Ab-1 anti-p53 mouse monoclonal antibody was from Oncogene Science. DePinho 2002; Vousden and Lu 2002). Activated p53 functions as a transcription factor to regulate the expression of many different downstream genes, whose products are implicated in cell cycle arrest, DNA repair, or apoptosis (Vousden and Lu 2002). To achieve proper function, p53 is tightly regulated by means of post-translational modifications, cofactor binding, and subcellular localization. The function of p53 is tightly controlled by Mdm2, an E3 ubiquitin ligase implicated in the inactivation of the tumor suppressor by accelerating its nuclear export and degradation by the 26S proteasome (Michael and Oren 2002). Phosphorylation of p53 within its amino-terminal domain facilitates p53 stabilization by disrupting p53-Mdm2 interaction (Wahl and Carr 2001; Michael and Oren 2002) and prevents its nucleocytoplasmic export (Zhang RU 24969 and Xiong 2001). Similar to nuclear DNA damage, stress conditions in other organelles are able to activate signal-transduction pathways leading to the induction of genes encoding for proteins that play key roles in damage sensing and apoptosis (Ferri and Kroemer 2001). For example, expression of mutant proteins, viral infection, energy or nutrient deprivation, extreme environmental conditions, or Ca2+ release from the lumen of the endoplasmic reticulum (ER) disrupt proper protein-folding activity in this organelle (Ferri and Kroemer 2001; Kaufman et al. 2002). This leads to the accumulation of unfolded proteins, which initiates transcriptional and translational-signaling pathways known as the unfolded protein response STAT6 (UPR; Ferri and Kroemer 2001; Kaufman et al. 2002). UPR is an adaptive response that involves the up-regulation of the expression, and thus function of ER-resident chaperons that augment ER-folding capacity (Ferri and Kroemer 2001; Kaufman et al. 2002). Also, UPR induces the expression of genes engaged in ER-associated protein degradation (Travers et al. 2000) and attenuates translation by inducing the phosphorylation of the subunit of translation initiation factor eIF2 through the activation of the pancreatic ER-resident kinase PERK (Harding et al. 2002). If these adaptive mechanisms are not sufficient to alleviate ER stress, then an apoptotic program is initiated through the activation of the JNK pathway and caspases 7, 12, and 3 (Ferri and Kroemer 2001; Harding et al. 2002; Kaufman et al. 2002). Given the role of p53 in stress sensing and proapoptotic signaling, we were interested to investigate whether p53 responds to ER stress. Herein, we report that ER stress induced by pharmacological or physiological means signals to p53. We demonstrate that ER stress induces the RU 24969 destabilization of p53 protein and prevents cells from p53-dependent apoptosis. This is mediated, at least in part, through the increased cytoplasmic localization of p53 as a result of phosphorylation at serines 315 and 376. We also demonstrate that ER stress induces glycogen synthase-3 (GSK-3) kinase activity, which phosphorylates p53 at serine 376 in vitro and mediates p53 phosphorylation at serines 315 and 376 in vivo. Furthermore, we show that GSK-3 interacts physically with p53 in the nucleus of ER-stressed cells, promotes the cytoplasmic localization of the protein, and prevents p53-mediated apoptosis. Our findings reveal a novel mechanism utilized by cells to adapt to ER stress through the inactivation of the tumor-suppressor protein by GSK-3. Results ER stress enhances the cytoplasmic localization of p53 We first noticed that ER stress induces the cytoplasmic localization of p53. Specifically, RU 24969 human diploid WI-38 cells (Fig. 1A) or human fibrosarcoma HT1080 cells (Fig. 1B) were treated with pharmacological inducers of ER stress, such as the protein glycosylation inhibitor tunicamycin (TM), the.