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Coiled coil domain

Introduction. Coiled coil domain. Stress signal e.g. H2O2. S S. S S. ASK1. ASK1. ASK1. ASK1. ASK1. ASK1. ASK1. ASK1. Poly ubiquitination, Degradation Cullin degradation. PP5. Conformational change. Trx1. Trx1. Trx1. Trx1. Thr845. P. P. Trans-autophosphorylation. Thr845.

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Coiled coil domain

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  1. Introduction Coiled coil domain Stress signal e.g. H2O2 S S S S ASK1 ASK1 ASK1 ASK1 ASK1 ASK1 ASK1 ASK1 Poly ubiquitination, Degradation Cullin degradation PP5 Conformational change Trx1 Trx1 Trx1 Trx1 Thr845 P P Trans-autophosphorylation Thr845 new interface

  2. Introduction PMX 464 (mM) Stress signal e.g. H2O2, TNFa ASK1 ASK1 ASK1 ASK1 ASK1 ASK1 Trx1 Trx1 P P38 ERK JNK Apoptosis

  3. ← total ASK1 ← Trx1 WCL DMSO DMSO 5μM 0.1mM 1mM 1mM AW464 H2O2 H2O2 H2O2 IP: Trx1 ← p54 phospho JNK ← p46 phospho JNK ← Trx1 DMSO DMSO 5μM 0.1mM 1mM 1mM AW464 H2O2 H2O2 H2O2 Does ROS cause dissociation of Trx from ASK? Are downstream kinases activated?

  4. Does knocking down Trx1 lead to activation of JNK/P38 or ASK? Initial evidence - HCT116, Trx KD doesn’t activate JNK/P38 Opposite effect on ASK activation. No effect on JNK

  5. Overexpression of ASK1 or ASK K709R GeneJuice ASKwt ASK K709R GeneJuice ASKwt ASK K709R HCT116 MCF7 ! phospho ASK1 (Top band) ! total ASK1 ! p54 phospho JNK ! p46 ! p54 total JNK ! p46 ! phospho P38 ! total P38 p42/44phospho ERK  p42/44total ERK  D AW H2O2D AW H2O2 D AW H2O2 D = DMSO AW = 5mM AW464, 3h H2O2 = 1mM H2O2, 1h D AW H2O2D AW H2O2 D AW H2O2 • Transfection of ASK1 does not activate JNK, P38, ERK • Transfection of ASK1 does not potentiate activation of JNK, P38 or ERK in response to quinol or H2O2 • Addition of quinol orH2O2 does not activate ASK1 as would be expected with the apparent dissociation of Trx1 from ASK1 • Unexpected activation of ERK in response toH2O2 seen in MCF7s - probably not via ASK1. • Catalytically inactive, dominant negative ASK K709R does not prevent the activation of JNK, P38 or ERK in response to quinol or H2O2 treatment

  6. Can Quinols dissociate Trx1/ASK1 directly? • HCT116 whole cell lysates (WCL) prepared • Lysates treated with 5mM AW464 for 3h • Immunopreciptiation carried out as before • Needs repeating! ASK1 WCL DMSO 5mM AW464 IP: Trx

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