As Noxa was first identified as a p53 target gene, the stabilization and activation of p53 would have been an attractive possibility for BAY 73-4506 apoptosis induction by PIs. However, PI-mediated tumor cell killing was also observed in p53-deficient cells and independently of NF-kB inhibition suggesting that other signaling pathways targeted by the proteasome are even more crucial for cell death induction by PIs. One of those might be instigated by members of the mitogen-activated protein kinase family, the c-Jun N-terminal kinases that were reproducibly found to be activated in PI-treated cells. More intriguingly, inhibition of JNK activity by either dominant-negative JNKs or by RNA interference rendered the cells resistant toward cell death induction by PIs. Thus, it appears that JNKs, in addition to several other pathways in which they were shown to contribute to apoptosis signaling, are also crucial players in PI-induced apoptosis. Three JNK isoforms with different splice variants are expressed either ubiquitously or preferentially in neuronal and heart tissues. They were originally identified by their ability to specifically phosphorylate and activate c-Jun, a constituent of the activator protein-1 transcription factor that is involved in the increased expression of several pro-apoptotic genes such as TNF-a, Fasligand, Bak and Bim. Although silencing of the c-Jun/AP-1 pathway conferred resistance to JNK-mediated apoptosis in several cellular systems, the observed stimulus- and cell typedependent manner of protection suggests participation of other downstream effectors. Indeed, JNKs appear to control apoptosis in quite a versatile manner as they not only phosphorylate and activate other pro-apoptotic transcriptions factors including p53 and c-Myc, but also several Bcl-2 family proteins causing inhibition of pro-survival members such as Bcl-2, Bcl-XL and Mcl-1 and activation of pro-apoptotic members such as Bim and Bad. However, although these phosphorylation events are consistent with the observation that JNKs are required for stress-induced activation of the mitochondrial death pathway, their contributions to apoptosis are controversially discussed. In addition, it is unknown whether JNK1 and JNK2 exhibit redundant or specific functions in PI-induced apoptosis and whether they are involved in the induction of Noxa. To elucidate these questions in more detail, we compared PIinduced apoptosis signaling of immortalized mouse embryonic fibroblasts that differ in their JNK1 and/or JNK2 status. In addition to our findings that JNK1 greatly accelerates de novo expression of Noxa and subsequent apoptosis, we also observed that both processes were strongly impaired in the presence of JNK2 implying oppositional roles for these isoforms in PI-induced apoptosis. Inhibition of the proteasome either on its own or in combination with other apoptotic stimuli is a Nutlin-3 powerful means to specifically eradicate tumor cells, but the underlying molecular pathways are only incompletely deciphered. JNKs and the BH3-only protein Noxa were reproducibly demonstrated in many diverse systems to be essential constituents of this process as inhibition of their activity and/or expression substantially protected cells from PI-induced apoptosis. However, these two pathways have never been connected before and the contributions of individual JNK isoforms to PI-mediated induction of Noxa and apoptosis were completely unknown.