Positive regulation of apoptosis signal-regulating kinase 1 by hD53L1.

Apoptosis signal-regulating kinase 1 (ASK1) is a mitogen-activated protein kinase kinase kinase family member that plays a central role in cytokine- and stress-induced apoptosis by activating c-Jun N-terminal kinase and p38 signaling cascades. ASK1-induced apoptotic activity is up-regulated by two cellular factors, Daxx and TRAF2, through direct protein-protein interactions. Daxx and TRAF2 are death receptor-associated proteins in Fas and tumor necrosis factor-alpha pathways, respectively. Recent studies suggest that calcium signaling may regulate ASK1 pathway. Here we report that human D53L1, a member of the tumor protein D52 family involved in cell proliferation and calcium signaling, up-regulates the ASK1-induced apoptosis. The human D53L1 physically interacts with the C-terminal regulatory domain of ASK1 and promotes ASK1-induced apoptotic activity by activating caspase signaling in mammalian cells. In luciferase reporter assays, hD53L1 activates c-Jun N-terminal kinase-mediated transactivation in the presence of ASK1. Expression of hD53L1 enhances autophosphorylation and kinase activity of ASK1 but has no effect on ASK1 oligomerization that is necessary for kinase activity and on binding of ASK1 to MKK6, a downstream factor of ASK1. Taken together, these results suggest that activation of ASK1 by hD53L1 may provide a novel mechanism for ASK1 regulation.

Apoptosis is a highly regulated process that controls normal development and homeostasis of multicellular organisms (1,2). The inability to control the tightly regulated apoptosis causes many human diseases such as cancer, autoimmune diseases, and various neurodegenerative disorders (3). Extensive studies in recent years have revealed that apoptotic cell death occurs through an orchestrated sequence of intracellular signaling cascades. In particular, the mitogen-activated protein kinase (MAPK) 1 signaling pathways have been known as highly conserved cascades for regulation of cell death and survival from yeast to humans (4,5). There are at least six independent MAPK signaling units in mammalian systems (6,7). Three of them, the extracellular signal-regulated kinase pathway, the c-Jun amino-terminal kinase (JNK; also known as stress-activated protein kinase (SAPK)) pathway, and the p38 pathway, have been extensively characterized. The extracellular signalregulated kinase signaling pathway is often stimulated by mitogens, whereas the JNK/SAPK and the p38 signaling pathways are responsive to proinflammatory cytokines (e.g. TNF-␣ and interleukin-1) and environmental stress-related stimuli, including UV irradiation, H 2 O 2 , ischemia and reperfusion, and removal of growth factors. Several lines of evidence suggest that the JNK/SAPK signaling cascade plays a role in apoptotic cell death induced by stress-related stimuli (4,5,8,9).
There are several antagonistic cellular partners for ASK1 including thioredoxin (Trx), glutaredoxin (Grx), 14-3-3 proteins, and protein serine/threonine phosphatase 5. Thioredoxin (Trx) was identified as a negative regulator of the ASK1-JNK/ p38 pathway through yeast two-hybrid screening for ASK1binding proteins (14). In resting cells, ASK1 constantly forms an inactive complex with Trx, but upon treatment of cells with TNF-␣ or reactive oxygen species such as H 2 O 2 , ASK1 is dissociated from Trx and activated by subsequent modifications, including oligomerization and auto-and/or cross-phosphorylation (10,14,15). It was recently reported that 14-3-3 proteins and Grx directly bind to ASK1, and that overexpression of 14-3-3 and Grx proteins blocked ASK1-induced apoptosis and JNK1 activity, respectively (16,17). In addition, protein serine/ threonine phosphatase 5 was identified as a negative regulator of the activated ASK1. Protein serine/threonine phosphatase 5 binds to and dephosphorylates ASK1 in response to H 2 O 2 , enabling inactivation of ASK1 by negative feedback (18). Moreover, intramolecular interaction, probably between the N-ter-minal and C-terminal domains of ASK1, may be required to maintain the inactive form of ASK1 (11). Thus, ASK1 is associated with the mechanism for apoptotic cell death. As a result, execution of apoptosis induced by ASK1 must be strictly regulated by intracellular partners. However, the molecular mechanism by which ASK1 activity is regulated in cells is not understood completely.
To better understand the mechanism for the regulation of the ASK1 activity, we searched for its binding partners using the yeast two-hybrid screening method. Among several positive clones, we have identified one of them as hD53L1, a novel hD53 splicing variant encoding 131 amino acids. The hD52 gene was originally identified through its elevated expression in human breast carcinoma. The hD52 gene encodes a 184-amino acid polypeptide including a potential glycosylation site and several potential phosphorylation sites (19). Studies of hD52 homologues from other species have indicated that hD52 may play roles in calcium-mediated signal transduction and cell proliferation (20). Two homologues of hD52, hD53 and hD54, have also been identified, demonstrating the existence of a novel protein family (21). The hD52-like protein sequences are all predicted to contain a coiled-coil domain, which is responsible for homo-and heteromeric interactions. Yeast two-hybrid screening showed that hD52 could specifically interact with hD52 as well as its family members (22). Thus, D52-like proteins appear to exert and regulate their activities through specific interactions with other hD52-like proteins (23).
In the present study, we show that hD53L1 physically interacts with ASK1 and acts as a positive regulator of ASK1. Activation of ASK1 by hD53L1 leads to the activation of the caspase-3-dependent apoptosis pathway. Expression of hD53L1 induces ASK1 autophosphorylation and JNK/p38 activation but has no effect on ASK1 self-oligomerization and binding to MKK6. The ASK1-activating action of hD53L1 appears to be a novel function of this tumor protein.
Antibodies-GST-tagged hD53L1 protein was overexpressed in E. coli and bound to glutathione Sepharose 4B beads. The bound GSTfused protein was treated with thrombin to elute hD53L1 from the beads. The eluted hD53L1 protein was used to immunize mice.
In Vivo and in Vitro Binding Assays-HEK 293 cells were co-transfected with ASK1 and hD53L1 expression plasmids. After 48 h of transfection, cells were washed twice with phosphate-buffered saline buffer and lysed in Nonidet P-40 lysis buffer containing 137 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1% Nonidet P-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, 1 g/ml pepstatin, and 1 g/ml aprotinin for 30 min at 4°C, followed by centrifugation at 13,000 rpm for 15 min (24). The soluble fractions were incubated with 30 l of anti-FLAG M2-agarose affinity gel (Sigma) or 20 l of glutathione-Sepharose 4B beads (Amersham Biosciences) for 5 h at 4°C with rotation. After binding, the beads were collected by centrifugation at 6,000 rpm for 1 min and washed five times in the lysis buffer. The bound proteins were eluted with the SDS-PAGE sample buffer and were separated by SDS-PAGE, followed by immunoblotting with anti-FLAG M2 antibody (1:2000 dilution; Sigma), an anti-GST antibody (1:2000 dilution; Sigma), or an anti-HA antibody (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The protein bands were visualized by the ECL detection system (Amersham Biosciences).
To examine interaction between the endogenous proteins, cell lysates were prepared from HEK 293 or HeLa cells as described above and subjected to immunoprecipitation with mouse preimmune IgG-agarose, mouse anti-ASK1-agarose (Santa Cruz Biotechnology), mouse preimmune serum, or mouse hD53L1-specific antiserum for 5 h at 4°C. The bound proteins were analyzed by immunoblot probed with anti-ASK1 antibody or hD53L1-specific antiserum.
The in vitro binding effect of ASK1 to MKK6 was analyzed by incubating the immunoprecipitated FLAG-ASK1 with GST-MKK6 protein purified from E. coli, followed by immunoblotting analysis with anti-GST antibody.
ASK1 Oligomerization and Phosphorylation Assays-To examine the effect of hD53L1 on the oligomerization of ASK1, HEK 293 cells were co-transfected with expression plasmids producing FLAG-ASK1 and HA-ASK1 along with HA-hD53L1. After 48 h of transfection, cell lysate was prepared as described above. The supernatants were incubated with 30 l of anti-FLAG M2-agarose affinity gel (Sigma) for 5 h at 4°C. The immunoprecipitates were washed five times with Nonidet P-40 lysis buffer, eluted with SDS-PAGE sample buffer, and transferred to a polyvinylidene difluoride membrane, followed by immunoblotting with rabbit anti-HA antibody. For phosphorylation assays, BOSC 23 cells were transfected as described above. After 48 h of transfection, cells were lysed in a buffer containing 20 mM Tris-HCl (pH 7.5), 12 mM ␤-glycerophosphate, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 1% Triton X-100, 0.5% deoxycholate, 3 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml aprotinin. Cell extracts were cleared by centrifugation, and the supernatants were immunoprecipitated with anti-FLAG M2 beads. The beads were washed once with the lysis buffer, twice with a solution containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 2 mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride, and then once with a solution containing 20 mM Tris-HCl (pH 7.5) and 20 mM MgCl 2 . The beads were then resuspended in kinase reaction buffer (20 mM Tris-HCl (pH 7.5), 20 mM MgCl 2 , 0.1 mM sodium orthovanadate, 1 mM dithiothreitol) containing 20 M ATP and 0.3 Ci of [␥-32 P]ATP with or without 2 g of GST-MKK6 for 30 min at 30°C. The phosphorylation reactions were resolved by SDS-PAGE and visualized by autoradiography.
Determination of Cell Viability-Cell viability of transfected HEK 293 cells was determined by trypan blue exclusion assay. Cells were washed twice with phosphate-buffered saline, trypsinized, and collected by centrifugation. The cell pellets were resuspended in 0.2% trypan blue in phosphate-buffered saline, and the proportion of living cells was counted. The relative number of surviving cells was determined by estimating the value of mock-transfected cells as 100%.
Measurement of Caspase-3 Activity-Caspase-3 activity was measured by an ApoProbe-3 caspase-3 fluorescent assay kit (Peptron) according to the manufacturer's instructions, in which a fluorogenic synthetic peptide DEVD-7-amino-4-methylcoumarin was used as a substrate. Approximately 2 ϫ 10 6 HEK 293 cells were transfected with both HA-ASK1 and FLAG-hD53L1 expression plasmids. After 24 h, transfected cells were exposed to serum-free Dulbecco's modified Eagle's medium for 16 h. The cells were collected by centrifugation and resuspended in 50 l of chilled lysis buffer including 25 mM HEPES, pH 7.5, 0.5% Nonidet P-40, 0.5 mM EDTA, 150 mM NaCl, 10 g/ml pepstatin, 10 g/ml leupeptin on ice for 20 min, centrifuged at 13,000 rpm at 4°C for 10 min. The supernatants were mixed with an equal volume of 2ϫ reaction buffer with freshly added 10 mM dithiothreitol. The mixture was incubated with 2 l of 2.5 mM fluorogenic substrate at 37°C for 40 min. The fluorescence of the released 7-amino-4-methylcoumarin was measured with an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
Apoptotic DNA Laddering Assay-The apoptotic DNA laddering as-say was performed with the protocol described by Duke (25) with the following modifications. Approximately 8 ϫ 10 6 cells were lysed with 500 l of lysis buffer containing 5 mM Tris-HCl, pH 7.5, 20 mM EDTA, and 0.5% Triton X-100 on ice for 20 min. Cell extracts were clarified by centrifugation at 13,000 rpm at room temperature for 5 min. The lysate was incubated with 0.1 mg/ml RNase A at 37°C for 20 min. DNA was purified by standard phenol-chloroform extraction and ethanol precipitation. Dry DNA pellets were then resuspended in 20 l of TE buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA) containing 0.02 mg/ml RNase A. Samples were loaded on a 1.5% agarose gel and visualized by ethidium bromide staining. Luciferase Reporter Assay-293T cells were grown to 50 -80% confluence in 60-mm plates and transfected with 1 g each of pFR-Luc and 0.5 g of pCMV/␤-gal along with the appropriate plasmids as indicated. Total amounts of DNA were equalized with empty plasmids. After 5 h of transfection in 2 ml of serum-free medium, an equal volume of medium containing 1% fetal bovine serum was added, and the cells were incubated for an additional 17 h. Medium was then changed with medium containing 0.5% fetal bovine serum and incubated for another 24 h before harvesting. The luciferase activity was determined using an assay system (Promega) with a luminometer. The relative -fold induction of luciferase activity was determined and normalized to ␤-galactosidase activity. All transfections were repeated at least three times.

ASK1 Physically
Interacts with hD53L1-In order to identify a new binding partner of ASK1, we carried out yeast twohybrid screening with a HeLa cDNA library using the C-terminal domain (residues 649 -1375) of ASK1 as a bait (26). The C-terminal part contains a kinase domain (residues 649 -940) and a regulatory domain (residues 941-1375). We have obtained and identified positive clones, some of which (e.g. MAP-KKK6 and 14-3-3 proteins) have already been reported to interact with ASK1 (17,27). One of the de novo positive clones was identified as a full-length protein that is a splicing variant of tumor protein D53 and termed as human D53-like 1 (hD53L1) (GenBank TM accession number AF208012). Sequence alignment of hD53L1 to hD53 shows that the C-terminal 76 amino acids of hD53 protein were deleted due to alternative splicing.
To confirm the interaction between ASK1 and hD53L1, we carried out interaction assays with transfected mammalian cell extract. HEK 293 cells were transiently co-transfected with FLAG-ASK1 and GST-hD53L1 expression plasmids. GST-hD53L1 protein was pulled down by glutathione beads, followed by immunoblotting with appropriate antibodies to detect ectopically expressed ASK1 in the pulled-down hD53L1 complexes (Fig. 1A). ASK1 was detected in the pulled-down hD53L1 complexes, whereas GST alone did not form a complex with ASK1. We also performed co-immunoprecipitation assays with expressed HA-ASK1 and FLAG-hD53L1 proteins and found HA-ASK1 in the immunoprecipitated FLAG-hD53L1 complex (data not shown), suggesting that ASK1 and hD53L1 interact each other, regardless of tags used. To test whether ASK1 could bring down hD53L1 in a reciprocal way, HA-hD53L1 and FLAG-ASK1 were expressed and ASK1 was immunoprecipitated by anti-FLAG beads, followed by immunoblotting with anti-HA antibody. HA-hD53L1 protein was detected in the pulled-down ASK1 complexes (Fig. 1B). The results showed that these two proteins interact with each other. To examine the interaction between the two endogenous proteins, we produced antiserum specific to hD53L1. The specificity of the antiserum was confirmed by immunoblotting analysis using cell extract from HEK 293 transfected with pFLAG-hD53L1 expression plasmid (data not shown). The two endogenous proteins, ASK1 and hD53L1, were detected in HEK 293 cells by immunoblotting with anti-ASK1 and anti-hD53L1 antibodies. Their interaction was examined by coimmunoprecipitation with either preimmune IgG-agarose or anti-ASK1-agarose, followed by immunoblotting with hD53L1-specific antiserum (Fig. 1C). We also detected hD53L1 in immunoprecipitated ASK1 complex from HeLa cell lysates, suggesting that the interaction is not cell type-specific (Fig. 1C). Immunoprecipitation of hD53L1 by hD53L1-specific antiserum and then immunoblotting with anti-ASK1 antibody also confirmed the interaction of the two endogenous proteins (Fig. 1D). In a subsequent experiment, we performed co-immunoprecipitation assays to determine which domain of ASK1 binds to hD53L1 in vivo. The C-terminal regulatory domain (residues 941-1375) of ASK1 was found to be responsible for the binding to hD53L1, whereas the kinase (residues 649 -940) domain showed no affinity to hD53L1 (Fig. 1E). Collectively, these data suggest that ASK1 specifically interacts with hD53L1 through its C terminus in mammalian cells.
Expression of hD53L1 Protein Potentiates ASK1-induced Apoptosis-Since hD53L1 interacts with ASK1, we next examined whether it could regulate ASK1-induced cell death. HEK 293 cells were co-transfected with ASK1 and hD53L1 expression plasmids. When cell death was determined by a trypan blue exclusion assay, ASK1-induced cell death was increased in the presence of hD53L1 in a dose-dependent manner ( Fig. 2A), suggesting that hD53L1 is involved in the ASK1-induced cell death. Whereas overexpression of hD53L1 alone induced cell death only slightly, co-expression of hD53L1 and ASK1 significantly enhanced ASK1-mediated cell death.
Based on the above observation, we further examined whether the hD53L1-ASK1 signal is accompanied by apoptotic features such as DNA fragmentation and induction of caspase-3 activity. DNA fragmentation is a process that appears in the later stage of apoptosis and results from the activation of endonucleases during the apoptotic program. These endonucleases degrade chromatin into 200-bp laddering of DNA fragments (28). Chromosomal DNA samples were prepared from HEK 293 cells co-transfected with appropriate expression plasmids and were analyzed by electrophoresis. As shown in Fig. 2B, DNA fragmentation was not detected in samples from mock-transfected, hD53L1-transfected, and catalytically inactive ASK1 K709R mutant-transfected cells, whereas ASK1-transfected and H 2 O 2 -treated cell samples displayed the apoptotic DNA laddering feature. Co-expression of ASK1 and hD53L1 gave strong laddering intensity in preference to ASK1 expression alone. Also, increasing concentration of hD53L1 elevated DNA fragmentation. On the other hand, expression of the catalytically inactive ASK1 K709R mutant failed to induce DNA fragmentation in the presence of expressed hD53L1, suggesting that a phosphorylation event is necessary for the apoptosis. These findings indicate that overexpression of ASK1 effectively induces apoptosis in HEK 293 cells through MAPK pathways and that ASK1-mediated apoptosis can be enhanced by the addition of hD53L1. These results, therefore, suggest that hD53L1 may contribute to activation of the proapoptotic activity of ASK1 by partly, if not completely, enhancing kinase activity of ASK1.
Overexpression of hD53L1 Activates Caspase-3 in an ASK1dependent Manner-To directly test whether hD53L1 can regulate the apoptotic activity of ASK1, we measured the ASK1induced caspase-3 activity in the presence of transiently expressed hD53L1. Caspase-3 has been implicated as a key protease activated during the early stage of apoptosis (29). Active caspase-3, found in cells undergoing apoptosis, consists of a heterodimer of 17-and 12-kDa subunits that are derived from the inactive 32-kDa proenzyme. Active caspase-3 proteolytically cleaves and activates other caspases. Since members of the caspase family are crucial mediators of apoptosis (30, 31), we examined the requirement of caspase-3 activities for ASK1induced apoptosis using DEVD-7-amino-4-methylcoumarin as ASK1-hD53L1 Interaction a substrate. Previous studies showed that caspase signaling is responsible for ASK1-induced apoptosis (32). HEK 293 cells were transfected with HA-ASK1 and FLAG-hD53L1 expression plasmids, followed by serum deprivation after 24 h of transfection. Following 16 h of serum starvation, ASK1-induced caspase-3 activity was measured in vitro (Fig. 3A). Whereas hD53L1-transfected cells gave no significant increase of caspase-3 activity, ASK1-expressing cells showed an apparent increase of caspase-3 activity. Cells co-transfected with ASK1 and hD53L1 expression plasmids show higher caspase-3 activity than cells transfected with the ASK1 expression plasmid alone, suggesting that hD53L1 up-regulates the ASK1induced caspase-3 activity. Co-immunoprecipitation assays were performed with anti-FLAG M2 affinity gel, followed by immunoblotting with an anti-HA antibody. Cell lysates were subjected to immunoblotting using anti-FLAG M2 (middle) and anti-HA (bottom) antibodies to show protein expression levels of transfected genes. IP, immunoprecipitation; IB, immunoblot. C, cell lysates from the untransfected HEK 293 or HeLa cells were immunoprecipitated with mouse preimmune IgG agarose or mouse anti-ASK1 antibody agarose. The immunopellets were subjected to SDS-PAGE and immunoblotted with hD53L1-specific antiserum. D, HEK 293 cell lysates were immunoprecipitated with mouse preimmune serum or mouse hD53L1-specific antiserum, followed by immunoblot analysis with anti-ASK1 antibody. E, domain localization of ASK1 for binding to hD53L1. Top panel, HA-ASKK (amino acids 649 -940) or HA-ASKC (amino acids 941-1375) expression plasmid was co-transfected into BOSC 23 cells along with either pEBG-hD53L1 or empty plasmid. Binding assays were performed with glutathione-Sepharose 4B beads, followed by immunoblotting with an anti-HA antibody. Bottom panel, immunoblot of expressed ASK1 mutants in cell extracts.

FIG. 2. Effect of hD53L1 on ASK1-dependent cell death.
A, HEK 293 cells were transfected with 1 g of HA-ASK1 and 1 g (ϩ) or 2 g (ϩϩ) of FLAG-hD53L1 plasmids as indicated using the calcium phosphate precipitation method. After 24 h of transfection, cells were washed with phosphate-buffered saline and then incubated for 40 h in serum-free medium. To minimize the influence of transfection reagents, mock-transfected cells were used as 100% viability. Cells exposed to 1 mM H 2 O 2 for 9 h were used as positive control. Cell viability was determined by trypan blue exclusion assay. Results shown are mean S. E. (n ϭ 3). B, transfections were done as described in A. Positive control cells were stimulated with 1 mM H 2 O 2 for 9 h. Laddering assay was performed as described under "Experimental Procedures." Soluble cleavage chromatins were detected by ethidium bromide staining. The first lane shows a 100-bp size marker.

ASK1-hD53L1 Interaction
To confirm that the increase of caspase-3 activity is directly related to the promotion of the in vivo caspase-3 activity, immunoblot analysis was performed to detect the protein level of intact PARP (Fig. 3B). Because caspase-3 is responsible for the proteolytic cleavage of PARP, PARP was chosen as an in vivo marker of caspase-3 activity. Intact PARP signal was decreased in an hD53L1 dose-dependent manner, suggesting that PARP was cleaved by activated caspase-3. This caspase-3 activity was further confirmed by Western blotting of a 17-kDa active caspase-3 fragment (Fig. 3C). With co-expression of hD53L1 and ASK1, detection of the active caspase-3 was more apparent than that of cells expressing ASK1 only.
The hD53L1 Protein Activates ASK1-mediated MAPK Pathway but Does Not Increase ASK1 Oligomerization and Substrate Binding for Activity-Since hD53L1 induces ASK1-mediated apoptosis through MAPK signaling cascades as described above, we performed reporter assays to determine the effect of hD53L1 on ASK1-mediated transactivation activities of c-Jun, which is a downstream target of JNK (Fig. 4). For these assays, the N-terminal region (amino acids 1-223) of c-Jun fused with the DNA binding domain of yeast GAL4 and a reporter plasmid (pFR-Luc) carrying 5ϫ GAL4 binding sequences in the promoter region that controls expression of the luciferase gene were used. ASK1 expression plasmid together with the GAL4-c-Jun expression plasmid in the presence or in the absence of hD53L1 expression plasmid was co-transfected into 293T cells. Whereas overexpression of ASK1 was sufficient for stimulations of c-Jun activity, co-expression of hD53L1 further enhanced its activity.
There have been several reports that oligomerization of ASK1 is necessary for its kinase activity (15,33). Since autophosphorylation of ASK1 by oligomerization is important for ASK1 kinase activity, we tested whether hD53L1 could enhance ASK1 autophosphorylation and its kinase activity. ASK1 was immunoprecipitated from the same cell extracts co-expressing hD53L1 and ASK1 and effects of hD53L1 on ASK1 activity in the immunocomplex were determined by autophosphorylation assays and in vitro kinase assays using GST-MKK6 as a substrate. Results show that ASK1 is more autophosphorylated and more active in the presence of hD53L1 than in the absence of hD53L1 (Fig. 5A). The effects of phosphorylation on ASK1 and MKK6 induced by the expression of hD53L1 are lower than those induced by the addition of 1 mM H 2 O 2 but similar to those induced by 100 M H 2 O 2 . Since   FIG. 3. Effect of hD53L1 on caspase-3 activity. A, activation of caspase-3 by hD53L1 in HEK 293 cells. After 44 h of transfection, cells were lysed, and the caspase-3 activity in the lysate were measured using DEVD-7-amino-4-methylcoumarin as a substrate. Cell lysates were subjected to immunoblotting using an anti-HA antibody for detection of ASK1 (middle) and anti-FALG M2 antibody for hD53L1 (bottom). B, effect of ASK1 and hD53L1 on the degradation of PARP. HEK 293 cells were transfected with 1 g of HA-ASK1 and 1 g (ϩ) or 2 g (ϩϩ) of FLAG-hD53L1 plasmids as indicated using the calcium phosphate precipitation method. After transfection into 293 cells, cells were lysed with Nonidet P-40 lysis buffer and immunoblotted with anti-PARP antibody (top). Cells treated with H 2 O 2 were used as a positive control. The middle and bottom panels show the protein levels of ASK1 and hD53L1, respectively. C, cell lysates were prepared as in B, and proproteins and cleaved caspase-3 proteins were detected by immunoblotting with an anti-caspase-3 antibody (top).

FIG. 4. Expression of hD53L1 enhances JNK1-mediated transactivation.
293T cells were transfected with LipofectAMINE using 1 g each of the reporter plasmid pFR-Luc containing 5ϫ GAL4 binding sites, together with other plasmids described. pCMV/␤-gal was included in all transfections. After 48 h of transfection, equal quantities of cell extracts were used for luciferase assays. The relative -fold induction of luciferase activity was determined and normalized to ␤-galactosidase activity. Results represent the activity relative to the basal activity of empty plasmid. All experiments were repeated at least three times with similar results. hD53L1 has no apparent kinase activity on ASK1, we conclude that ASK1 is autophosphorylated at a higher level in the presence of hD53L1 than in the absence of hD53L1. Since activation of ASK by hD53L1 may result from the enhanced ASK1 oligomerization, we then tested whether hD53L1 induces ASK1 dimerization to increase ASK1 activity. HEK 293 cells expressing HA-ASK1 and FLAG-ASK1 in the presence or absence of hD53L1 were lysed, and then cell extracts were analyzed by immunoprecipitation, followed by immunoblotting. As shown in Fig. 5B, there is no apparent increase of ASK1 dimerization regardless of the hD53L1 expression level, suggesting that hD53L1-dependent activation of ASK1 is not induced by the modulated oligomerization of ASK1.
Activation of ASK1 by hD53L1 could result from the increased interaction between ASK1 and its substrate. This was tested in in vitro binding experiments using the immunoprecipitated FLAG-ASK1 and GST-MKK6 protein. After incuba-tion of the immunoprecipitated ASK1 with GST-MKK6, the complexes were washed and subjected to SDS-PAGE, followed by immunoblot with anti-GST antibody. No evidence of increased interaction in the presence of hD53L1 was observed (data not shown), indicating that activation of ASK1 by hD53L1 is not due to change of ASK1 affinity to its substrate. DISCUSSION Recent extensive studies on ASK1-mediated signaling pathway have identified three important signaling routes that could activate ASK1 kinase activity. TNF-␣, Fas, and endoplasmic reticulum stress signals activate ASK1 kinase activity through direct interactions between ASK1 and receptor-associated proteins (TRAF2 for TNF and endoplasmic reticulum stress signals and Daxx for Fas signal) (11,12,34).
However, there are several recent reports that other regulatory mechanisms such as Ca 2ϩ signaling may be involved in the regulation of ASK1. In neuronal cells of C. elegans, NSY-1, which is the homolog of the human ASK1, is activated by calcium/calmodulin-dependent protein kinase II, UNC-43, suggesting that ASK1 is controlled by calcium influx to regulate cell fate (35). In addition, the report that the ASK1-MKK4-JNK pathway is activated by Ca 2ϩ -permeable ␣-amino-3-hydroxy-5methyl-4-isoxazolepropionate receptors in the hippocampal CA1 region after ischemia provides further evidence that ASK1 may be regulated by calcium influx (36). It also has been reported that D52 tumor proteins are involved in Ca 2ϩ signaling to regulate proliferation and Ca 2ϩ -stimulated secretory activity (37,38). Recently, it has been reported that calcium/ calmodulin-dependent protein kinase II phosphorylates a Ca 2ϩ -regulated heat-stable protein of 28 kDa (CRHSP-28; a member of the tumor protein D52 family) in cultured mucosal T84 cells (39). Thus, our finding that hD53L1, a member of the tumor protein D52 family, activates the ASK1 pathway suggests that it may play a role in the Ca 2ϩ -dependent ASK1regulating pathway. The functional and biochemical basis of D52 family proteins, however, remains unknown. D52-like proteins have been found to interact with MAL2, a novel member of the MAL phospholipid family, as well as annexin VI (40,41). Both D52-interacting proteins are found in membrane fractions, whereas D52 proteins are present as peripheral membrane proteins that are recovered in the soluble fraction after alkaline treatment. In addition, binding of D52 to annexin VI is Ca 2ϩ -dependent, whereas binding to MAL2 is not. Interestingly, the D52 orthologue R10 in Japanese quail was identified as a chimeric cellular cDNA in proliferation-stimulated quail neuroretina cultures infected with RAV-1 retrovirus (42). This report and our results suggest that D52 proteins may participate in an unidentified signaling pathway that is involved in cell proliferation, despite the lack of evidence for what is located upstream of D52-ASK1 pathway.
It is worth noting that both hD53L1 and ASK1 contain coiled-coil motifs for protein-protein interactions. The C-terminal coiled-coil motif (residues 1236 -1293) of ASK1 is conserved among ASK1s from different species and involved in the ASK1 oligomerization and activation. The coiled-coil domain of hD53L1 may participate in binding to that of ASK1. However, we found that oligomerization of ASK1 was not altered by expression of hD53L1, suggesting that hD53L1 activates ASK1 without inducing oligomerization. TRAF2, an activator of ASK1, enhances ASK1 oligomerization probably by mediating the reactive oxygen species-mediated dissociation of Trx from ASK1 (43). Therefore, we could conclude that the activation mechanism of ASK1 by hD53L1 is different from the TRAF2mediated ASK1 activation mechanism. One possible explanation for this observation is that hD53L1 acts on the preformed oligomer of ASK1 to induce conformational change to lead to FIG. 5. Expression of hD53L1 activates ASK1 kinase activity but has no effect on oligomerization of ASK1. A, BOSC 23 cells were co-transfected using 2 g of FLAG-ASK1 plasmid with or without 5 g of HA-hD53L1 expression plasmid. After 48 h of transfection, cell lysates were prepared and analyzed as described under "Experimental Procedures." Autophosphorylation activity and in vitro kinase activity relative to the amount of ASK1 protein are shown as -fold increase relative to that of FLAG-ASK1 protein from transfected cells. Cells treated with 100 M H 2 O 2 for 30 min (ϩ) or 1 mM H 2 O 2 for 1 h (ϩϩ) were used as positive controls. The experiments were repeated at least three times with similar results. Top, autophosphorylation assay of ASK1; middle, in vitro kinase assay using GST-MKK6 as a substrate; bottom, immunoblotting of ASK1 with anti-FLAG M2 antibody. B, HEK 293 cells were co-transfected with 1 g (ϩ) or 2 g (ϩϩ) of hD53L1 expression plasmid. After 48 h of transfection, cells were lysed and immunoprecipitated with anti-FLAG M2 affinity gel. Immunoprecipitates were subjected to SDS-PAGE and then immunoblotted with an anti-HA antibody. the activation of ASK1-mediated apoptosis. In fact, it has been reported that ASK1 forms a silent homo-oligomer in nonstressed cells and then undergoes conformational change upon exposure to H 2 O 2 (33). In addition, binding of hD53L1 to ASK1 did not alter the binding affinity of ASK1 to its substrate, MKK6, which suggests that hD53L1 regulates ASK1 through a mechanism independent of physical interaction between ASK1 and MKK6.
Interestingly, hD53L1 did not enhance the kinase activity of ASK1 as strong as other adapter activators such as Daxx of Fas signaling and TRAF2 of TNF signaling (11,12). It is possible that hD53L1 may act as a competitive inhibitor of 14-3-3 or Grx proteins that bind to the C-terminal portion of ASK1 and suppress its activation (16,17,44), resulting in prevention of 14-3-3 proteins or Grx from binding to ASK1 in a concentrationdependent manner. This possible role of hD53L1 as an inhibitor of ASK1-inhibiting proteins may explain why the kinase activity of ASK1 is not induced significantly. Whereas little is known of hD52 family protein function, a recent report that hD53 (TPD 52L1) and hD54 (TPD52L2) contain 14-3-3 binding motifs suggests possible roles of hD52 family members in 14-3-3-regulated cellular processes (45). However, the fact that hD52 and hD53L1 lack 14-3-3 binding motifs indicates that there are functional differences among D52 family proteins in modulating cellular processes. It is also possible that hD53L1 may require other cellular factor(s) or external stimuli to gain full activity. Calcium may also be involved in the regulation, since calcium/calmodulin-dependent protein kinase II acts on both ASK1 and D52 family (35,39). These possibilities suggest that the regulation mechanism of ASK1 by hD53L1 may be different from the mechanisms of ASK1 by Daxx and TRAF2. Alternatively, ASK1-induced apoptosis by hD53L1 may not be mediated by the activation of ASK1 kinase activity. Roles of D52 family proteins in the regulation of vesicle trafficking and exocytotic secretion may be more directly linked to apoptosis. However, inability of the catalytically inactive ASK1 K709R mutant to enhance DNA fragmentation in the presence of hD53L1 suggests that the kinase activity of ASK1 is important for hD53L1-ASK1-apoptosis signaling.
When we compared the ability of hD53L1 to induce ASK1 kinase activity by autophosphorylation assays and in vitro phosphorylation assays using MKK6 with the effects of H 2 O 2 on ASK1, ASK1 activity induced by hD53L1 was lower than the activity induced by the addition of 1 mM H 2 O 2 . This stronger effect by H 2 O 2 may be mainly due to multiple roles of H 2 O 2 for regulation of ASK1. There are two identified pathways for activation of ASK1 by H 2 O 2 : glutathione-dependent Grx-ASK1 and glutathione-independent Trx-ASK1 pathways (14,16,44). Those two H 2 O 2 -sensing proteins, Trx and Grx, bind and inhibit ASK1 under reduced conditions. Upon exposure to H 2 O 2 , both Trx and Grx are oxidized and dissociated from ASK1, which results in activation of ASK1. In addition, H 2 O 2 induces ASK1 to undergo conformational change and create a new interface for autophosphorylation, which leads to enhancement of ASK1 activity (33). Whereas hD53L1 does not increase ASK1 activity as strongly as H 2 O 2, the protein still induces apoptosis as strongly as H 2 O 2 . This may suggest that hD53L1 keeps ASK1 moderately active and therefore leads to the accumulation of apoptotic factors. Further studies at the molecular level will be necessary to understand this regulatory effect of hD53L1 on ASK1 and apoptosis.