Figure 4.

JNK1/2 phosphorylate Bid at Thr59 both in vitro and in vivo. (a) Levels of Bid and p-Bid (Thr59) in PC-3 cells in response to ALDH1L1. (b) Phosphorylation of Bid at Thr59 by JNK1 and JNK2 (in vitro kinase assay). (c) The phosphorylation of recombinant Bid by JNK2 (in vitro kinase assay) prevents its cleavage by caspase-8; the treatment of p-Bid (Thr59) with λ-phosphatase dephosphorylates Thr59 and enables the cleavage by caspase-8. (d) Treatment of PC-3 cells with specific JNK inhibitor SP600125 prevents both Bid phosphorylation at Thr59 and its accumulation in response to ALDH1L1. (e) Cell-permeable caspase-8 inhibitor increases levels of Bid in the absence of the protein phosphorylation. (f) Levels of transiently expressed wild-type (WT) Bid and its mutants in PC-3 cells treated with 50 μM cycloheximide (CHX). (g) Co-immunoprecipitation of activated JNK1 and JNK2 (pJNK1 and pJNK2) using Bid-specific antibody at different times of ALDH1L1 expression. (h) Activated JNK1/2, pulled down from ALDH1L1-expressing PC-3 cells using phospho-JNK-specific antibody, phosphorylates recombinant Bid at Thr59