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Supplementary Figures Supplementary Figure 1. T183 of JNK and WW domain of Pin1 are involved in the interaction. (a) HEK 293 cells transfected with plasmids.

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Presentation on theme: "Supplementary Figures Supplementary Figure 1. T183 of JNK and WW domain of Pin1 are involved in the interaction. (a) HEK 293 cells transfected with plasmids."— Presentation transcript:

1 Supplementary Figures Supplementary Figure 1. T183 of JNK and WW domain of Pin1 are involved in the interaction. (a) HEK 293 cells transfected with plasmids as indicated were treated with 1 mM H 2 O 2 for 1 h or left untreated. Immunoprecipitation and immunoblotting were performed. (b) HEK 293 cells were co-transfected with FLAG-tagged Pin1 WT or two deletion mutants of Pin1, WW or PPIase (amino acid residues 1-54 or , respectively) expression plasmids along with the HA-JNK1 plasmid, and exposed to 1 mM H 2 O 2 for 1 h. Immunoprecipitation and immunoblotting were performed. FLAG-tagged Pin1 WW domain was not shown due to its small size. (c) HEK 293 cells were co-transfected with FLAG-tagged Pin1 WT or two point mutants of Pin1 (W34A or C113A) expression plasmids along with the HA-JNK1 plasmid, and exposed to 1 mM H 2 O 2 for 1 h. Immunoprecipitation and immunoblotting were performed. Supplementary Figure 2. H 2 O 2 has no effect on Pin1 activity. Pin1 -/- MEF cells were transiently transfected with FLAG-only or FLAG-Pin1 expression plasmids and then lysates from H 2 O 2 -treated for 1 h or untreated Pin1 -/- MEF cells were immunoprecipitated with anti-FLAG beads. The immunoprecipitates were incubated with active JNK1, c-Jun, and [γ- 32 P] ATP for 10 min at 30°C. Supplementary Figure 3. (a) Pin1does not interact with TCF 1. After transfection, HEK 293 cells were treated with 1 mM H 2 O 2 for 1 h. Lysates were immunoprecipitated and immunoblotted with appropriate antibodies. (b) JNK1 phosphorylates Ser-232 of TCF 1. Active recombinant JNK1 was incubated with TCF 1 (WT, T242A, or S232/T242A) in 20 µl of kinase reaction buffer. The products of kinase reactions were separated by SDS-PAGE. The gels were dried and exposed to film. Supplementary Figure 4. The data shown in Figure 4b were quantified as a percentage of phospho-JNK1.

2 Supplementary Figure 5. Pin1-induced JNK1 activity is stable. GST, GST-Pin1, or GST-Pin1 (C113A) proteins were pulled down with GST beads and then each bead complex was incubated with active JNK1 for 1 h at room temperature. After removal of GST-fusion proteins by centrifugation, the supernatants were subjected to SDS- PAGE and then immunoblotted with antibodies as indicated. The same supernatant samples were also subjected to in vitro kinase assays. Supplementary Figure 6. Pin1 enhances JNK1/TCFβ1-mediated IL-2 production. After 16 h stimulation with PMA/ionomycin, supernatants were analyzed for IL-2 production using an ELISA assay. The results presented are representative of three independent experiments. Error bars indicate ±SD. Statistical significance was determined by the Student t test. *P <0.05 and **P <0.01 compared with control (TCFβ1 WT alone). #P <0.05 compared with control (TCFβ1 WT + JNK1). Supplementary Figure 7. Hypothetical model for the role of Pin1 in JNK1 activation. The docking domain and the catalytic pocket of inactive JNK1 are depicted by rectangular and skewed triangular indentations, respectively. First, JNK1 is phosphorylated by several stresses, including UV, TNF-α and oxidative stress (H 2 O 2 ), and partially activated by phosphorylation-induced structural changes in the docking domain and the catalytic pocket. The docking domain and the catalytic pocket of partially active JNK1 are indicated by oval and triangular indentations, respectively. Then, Pin1 binds to the pThr 183-Pro motif of JNK1 and induces subsequent cis-trans isomerization of the peptidyl-prolyl bond in the motif (inset). This isomerization induces further conformational change in the docking domain for substrate binding to become fully active. The docking domain of fully active JNK1 is depicted by pentagonal indentation. The JNK1 recognition site and the phosphoacceptor region of the target substrate are indicated by pentagonal and triangular extrusions, respectively. Double arrows denote up-regulation.

3 c W34A IP: FLAG lysate IB: HA JNK1 HA-JNK FLAG-Pin1 WT C113A IB: FLAG Pin1 IB: FLAG Pin1 p-JNK1 IB: p-JNK a T183A Y185F lysate IB: HA IB: FLAG HA-JNK IB: HA WT T183A WT Pin1 JNK1 Y185F IB: p-JNK p-JNK1 FLAG-Pin1 H 2 O 2 IB: FLAG Pin1 IP: FLAG b lysate IB: HA JNK1 HA-JNK1 + + FLAG-Pin1 - WT WW PPIase IB: FLAG Pin1 IB: FLAG Pin1 Supplementary Figure 1

4 a lysate IB: HA Pin1 c-Jun Pin1 IB: FLAG TCF 1 FLAG-c-Jun HA-Pin1 FLAG- TCF 1 (WT) FLAG- TCF 1 (T242A) IP: FLAG H 2 O IB: FLAG c-Jun TCF 1 b [ 32 P]- TCF 1 TCF 1 JNK kinase assay S232A, T242A T242A WT Coomassie staining [ 32 P]-c-Jun FLAG-Pin1 H 2 O Fold: IB: FLAG Pin1 IB: tubulin tubulin Supplementary Figure 2 Supplementary Figure 3

5 Relative p-JNK level (%) (min) Supplementary Figure 4

6 supernatant Pin1 WTPin1 C113AGST (min) [ 32 P]-c-Jun IB : JNK IB : GST Fold IB : p-JNK input Supplementary Figure 5

7 IL-2 (pg/ml) TCF 1 WT JNK1 Pin1 WT TCF 1 (T242A) TCF 1 (S232A/T242A) Pin1 (C113A) Pin1 (W34A) TCF 1 (S232A) * ** # Supplementary Figure 6

8 JNK1 P Pin1 Inactive Isomerization Binding affinity JNK signaling TNF-α, oxidative stress, and UV JNK1 conformational change and full activation Target Partially active Weak interaction JNK1 P Target Pin1 JNK1 P P P Supplementary Figure 7


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