrk1A becomes activated during cell death. We found that Dyrk1A is linked to JNK signaling pathway and acts as the upstream kinase of JNK. Additionally, Dyrk1A interacts with and positively regulates ASK1 under various cell death conditions. These finding suggest that Dyrk1A plays an important role in ASK1-mediated transmission of cell death signals.
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), LipofectAMINE PLUS reagent, anti-Xpress, horseradish peroxidase-conjugated anti-rabbit, and anti-mouse IgGs were purchased from Invitrogen (Carlsbad, CA, USA). Enhanced chemiluminescence (ECL) reagents and [γ-32P]ATP were purchased from Perkin-Elmer Life and Analytical Sciences (Waltham, MA, USA). Glutathione-Sepharose 4B and Protein A-Sepharose were obtained from Amersham Biosciences (Piscataway, NJ, USA). Anti-phosphoASK1 (Thr-845), anti-phosphoJNK, and anti-Dyrk1A antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-HA, anti-ASK1, anti-JNK, anti-Hsp90, mouse immunoglobulins (IgGs) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-Dyrk1A antibody was purchased from Abnova Corporation (Taipei City, Taiwan). Anti-phosphotyrosine antibody was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Anti-Flag antibodies and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Mammalian constructs encoding wild-type and kinase-inactive rat Dyrk1A tagged with hemagglutinin (HA) (pSVL-HA-Dyrk1A WT and K188R) were a kind gift from W. Becker (Institut fur Pharmakologie und Toxikologie, Universitatsklinikum der RWTH, Germany). Plasmids encoding 6xHis-Xpress-tagged wild-type and K188R mutant Dyrk1A (pcDNA4/HisMax-Dyrk1A WT and K188R) were generated as described previously (Park et al., 2007). Constructs encoding HA-tagged forms of ASK1, ASK1ΔN, ASK1ΔC, ASK1-NT, and Flag-ASK1 was kindly provided by E.J. Choi (Korea University, Seoul, Korea).
Human embryonic kidney 293 (HEK293) cells were maintained in DMEM containing 10% FBS and 100 unit/ml penicillin-streptomycin. The cells were transfected with various expression vectors using LipofectAMINE PLUS reagent, according to the manufacturer's protocols. In order to prepare cell lysates, the cells were rinsed twice with ice-cold phosphate-buffered saline, and then solubilized in lysis buffer (50 mM Tris, pH 7.5, containing 1.0% Nonidet P-40, 150 mM NaCl, 10% glycerol, 1 mM Na3VO4, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM EGTA, 1 mM EDTA,10 mM NaF, and 0.2 mM phenylmethylsulfonyl fluoride). The cells were scraped, and the supernatants were collected after centrifugation for 20 min at 14,000×g at 4℃.
One microgram of suitable antibodies was incubated with 0.5 to 1 mg of cell extracts prepared in cell lysis buffer overnight at 4℃. Fifty microliter of a 1:1 suspension of Protein A-Sepharose beads was added and incubated for 2 h at 4℃ with gentle rotation. After beads were pelleted and washed extensively with cell lysis buffer, the immunocomplexes were dissociated by boiling in SDS-PAGE sample buffer, separated onto SDS-PAGE gel, and transferred to a nitrocellulose membrane (Millipore, Japan). Membranes were then blocked in TBST buffer (20 mM Tris, pH 7.5, 137 mM NaCl, and 0.1% Tween 20) plus 5% nonfat dry milk for 1 h at room temperature, and incubated overnight at 4℃ in TBST buffer with 3% nonfat dry milk and the appropriate primary antibodies. Membranes were washed three times in TBST, and then incubated with appropriate secondary IgG-coupled horseradish peroxidase antibodies for 1 h at room temperature. The membranes were washed three times with TBST and visualized with ECL reagent.
After cells were harvested and lysed in lysis buffer, the protein extracts (~500 µg) were incubated with appropriate antibody for overnight at 4℃. The immunocomplexes were mixed with protein A-Sepharose beads, and washed three times in lysis buffer. For JNK assay, the kinase reaction was carried out at 30℃ for 30 min in 20 µl of kinase buffer (25 mM HEPES, pH 7.4, 25 mM glycerophosphate, 25 mM MgCl2, 100 µM sodium orthovanadate) containing 10 µM ATP, 5 µCi of [γ-32P] ATP, and 5 µg GST-c-Jun as a substrate. For Dyrk1A kinase assay, immunocomplexes were resuspended in the kinase reaction buffer (20 mM Tris-HCl, pH 7.5, 20 mM MgCl2) containing 10 µM ATP, 5 µCi [γ-32P]ATP, and incubated for 15 min at 30℃ in the presence of 5 µg of GST-ASK1 as a substrate. The reactions were stopped by adding SDS-sample buffer, and analyzed by SDS-PAGE followed by autoradiography.
In a previous report we have shown that the addition of etoposide to embryonic hippocampal H19-7 cells causes the activation of Dyrk1A, which facilitates the interaction between Hip-1 and caspase-3 and then leads to the cell death (Kang et al., 2005). In addition to etoposide, we firstly examined whether Dyrk1A becomes activated upon the stimulation with other toxic stimuli, such as tumor necrosis factor-α (TNFα) and hydrogen peroxide (H2O2). After HEK293 cells were incubated with hydrogen peroxide or TNFα, cell lysates were subjected to immunoprecipitation with anti-Dyrk1A, followed by immunoblotting with anti-phosphotyrosine. As shown in Fig. 1, the auto-phosphorylation of Dyrk1A was significantly increased upon the exposure to these two stimuli, reaching the maximum value of Dyrk1A activity at 2 hr and sustained thereafter until 4 hr of post-stimuli treatment. The quantification of the phospho-Dyrk1A bands revealed a 14.92- and 1.81-fold increase of Dyrk1A activity in response to TNFα and H2O2, respectively (Fig. 1). These results suggest that Dyrk1A becomes activated in response to toxic stimuli.
Based on the finding that intracellular JNK signaling pathway is activated by exposure with pro-inflammatory cytokines and cellular stress stimuli, such as etoposide and H2O2 (Ip and Davis, 1998), we next examined whether Dyrk1A is linked to JNK signaling pathway, and if it occurs, how they interact together in a biochemical and functional mode. After HEK293 cells were mock-transfected or transfected with Xpress-Dyrk1A for 24 hr followed by treatment with etoposide, cell lysates were immunoblotted with anti-phosphoJNK. As shown in Fig. 2A, while the addition of etoposide caused JNK activation, transfection of wild type Dyrk1A led to a synergistic increase in the level of phospho-JNK in a dose-dependent manner. To further check whether Dyrk1A causes the JNK activation,
To further characterize how Dyrk1A is linked to JNK pathway, we examined the possibility that Dyrk1A directly interacts with JNK. After HEK293 cells were transfected with Xpress-Dyrk1A or/and Flag-JNK alone or together, cell lysates were immunoprecipitated with anti-Flag followed by immunoblotting with anti-Xpress. As shown in Fig. 3, the anti-Flag-JNK immunoprecipitates did not include Xpress-tagged Dyrk1A, suggesting that Dyrk1A does not bind with JNK directly, whereas it is related to JNK signaling pathway.
Like as other MAP kinase family member, ERK and p38, JNK-mediated signaling pathway consists of three components, such as upstream MAP3K (ASK1), MAP2K (MKK4/7 or SEK1), and JNK/SAPK, (JNK1 and JNK2) (Kanamoto et al., 2000). Next we examined whether Dyrk1A interacts with ASK1. After HEK293 cells were transfected with HA-ASK1 or/and Xpress-Dyrk1A alone or together, cell lysates were immunoprecipitated with anti-HA and immunoblotted with anti-Xpress. As shown in Fig. 4A, when the cells were co-transfected with Dyrk1A and ASK1, Dyrk1A binds to ASK1. We further investigated whether the kinase activity of Dyrk1A is necessary for its binding to ASK1. As shown in Fig. 4B, HEK293 cells were transfected with a plasmid encoding either Xpress-tagged wild type or kinase-defective Dyrk1A alone or together with Flag-ASK1. Co-immunoprecipitation assay revealed that ASK1 interacts with kinase-deficient Dyrk1A as well as with wild type (Fig. 4B). These data suggest that the kinase activity of Dyrk1A appears not to be required for the interaction between Dyrk1A and ASK1.
Next we examined whether the mutual interaction between endogenous Dyrk1A and endogenous ASK1 still occurs. After HEK293 cell lysates were immunoprecipitated with mouse anti-Dyrk1A, followed by immunoblotting with anti-ASK1 and anti-Dyrk1A, Dyrk1A well interacts with ASK1 endogenously (Fig. 4C). Immunoprecipitation of the same lysates with mouse monoclonal anti-ASK1 antibody with followed by immunoblotting with anti-Dyrk1A in a reverse order produced the same binding band (Fig. 4C). As a control, the immunoprecipitation with mouse preimmune anti-IgG produced no band with anti-Dyrk1A or anti-ASK1, confirming the validity of assay (Fig. 4C). This data confirms that the mutual interaction between ASK1 and Dyrk1A is not an artifact arising from the DNA transfection, and still occurs inside cells.
To examine whether the interaction between Dyrk1A and ASK1 is being affected by toxic stimuli, including H2O2, TNFα, or etoposide, HEK293 cells were incubated with each stimuli for 1 hr. When cell lysates were immunoprecipitated with anti-ASK1, followed by immunoblotting with anti-Dyrk1A, the binding of Dyrk1A to ASK1 was significantly increased by the exposure with these toxic stimuli (Fig. 4D). While the addition of TNFα caused the most remarkable increased binding of Dyrk1A to ASK1, H2O2 and etoposide had less effect (Fig. 4D). These results suggest that toxic stimuli facilitate interaction between Dyrk1A and ASK1.
To identify the specific Dyrk1A-binding region within ASK1, several ASK1 constructs encoding its deletion fragments, such as HA-tagged wild type ASK1, ASK1-ΔN, ASK1-ΔC, and ASK1-NT, were generated (Fig. 5A). After HEK293 cells were transfected with expression plasmid encoding HA-tagged wild type ASK1, ASK1ΔN, ASK1ΔC, or ASK1-NT alone or together with Xpress-Dyrk1A, cell lysates were then subjected to immunoprecipitation with anti-HA. Immunoblotting of anti-HA-ASK1 complexes with anti-Xpress revealed that Dyrk1A still binds to ASK1-ΔN as well as the full length of ASK1 (Fig. 5B). However, Dyrk1A did not interact with ASK1-ΔC and ASK1-NT (Fig. 5B). These results suggest that the C-terminal domain of ASK1 spanning 937~1145th amino acid is critical for the interaction with Dyrk1A.
Based on the finding that Dyrk1A is linked to JNK signaling pathway and interacts with ASK1, we next examined how Dyrk1A is linked to ASK1 and/or JNK signaling cascades. To firstly determine whether the activation of Dyrk1A is mediated by ASK1, HEK293 cells were mock-transfected or transfected with Flag-tagged kinase defective ASK1 for 24 hr. After treatment with TNFα, cell lysates were then immunoprecipitated with anti-Dyrk1A, followed by immunoblotting with anti-phosphotyrosine. As shown in Fig. 6A, the exposure of TNFα caused the activation of Dyrk1A as well as JNK, and the presence of kinase-defective ASK1 resulted in the significant reduction of JNK activity, suggesting that ASK1 is present upstream of JNK. Unlike to JNK, the levels of phospho-Dyrk1A levels were not changed remarkably by kinase-inactive ASK1, compared to the cells in the absence of ASK1 mutants (Fig. 6A). When the same experiments were performed under the condition of H2O2- and etoposide-induced cell death, the similar activation pattern of JNK and Dyrk1A was observed in HEK293 cells, whereas these two stimuli caused much less activation of Dyrk1A than TNFα (Fig. 6A). However, the presence of kinase-defective ASK1 did not affect the activation of Dyrk1A induced by H2O2 and etoposide (Fig. 6A). Taken together, these results suggest that ASK1 does not affect Dyrk1A activity.
We next examined whether Dyrk1A acts as an upstream regulator of ASK1-JNK signaling. After HEK293 cells were co-transfected with HA-ASK1 alone or together with kinase-defective Xpress-Dyrk1A-K188R, the activation pattern of ASK1 and JNK under the condition of toxin-induced cell death was determined by immunoblot assay with anti-phospho ASK1 and anti-phospho JNK. As shown Fig. 6B, when the cells were co-transfected with ASK1 plus kinase-defective Dyrk1A, the activation of ASK1 and JNK was significantly inhibited by TNFα, H2O2, and etoposide, compared with the cells transfected with ASK1 alone. These data indicated that Dyrk1A as a upstream regulator positively modulates ASK1-JNK signaling during toxin-induced cell death.
To examine Dyrk1A directly phosphorylates ASK1,
Dyrk1A activity is involved in cell proliferation and differentiation (Dierssen and de Lagran, 2006). For example, while Ras-dependent signaling is required for promoting or maintaining neuronal differentiation, Dyrk1A modulates ERK activation by interacting with Ras, B-Raf, and MEK1 and by facilitating the formation of a Ras/B-Raf/MEK1 multi-protein complex (Kelly and Rahmani, 2005). Recently, many studies implicated a potential role of Dyrk1A during cell death. For example, Dyrk1A caused the formation of abnormal protein aggregates through the phosphorylation of α-synuclein, APP, and tau (Kim et al., 2006; Park et al., 2007; Ryoo et al., 2008). Furthermore, these cells show a marked increase of apoptotic cell death (Park et al., 2007), indicating that the overexpression of Dyrk1A induces cell death. Moreover, Dyrk1A seems to participate in the pathogenesis of Huntington disease by modulating the interaction of toxic huntingtin and Hip-1 in hiipocampal neuronal cells (Kang et al., 2005). However, the functional role of Dyrk1A during cell death and its signal transduction pathway have not been elucidated yet. Presently, we demonstrated that the activation of Dyrk1A occurs during toxin-induced cell death, including TNFα, hydrogen peroxide, and etoposide. In addition, while the activation of Dyrk1A is linked to and positively stimulates JNK-signaling, Dyrk1A did not directly bind to JNK.
Pro-apoptotic ASK1 mediates the induction of apoptosis under the stimulation of diverse stress through the activation of JNK signaling pathway. This hypothesis was confirmed by the current finding. We demonstrated that Dyrk1A physically interacts with ASK1 and enhances ASK1 and acts as a positive regulator of ASK1. Moreover, Dyrk1A appears to directly phosphorylate the C-terminal domain of ASK1. Similar to Dyrk1A, several proteins were reported to positively regulate ASK1 activity. For example, TRAF family and Gemin5 promoted the homo-oligomerization of ASK1 (Nishitoh et al., 1998; Kim et al., 2007), and CaMKII induces the phosphorylation of ASK1 in Ca2+-influx condition (Takeda et al., 2004). Further study is required how Dyrk1A positively regulates ASK1.
The Dyrk family consists of five mammalian members (Dyrk1A, Dyrk1B, Dyrk2, Dyrk3, and Dyrk4). Among these family members, Dyrk1A is the only member located on chromosome 21. Dyrk1A contains multiple domains, including a nuclear localization signal at the N-terminus, a kinase domain, a PEST domain for protein degradation, a 13-consecutive-histidine repeat, and an S/T rich region. Even though Dyrk1A has a nuclear localization signal and 13 histidine repeat for nuclear speckle targeting, Dyrk1A has been detected in the soma and dendrites of neurons. Therefore, it is not surprising that Dyrk1A substrates comprise both nuclear and cytosolic proteins, including transcriptional factors (CREB, NFAT, STAT3, FKHR, Gli1), splicing factors (cyclin L2, SF2, SF3), a translation factor (eIF2Bε), and synaptic proteins (dynamin I, amphiphysin I, synaptojanin I). Expanding the substrate diversity and pleiotropic roles for Dyrk1A, the current finding demonstrates that Dyrk1A interacts with and phosphorylates other cytosolic protein kinase, ASK1.
Although Dyrk1A is linked to JNK signal transduction pathway during cell death, the direct effect of Dyrk1A on cell viability or cell death/survival was not assessed yet. The current finding that Dyrk1A enhances the activities of ASK1 and JNK1, it could act as a pro-apoptotic player. This speculation was further supported by the previous reports. For example, the activation of Dyrk1A has been reported in several neurodegenerative diseases, such as DS and AD. For example, Ts65Dn mouse, a well-known mouse model of DS with abnormal 1.5-fold accumulation of Dyrk1A, exhibits hippocampal hypocellularity in early development and adult (Lorenzi and Reeves, 2006), and increased mitochondrial superoxide level (Schuchmann and Heinemann, 2000). Besides, the susceptibility of cell death has been increased in various types of cells from DS patients (Busciglio and Yankner, 1995; Roat et al., 2007). Furthermore, the cells overexpressing Dyrk1A exhibited abnormal processing of several AD-related pathogenic proteins (Park et al., 2007). Like to Dyrk1A, ASK1 activity also contributes to the cell death in the neurodegenerative diseases, including AD and HD. The accumulation of intracellular aggregates induced cellular stress, which subsequently leads to neuronal cell death in an ASK1-dependent manner in HD (Nishitoh et al., 2002). ROS-induced activation of ASK1 is also a key mechanism for β-amyloid-induced neurotoxicity in AD (Hashimoto et al., 2003; Kadowaki et al., 2005). Based on these previous reports, it would be interesting to examine whether and how Dyrk1A leads to cell death through ASK1-JNK signal transduction.
In conclusion, the current study demonstrated that Dyrk1A positively regulates ASK1-JNK activation during toxin-induced cell death.