Bidirectional signals transduced by DAPK?ERK interaction promote the apoptotic effect of DAPK
2004; Springer Nature; Volume: 24; Issue: 2 Linguagem: Inglês
10.1038/sj.emboj.7600510
ISSN1460-2075
AutoresChun-Hau Chen, Won‐Jing Wang, Jean‐Cheng Kuo, Hsiao-Chien Tsai, Jia‐Ren Lin, Zee‐Fen Chang, Ruey‐Hwa Chen,
Tópico(s)Melanoma and MAPK Pathways
ResumoArticle16 December 2004free access Bidirectional signals transduced by DAPK–ERK interaction promote the apoptotic effect of DAPK Chun-Hau Chen Chun-Hau Chen Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Won-Jing Wang Won-Jing Wang Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Jean-Cheng Kuo Jean-Cheng Kuo Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Hsiao-Chien Tsai Hsiao-Chien Tsai Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Jia-Ren Lin Jia-Ren Lin Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Zee-Fen Chang Zee-Fen Chang Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Ruey-Hwa Chen Corresponding Author Ruey-Hwa Chen Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Chun-Hau Chen Chun-Hau Chen Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Won-Jing Wang Won-Jing Wang Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Jean-Cheng Kuo Jean-Cheng Kuo Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Hsiao-Chien Tsai Hsiao-Chien Tsai Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Jia-Ren Lin Jia-Ren Lin Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Zee-Fen Chang Zee-Fen Chang Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Ruey-Hwa Chen Corresponding Author Ruey-Hwa Chen Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan Search for more papers by this author Author Information Chun-Hau Chen1, Won-Jing Wang1, Jean-Cheng Kuo1, Hsiao-Chien Tsai1, Jia-Ren Lin1, Zee-Fen Chang2 and Ruey-Hwa Chen 1 1Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan 2Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan *Corresponding author. Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan. Tel.: +886 2 231 234 56 Ext. 5700; Fax: +886 2 239 578 01; E-mail: [email protected] The EMBO Journal (2005)24:294-304https://doi.org/10.1038/sj.emboj.7600510 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Death-associated protein kinase (DAPK) is a death domain-containing serine/threonine kinase, and participates in various apoptotic paradigms. Here, we identify the extracellular signal-regulated kinase (ERK) as a DAPK-interacting protein. DAPK interacts with ERK through a docking sequence within its death domain and is a substrate of ERK. Phosphorylation of DAPK at Ser 735 by ERK increases the catalytic activity of DAPK both in vitro and in vivo. Conversely, DAPK promotes the cytoplasmic retention of ERK, thereby inhibiting ERK signaling in the nucleus. This reciprocal regulation between DAPK and ERK constitutes a positive feedback loop that ultimately promotes the apoptotic activity of DAPK. In a physiological apoptosis system where ERK–DAPK interplay is reinforced, downregulation of either ERK or DAPK suppresses such apoptosis. These results indicate that bidirectional signalings between DAPK and ERK may contribute to the apoptosis-promoting function of the death domain of DAPK. Introduction Apoptosis is essential for normal development and maintenance of tissue homeostasis and is controlled by a complex interplay between pro- and antiapoptotic proteins. The death domain superfamily, composed of the death domain (DD), death effector domain and caspase recruitment domain family of proteins, has emerged as the pivotal mediator of protein–protein interactions required for the transmission and regulation of apoptotic signals (Weber and Vincenz, 2001). Death-associated protein kinase (DAPK), a DD-containing, calmodulin (CaM)-regulated serine/threonine kinase, exerts proapoptotic activity and functions as a positive mediator of apoptosis induced by a variety of stimuli, such as interferon-γ, Fas, TNF-α, TGF-β, ceramide and oncogenes c-myc and E2F (Deiss et al, 1995; Cohen et al, 1997, 1999; Inbal et al, 1997; Raveh et al, 2001; Jang et al, 2002; Pelled et al, 2002). DAPK displays a multidomain structure, comprising a kinase domain, a CaM-binding motif, eight ankyrin repeats, a cytoskeleton binding region and a DD, in which the kinase domain and DD are both important for its proapoptotic activity (Cohen et al, 1997, 1999). Emerging evidence reveals the role of DAPK in tumor suppression. DAPK expression is frequently lost in tumors due to hypermethylation of the DAPK gene (Cohen and Kimchi, 2001). Furthermore, DAPK is capable of suppressing oncogenic transformation induced by c-myc and E2F in vitro (Raveh et al, 2001), and blocking tumor metastasis in vivo (Inbal et al, 1997). The mechanism underlying the proapoptotic function of DAPK has not been completely unraveled. Activation of a p53-mediated apoptotic pathway was found to account for its apoptosis inducibility (Raveh et al, 2001). Accordingly, several p53-deficient cell lines escape from apoptosis but undergo autophagic death in response to DAPK overexpression (Inbal et al, 2002). Recently, we demonstrated that DAPK inactivates integrin through an inside-out mechanism, which subsequently blocks matrix survival signal, thereby activating p53 and inducing an anoikis-type apoptosis (Wang et al, 2002). The catalytic activity of DAPK is absolutely required for all the biological functions of this kinase, including anoikis, whereas deletion of DD greatly impairs but not completely abolishes the death-promoting function of DAPK (Cohen et al, 1999; Cohen and Kimchi, 2001; Kuo et al, 2003). Notably, overexpression of DD alone protects cells from apoptosis induced by the full-length protein (Raveh et al, 2000), suggesting that protein–protein interactions through this domain could positively regulate the proapoptotic activity of DAPK. However, the interacting partner of the DD of DAPK has not been identified. The extracellular signal-regulated kinase (ERK) family of mitogen-activated kinase (MAPK) is activated by mitogens through a pathway involving Ras, Raf and MEK (Cobb, 1999; Schaeffer and Weber, 1999; Chang and Karin, 2001). Once activated, ERK translocates from the cytoplasm to the nucleus, which is critical for the fulfillment of many cellular functions of ERK, such as gene transcription, cell proliferation and differentiation (Howe et al, 2002). Thus, subcellular localization of ERK is a significant factor in determining the biological responses of ERK pathway. ERK subcellular localization is regulated by interactions with various proteins. For example, MEK1 is responsible for both cytoplasmic retention of ERK prior to its activation and nuclear export after its dephosphorylation (Fukuda et al, 1997; Adachi et al, 2000). Furthermore, interactions with several cytoplasmic and nuclear proteins promote ERK cytoplasmic and nuclear retentions, respectively (Volmat and Pouyssegur, 2001). In addition, ERK nuclear translocation is also regulated by cell adhesion status (Danilkovitch et al, 2000; Aplin et al, 2001) through an integrin-dependent mechanism (Aplin et al, 2001). The biological consequence of ERK activation in a given cell is determined in part by the cell-specific combination of downstream substrates. Substrate recognition by ERK and other MAPKs is achieved by interactions with conserved docking sequences in the substrates. These sequences, which are distinct from the phosphoacceptor residues, are responsible for increasing substrate phosphorylation efficiency and for providing specificity (Holland and Cooper, 1999; Sharrocks et al, 2000). One class of the docking sites, termed D-domain, is also found in MAPK upstream kinases, MAPK phosphatases and MAPK scaffolds (Sharrocks et al, 2000; Enslen and Davis, 2001). Mutagenesis studies reveal that a conserved domain (termed CD domain), which lies in the near C-terminal region outside of the catalytic domain of ERK, is required for binding D-domain-containing proteins (Tanoue et al, 2000). In addition, studies on the structures of p38 MAPK (Chang et al, 2002) and ERK2 (Lee et al, 2004) bound to D-domain peptides reveal a docking groove in these kinases responsible for interacting with the D-domain. Here we report that DAPK interacts with ERK through a D-domain within its DD and is a substrate of ERK. Phosphorylation of DAPK by ERK increases the catalytic activity of DAPK. Conversely, DAPK promotes the cytoplasmic retention of ERK, thereby inhibiting ERK signaling in the nucleus. This reciprocal regulation between ERK and DAPK plays a positive and physiological role in apoptosis regulation. Together, our results suggest that bidirectional signaling between DAPK and ERK may be one mechanism that contributes to the apoptosis-promoting function of the DD of DAPK. Results DAPK interacts with ERK through the DD of DAPK In a search for potential partner of the DAPK DD, we carried out a yeast two-hybrid screen of a human placenta cDNA library using the DD of DAPK as bait. Both ERK1 and ERK2 were recovered from this screen as strongly positive clones. The specificity of interaction between ERK and DAPK DD was verified by one-on-one transformation (Figure 1A). To examine this interaction further, we expressed the DD of DAPK as a GST fusion protein (GST-DD) and tested its ability to pull down in vitro-translated ERK1 and ERK2. GST-DD, but not GST, efficiently interacted with ERK1 and ERK2 (Figure 1B), suggesting direct associations of ERK1/2 with this DD. To determine if full-length DAPK interacts with ERK, 293T cells were transfected with Flag-tagged DAPK together with ERK1 or ERK2. Western blotting of the anti-Flag immunoprecipitates from lysates of transfected cells revealed co-precipitations of ERK1 and ERK2 with DAPK (Figure 1C). Furthermore, the interaction of endogenous DAPK with endogenous ERK in 293T cells (Figure 1D) and HeLa cells (data not shown) was detected by a reverse immunoprecipitation assay with the anti-ERK antibody followed by Western blotting with the anti-DAPK antibody. To examine whether the interaction of DAPK with ERK involves a region other than the DD of DAPK, we generated a series of DAPK deletion mutants fused with GST. Pull-down analysis revealed that only the DD segment was capable of interacting with ERK (Figure 2A). Accordingly, deletion of DD almost completely abolished the interaction between DAPK and ERK, as determined by co-immunoprecipitation assays (Figure 2B). In contrast, the kinase activity of DAPK is not required for ERK interaction, as both DAPK active (ΔCaM) and kinase-dead (K42A) mutants could still bind ERK (Figure 2B). Together, our results identify ERK as a DAPK-interacting protein, and the DD of DAPK is both sufficient and necessary for this interaction. Figure 1.Identification of ERK1/2 as DAPK-binding proteins. (A) Yeast strain L40 cotransformed with Gal- and LexA-based fusion constructs was assayed for β-gal activity or His3 phenotype (−His). (B) In vitro-translated, 35S-labeled ERK1 or ERK2 was incubated with either GST or GST-DD. The pull-down products and 5% amount of input ERK1/2 were analyzed by autoradiography (upper panel). The lower panel indicates the equal input of GST and GST-DD in pull-down reactions. (C) 293T cells were transfected with ERK1 or ERK2, together with Flag-DAPK (F-DAPK) or a control plasmid (−). Cell lysates were subjected to immunoprecipitations followed by Western blot with antibodies as indicated. The amounts of ERK1/2 in cell lysates are shown on the bottom. As the ERK antibody used in this study detects ERK2 more efficiently than ERK1, a strong ERK2 band, corresponding to the endogenous ERK2, was seen in the ERK1 transfectants. (D) DAPK and ERK interact endogenously. Lysates of 293T cells were used for immunoprecipitations with control IgG or anti-ERK and followed by immunoblot with anti-DAPK or anti-ERK. Download figure Download PowerPoint Figure 2.Mapping the ERK binding region in DAPK. (A) Lysates of 293T cells were incubated with GST-DAPK deletion mutants as illustrated in the upper panel. The pull-down products were assayed by Western blot with anti-ERK. The equal inputs of GST fusion proteins are shown on the bottom (input). (B) 293T cells transfected with various Flag-tagged DAPK constructs were subjected to immunoprecipitations and Western blot with antibodies as indicated. The amounts of ERK and Flag-DAPK proteins in lysates are shown on the bottom. (C) Alignment of the ERK docking sequences found in DAPK DD and several other ERK substrates. In the sequence of DAPK, the first amino acid listed is numbered. Conserved residues are highlighted. The feature of consensus D-domain sequence is indicated on the bottom; φ and X represent a hydrophobic amino acid and any amino acid, respectively. (D) GST fusion proteins immobilized on beads were incubated with equal amounts of 293 lysates. The pull-down products were analyzed by Western blot with anti-ERK2 antibody. The lower panel shows an equal input of the GST and GST fusion proteins in pull-down reactions. Download figure Download PowerPoint Recruitment of ERK to its binding partners often requires conserved docking sequences (Holland and Cooper, 1999; Sharrocks et al, 2000; Enslen and Davis, 2001). Examination of the sequence of DAPK DD revealed the existence of such sequence (D-domain) (Figure 2C). To determine whether the integrity of D-domain is required for ERK–DAPK interaction, GST pull-down analysis was performed using wild-type or mutant GST-DD proteins. In GST-DDm1 and GST-DDm2 mutants, the consensus LXL and RR residues were replaced by AXA and AA, respectively, whereas GST-DDm1/2 carried mutations in both LXL and RR sequences. Compared to wild-type DAPK DD, mutation of either RR or LXL residues markedly reduced binding to ERK, and mutation of both sequences completely abrogated binding (Figure 2D). Thus, the ERK–DAPK interaction is mediated through a specific ERK docking sequence present in the DD of DAPK. ERK stimulates the catalytic activity of DAPK Having identified a physical interaction between DAPK and ERK, we next determined their functional relationships. Our previous studies revealed an elevation of DAPK catalytic activity in response to serum stimulation (Kuo et al, 2003). To test whether this effect involves ERK, 293T cells expressing Flag-DAPK were pretreated with the MEK inhibitor PD98059 followed by serum stimulation, and DAPK catalytic activity was assessed by its ability to phosphorylate the regulatory light chain of myosin II (MLC), a DAPK substrate both in vitro and in vivo (Kuo et al, 2003; Bialik et al, 2004). In vitro kinase assay using the Flag-DAPK immunoprecipitated from cell extract revealed that PD98059 blocked serum-induced activation of DAPK (Figure 3A). Similar results were obtained by coexpressing Flag-DAPK with myc-MKP3 (Figure 3A), a dual-specificity phosphatase specifically deactivating ERK1/2 (Muda et al, 1996). The steady-state kinetic parameters for DAPK isolated from differentially treated cells were determined by measuring the rate constant of phosphorylation at various concentrations of substrate. Although the Vmax and Kcat values were comparable at each condition, serum stimulation led to a two-fold decrease of the Km value, which was reversed by either PD98059 or MKP3 (Figure 3B). To demonstrate directly the induction of DAPK catalytic activity by ERK, 293T cells were cotransfected with Flag-DAPK and a constitutively active MEK1 mutant followed by serum starvation. Expression of this MEK1 mutant indeed induced an increase in the DAPK catalytic activity (Figure 3C and Supplementary Figure S1). However, this activation of DAPK was not observed with a DAPK mutant lacking its DD (DAPKΔDD) (Supplementary Figure S1). As DD mediates the interaction between DAPK and ERK, this result suggests that activation of DAPK by ERK requires their direct association. Figure 3.ERK upregulates DAPK catalytic activity. (A) Serum-starved 293T cells transfected with Flag-DAPK and/or MKP3 were pretreated with 50 μM PD98059 and/or stimulated with or without serum for 15 min. Lysates with equal amounts of proteins were subjected to immunoprecipitations followed by in vitro kinase assays with MLC as a substrate or by Western blot with anti-Flag. Cell lysates were also used for Western blot analysis to detect the expression of various proteins (bottom panels). (B) Double reciprocal (Lineweaver–Burk) plots for analyzing the kinetic parameters of MLC phosphorylation catalyzed by DAPK or DAPKS735D isolated from transfected 293T cells at various conditions. The initial velocities (V0) were measured using 1 μg DAPK at increasing substrate concentrations, and kinetic analysis was performed as described in Materials and methods. (C) 293T cells transfected with various constructs were serum-starved and then lysed. DAPK kinase activity was assayed as in (A). The expression of various proteins in cell lysates is shown in the bottom panels. Download figure Download PowerPoint DAPK is a substrate of ERK Next, we investigated the mechanism by which ERK induces DAPK activity. As this effect presumably requires a direct interaction of the two proteins, one possibility is that phosphorylation of DAPK by ERK enhances the kinase activity of DAPK. Notably, DAPK possesses a serine-containing peptide (PPSP, at amino acids 733–736) that resembles the reported consensus site PXS/TP for phosphorylation by ERK, and this peptide is conserved in mouse DAPK (Jin et al, 2001). To test the possible phosphorylation of DAPK by ERK, we performed in vitro kinase assays using purified and active ERK2 as the enzyme and various recombinant DAPK proteins purified from baculovirus as the substrates. To distinguish DAPK phosphorylation by ERK from DAPK autophosphorylation, the kinase-dead K42A mutant was used. As shown in Figure 4A, the K42A mutant was phosphorylated by ERK2 in a dose-dependent manner. This phosphorylation, however, was greatly impaired in the K42A/S735A double mutant. This result not only identifies DAPK as a direct substrate of ERK but also indicates Ser 735 as the principal site for phosphorylation by ERK. The time-course and stoichiometry analysis of phosphorylation for DAPK by ERK2 indicates that up to 73% of the K42A mutant was phosphorylated by ERK2 in this kinase assay (Supplementary Figure S2). To demonstrate DAPK as an in vivo substrate of ERK, we generated an antiserum to a phosphopeptide corresponding to the region around Ser 735 of DAPK, and used it to detect phosphorylation of DAPK at Ser 735 in vivo. As shown in Figure 4B, DAPK or S735A mutant expressed in quiescent cells showed little reactivity to the anti-phospho-DAPK antibody. Activation of ERK signaling by introducing the active MEK1 significantly enhanced the recognition of wild-type DAPK, but not S735A, by this antibody, suggesting a role of ERK in phosphorylating DAPK at Ser 735 in vivo. Furthermore, the level of phosphorylated DAPK was elevated by serum stimulation, whereas either PD98059 or MKP3 was capable of blocking serum-induced DAPK phosphorylation at Ser 735 (Figure 4C). Together, these data indicate that DAPK is a substrate of ERK both in vitro and in vivo. Figure 4.ERK activates DAPK kinase activity through a direct phosphorylation at Ser 735. (A) DAPK proteins as indicated were purified from baculovirus and used as substrates in the in vitro kinase reactions with increasing amounts of purified and active ERK2. The reactions were analyzed by autoradiography (upper) or Coomassie blue staining (lower). (B, C) 293T cells transfected with plasmids as indicated were serum-starved (B), or pretreated with or without PD98059 (PD) and stimulated with or without serum for 15 min (C). Cell lysates were subjected to immunoprecipitations with anti-Flag followed by immunoblot with anti-DAPK or anti-phospho-DAPK(S735). The expression levels of various proteins in cell lysates are shown on the bottom. (D) In vitro phosphorylation of DAPK by ERK enhances the kinase activity of DAPK. Purified DAPK or its mutants immobilized on beads were subjected to in vitro kinase reactions with or without active ERK2 as described in Materials and methods. The phosphorylated and unphosphorylated DAPK or its mutants were assayed for their kinase activities using MLC as a substrate. [γ-32P]ATP was included only in the second kinase reaction. The final reaction products were analyzed by autoradiography (upper) or Coomassie blue staining (lower). (E) 293 cells transfected with plasmids as indicated were serum-starved and lysed. Cell lysates with equal amounts of proteins were used for Western blot with antibodies as indicated. Download figure Download PowerPoint Phosphorylation of DAPK by ERK stimulates the kinase activity of DAPK To assess the functional consequence of DAPK phosphorylation by ERK, we compared the kinase activity of ERK-phosphorylated DAPK to that of unphosphorylated DAPK. To this end, purified DAPK or its mutants was introduced into a phosphorylation reaction in the presence or absence of purified and active ERK2. After incubation, these DAPK proteins were subjected to the second phosphorylation reaction for testing their capabilities to phosphorylate MLC in vitro. As shown in Figure 4D, the ERK-phosphorylated DAPK displayed an increased activity in MLC phosphorylation, compared to the unphosphorylated DAPK. Mutation of the Ser 735 residue of DAPK to Ala (S735A), however, abrogated this activation by ERK. The observed MLC phosphorylation was specific to DAPK, as the K42A mutant, either phosphorylated or unphosphorylated by ERK, did not result in MLC phosphorylation. To ascertain that the induction of DAPK activity by ERK indeed requires ERK phosphorylation of DAPK at Ser 735, the S735A mutant was expressed in 293T cells in the presence or absence of active MEK1, and the kinase activity of this mutant was determined by in vitro kinase assays. In contrast to the wild-type DAPK, the activity of S735A mutant could no longer be upregulated in response to ERK activation (Figure 3C). Furthermore, a phosphorylation-mimicking mutant S735D displayed a higher basal activity than the wild-type protein, and could not be further activated by active MEK1 (Figure 3C). Steady-state kinetic analysis revealed that the phosphorylation-mimicking mutation led to an ∼3.5-fold decrease of the Km value without significantly affecting the Vmax and Kcat values (Figure 3B). Thus, both ERK activation and S735D mutation resulted in a similar mode of alterations in DAPK kinetic parameters, which provides additional evidence for Ser 735 phosphorylation as the prime mechanism of ERK-induced DAPK activation. Finally, we investigated whether ERK phosphorylation of DAPK at Ser 735 stimulates DAPK catalytic activity in vivo. Antibody specifically recognizing MLC phosphorylated at Thr 18 and Ser 19 (Ratcliffe et al, 1999) was used to monitor DAPK activity in vivo, as MLC has been the only known in vivo substrate of DAPK (Kuo et al, 2003; Bialik et al, 2004). As expected, expression of DAPK in quiescent cells increased the level of phosphorylated MLC. This induction of phosphorylated MLC was further augmented by introducing the active MEK1 mutant, demonstrating the ability of ERK to promote DAPK kinase activity in vivo. However, this enhancement of DAPK in vivo activity by ERK was not observed with the S735A mutant, even though in the absence of active MEK1, this mutant induced a comparable level of phospho-MLC to the wild-type protein (Figure 4E). In conclusion, these data provide substantial evidence that phosphorylation of DAPK at Ser 735 by ERK promotes the catalytic activity of DAPK. DAPK inhibits ERK nuclear translocation Having demonstrated the activation of DAPK by ERK, we next explored the influence of DAPK on ERK function. Expression of DAPK, its active (ΔCaM) or dominant-negative (K42A) mutant in quiescent 293 cells (Supplementary Figure S3) or NIH3T3 cells (data not shown) did not lead to ERK activation, and did not affect MEK1-induced activation of ERK. However, DAPK attenuated MEK1-induced transcriptional activation of Elk-1 (Figure 5A) and phosphorylation of Elk-1 at Ser 383 (Figure 5B). As phosphorylation and activation of Elk-1 by ERK requires ERK nuclear translocation, we assessed whether DAPK impairs ERK translocation. In NIH3T3 cells stimulated with serum for 2 h, a significant portion of ERK was localized in the nucleus. However, in cells overexpressing DAPK, ERK was largely confined to the cytosol (Figure 5C). Indeed, in the population of cells overexpressing DAPK, only 33% displayed nuclear staining of ERK, which is in sharp contrast with the 82% observed in the population without overexpressing DAPK (Figure 5C, bottom panel). These results not only indicate that increased expression of DAPK impairs ERK nuclear translocation and nuclear signaling, but also establish a reverse direction of regulation between ERK and DAPK. Figure 5.DAPK blocks ERK nuclear translocation and nuclear signaling. (A) DAPK blocks ERK signaling to Elk-1 activation. NIH3T3 cells were transfected with Gal4-Elk-1, Gal4-luciferase, pRK5β-Gal, and AcMEK1 (+) or a control vector (−), together with increasing amounts of Flag-DAPK or a control vector (−). Cells were serum-starved and lysed for luciferase and β-gal assays and Western blot analysis. (B) NIH3T3 cells transfected with plasmids as indicated were serum-starved. Cell lysates were subjected to immunoprecipitations and Western blot with antibodies as indicated. The levels of AcMEK1 or DAPK in cell lysates are shown on the bottom. (C) NIH3T3 cells transfected with myc-tagged DAPK or its mutants as indicated were serum-starved and then stimulated with serum for 2 h. Cells were triple stained with anti-myc, anti-ERK and Hoechst 33258, and visualized by confocal microscopy. The percent of cells showing stronger ERK nuclear staining than the cytoplasmic staining was quantitated and is listed on the bottom. The values shown are means±s.d. from three independent experiments, and at least 300 cells were counted for each population in each experiment. (D) NIH3T3 cells were cotransfected with Gal4-Elk-1, Gal4-luciferase, pRK5β-Gal, together with various DAPK constructs and/or AcMEK1. Transfectants were serum-starved or stimulated and then lysed for Elk-1 reporter assays as described in (A) and Western blot analysis (upper panel). Download figure Download PowerPoint Next, we explored the mechanism by which DAPK interferes with ERK nuclear translocation. We reasoned that the tight association of ERK with cytoplasmically localized DAPK might prevent ERK nuclear transport. However, the noninteracting mutant DAPKΔDD could still inhibit serum-induced ERK nuclear translocation (Figure 5C) and downregulate MEK1-induced Elk-1 activation (Figure 5D, left panel), albeit with a lower efficiency, suggesting the existence of an additional mechanism. As cell adhesion is also a determining factor in the nuclear translocation of active ERK (Aplin et al, 2001), the antiadhesion function of DAPK, which requires its catalytic activity (Wang et al, 2002), may in part contribute to the ERK sequestration effect of DAPK. Indeed, the kinase-dead K42A mutant partially alleviated the inhibition of Elk-1 activation, and the K42A/ΔDD double mutant almost completely relieved this blockage (Figure 5D, left panel). Consistently, confocal analysis revealed that the K42A/ΔDD mutant could no longer sequester ERK in the cytoplasm in serum-stimulated cells (Figure 5C). Consequently, serum-induced Elk-1 activation was not affected by expression of K42A/ΔDD, in contrast to the effect of wild-type protein (Figure 5D, right panel). Together, these results suggest that DAPK-induced cytoplasmic retention of ERK involves both interaction-dependent and kinase activity-dependent mechanisms. ERK promotes the anoikis inducibility of DAPK The inhibition of ERK nuclear translocation by DAPK, combined with the upregulation of DAPK kinase activity by ERK, establishes a bidirectional regulatory circuit between the two proteins. We hypothesize that this ERK–DAPK interplay would constitute a positive feedback loop to promote the function of DAPK via two mechanisms. First, blockage of ERK nuclear translocation would increase the cytoplasmic level of active ERK, thereby further potentiating the catalytic activity and subsequently apoptotic effect of DAPK. Second, although ERK signaling is generally considered to be antiapoptotic, DAPK might interfere with ERK survival signal by blocking the phosphorylation of its nuclear targets. In these regards, increased expression of DAPK would stimulate the formation of this fe
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