1. ASK1 Is Essential for JNK/SAPK Activation by TRAF2
Hideki Nishitoh, Masao Saitoh, Yoshiyuki Mochida, Kohsuke Takeda, Hiroyasu Nakano, Mike Rothe, Kohei Miyazono, Hidenori Ichijo 
Molecular Cell 
Volume 2, Issue 3, Pages (September 1998)
DOI: /S (00)80283-X

2. Figure 1 Activation and Interaction of ASK1 with TRAF Family Proteins
(A) Activation of ASK1 by TRAF proteins. HA-tagged ASK1 expression plasmid (pcDNA3-HA-ASK1) (1 μg; lanes 2–7) was transiently cotransfected with Flag-tagged TRAF expression plasmid (pRK-Flag-TRAF1 and -TRAF2, and pCR3-Flag-TRAF3, -TRAF5, and -TRAF6) (1 μg; lanes 3–7) into 293 cells. After 12 hr, ASK1 was immunoprecipitated by anti-HA antibody. The immune complex was incubated with GST-MKK6, and then the kinase activity was measured with the substrate GST-p38γKN. Samples were analyzed by SDS-PAGE (8.5%) and an image analyzer. Top, in vitro kinase assay (IVK) for ASK1 activity. Bottom, immunoblotting (WB) of immunoprecipitated HA-ASK1 in the same sample. Kinase activity relative to the amount of ASK1 protein was calculated, and the activity is shown as fold increase relative to that of HA-ASK1 from TRAF-negative cells (lane 2).
(B) Interaction of ASK1 with TRAF proteins. pcDNA3-HA-ASK1 (1 μg; lanes 2, 4, 6, 8, 10, and 12) was transiently cotransfected with each TRAF expression vector (1 μg; lanes 3–12) into 293 cells. After 36 hr, transfected cells were extracted with lysis buffer and immunoprecipitated (IP) with anti-Flag antibody, and immunoblotted (WB) with anti-HA antibody (top). The presence of Flag-TRAF (middle) and HA-ASK1 (bottom) in the same lysates is shown. Markers of molecular mass are shown on the left.
(C) Specific interaction of ASK1 with TRAF cells were transiently cotransfected with pcDNA3-HA-ASK1 (1 μg; lanes 1–4) and pRK-Flag-TRAF2 (1 μg; lanes 2 and 4). Lysates were divided and immunoprecipitated with anti-Flag antibody (lanes 1 and 2) or control antibody (lanes 3 and 4). The interaction was detected by immunoblotting with anti-HA antibody and anti-Flag antibody. The presence of HA-ASK1 and Flag-TRAF2 in the same lysates was verified by immunoblotting.
Molecular Cell 1998 2, DOI: ( /S (00)80283-X)

3. Figure 2 Binding Sites between TRAF2 and ASK1
(A) Schematic representation of wild-type and mutant TRAF2 proteins. Their abilities to interact with and activate ASK1 when cotransfected into 293 cells are shown. (+), activation of or interaction with ASK1. (−), lack of such activities.
(B) Interaction of ASK1 with wild-type and mutant TRAF2. pcDNA3-HA-ASK1 (1 μg; lanes 2, 4, 6, and 8) was transiently cotransfected into 293 cells with expression plasmids for Flag-tagged wild-type and mutant TRAF2 (1 μg; lanes 3–10). Lysates were immunoprecipitated and immunoblotted as described in Figure 1B.
(C) Schematic representation of wild-type and mutant ASK1 proteins. The kinase domain is shown by the hatched boxes. ASK1KM represents a catalytically inactive mutant in which Lys-709 has been replaced by Met. Positive interaction with wild-type TRAF2 in 293 cells is shown by a (+).
(D) Interaction of TRAF2 with wild-type and mutant ASK1. pRK-Flag-TRAF2 (1 μg; lanes 2, 4, 6, 8, and 10) was transiently cotransfected into 293 cells with HA-tagged wild-type and mutant ASK1 (1 μg; lanes 3–10). Lysates were immunoprecipitated and immunoblotted as described in Figure 1B. Markers of molecular mass are shown on the left.
Molecular Cell 1998 2, DOI: ( /S (00)80283-X)

4. Figure 3
Dominant-Negative Effects of TRAF2 and ASK1 Mutants in TNF-Induced JNK Pathway
(A) Inhibition of the TNF-induced activation of ASK1 by a dominant-negative TRAF2 mutant. Indicated amounts of pRK-Flag-TRAF2(87–501) were transiently cotransfected into 293 cells with pcDNA3-HA-ASK1 (1 μg). After 12 hr, the cells were incubated with (+) or without (−) TNF (200 ng/ml) for 15 min. ASK1 was immunoprecipitated with anti-HA antibody and assayed for kinase activity as described in Figure 1A. Top, phosphorylation of GST-p38γKN. Middle, immunoblotting of immunoprecipitated HA-ASK1 in the same sample. Bottom, immunoblotting of Flag-TRAF2(87–501) in the same lysate. Kinase activity relative to the amount of ASK1 protein is shown as fold increase relative to that of HA-ASK1 from TRAF2(87–501)- and TNF-negative cells (lane 2).
(B) ASK1KM inhibits TNF-induced JNK activation. HA-tagged JNK expression plasmid (pcDNA3-HA-JNK; 1 μg; lane 2–6) was transiently cotransfected into 293 cells with the indicated amounts of ASK1KM expression plasmid (pcDNA3-ASK1KM; lane 3, 5, and 6). After 12 hr, cells were treated with TNF (200 ng/ml) for 15 min. Immunoprecipitated HA-JNK activity was measured by GST-cJUN phosphorylation as a direct substrate for JNK. Samples were analyzed by SDS-PAGE (10%) and an image analyzer. Expression of ASK1KM was verified by the immunoblotting with anti-ASK1 antiserum (DAV). Top, phosphorylation of GST-cJUN. Middle, immunoblotting of HA-JNK in the same sample. Bottom, expression of ASK1KM in the same lysate. Kinase activity relative to the amount of JNK protein is shown as fold increase relative to that of HA-JNK from ASK1KM- and TNF-negative cells (lane 2).
(C) ASK1KM inhibits TRAF2-induced JNK activation. 293 cells were transiently cotransfected with pcDNA3-HA-JNK (2 μg; lane 2–7), pcDNA3-Flag-TRAF2 (0.25 μg; lane 4–7), and the indicated amounts of pcDNA3-ASK1KM (lane 3, 5–7). The relative kinase activity of JNK was determined by immune complex kinase assay as described in (B).
Molecular Cell 1998 2, DOI: ( /S (00)80283-X)

5. Figure 4 TNF-Dependent Endogenous Interaction between ASK1 and TRAF2
(A) Identification of endogenous ASK1 protein in mouse L929 cells. Lysate from L929 cells (2 × 108) was immunoprecipitated with the polyclonal anti-ASK1 antiserum (DAV) with (+) or without (−) blocking peptide (5 μg/ml) against the antiserum, and immunoblotted with DAV. Arrowheads indicate two major ASK1 proteins. Another specific band with an apparent size of 120 kDa may represent a degradation product of ASK1 or an unidentified ASK1-related molecule. Markers of molecular mass are shown on the left.
(B) TNF-induced interaction of ASK1 with TRAF2. L929 cells (2 × 108) were treated with TNF (200 ng/ml) for the indicated time periods (lanes 1 and 3–6) or left untreated (lane 2). Cell lysates were immunoprecipitated with the nonimmune antiserum (lane 1) or the polyclonal anti-TRAF2 antiserum (lanes 2–6). Copurified ASK1 proteins were detected by immunoblotting with anti-ASK1 antiserum (top). Arrowheads indicate ASK1 proteins that specifically associated with endogenous TRAF2. Consistent expression of ASK1 (middle) and TRAF2 (bottom) after TNF treatment was confirmed by immunoblotting using similarly treated cell lysates. This experiment was performed four times with similar results.
(C) TNF-specific interaction of ASK1 with TRAF2. L929 cells (2 × 108) were exposed for 15 min to TNF (200 ng/ml; lane 2), IL-1α (100 ng/ml; lane 3), or H2O2 (1 mM; lane 4). Coimmunoprecipitated ASK1 (top) with TRAF2 was determined as described in Figure 5B. The presence of ASK1 and TRAF2 proteins in the same lysate was verified by the immunoblotting. The endogenous ASK1 from similarly treated cells was immunoprecipitated by DAV, and the kinase activity was measured (bottom) as described in Figure 1A.
Molecular Cell 1998 2, DOI: ( /S (00)80283-X)

6. Figure 5 Schematic Representation of the Role of ASK1 in TNF Signaling
See text for details.
Molecular Cell 1998 2, DOI: ( /S (00)80283-X)