Philippos Peidis

Phosphorylation of eIF2α at Serine 51 Is an Important Determinant of Cell Survival and Adaptation to Glucose Deficiency

Glucose deficiency leads to the induction of eIF2α phosphorylation at serine 51, which results in a global inhibition of protein synthesis. Phosphorylation of eIF2α is an adaptive process that establishes a cytoprotective state in glucose-deficient cells, with possible implications in biological responses that interfere with glucose metabolism.

Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death.

The eukaryotic cell responds to various forms of environmental stress by adjusting the rates of mRNA translation thus facilitating adaptation to the assaulting stress. One of the major pathways that control protein synthesis involves the phosphorylation of the α-subunit of eukaryotic initiation factor eIF2 at serine 51. Different forms of DNA damage were shown to induce eIF2α phosphorylation by using PERK, GCN2 or PKR. However, the specificity of the eIF2α kinases and the biological role of eIF2α phosphorylation pathway in the DNA damage response (DDR) induced by chemotherapeutics are not known. Herein, we show that PKR is the eIF2α kinase that responds to DDR induced by doxorubicin. We show that activation of PKR integrates two signaling pathways with opposing biological outcomes. More specifically, induction of eIF2α phosphorylation has a cytoprotective role, whereas activation of c-jun N-terminal kinase (JNK) by PKR promotes cell death in response to doxorubicin. We further show that the proapoptotic effects of JNK activation prevail over the cytoprotection mediated by eIF2α phosphorylation. These findings reveal that PKR can be an important inducer of cell death in response to chemotherapies through its ability to act independently of eIF2α phosphorylation.

The PERK-eIF2α phosphorylation arm is a pro-survival pathway of BCR-ABL signaling and confers resistance to imatinib treatment in chronic myeloid leukemia cells

Activation of adaptive mechanisms plays a crucial role in cancer progression and drug resistance by allowing cell survival under stressful conditions. Therefore, inhibition of the adaptive response is considered as a prospective therapeutic strategy. The PERK-eIF2α phosphorylation pathway is an important arm of the unfolded protein response (UPR), which is induced under conditions of endoplasmic reticulum (ER) stress. Our previous work showed that ER stress is induced in chronic myeloid leukemia (CML) cells. Herein, we demonstrate that the PERK-eIF2α phosphorylation pathway is upregulated in CML cell lines and CD34+ cells from CML patients and is associated with CML progression and imatinib resistance. We also show that induction of apoptosis by imatinib results in the downregulation of the PERK-eIF2α phosphorylation arm. Furthermore, we demonstrate that inactivation of the PERK-eIF2α phosphorylation arm decreases the clonogenic and proliferative capacities of CML cells and sensitizes them to death by imatinib. These findings provide evidence for a pro-survival role of PERK-eIF2α phosphorylation arm that contributes to CML progression and development of imatinib resistance. Thus, the PERK-eIF2α phosphorylation arm may represent a suitable target for therapeutic intervention for CML disease.

eIF2α Kinase PKR Modulates the Hypoxic Response by Stat3-Dependent Transcriptional Suppression of HIF-1α

Hypoxia within the tumor microenvironment promotes angiogenesis, metabolic reprogramming, and tumor progression. In addition to activating hypoxia-inducible factor-1α (HIF-1α), cells also respond to hypoxia by globally inhibiting protein synthesis via serine 51 phosphorylation of translation eukaryotic initiation factor 2α (eIF2α). In this study, we investigated potential roles for stress-activated eIF2α kinases in regulation of HIF-1α. Our investigations revealed that the double-stranded RNA-dependent protein kinase R (PKR) plays a significant role in suppressing HIF-1α expression, acting specifically at the level of transcription. HIF-1α transcriptional repression by PKR was sufficient to impair the hypoxia-induced accumulation of HIF-1α and transcriptional induction of HIF-1α-dependent target genes. Inhibition of HIF-1A transcription by PKR was independent of eIF2α phosphorylation but dependent on inhibition of the signal transducer and activator of transcription 3 (Stat3). Furthermore, HIF-1A repression required the T-cell protein tyrosine phosphatase, which acts downstream of PKR, to suppress Stat3. Our findings reveal a novel tumor suppressor function for PKR, which inhibits HIF-1α expression through Stat3 but is independent of eIF2α phosphorylation.

Functional potential of P2P-R: a role in the cell cycle and cell differentiation related to its interactions with proteins that bind to matrix associated regions of DNA?

P2P‐R is the alternately spliced product of the P2P‐R/PACT gene in that P2P‐R lacks one exon encoding 34 amino acids. The 250 kDa P2P‐R protein is the predominate product expressed in multiple murine cell lines. It is a highly basic protein that contains multiple domains including an N‐terminal RING type zinc finger, a proline rich domain, an RS region, and a C‐terminal lysine‐rich domain. P2P‐R binds the p53 and the Rb1 tumor suppressors and is phosphorylated by the cdc2 and SRPK1a protein kinases. P2P‐R also interacts with scaffold attachment factor‐B (SAF‐B), a well characterized MARs (for matrix attachment regions) binding factor, and may interact with nucleolin, another MARs binding factor. In addition, P2P‐R binds single strand DNA (ssDNA). The expression of P2P‐R is regulated by differentiation and cell cycle events. P2P‐R mRNA is markedly repressed during differentiation, whereas immunoreactive P2P‐R protein levels are >10‐fold higher in mitotic than in G0 cells. The localization of P2P‐R also is modulated during the cell cycle. During interphase, P2P‐R is present primarily in nucleoli and nuclear speckles whereas during mitosis, P2P‐R associates with the periphery of chromosomes. Overexpression of near full length P2P‐R induces mitotic arrest in prometaphase and mitotic apoptosis, and overexpression of selected P2P‐R segments also can promote apoptosis. This compendium of data supports the possibility that P2P‐R may form complexes with the Rb1 and/or p53 tumor suppressors and MARs‐related factors, in a cell cycle and cell differentiation‐dependent manner, to influence gene transcription/expression and nuclear organization. J. Cell. Biochem. 90: 6–12, 2003.

Systems genetics analyses predict a transcription role for P2P-R: Molecular confirmation that P2P-R is a transcriptional co-repressor

BackgroundThe 250 kDa P2P-R protein (also known as PACT and Rbbp6) was cloned over a decade ago and was found to bind both the p53 and Rb1 tumor suppressor proteins. In addition, P2P-R has been associated with multiple biological functions, such as mitosis, mRNA processing, translation and ubiquitination. In the current studies, the online GeneNetwork system was employed to further probe P2P-R biological functions. Molecular studies were then performed to confirm the GeneNetwork evaluations.ResultsGeneNetwork and associated gene ontology links were used to investigate the coexpression of P2P-R with distinct functional sets of genes in an adipocyte genetic reference panel of HXB/BXH recombinant strains of rats and an eye genetic reference panel of BXD recombinant inbred strains of mice. The results establish that biological networks of 75 and 135 transcription-associated gene products that include P2P-R are co-expressed in a genetically-defined manner in rat adipocytes and in the mouse eye, respectively. Of this large set of transcription-associated genes, >10% are associated with hormone-mediated transcription. Since it has been previously reported that P2P-R can bind the SRC-1 transcription co-regulatory factor (steroid receptor co-activator 1, [Ncoa1]), the possible effects of P2P-R on estrogen-induced transcription were evaluated. Estrogen-induced transcription was repressed 50-70% by the transient transfection of P2P-R plasmid constructs into four different cell types. In addition, knockdown of P2P-R expression using an antisense oligonucleotide increased estrogen-mediated transcription. Co-immunoprecipitation assays confirmed that P2P-R interacts with SRC-1 and also demonstrated that P2P-R interacts with estrogen receptor α.ConclusionsThe findings presented in this study provide strong support for the value of systems genetics, especially GeneNetwork, in discovering new functions of genes that can be confirmed by molecular analysis. More specifically, these data provide evidence that the expression of P2P-R co-varies in a genetically-defined manner with large transcription networks and that P2P-R can function as a co-repressor of estrogen-dependent transcription.

SAFB1 interacts with and suppresses the transcriptional activity of p53

SAFB2 physically interacts with p53 : shown by pull down ( view interaction )

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.

Abstract C51: eIF2alpha phosphorylation determines cell susceptibility to oxidative stress via Akt activation.

Eukaryotic cells respond to various forms of stress by blocking mRNA translation initiation via the phosphorylation of the alpha (α) subunit of eIF2 at serine 51 (S51) (eIF2αP). Herein, we demonstrate that increased eIF2αP facilitates the adaptation of cells to oxidative stress through the regulation of Akt. Specifically, genetic inactivation of either eIF2αP or the ER-resident kinase PERK in primary mouse or human fibroblasts increases reactive oxygen species (ROS) production leading to increased DNA damage, Akt hyperactivation and induction of senescence. Contrary to the primary cells, immortalized and tumor cells are tolerant to elevated levels of intrinsic ROS caused by eIF2αP inactivation. Nevertheless, eIF2αP-deficient immortalized or tumor cells are more susceptible than eIF2αP-proficient cells to extrinsic oxidative stress caused by hydrogen peroxide or doxorubicin treatment. Extrinsic oxidative stress leads to the induction of either senescence or death in eIF2αP-deficient tumor cells in vitro and in vivo via impaired Akt activation. Our work concludes that Akt acts downstream of eIF2αP to convey either a pro-senescent or a pro-survival role in a cell-context dependent manner in response to oxidative stress. Also, eIF2αP is a potential pharmacological target for tumor treatment in combinational therapies with drugs that induce oxidative stress. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C51. Citation Format: Antonis E. Koromilas, Rajesh Kamindla, Andreas I. Papadakis, Urszula Kazimierczak, Philippos Peidis, Shuo Wang, Clara Tenkerian, Jothi-Latha Krishnamoorthy, Maria Hatzoglou, Fawaz G. Haj, Gerardo Ferbeyre, Randal J. Kaufman. eIF2alpha phosphorylation determines cell susceptibility to oxidative stress via Akt activation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C51.