Murine protein serine/threonine kinase 38 activates apoptosis signal-regulating kinase 1 via Thr 838 phosphorylation

Abstract

Murine protein serine/threonine kinase 38 (MPK38) is a member of the AMP-activated protein kinase-related serine/threonine kinase family that plays an important role in various cellular processes, including cell cycle, signaling pathways, and self-renewal of stem cells. Here we demonstrate a functional association between MPK38 and apoptosis signal-regulating kinase 1 (ASK1). The physical association between MPK38 and ASK1 was mediated through their carboxyl-terminal regulatory domains and was increased by H(2)O(2) or tumor necrosis factor alpha treatment. The use of kinase-dead MPK38 and ASK1 mutants revealed that MPK38-ASK1 complex formation was dependent on the activities of both kinases. Ectopic expression of wild-type MPK38, but not kinase-dead MPK38, stimulated ASK1 activity by Thr(838) phosphorylation and enhanced ASK1-mediated signaling to both JNK and p38 kinases. However, the phosphorylation of MKK6 and p38 by MPK38 was not detectable. In addition, MPK38-mediated ASK1 activation was induced through the increased interaction between ASK1 and its substrate MKK3. MPK38 also stimulated H(2)O(2)-mediated apoptosis by enhancing the ASK1 activity through Thr(838) phosphorylation. These results suggest that MPK38 physically interacts with ASK1 in vivo and acts as a positive upstream regulator of ASK1.

FIGURE 1.

Direct interaction of MPK38 with ASK1 in vivo and in vitro. A, regulation of MPK38 and ASK1 kinase activity by H₂O₂. 293T cells transiently transfected with wild-type GST-MPK38 were incubated with increasing amounts of H₂O₂ for 30 min, and MPK38 was purified on glutathione-Sepharose beads. The GST precipitates were subjected to an in vitro kinase assay using ZPR9 as a substrate, followed by SDS-PAGE and autoradiography (left). For H₂O₂ time-dependent regulation of MPK38 and ASK1 kinase activity, 293T cells were treated with 2 mm H₂O₂ for the indicated times. Cell lysates were immunoprecipitated by the indicated antibodies, and the kinase activities of MPK38 and ASK1 were determined by in vitro kinase assays using ZPR9 and MKK6(K82A) as substrates (right). The circled P-ZPR9 and P-MKK6(K82A) indicate the phosphorylated ZPR9 and MKK6(K82A), respectively. The relative level of kinase activity was quantitated by densitometric analyses, and fold increase relative to untreated samples was calculated. IP, immunoprecipitation; WB, Westernblot. B, invivo and invitro association of MPK38 with ASK1. GST alone and GST-MPK38 were cotransfected with FLAG-ASK1 into 293T cells. GST fusion proteins were purified on glutathione-Sepharosebeads (GST Purification), and the amounts of complex formation and FLAG-ASK1 used for the in vivo binding assay were determined by anti-FLAG antibody immunoblot (left). Cell lysates from HEK293 cells were subjected to immunoprecipitation using either rabbit preimmune serum (Preimm.) or anti-MPK38 antibody (α-MPK38), followed by immunoblot analysis using an anti-ASK1 antibody to determine the complex formation between endogenous MPK38 and ASK1 (middle). As a control, the expression levels of MPK38 and ASK1 in the total cell lysate were analyzed by immunoblot using anti-MPK38 and anti-ASK1 antibodies, respectively. For native PAGE (8%) of the MPK38-ASK1 complex (right), purified recombinant MPK38 was autophosphorylated as described under “Materials and Methods.” Autophosphorylated MPK38 (2–3 μg) was incubated with unlabeled recombinant GST or GST-ASK1(K709R) (each 5 μg) at room temperature for 1 h. C, effect of MPK38 and ASK1 kinase activities on MPK38-ASK1 association. HEK293 cells were transiently transfected with the appropriate expression plasmids, and MPK38-ASK1 complex formation was determined by Western blot analysis using anti-HA or anti-FLAG antibody as described in B. D, modulation of MPK38-ASK1(K709R), MPK38(K40R)-ASK1, or MPK38(K40R)-ASK1(K709R) complex formation by H₂O₂. HEK293 cells transfected with the indicated expression vectors were incubated with or without 2 mm H₂O₂ for 30 min. The level of MPK38-ASK1 complex was analyzed by immunoblot using an anti-HA antibody. E, H₂O₂ and TNF-α modulation of MPK38-ASK1 interaction. HEK293 cells transfected with the expression vectors indicated were treated with 2 mm H₂O₂ for 30 min or 500 ng/ml TNF-α for 30 min. Cell lysates were purified on glutathione-Sepharose beads (GST Purification) and immunoblotted with an anti-FLAG antibody (upper). The endogenous level of MPK38-ASK1 complex in the presence or absence of H₂O₂ (or TNF-α) was also analyzed by immunoblot using an anti-ASK1 antibody (lower). The relative level of MPK38-ASK1 complex formation was quantitated by densitometric analyses and fold increase relative to untreated samples expressing wild-type MPK38 and ASK1 was calculated (C–E).

FIGURE 1.

Direct interaction of MPK38 with ASK1 in vivo and in vitro. A, regulation of MPK38 and ASK1 kinase activity by H₂O₂. 293T cells transiently transfected with wild-type GST-MPK38 were incubated with increasing amounts of H₂O₂ for 30 min, and MPK38 was purified on glutathione-Sepharose beads. The GST precipitates were subjected to an in vitro kinase assay using ZPR9 as a substrate, followed by SDS-PAGE and autoradiography (left). For H₂O₂ time-dependent regulation of MPK38 and ASK1 kinase activity, 293T cells were treated with 2 mm H₂O₂ for the indicated times. Cell lysates were immunoprecipitated by the indicated antibodies, and the kinase activities of MPK38 and ASK1 were determined by in vitro kinase assays using ZPR9 and MKK6(K82A) as substrates (right). The circled P-ZPR9 and P-MKK6(K82A) indicate the phosphorylated ZPR9 and MKK6(K82A), respectively. The relative level of kinase activity was quantitated by densitometric analyses, and fold increase relative to untreated samples was calculated. IP, immunoprecipitation; WB, Westernblot. B, invivo and invitro association of MPK38 with ASK1. GST alone and GST-MPK38 were cotransfected with FLAG-ASK1 into 293T cells. GST fusion proteins were purified on glutathione-Sepharosebeads (GST Purification), and the amounts of complex formation and FLAG-ASK1 used for the in vivo binding assay were determined by anti-FLAG antibody immunoblot (left). Cell lysates from HEK293 cells were subjected to immunoprecipitation using either rabbit preimmune serum (Preimm.) or anti-MPK38 antibody (α-MPK38), followed by immunoblot analysis using an anti-ASK1 antibody to determine the complex formation between endogenous MPK38 and ASK1 (middle). As a control, the expression levels of MPK38 and ASK1 in the total cell lysate were analyzed by immunoblot using anti-MPK38 and anti-ASK1 antibodies, respectively. For native PAGE (8%) of the MPK38-ASK1 complex (right), purified recombinant MPK38 was autophosphorylated as described under “Materials and Methods.” Autophosphorylated MPK38 (2–3 μg) was incubated with unlabeled recombinant GST or GST-ASK1(K709R) (each 5 μg) at room temperature for 1 h. C, effect of MPK38 and ASK1 kinase activities on MPK38-ASK1 association. HEK293 cells were transiently transfected with the appropriate expression plasmids, and MPK38-ASK1 complex formation was determined by Western blot analysis using anti-HA or anti-FLAG antibody as described in B. D, modulation of MPK38-ASK1(K709R), MPK38(K40R)-ASK1, or MPK38(K40R)-ASK1(K709R) complex formation by H₂O₂. HEK293 cells transfected with the indicated expression vectors were incubated with or without 2 mm H₂O₂ for 30 min. The level of MPK38-ASK1 complex was analyzed by immunoblot using an anti-HA antibody. E, H₂O₂ and TNF-α modulation of MPK38-ASK1 interaction. HEK293 cells transfected with the expression vectors indicated were treated with 2 mm H₂O₂ for 30 min or 500 ng/ml TNF-α for 30 min. Cell lysates were purified on glutathione-Sepharose beads (GST Purification) and immunoblotted with an anti-FLAG antibody (upper). The endogenous level of MPK38-ASK1 complex in the presence or absence of H₂O₂ (or TNF-α) was also analyzed by immunoblot using an anti-ASK1 antibody (lower). The relative level of MPK38-ASK1 complex formation was quantitated by densitometric analyses and fold increase relative to untreated samples expressing wild-type MPK38 and ASK1 was calculated (C–E).

FIGURE 2.

Mapping of the ASK1 and MPK38 interaction domains. The schematic structures of wild-type and deletion constructs of ASK1 (A) and MPK38 (B) are indicated. Numbers indicate the amino acid residues corresponding to the domain boundaries. 293T cells transiently transfected with the indicated expression vectors were lysed and then precipitated using glutathione-Sepharose beads (GST Purification), and immunoblotted with an anti-HA antibody to determine the level of MPK38-ASK1 complex formation. The same blot was re-probed with an anti-GST antibody to confirm the expression of GST fusion proteins in the coprecipitates, and the expression level of ASK1 in total cell lysates was determined by immunoblot analysis using an anti-HA antibody. These experiments were independently performed at least three times with similar results. WB, Western blot.

FIGURE 3.

Enhancement of ASK1 kinase activity by MPK38. A, stimulation of ASK1 kinase activity by MPK38. 293T cells were transiently transfected with HA-ASK1 in the presence or absence of GST-MPK38. Cell lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody, and the HA immunoprecipitates were analyzed for ASK1 kinase activity by an in vitro kinase assay using recombinant MKK6(K82A) as a substrate. The circled P-MKK6(K82A) indicates phosphorylated MKK6(K82A). The same blot was stripped and re-probed with an anti-HA antibody to determine the expression level of immunoprecipitated ASK1 (left, 2nd panel), and the expression level of GST-MPK38 in total cell lysates was examined by immunoblot analysis using an anti-GST antibody. (left, 3rd panel). The presence of equivalent amounts of substrate was verified by immunoblotting with an anti-GST antibody (left, bottom panel). 5 μg of recombinant ASK1-K (re.ASK1-K) or wild-type ASK1 (re.ASK1) proteins were incubated at room temperature for 1 h with the indicated amount of recombinant MPK38 (re.MPK38) in 50 μl of 25 mm HEPES buffer, pH 7.4, and then analyzed for ASK1 kinase activity by an in vitro kinase assay using recombinant MKK6(K82A) as a substrate (middle and right). WB, Western blot. B, requirement of MPK38 activity for MPK38-induced stimulation of ASK1 kinase activity. After 48 h of transfection with the indicated expression vectors, 293T cell lysates were subjected to immunoprecipitation with an anti-HA antibody and then analyzed by an in vitro kinase assay with recombinant MKK6(K82A) substrate (top panel). ASK1 Thr⁸³⁸ phosphorylation in the HA immunoprecipitates was determined by immunoblot analysis using an anti-phospho-ASK1(T845) antibody (2nd panel). The expression levels of immunoprecipitated ASK1 and wild-type and kinase-dead MPK38 in total cell lysates were analyzed using anti-HA and anti-GST antibodies, respectively (3rd and 4th panels). The relative level of ASK1 kinase activity was quantitated by densitometric analyses, and fold increase relative to control expressing ASK1 or ASK1-K alone was calculated. re., recombinant.

FIGURE 4.

Phosphorylation of ASK1 by MPK38. A, for an in vitro kinase assay, ∼3–4 μg of recombinant ASK1(K709R) or wild-type ASK1 was mixed with 10 μm ATP, 5 μCi of [γ-³²P]-ATP, and 10 mm MgCl₂ in 20 μl of kinase buffer (see “Materials and Methods”) and incubated with the precipitated MPK38 for 15 min at 37 °C with frequent gentle mixing (top panel). ASK1 Thr⁸³⁸ phosphorylation was determined by immunoblot analysis using an anti-phospho-ASK1(T845) antibody (2nd panel). The position and expression levels of GST-MPK38, re.ASK1(K709R), and re.ASK1(WT) were monitored by immunoblotting with an anti-GST antibody (3rd and bottom panels). The relative level of kinase activity was quantitated by densitometric analyses, and fold increase relative to control containing recombinant wild-type ASK1 alone was calculated. WB, Western blot. B, identification of MPK38 phosphorylation sites on ASK1. An in vitro kinase assay was performed with 5 μg of recombinant ASK1(K709R) or one of its substitution mutants (ASK1(S83A), ASK1(T838A), ASK1(S967A), or ASK1(S1034A)), as well as recombinant wild-type MPK38 (10 μg), as described in A (top panel). The phosphorylation of ASK1 Thr⁸³⁸ and expression of recombinant MPK38 and ASK1 were determined by immunoblotting with anti-phospho-ASK1(T845) and anti-GST antibodies, respectively (2nd to bottom panels). The relative level of kinase activity was quantitated by densitometric analyses, and fold increase relative to control samples containing ASK1(K709R) was calculated. C, after 48 h of transfection with GST-MPK38, HEK293 cell lysates were subjected to precipitation with glutathione-Sepharose beads (GST Purification) and then analyzed by in vitro kinase assays with recombinant ASK1(K709R), MKK6(K82A), and p38 as substrates. The circled P-ASK1(K709R) and circled P-MPK38 indicate the phosphorylated ASK1(K709R) and autophosphorylated MPK38, respectively. re., recombinant.

FIGURE 5.

MPK38-mediated modulation of the association between ASK1 and MKK3. A–C, HEK293 cells were transfected with the indicated combinations of HA-MKK3, FLAG-ASK1, FLAG-ASK1(T838A), GST-MPK38, and GST-MPK38(K40R). Cell lysates were then subjected to immunoprecipitation (IP) with an anti-HA antibody, and the resulting immunoprecipitates were analyzed by immunoblot analysis with an anti-FLAG antibody to determine the association of MKK3 with ASK1 in the presence or absence of 15 mm Nac for 30 min. The expression levels of ASK1 and MPK38 in total cell lysates were analyzed by immunoblot using anti-FLAG and anti-GST antibodies. D, effect of MPK38 knockdown on the association between ASK1 and MKK3 was determined by immunoblotting with the indicated antibodies using HEK293 cells transiently transfected with MPK38 siRNA 1 (left) or HaCaT cells (MPK38(KD)) stably expressing MPK38 shRNA (right). MPK38(KD) cells were transfected with 4 μg of plasmid (MPK38(W)) containing a wobble mutant cDNA encoding MPK38 with three synonymous point mutations within the siRNA target sequence (right). The relative level of ASK1-MKK3 complex formation was quantitated by densitometric analyses, and fold increase relative to control expressing ASK1 and MKK3 in the absence of MPK38 was calculated. WB, Western blot.

FIGURE 6.

Effect of MPK38 on ASK1 downstream signaling. A, MPK38 stimulates ASK1 downstream signaling. Parental SK-N-BE(2)C cells or MPK38 stable transfectants (SK-MPK38) were incubated in the presence or absence of 2 mm H₂O₂ for 30 min. Cell lysates were subjected to immunoprecipitation (IP) using the indicated antibodies, and the immunoprecipitates were assayed for activities of ASK1, MKK3, or p38 by in vitro kinase assays using the indicated substrates. The expression levels of ASK1, MKK3, and p38 in the immunoprecipitates were analyzed by immunoblot using the indicated antibodies. The presence of equal amounts of each substrate in these assays was confirmed by immunoblotting with an anti-GST antibody (upper, bottom panels). WB, Western blot. B, modulation of ASK1 downstream signaling by knockdown of endogenous MPK38. Parental HaCaT cells or HaCaT cells stably expressing MPK38 shRNA (MPK38(KD)) were treated with 2 mm H₂O₂ for 30 min. The immunoprecipitates were also subjected to in vitro kinase assays as described in A. ASK1 Thr⁸³⁸, MKK3/6, p38, and ATF2 phosphorylation were determined by immunoblot analysis using the indicated phospho-specific antibodies (A and B, lower, left panels). The expression levels of ASK1, MKK3, p38, ATF2, and MPK38 in total cell lysates were analyzed by immunoblot using the indicated antibodies (A and B, lower, right panels). The relative level of kinase activity was quantitated by densitometric analyses, and fold increase relative to untreated samples in parental cells was calculated.

FIGURE 7.

Up-regulation of JNK-mediated transcription by MPK38. A, 293T cells were transfected with 0.2 μg of AP-1 luciferase plasmid and increasing amounts of wild-type (WT) and kinase-dead (K40R) forms of MPK38 (6 and 9 μg) and MPK38 siRNA 1 (50 and 200 nm) as indicated in the presence or absence of c-Fos (0.6 μg). Data shown are means (± S.E.) of three independent experiments. p < 0.05 relative to control; significance calculated by Student's t test. B, enhancement of JNK activity by MPK38. HEK293 cells were cotransfected using GST-JNK (1.5 μg) and HA-ASK1 (2 μg) in the presence or absence of increasing amounts of wild-type (WT) and kinase-dead (K40R) forms of MPK38 (6 and 9 μg) or MPK38 siRNA 1 (100 and 200 nm). An in vitro kinase assay for JNK activity was performed as described under “Materials and Methods.” The amounts of precipitated JNK and the expression level of ASK1 and MPK38 in total cell lysates were determined by immunoblot analysis using anti-GST and anti-HA antibodies. MPK38(KD) cells transfected with GST-JNK and HA-ASK1 were also precipitated using glutathione-Sepharose beads (GST Purification), and the precipitates were subjected to an in vitro kinase assay using c-Jun as a substrate to determine JNK activity (right). The relative level of JNK activity was quantitated by densitometric analyses, and fold increase relative to control HEK293 cells expressing JNK alone or untreated HaCaT cells expressing JNK and ASK1 was calculated. WB, Western blot.

FIGURE 8.

Effect of MPK38 on H₂O₂-mediated apoptosis. HEK293 cells were transiently transfected with increasing amounts of wild-type MPK38 (0.8 and 1.6 μg) and ASK1 (0.5 and 1 μg) or MPK38 and ASK1 siRNAs (100 and 200 nm) in the presence or absence of H₂O₂. Apoptotic cell death was determined using the GFP expression system (GFP) or TUNEL. Cells exposed to 1 mm H₂O₂ for 9 h was used as a positive control. Data shown are means (±S.E.) of three independent experiments. p < 0.05 relative to control; significance calculated by Student's t test.