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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. Untransfected ASKwt ASK DN K709R Untransfected ASKwt ASK DN K709R Untransfected ASKwt ASK DN K709R Untransfected ASKwt ASK DN K709R Untransfected ASKwt ASK DN K709R Untransfected ASKwt ASK DN K709R Untransfected ASKwt ASK DN K709R C AW H2O2 C AW H2O2C AW H2O2 C AW H2O2 C AW H2O2C AW H2O2 C AW H2O2 C AW H2O2C AW H2O2 C AW H2O2 C AW H2O2C AW H2O2 C AW H2O2 C AW H2O2C AW H2O2 C AW H2O2 C AW H2O2C AW H2O2 C AW H2O2 C AW H2O2C AW H2O2 Effects of AW464/H2O2 on ASK Phosphorylation HEK293 HCT116 MCF7 050207 180107 180107 200407 200407 160507 160507

  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. MCF7

  6. GeneJuice ASKwt ASK K709R GeneJuice ASKwt ASK K709R ! phospho ASK1 (Top band) ! total ASK1 ! p54 phospho JNK ! p46 ! p54 total JNK ! p46 ! phospho P38 ! total P38 D AW H2O2D AW H2O2 D AW H2O2 p42/44phospho ERK  p42/44total ERK  D AW H2O2D AW H2O2 D AW H2O2 Effects of

  7. Untransfected ASKwt ASK DN K709R Untransfected ASKwt ASK DN K709R C AW H2O2 C AW H2O2C AW H2O2 C AW H2O2 C AW H2O2C AW H2O2 HCT116 180107 200407 160507

  8. Untransfected ASKwt ASK DN K709R MCF7 180107 200407 160507 GeneJuice ASK1 ASK1 wt K709R ! phospho ASK1 (Top band) ! total ASK1 ! P54 phospho JNK ! P46 ! P54 total JNK ! P46 ! phospho P38 ! total P38 m m m DMSO 5 M 1mM DMSO 5 M 1mM DMSO 5 M 1mM AW464 H O AW464 H O AW464 H O 2 2 2 2 2 2 GeneJuice ASK1 ASK1 wt K709R ! phospho ASK1 ! total ASK1 ! P54 phospho JNK ! P46 ! P54 total JNK ! P46 ! phospho P38 ! total P38 H phospho p42/44 ERK1/2 H total p42/44 ERK1/2 C AW H2O2 C AW H2O2C AW H2O2 m m m DMSO 5 M 1mM DMSO 5 M 1mM DMSO 5 M 1mM AW464 H O AW464 H O AW464 H O 2 2 2 2 2 2

  9. 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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