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. 2001 Feb;21(3):893-901.
doi: 10.1128/MCB.21.3.893-901.2001.

Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1

A H Kim  1 , G Khursigara, X Sun, T F Franke, M V Chao
Affiliations

Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1

A H Kim et al. Mol Cell Biol. 2001 Feb.

Abstract

The Akt family of serine/threonine-directed kinases promotes cellular survival in part by phosphorylating and inhibiting death-inducing proteins. Here we describe a novel functional interaction between Akt and apoptosis signal-regulating kinase 1 (ASK1), a mitogen-activated protein kinase kinase kinase. Akt decreased ASK1 kinase activity stimulated by both oxidative stress and overexpression in 293 cells by phosphorylating a consensus Akt site at serine 83 of ASK1. Activation of the phosphoinositide 3-kinase (PI3-K)/Akt pathway also inhibited the serum deprivation-induced activity of endogenous ASK1 in L929 cells. An association between Akt and ASK1 was detected in cells by coimmunoprecipitation. Phosphorylation by Akt inhibited ASK1-mediated c-Jun N-terminal kinase and activating transcription factor 2 activities in intact cells. Finally, activation of the PI3-K/Akt pathway reduced apoptosis induced by ASK1 in a manner dependent on phosphorylation of serine 83 of ASK1. These results provide the first direct link between Akt and the family of stress-activated kinases.

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Figures

FIG. 1
FIG. 1
Akt phosphorylates ASK1 in cells and in vitro. (A) ASK1 phosphorylation in cells stimulated by serum. 293 cells transiently transfected for 36 h with 1 μg each of the indicated constructs were deprived of phosphate and serum for 1 h, incubated with or without LY294002 (20 μM) for 1 h, and exposed to [32P]orthophosphate and dialyzed serum for 2 h. Immunoprecipitated ASK1 was assessed for 32P labeling (upper panel) and protein levels with anti-HA 3F10 (lower panel). The histogram shows mean densitometric ratios (32P-labeling/protein) plus standard errors of the mean (SEM) from three independent experiments. (B) A consensus Akt phosphorylation site is found in ASK1. Several known Akt substrates are shown. The phosphorylated serine/threonine site is highlighted. (C) Akt phosphorylates ASK1 on serine 83 in IGF-1-stimulated cells. Samples were processed as for panel A except that cells were stimulated for 10 min with 100 ng of IGF-1/ml after [32P]orthophosphate incubation. Dialyzed serum was not present during labeling. Data are representative of three independent experiments. IP, immunoprecipitation; W, Western blot. (D) In vitro phosphorylation of ASK1 by Akt. GST-ASK1 (aa 20 to 118) and GST-ASK1S83A were used as substrates for preactivated recombinant human Akt1 (Akt). [γ-32P]ATP incorporation (upper panel) and protein levels determined by Coomassie blue staining for substrates (middle panel) and Akt (lower panel) are shown. Data are representative of three independent experiments.
FIG. 2
FIG. 2
Akt interacts with ASK1 in cells. (A) Endogenous association of Akt and ASK1 in L929 cells. Cells were serum starved for 36 h and then treated with or without 100 ng of IGF-1/ml for 7 min. Endogenous Akt was immunoprecipitated (IP) from lysates with anti-Akt, and proteins were detected with anti-ASK1 H-300 and anti-Akt. To confirm that both active and inactive Akt were immunoprecipitated, membranes were stripped and reprobed with anti-phospho-Akt(S473) (anti-pAkt). As a positive control, crude lysate (left) was probed for ASK1 and Akt by immunoblotting. Aktp represents immunoprecipitation with anti-Akt antibodies preincubated with an Akt peptide. All data are representative of three independent experiments. W, Western blot. (B) Akt and ASK1 associate independently of ASK1 serine 83 phosphorylation in transfected 293 cells. Cells were transfected with 1 μg each of the indicated constructs for 36 h, and lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. (C) Akt and ASK1 binding occurs independently of kinase activity in transfected 293 cells. Samples were processed as for panel B.
FIG. 2
FIG. 2
Akt interacts with ASK1 in cells. (A) Endogenous association of Akt and ASK1 in L929 cells. Cells were serum starved for 36 h and then treated with or without 100 ng of IGF-1/ml for 7 min. Endogenous Akt was immunoprecipitated (IP) from lysates with anti-Akt, and proteins were detected with anti-ASK1 H-300 and anti-Akt. To confirm that both active and inactive Akt were immunoprecipitated, membranes were stripped and reprobed with anti-phospho-Akt(S473) (anti-pAkt). As a positive control, crude lysate (left) was probed for ASK1 and Akt by immunoblotting. Aktp represents immunoprecipitation with anti-Akt antibodies preincubated with an Akt peptide. All data are representative of three independent experiments. W, Western blot. (B) Akt and ASK1 associate independently of ASK1 serine 83 phosphorylation in transfected 293 cells. Cells were transfected with 1 μg each of the indicated constructs for 36 h, and lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. (C) Akt and ASK1 binding occurs independently of kinase activity in transfected 293 cells. Samples were processed as for panel B.
FIG. 3
FIG. 3
Regulation of ASK1 kinase activity by Akt is dependent on ASK1 serine 83. (A) H2O2-stimulated ASK1 activity is inhibited by Akt. 293 cells were transfected with 0.25 μg of ASK1-HA or ASK1S83A-HA with or without 1 μg of myc-Akt for 20 h. Where indicated, cells were then exposed to 300 μM H2O2 for 10 min. ASK1-HA was immunoprecipitated with anti-HA and subjected to a coupled in vitro kinase assay. After samples were resolved by SDS-PAGE, kinase activity was visualized by PhosphorImager analysis and normalized to ASK1 protein levels in the same sample by anti-HA immunoblotting. Values from single transfections were normalized to ASK1-HA without H2O2 (=1.0), and those from double transfections were normalized to ASK1-HA and myc-Akt cotransfection without H2O2 (=1.0). The histogram shows the means plus the SEM from three to five independent experiments. (B) The activity of high ectopic ASK1 is diminished by Akt. 293 cells were transiently transfected with 1 μg each of the indicated constructs for 20 h. Cells were processed for the ASK1 in vitro kinase assay as for panel A. The histogram shows the means plus the SEM from five independent experiments. All values were calculated in reference to ASK1 transfection alone (=1.0). The asterisk indicates a significant difference from ASK1 at P < 0.01 by one-way analysis of variance (ANOVA) followed by the Bonferroni t test. (C) Activation of the P13-K/Akt pathway inhibits serum deprivation-induced ASK1 activity. L929 cells were deprived of serum for 20 min (-S) with or without 100 ng of IGF-1/ml. Where indicated, cells were treated with wortmannin (W)(200 nM) 10 min prior to and during serum deprivation. Wortmannin treatment alone was carried out in DMEM plus 10% FBS. Fold activation was determined in reference to control cells (=1.0). Immunoprecipitated (IP) ASK1 protein in the kinase assay samples was detected with anti-ASK1. To verify the effects of pharmacological treatments, Akt activity was detected with anti-phospho-Akt(S473) (anti-pAkt), and Akt protein was identified with anti-Akt. Data are representative of three independent experiments.
FIG. 3
FIG. 3
Regulation of ASK1 kinase activity by Akt is dependent on ASK1 serine 83. (A) H2O2-stimulated ASK1 activity is inhibited by Akt. 293 cells were transfected with 0.25 μg of ASK1-HA or ASK1S83A-HA with or without 1 μg of myc-Akt for 20 h. Where indicated, cells were then exposed to 300 μM H2O2 for 10 min. ASK1-HA was immunoprecipitated with anti-HA and subjected to a coupled in vitro kinase assay. After samples were resolved by SDS-PAGE, kinase activity was visualized by PhosphorImager analysis and normalized to ASK1 protein levels in the same sample by anti-HA immunoblotting. Values from single transfections were normalized to ASK1-HA without H2O2 (=1.0), and those from double transfections were normalized to ASK1-HA and myc-Akt cotransfection without H2O2 (=1.0). The histogram shows the means plus the SEM from three to five independent experiments. (B) The activity of high ectopic ASK1 is diminished by Akt. 293 cells were transiently transfected with 1 μg each of the indicated constructs for 20 h. Cells were processed for the ASK1 in vitro kinase assay as for panel A. The histogram shows the means plus the SEM from five independent experiments. All values were calculated in reference to ASK1 transfection alone (=1.0). The asterisk indicates a significant difference from ASK1 at P < 0.01 by one-way analysis of variance (ANOVA) followed by the Bonferroni t test. (C) Activation of the P13-K/Akt pathway inhibits serum deprivation-induced ASK1 activity. L929 cells were deprived of serum for 20 min (-S) with or without 100 ng of IGF-1/ml. Where indicated, cells were treated with wortmannin (W)(200 nM) 10 min prior to and during serum deprivation. Wortmannin treatment alone was carried out in DMEM plus 10% FBS. Fold activation was determined in reference to control cells (=1.0). Immunoprecipitated (IP) ASK1 protein in the kinase assay samples was detected with anti-ASK1. To verify the effects of pharmacological treatments, Akt activity was detected with anti-phospho-Akt(S473) (anti-pAkt), and Akt protein was identified with anti-Akt. Data are representative of three independent experiments.
FIG. 4
FIG. 4
Akt diminishes ASK1-mediated JNK and ATF-2 activation. (A) Akt activity decreased ASK1-dependent JNK activity. 293 cells were transiently transfected for 24 h with 1 μg each of the indicated constructs, serum starved for 6 h, and treated with 100 ng of IGF-1/ml for 15 min. GST-JNK3 was immunoprecipitated (IP) from cell lysates and immunoblotted (W) with anti-phospho-JNK (anti-pJNK). After immunoblots were stripped, GST-JNK3 protein was identified with anti-GST. Crude lysates were probed with anti-HA to determine ASK1 protein levels and with anti-Akt to determine Akt protein levels. Data are representative of three independent experiments. (B) Akt decreased ASK1-induced ATF-2 activity. 293 cells were transiently transfected with ATF-2 luciferase plasmids and the indicated constructs for 24 h and then incubated in 1% FBS for 24 h. Cells were lysed and assayed for luciferase activity. The fold activation was determined in reference to the vector (pcDNA3) alone (=1.0). All bars represent means plus SEM from three to five independent experiments. The asterisk indicates a significant difference from ASK1 at P < 0.01 by one-way ANOVA followed by the Bonferroni t test.
FIG. 5
FIG. 5
Activation of the PI3-K/Akt pathway decreases ASK1-mediated apoptosis. (A) 293 cells were cotransfected with 1.75 μg of lacZ or HA-tagged ASK1 constructs with 0.1 μg of pDsRed as a marker for 24 h. Cells were incubated with or without LY294002 (10 μM) for 1 h, and then all cells were exposed to 10 ng of IGF-1/ml for 36 h. Cell death was assessed by TUNEL-FITC staining and flow cytometry for both TUNEL positivity and DsRed positivity to assay transfected cells only (upper panel). All bars represent means plus SEM from four to seven independent experiments. The asterisk indicates a significant difference from ASK1 at P < 0.05 by one-way ANOVA followed by the Bonferroni t test. In parallel, sister cultures were transfected with solutions identical to those used in cultures later assessed for cell death. Cells were lysed in 1% NP-40 buffer after 24 h, and lysates were probed for expression of ASK1 constructs with anti-HA (bottom panel). (B) HeLa cells were cotransfected with 0.75 μg of the indicated ASK1 constructs plus 0.25 μg of EGFP-IRES-HA-Akt (E40K) (active Akt) or EGFP-IRES (Vector) for 24 h and then serum starved for 24 h. Cells were labeled with Hoechst 33342, and EGFP-positive cells were scored for apoptosis by nuclear morphology. In each experiment, 100 EGFP-positive cells were counted per condition. The histogram represents means plus SEM from four independent experiments. As described for panel A, sister cultures were transfected with solutions identical to those used in cultures assessed for cell death. Cell lysates were probed for expression of ASK1 constructs and active Akt with anti-HA (bottom panel).
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