Dionissios Baltzis

The eIF2α Kinases PERK and PKR Activate Glycogen Synthase Kinase 3 to Promote the Proteasomal Degradation of p53

Phosphorylation of eukaryotic initiation factor 2α (eIF2α) is mediated by a family of kinases that respond to various forms of environmental stress. The eIF2α 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 eIF2α kinase PERK mediates the proteasomal degradation of p53 independently of translational control. This role is not specific for PERK, because the eIF2α kinase PKR also promotes p53 degradation in response to double-stranded RNA. We further establish that the eIF2α 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 eIF2α kinases and p53 with implications in cell proliferation and tumorigenesis.

Endoplasmic Reticulum Stress Accelerates p53 Degradation by the Cooperative Actions of Hdm2 and Glycogen Synthase Kinase 3β

ABSTRACT 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 3β, 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.

PKR and PKR-like Endoplasmic Reticulum Kinase Induce the Proteasome-dependent Degradation of Cyclin D1 via a Mechanism Requiring Eukaryotic Initiation Factor 2α Phosphorylation

Cyclin D1 plays a critical role in controlling the G1/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 2α (eIF2α) kinases. We examined the effect of activation of the eIF2α 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 eIF2α 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 3β and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2α phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2α phosphorylation during short, but not prolonged, periods of stress.

Resistance to Vesicular Stomatitis Virus Infection Requires a Functional Cross Talk between the Eukaryotic Translation Initiation Factor 2α Kinases PERK and PKR

ABSTRACT Phosphorylation of the alpha (α) 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 eIF2α kinase PKR has been shown to play an essential role against vesicular stomatitis virus (VSV) infection. We demonstrate here that another eIF2α 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 eIF2α phosphorylation. We also show that VSV-infected PERK−/− MEFs are unable to fully activate PKR, suggesting a cross talk between the two eIF2α 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.

PKR and PERK induce the proteasome-dependent degradation of cyclin D1 via a mechanism requiring eIF2α phosphorylation

Cyclin D1 plays a critical role in controlling the G1/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 2α (eIF2α) kinases. We examined the effect of activation of the eIF2α 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 eIF2α 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 3β and mitogen-activated protein kinase pathways as described in previous studies. Our study reveals a novel functional cross-talk between eIF2α phosphorylation and the proteasomal degradation of cyclin D1 and that this degradation is dependent upon eIF2α phosphorylation during short, but not prolonged, periods of stress.

Interferons induce tyrosine phosphorylation of the eIF2α kinase PKR through activation of Jak1 and Tyk2

The interferon (IFN)‐inducible, double‐stranded RNA activated protein kinase (PKR) is a dual‐specificity kinase, which has an essential role in the regulation of protein synthesis by phosphorylating the translation eukaryotic initiation factor 2 (eIF2). Here, we show the tyrosine (Tyr) phosphorylation of PKR in response to type I or type II IFNs. We show that PKR physically interacts with either Jak1 or Tyk2 in unstimulated cells and that these interactions are increased in IFN‐treated cells. We also show that PKR acts as a substrate of activated Jaks, and is phosphorylated at Tyr 101 and Tyr 293 both in vitro and in vivo. Moreover, we provide strong evidence that both the induction of eIF2α phosphorylation and inhibition of protein synthesis by IFN are impaired in cells lacking Jak1 or Tyk2, which corresponds to a lack of induction of PKR tyrosine phosphorylation. We conclude that PKR tyrosine phosphorylation provides an important link between IFN signalling and translational control through the regulation of eIF2α phosphorylation.

IRES-mediated translational control of AMAP1 expression during differentiation of monocyte U937 cells

Global control of mRNA translation plays key roles in cell regulation, including growth, differentiation and apoptosis. Human monocyte-like U937 cells differentiate into macrophage-like cells upon 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment, a process which is known to be accompanied with a large decrease in general protein synthesis. Here, we found that protein levels of AMAP1 (also called ASAP1 or DDEF1), a GTPase-activating protein for Arf GTPases, increase several fold during U937 cell differentiation. This increase was not accompanied with a notable increase in the AMAP1 gene transcript, nor seemed to be due to 5’-Cap-dependent mRNA translational activities in differentiated U937 cells. We identified the 5’-untranslated region (5’-UTR) of AMAP1 mRNA, and found that this 5’-UTR exhibits significant internal ribosome entry site (IRES)-dependent translational activity in differentiated U937 cells, but not in undifferentiated cells. Our results indicate that monocyte differentiation involves enhancement of IRES activity, by which protein levels of AMAP1 are primarily upregulated.

Development of transgenic mice expressing a conditionally active form of the eIF2α kinase PKR

Phosphorylation of the alpha (α) subunit of the eukaryotic initiation factor 2 (eIF2) at serine 51 is an important mechanism of translational control in response to various forms of environmental stress. In metazoans, eIF2α phosphorylation is mediated by four kinases each of which becomes activated by distinct stimuli. Previous work established that expression of a chimera protein comprising of the bacteria Gyrase B N‐terminal (GyrB) domain fused to the kinase domain (KD) of the eIF2α kinase PKR is capable of inducing eIF2α phosphorylation in cultured cells after treatment with the antibiotic coumermycin. Herein, we report the development of transgenic mice expressing the fusion protein GyrB.PKR ubiquitously. Treatment of mice with coumermycin induces eIF2α phosphorylation in vivo as demonstrated by immunoblotting and immunoshistochemistry of mouse tissues. The GyrB.PKR transgene represents a useful model system to investigate the biological effects of the conditional induction of eIF2α phosphorylation in vivo in the absence of parallel signaling pathways that are elicited in response to stress. genesis 49:743–749, 2011.