1. Volume 36, Issue 5, Pages 805-818 (December 2009)
Ubiquitin-like Sequence in ASK1 Plays Critical Roles in the Recognition and Stabilization by USP9X and Oxidative Stress-Induced Cell Death 
Hiroaki Nagai, Takuya Noguchi, Kengo Homma, Kazumi Katagiri, Kohsuke Takeda, Atsushi Matsuzawa, Hidenori Ichijo 
Molecular Cell 
Volume 36, Issue 5, Pages (December 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Oxidative Stress-Dependent Recruitment of USP9X to the ASK1 Signalosome
(A) HEK293 cells stably expressing FLAG-ASK1 were treated with or without 1 mM H2O2 for 15 min, and ASK1 complexes were affinity purified with FLAG antibody, followed by SDS-PAGE and silver staining. Open arrowheads indicate H2O2-dependent ASK1-interacting proteins.
(B) Extracts from RAW264.7 cells treated with or without 10 mM H2O2 for 5 min were immunoprecipitated with ASK1 antibody or control rat IgG. Coimmunoprecipitated USP9X with ASK1 was detected by USP9X antibody (top panel). The immunoprecipitated ASK1 was confirmed by immunoblotting with ASK1 antibody (bottom panel).
(C) HEK293A cells were transiently transfected with FLAG-ASK1WT. After 24 hr, cells were treated with or without 3 mM H2O2 for 20 min, and then the cell extracts were prepared as described in the Supplemental Experimental Procedures. After fractionation on a Superose6 10/300 GL column, each fraction was immunoprecipitated with FLAG antibody and immunoblotted with the indicated antibodies. Apparent molecular mass was evaluated after column calibration with standard proteins: thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), and aldolase (158 kDa). The elution positions of these proteins are indicated at the top of the figure.
(D and E) HEK293A cells were transiently transfected with FLAG-ASK1WT. After 24 hr, extracts from the cells treated with 1 mM or the indicated concentrations of H2O2 for the indicated periods were immunoprecipitated with FLAG antibody and immunoblotted with the indicated antibodies. The lower two panels (input) show that equal amounts of proteins were used to perform the immunoprecipitation.
(F) HEK293A cells were transiently transfected with FLAG-ASK1WT, FLAG-ASK1KM (kinase negative mutant: K709M), FLAG-ASK1T838A (kinase negative mutant: T838A), or empty vector. After 24 hr, extracts from the cells treated with or without 3 mM H2O2 for 5 min were immunoprecipitated with FLAG antibody and immunoblotted with the indicated antibodies. IB, immunoblotting; IP, immunoprecipitation. Data are representative of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
The GG Motif of ASK1 Is Essential for the Interaction with USP9X
(A) Schematic representation of human ASK1 and its deletion mutants analyzed in Figure 2. ASK11295–1374 contains the identical sequence to the ubiquitin C terminus: LRLRGG (amino acid 1352–1357 in human ASK1). CC, coiled-coil domain.
(B) HEK293A cells were transiently transfected with the indicated FLAG-ASK1 constructs. After 24 hr, cell extracts treated with or without 1 mM H2O2 for 5 min were immunoprecipitated (IP) with FLAG antibody and immunoblotted (IB) with the indicated antibodies. The lower two panels (input) show that equal amounts of proteins were used to perform the immunoprecipitation.
(C, D and E) HEK293A cells were transiently transfected with the indicated FLAG-ASK1 constructs. After 24 hr, extracts from the cells treated with or without 1 mM H2O2 for 5 min were analyzed as described in (B). Data are representative of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 USP9X Is Required to Maintain the Activation Status of ASK1
(A) HEK293A cells were transfected with the indicated FLAG-ASK1 constructs. After 48 hr, the cells were treated with 1 mM H2O2 for the indicated periods. The cell extracts were subjected to immunoblotting (IB) with FLAG antibody and phospho-ASK1 antibody.
(B) HeLa cells were transfected with siRNAs as a negative control or to target ASK1 or USP9X. After 48 hr, the cells were treated with 1 mM H2O2 for the indicated periods. The cell extracts were subjected to immunoblotting with the indicated antibodies.
(C) HEK293A cells were transfected with FLAG-ASK1WT or ASK1ΔGG. After 48 hr, the cells were pretreated with 100 μM MG132 for 2 hr and then treated with 1 mM H2O2 for the indicated periods. The cell extracts were subjected to immunoblotting with FLAG antibody and phospho-ASK1 antibody. Data are representative of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
USP9X Is Required for Stabilization of ASK1 under Oxidative Stress
(A) HEK293A cells transiently transfected with FLAG-ASK1 were pulse-labeled with 35S-methionine/cysteine, and chased in the presence or absence of 0.5 mM H2O2 for the indicated periods. The cell extracts were immunoprecipitated with FLAG antibody, subjected to SDS-PAGE, and then analyzed by autoradiography (left). In panels (A) to (D), relative protein levels of ASK1 were quantified from the results of three independent experiments and graphed (right). ARG, autoradiography.
(B) HEK293A cells transiently transfected with FLAG-ASK1 were treated with 5 μg/ml cycloheximide (CHX) and 0.5 mM H2O2 for the indicated periods in the presence of 10 μM MG132 or DMSO. The cell extracts were subjected to immunoblotting with FLAG antibody and α-tubulin antibody as a loading control (left).
(C) HEK293A cells were transfected with siRNAs as a negative control or to target USP9X. After 48 hr, cells were treated with 5 μg/ml CHX and 0.5 mM H2O2 for the indicated periods. The cell extracts were subjected to immunoblotting with the indicated antibodies (left).
(D) HEK293A cells transiently transfected with FLAG-ASK1WT or FLAG-ASK1ΔGG were treated with 5 μg/ml CHX and 0.5 mM H2O2 for the indicated periods. The cell extracts were subjected to immunoblotting with FLAG antibody and α-tubulin antibody as a loading control (left). Error bars indicate SEM (∗p < 0.05; ∗∗p < 0.01, Student's t test).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 The C-Terminal Region of ASK1 Is Ubiquitinated
(A) HeLa cells were treated with 1 mM H2O2 for the indicated periods and then were lysed in IP lysis buffer supplemented with 10 mM N-ethylmaleimide (NEM). The cell extracts were immunoprecipitated (IP) with ASK1 antibody or control rat IgG followed by immunoblotting (IB) with the indicated antibodies. The bottom panel (input) shows that equal amounts of proteins were used to perform the immunoprecipitation.
(B, D, and E) HEK293A cells cotransfected with the indicated constructs were treated with 1 mM H2O2 for 15 min. The cell extracts were subjected to in vivo ubiquitination assays as described in Experimental Procedures and then to immunoblotting with the indicated antibodies. Open arrowheads in (D) indicate monoubiquitinated ASK1. Re-IP, reimmunoprecipitation.
(C) Schematic representation of human ASK1 and its deletion mutants analyzed in Figure 5.
(F) HEK293A cells were transfected with FLAG-ASK1WTor ASK115KR. After 48 hr, the cells were treated with 1 mM H2O2 for the indicated periods. The cell extracts were subjected to immunoblotting with FLAG antibody and phospho-ASK1 antibody. Data are representative of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 USP9X Cleaves Ubiquitin from the ASK1 C-Terminal
(A) USP9X and FLAG-ASK1 independently purified from HEK293A cells were subjected to in vitro deubiquitination assay. Ubiquitinated ASK1 was incubated with purified USP9X or control for 2 hr at room temperature. The reaction was terminated by the addition of SDS sample buffer and was immunoblotted with the indicated antibodies. The bottom panel shows that equal amounts of proteins were used to perform the in vitro deubiquitination assay.
(B) HeLa cells were transfected with siRNAs as a negative control or to target USP9X. After 72 hr, cells were treated with or without 1 mM H2O2 for the indicated periods and then were lysed in IP lysis buffer supplemented with 10 mM NEM. The cell extracts were immunoprecipitated with ASK1 antibody or control rat IgG and were immunoblotted with the indicated antibodies.
(C and D) HEK293A cells cotransfected with the indicated constructs were treated with 1 mM H2O2 for 15 min. The cell extracts were subjected to in vivo ubiquitination assays and then to immunoblotting with the indicated antibodies.
(E) HEK293A cells transiently transfected with the indicated FLAG-ASK1 constructs were treated with 5 μg/ml CHX and 0.5 mM H2O2 for the indicated periods. The cell extracts were subjected to immunoblotting with FLAG antibody and α-tubulin antibody as a loading control (left). Relative protein levels of ASK1 were quantified from the results of three independent experiments and graphed (right). Error bars indicate SEM (∗∗p < 0.01, Student's t test). Data are representative of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

8. Figure 7
USP9X Is Required for Oxidative Stress-Induced MAPK Activation and Cell Death
(A) HeLa cells were transfected with siRNAs as a negative control or to target ASK1 or USP9X. After 48 hr, the cells were treated with 1 mM H2O2 for the indicated periods. The cell extracts were subjected to immunoblotting with the indicated antibodies (upper panels). The relative activities of p38 and JNK (p38 activity at 30 min and JNK activity at 120 min in siCtrl cells were set to 100%) are shown in the graph (lower panels). Error bars indicate SEM.
(B) HeLa cells were transfected with siRNAs as a negative control or to target ASK1 or USP9X. After 72 hr, the cells were treated with 3 mM H2O2 for the indicated periods. H2O2-induced cell death was quantified by LDH assay. Error bars indicate SEM (∗p < 0.05; ∗∗p < 0.01, versus control siRNA, Student's t test).
(C) Immunoblot analysis of phospho-p38 and -JNK in extracts from wild-type (WT) and USP9X knockout (KO) ES cells treated with 0.5 mM H2O2 for the indicated periods.
(D) WT and USP9X KO ES cells were treated with 1 mM H2O2 for 12 hr, and cell death was quantified by LDH assay as in (B). Error bars indicate SEM (∗∗p < 0.01, versus H2O2-treated WT, Student's t test).
(E) A proposed model for USP9X-mediated stabilization of activated ASK1. See Discussion for details. Data are representative of at least three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions