Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor eIF2alpha

Hypoxia profoundly influences tumor development and response to therapy. While progress has been made in identifying individual gene products whose synthesis is altered under hypoxia, little is known about the mechanism by which hypoxia induces a global downregulation of protein synthesis. A critical step in the regulation of protein synthesis in response to stress is the phosphorylation of translation initiation factor eIF2alpha on Ser51, which leads to inhibition of new protein synthesis. Here we report that exposure of human diploid fibroblasts and transformed cells to hypoxia led to phosphorylation of eIF2alpha, a modification that was readily reversed upon reoxygenation. Expression of a transdominant, nonphosphorylatable mutant allele of eIF2alpha attenuated the repression of protein synthesis under hypoxia. The endoplasmic reticulum (ER)-resident eIF2alpha kinase PERK was hyperphosphorylated upon hypoxic stress, and overexpression of wild-type PERK increased the levels of hypoxia-induced phosphorylation of eIF2alpha. Cells stably expressing a dominant-negative PERK allele and mouse embryonic fibroblasts with a homozygous deletion of PERK exhibited attenuated phosphorylation of eIF2alpha and reduced inhibition of protein synthesis in response to hypoxia. PERK(-/-) mouse embryo fibroblasts failed to phosphorylate eIF2alpha and exhibited lower survival after prolonged exposure to hypoxia than did wild-type fibroblasts. These results indicate that adaptation of cells to hypoxic stress requires activation of PERK and phosphorylation of eIF2alpha and suggest that the mechanism of hypoxia-induced translational attenuation may be linked to ER stress and the unfolded-protein response.     

Purification and characterization of a dimer form of the cAMP-dependent protein kinase from mouse liver cytosol

A protein kinase that phosphorylates histones and polysomal proteins was partially purified from mouse liver cytosol. The active enzyme has a molecular mass of 100 kDa and a phosphorylatable subunit of 54 kDa. Biochemical as well as immunological data suggest that the enzyme is a heterodimer composed of the catalytic subunit of cyclic AMP-dependent protein kinase and the R_II regulatory subunit. This RC form does not seem to dissociate upon activation with 3, 5 cyclic AMP and exhibits identical specificity as the classical cAMP-dependent protein kinase (2.7.1.37). The enzyme is affected by the 3, 5 cyclic phosphates of adenosine mainly, but also of guanosine, uridine and cytidine in a substrate-dependent manner. Cyclic nucleotides slightly stimulate phosphate incorporation into histones, while phosphorylation of polysomal proteins in intact polysomes is dramatically increased. The substrate- specific stimulatory effects of 3, 5 cyclic nucleotides are due to repression of the inhibition exerted upon the reaction, by negatively charged macromolecules such as RNA, DNA and to a lesser extent heparin.     

PLEKHA7 defines an apical junctional complex with cytoskeletal associations and miRNA-mediated growth implications

E-cadherin-p120 catenin complexes are essential for adherens junction (AJ) formation and for the maintenance of the normal epithelial phenotype. PLEKHA7 was originally identified as a member of this complex that tethers microtubules to the AJs and supports their overall integrity. Recently, we revealed that PLEKHA7 regulates cellular behavior via miRNAs by associating with the microprocessor complex at the apical zonula adherens (ZA). We have also identified a new set of PLEKHA7 interacting partners at the apical ZA, via proteomics. Our analysis shows that the main groups of proteins associating with PLEKHA7 are cytoskeletal-related and RNA-binding proteins. Here, we provide extended evidence for association of PLEKHA7 with several of these proteins. We also show that PLEKHA7 loss activates the actin regulator cofilin in a p120-dependent manner, providing an explanation for the effects of PLEKHA7 on the cortical actin ring. Interestingly, PLEKHA7 regulates the levels and associates with PP1α, a phosphatase responsible for cofilin activation. Finally, we clarify the mode of regulation of the oncogenic miR-19a by PLEKHA7. Overall, our findings support a multi-layered role of PLEKHA7 in converging cytoskeletal dynamics and miRNA-mediated growth regulation at the ZA, with potentially critical implications in cancer that warrant further investigation.     

Hidden diversity uncovered in Hygrophorus sect. Aurei (Hygrophoraceae), including the Mediterranean H. meridionalis and the North American H. boyeri, spp. nov.

For many years, the binomial Hygrophorus hypothejus was widely applied to collections from various geographical regions in different continents, assuming a circum-boreal and circum-mediterranean distribution for this species. This hypothesis, however, had never been put to the test. To assess the diversity and species-limits within this complex of yellow-coloured waxcaps, a phylogenetic, morphological and taxonomical investigation into Hygrophorus sect. Aurei and similar species in sect. Olivaceoumbrini was carried out, including material of pan-European origin, as well as the east and west coasts of North America. Following sequencing of the ITS rDNA locus, nine lineages are confirmed in sect. Aurei, most of them highly continentalised. Of these, two are new to science, introduced here as Hygrophorus boyeri sp. nov., from Pinus banksiana and P. rigida forests in eastern North America and from P. muricata and P. contorta forests in western North America, and Hygrophorus meridionalis sp. nov., from Pinus brutia and Pinus halepensis forests in the island of Cyprus and mainland Greece. H. hypothejus is lectotypified and epitypified, and here resolved as a strictly European species, with the old forgotten taxon Hygrophorus siccipes revived as its North American vicariant. The placement of Hygrophorus fuligineus in sect. Aurei is phylogenetically confirmed and detailed comparisons between morphologically similar and phylogenetically affiliated taxa in sect. Aurei and sect. Olivaceoumbrini are provided. The chronic confusion associated with Hygrophorus fuscoalbus, a highly controversial taxon described from Germany nearly two centuries ago and variously interpreted since, is discussed, concluding that this name is too ambiguous to be applied to any currently recognized species.     

The role of corticotropin-releasing hormone in blastocyst implantation and early fetal immunotolerance.

During blastocyst implantation, the maternal endometrial response to the invading semi-allograft has characteristics of an acute, aseptic inflammatory response. However, once implanted, the embryo suppresses this response and prevents rejection. Simultaneously, the mother's immune system prevents a graft VS. host reaction deriving from the fetal immune system. We have shown that embryonic trophoblast and maternal decidua cells, i.e., cells located in the interface between the fetal placenta and the maternal endometrium, produce corticotropin-releasing hormone (CRH) and express Fas ligand. CRH may play a crucial role in the implantation and the anti-rejection process that protects the fetus from the maternal immune system, primarily by killing activated T cells through the Fas-FasL interaction. In experimental animals, type 1 CRH receptor (CRH-R1) blockade by antalarmin, a specific type 1 CRH receptor antagonist, decreased implantation sites by approximately 70%. CRH is also involved in controlled trophoblast invasion, by downregulating the synthesis of the carcinoembryonic antigen-related cell adhesion molecule 1 by extravillous trophoblast cells. IN VITRO findings showed that CRH-R1 blockade by antalarmin increased trophoblast invasion by approximately 60%. Defective uterine CRH/CRH-R1 system during early pregnancy may be implicated in the pathophysiology of recurrent miscarriage, placenta accreta, and preeclampsia.     

Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation

Mouse embryonic endothelial progenitor cells (eEPCs) acquire a mature phenotype after treatment with cyclic adenosine monophosphate (cAMP), suggesting an involvement of Raf serine/threonine kinases in the differentiation process. To test this idea, we investigated the role of B-Raf and C-Raf in proliferation and differentiation of eEPCs by expressing fusion proteins consisting of the kinase domains from Raf molecules and the hormone binding site of the estrogen receptor (ER), or its variant, the tamoxifen receptor. Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation. In contrast, the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than B-Raf. In a complimentary approach, we used siRNA silencing to knockdown endogenously expressed B-Raf and C-Raf in eEPCs. In this experimental setting, we found that eEPCs lacking B-Raf failed to differentiate, whereas loss-of C-Raf function primarily slowed cell growth without impairing cAMP-induced differentiation. These findings were further corroborated in B-Raf null eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro. Thus, gain- and loss-of-function experiments point to distinct roles of B-Raf and C-Raf in regulating growth and differentiation of endothelial progenitor cells, which may harbour therapeutic implications.     

Interferons induce tyrosine phosphorylation of the eIF2alpha 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 eIF2alpha 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 eIF2alpha phosphorylation.