The tumor suppressor p53, a sensor of multiple forms of cellular stress, is regulated by post-translational mechanisms to induce cell-cycle arrest, senescence, or apoptosis. We demonstrate that endoplasmic reticulum (ER) stress inhibits p53-mediated apoptosis. The mechanism of inhibition involves the increased cytoplasmic localization of p53 due to phosphorylation at serine 315 and serine 376, which is mediated by glycogen synthase kinase-3 ␤ (GSK-3␤). ER stress induces GSK-3␤ binding to p53 in the nucleus and enhances the cytoplasmic localization of the tumor suppressor. Inhibition of apoptosis caused by ER stress requires GSK-3␤ and does not occur in cells expressing p53 with mutation(s) of serine 315 and/or serine 376 to alanine(s). As a result of the increased cytoplasmic localization, ER stress prevents p53 stabilization and p53-mediated apoptosis upon DNA damage. It is concluded that inactivation of p53 is a protective mechanism utilized by cells to adapt to ER stress.[Keywords: Endoplasmic reticulum stress; p53; glycogen synthase kinase-3␤; protein phosphorylation; protein localization; apoptosis] Supplemental material is available at http://www.genesdev.org.

Inactivation of the tumor suppressor p53 by degradation is a mechanism utilized by cells to adapt to endoplasmic reticulum (ER) stress. However, the mechanisms of p53 destabilization by ER stress are not known. We demonstrate here that the E3 ubiquitin-ligase Hdm2 is essential for the nucleocytoplasmic transport and proteasome-dependent degradation of p53 in ER-stressed cells. We also demonstrate that p53 phosphorylation at S315 and S376 is required for its nuclear export and degradation by Hdm2 without interfering with the ubiquitylation process. Furthermore, we show that p53 destabilization in unstressed cells utilizes the cooperative action of Hdm2 and glycogen synthase kinase 3␤, a process that is enhanced in cells exposed to ER stress. In contrast to other stress pathways that stabilize p53, our findings further substantiate a negative role of ER stress in p53 activation with important implications for the function of the tumor suppressor in cells with a dysfunctional ER.The p53 gene encodes for a protein whose loss of function is associated with the majority of human cancers (17). The p53 protein primarily functions as a transcription factor and mediates several biological effects including growth arrest, senescence, and apoptosis in response to diverse forms of stress (50). In the absence of stress, p53 is a short-lived protein whose activity is maintained at low levels. Upon exposure to a variety of stresses, p53 becomes stabilized and accumulates in the nucleus, where it resumes its transcriptional function. The levels and localization of p53 are tightly controlled through several posttranslational mechanisms, including protein stability, phosphorylation, and subcellular localization (3,42,46). Although several factors influence p53 function, the Hdm2 (human Mdm2) protein plays an essential role in regulating p53 protein levels in unstressed cells. Hdm2 is a nucleoplasmic and nucleolar RING-finger protein that promotes p53 nuclear export and degradation through its specific E3 ubiquitin ligase activity (2, 40). Other studies have revealed various Hdm2-independent pathways that impinge on p53 turnover, including c-Jun N-terminal kinase (10), COP1 (9), and Pirh2 (32). However, little is known about how and in what cellular context these pathways act.The current model places both p53 and Hdm2 in an autoregulatory feedback loop where p53 induces the transcription of Hdm2 gene. The Hdm2 protein then binds to and ubiquitylates p53 in the nucleus, a process that allows the nuclear export and cytoplasmic proteasome-dependent degradation of the tumor suppressor (40, 56). The importance of this autoregulatory loop was demonstrated when the lethality of mdm2-null mice was rescued by the deletion of the p53 gene (22, 41). In this model, Hdm2 does not physically shuttle p53 out of the nucleus since the nuclear export sequence (NES) of Hdm2 is not necessary for p53 degradation, as opposed to its RING domain, which is important for p53 ubiquitylation and degradation (5,11,43). It has also been proposed that low Hdm2 l...

Phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) is mediated by a family of kinases that respond to various forms of environmental stress. The eIF2␣ kinases are critical for mRNA translation, cell proliferation, and apoptosis. Activation of the tumor suppressor p53 results in cell cycle arrest and apoptosis in response to various types of stress. We previously showed that, unlike the majority of stress responses that stabilize and activate p53, induction of endoplasmic reticulum stress leads to p53 degradation through an Mdm2-dependent mechanism. Here, we demonstrate that the endoplasmic reticulum-resident eIF2␣ kinase PERK mediates the proteasomal degradation of p53 independently of translational control. This role is not specific for PERK, because the eIF2␣ kinase PKR also promotes p53 degradation in response to double-stranded RNA. We further establish that the eIF2␣ kinases induce glycogen synthase kinase 3 to promote the nuclear export and proteasomal degradation of p53. Our findings reveal a novel cross-talk between the eIF2␣ kinases and p53 with implications in cell proliferation and tumorigenesis.The tumor suppressor p53 is a transcription factor mutated in ϳ50% of human cancers (1). In normal cells, p53 plays a pivotal role in controlling cell cycle, apoptosis, and DNA repair in response to various forms of genotoxic stress (2, 3). The regulation of p53 is complex and occurs mainly at the post-translational level (4). This is mediated by various post-translational modifications, such as phosphorylation and acetylation, which contribute to its stabilization and activation (5). The stability of p53 is regulated by its interaction with Hdm2 (human Mdm2), an E3-ubiquitin ligase that acts as an antagonist limiting p53 tumor suppressor function (6). Both p53 and Hdm2 are in an autoregulatory feedback loop in which p53 induces Hdm2 expression at the transcriptional level. The Hdm2 protein then binds to and ubiquitinates p53 in the nucleus, a process that allows the nuclear export and the cytoplasmic proteasome-dependent degradation of the tumor suppressor (6). In addition to Hdm2, other ubiquitin ligases, such as COP1 (7) and Pirh2 (8), have been shown to disrupt p53 stability. However, compared with Hdm2, little is currently known about how these ligases act on p53 (9).The majority of stress responses that activate p53 require its nuclear accumulation and function (10). This is mediated mainly through inactivation of the Hdm2-dependent degradation pathway as well as through interactions with nuclear proteins that promote post-translational modifications of p53 leading to its stabilization and activation (10). The current interest in p53 is underscored by the tremendous therapeutic benefits of its reactivation in cancer cells. Small molecules or peptides that restore the function of mutant p53 proteins have a great anti-tumor potential by enhancing the apoptotic sensitivity of tumor cells (11-13). Because p53 activity is influenced by many factors, targeting of proteins that regulate p53 function may...

Phosphoinositide-3 kinase (PI3K) plays an important role in signal transduction in response to a wide range of cellular stimuli involved in cellular processes that promote cell proliferation and survival. Phosphorylation of the alpha subunit of the eukaryotic translation initiation factor eIF2 at Ser51 takes place in response to various types of environmental stress and is essential for regulation of translation initiation. Herein, we show that a conditionally active form of the eIF2alpha kinase PKR acts upstream of PI3K and turns on the Akt/PKB-FRAP/mTOR pathway leading to S6 and 4E-BP1 phosphorylation. Also, induction of PI3K signaling antagonizes the apoptotic and protein synthesis inhibitory effects of the conditionally active PKR. Furthermore, induction of the PI3K pathway is impaired in PKR(-/-) or PERK(-/-) mouse embryonic fibroblasts (MEFs) in response to various stimuli that activate each eIF2alpha kinase. Mechanistically, PI3K signaling activation is indirect and requires the inhibition of protein synthesis by eIF2alpha phosphorylation as demonstrated by the inactivation of endogenous eIF2alpha by small interfering RNA or utilization of MEFs bearing the eIF2alpha Ser51Ala mutation. Our data reveal a novel property of eIF2alpha kinases as activators of PI3K signaling and cell survival.

Phosphorylation of the alpha (␣) subunit of the eukaryotic translation initiation factor 2 (eIF2) leads to the inhibition of protein synthesis in response to diverse stress conditions, including viral infection. The eIF2␣ kinase PKR has been shown to play an essential role against vesicular stomatitis virus (VSV) infection. We demonstrate here that another eIF2␣ kinase, the endoplasmic reticulum-resident protein kinase PERK, contributes to cellular resistance to VSV infection. We demonstrate that mouse embryonic fibroblasts (MEFs) from PERK ؊/؊ mice are more susceptible to VSV-mediated apoptosis than PERK ؉/؉ MEFs. The higher replication capacity of VSV in PERK ؊/؊ MEFs results from their inability to attenuate viral protein synthesis due to an impaired eIF2␣ phosphorylation. We also show that VSV-infected PERK ؊/؊ MEFs are unable to fully activate PKR, suggesting a cross talk between the two eIF2␣ kinases in virus-infected cells. These findings further implicate PERK in virus infection, and provide evidence that the antiviral and antiapoptotic roles of PERK are mediated, at least in part, via the activation of PKR.

Cyclin D1 plays a critical role in controlling the G 1 /S transition via the regulation of cyclin-dependent kinase activity. Several studies have indicated that cyclin D1 translation is decreased upon activation of the eukaryotic initiation factor 2␣ (eIF2␣) kinases. We examined the effect of activation of the eIF2␣ kinases PKR and PKR-like endoplasmic reticulum kinase (PERK) on cyclin D1 protein levels and translation and determined that cyclin D1 protein levels decrease upon the induction of PKR and PERK catalytic activity but that this decrease is not due to translation. Inhibition of the 26 S proteasome with MG132 rescued cyclin D1 protein levels, indicating that rather than inhibiting translation, PKR and PERK act to increase cyclin D1 degradation. Interestingly, this effect still requires eIF2␣ phosphorylation at serine 51, as cyclin D1 remains unaffected in cells containing a non-phosphorylatable form of the protein. This proteasome-dependent degradation of cyclin D1 requires an intact ubiquitination pathway, although the ubiquitination of cyclin D1 is not itself affected. Furthermore, this degradation is independent of phosphorylation of cyclin D1 at threonine 286, which is mediated by the glycogen synthase kinase 3␤ and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2␣ phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2␣ phosphorylation during short, but not prolonged, periods of stress.

Phosphorylation of the ␣-subunit of translation eukaryotic initiation factor-2 (eIF2) leads to the inhibition of protein synthesis in response to diverse conditions of stress. Serine͞threonine RNAdependent protein kinase (PKR) is an eIF2␣ kinase family member induced by type I IFN and activated in response to dsRNA or virus infection. Herein, we demonstrate that human PKR is a dual specificity kinase phosphorylated at Y101, Y162 and Y293 in vitro and in vivo. Site-specific tyrosine phosphorylation is essential for efficient dsRNA-binding, dimerization, kinase activation and eIF2␣ phosphorylation of PKR. Biologically, tyrosine phosphorylation of PKR mediates the antiviral and antiproliferative properties of the kinase through its ability to control translation. Our data demonstrate an important role of tyrosine phosphorylation in biochemical and biological processes caused or mediated by the activation of the eIF2␣ kinase PKR.cell proliferation ͉ protein phosphorylation ͉ translational control ͉ virus infection

Reverse transcription-PCRs demonstrate that SFmPKR mRNA is expressed in several normal mouse tissues, and appears to be under developmental control during embryogenesis. Our data demonstrate that both PKR ؊/؊ models are incomplete knockouts, and expression of the PKR variants may account, at least in part, for the significant signaling differences between cells from the two PKR ؊/؊ mice.