Divergent Effects of PERK and IRE1 Signaling on Cell Viability

Protein misfolding in the endoplasmic reticulum (ER) activates a set of intracellular signaling pathways, collectively termed the Unfolded Protein Response (UPR). UPR signaling promotes cell survival by reducing misfolded protein levels. If homeostasis cannot be restored, UPR signaling promotes cell death. The molecular basis for the switch between prosurvival and proapoptotic UPR function is poorly understood. The ER-resident proteins, PERK and IRE1, control two key UPR signaling pathways. Protein misfolding concomitantly activates PERK and IRE1 and has clouded insight into their contributions toward life or death cell fates. Here, we employed chemical-genetic strategies to activate individually PERK or IRE1 uncoupled from protein misfolding. We found that sustained PERK signaling impaired cell proliferation and promoted apoptosis. By contrast, equivalent durations of IRE1 signaling enhanced cell proliferation without promoting cell death. These results demonstrate that extended PERK and IRE1 signaling have opposite effects on cell viability. Differential activation of PERK and IRE1 may determine life or death decisions after ER protein misfolding.     

The protein kinase/endoribonuclease IRE1alpha that signals the unfolded protein response has a luminal N-terminal ligand-independent dimerization domain

In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), cells activate an intracellular signal transduction pathway called the unfolded protein response (UPR). IRE and PERK are the two type-I ER transmembrane protein kinase receptors that signal the UPR. The N-terminal luminal domains (NLDs) of IRE1 and PERK sense ER stress conditions by a common mechanism and transmit the signal to regulate the cytoplasmic domains of these receptors. To provide an experimental system amenable to detailed biochemical and structural analysis to elucidate the mechanism of ER-transmembrane signaling mechanism mediated by the NLD, we overexpressed the soluble luminal domain of human IRE1alpha in COS-1 cells by transient DNA transfection. Here we report the expression, purification, and characterization of the soluble NLD. The biological function of the NLD was confirmed by its ability to associate with itself and to interact with both the membrane-bound full-length IRE1alpha receptor and the ER chaperone BiP. Functional and spectral studies suggested that the highly conserved N-linked glycosylation site is not required for proper protein folding and self-association. Interestingly, we demonstrated that the NLD forms stable dimers linked by intermolecular disulfide bridges. Our data support that the luminal domain represents a novel ligand-independent dimerization domain.     

JAB1 participates in unfolded protein responses by association and dissociation with IRE1

Recent papers have reported that neuronal death in patients with Alzheimer's disease, Parkinson's disease, and cerebral ischemia has its origin in the endoplasmic reticulum (ER). IRE1alpha is one of the ER stress transducers that detect the accumulation of unfolded proteins in the ER. IRE1alpha mediates two major cellular responses, which are the unfolded protein response (UPR), a defensive response, and apoptosis that leads to cell death. However, little is known about the regulatory mechanisms that select between the UPR and apoptosis. We identified Jun activation domain-binding protein-1 (JAB1) as a molecule that interacts with IRE1alpha using a yeast two-hybrid system. We demonstrated that JAB1 binds to IRE1alpha in the absence of stress, but that binding is decreased by ER stress inducers. Moreover, mutant JAB1 down-regulates the UPR signaling pathway through tight binding with IRE1alpha. These results suggested that JAB1 may act as a key molecule in selecting the UPR or cell death by association and dissociation with IRE1alpha.     

Ligand-independent dimerization activates the stress response kinases IRE1 and PERK in the lumen of the endoplasmic reticulum

IRE1 and PERK are type I transmembrane serine/threonine protein kinases that are activated by unfolded proteins in the endoplasmic reticulum (ER) to signal adaptive responses. IRE1 is present in all eukaryotic cells and signals the unfolded protein response through its kinase and endoribonuclease activities. PERK signals phosphorylation of a translation initiation factor to inhibit protein synthesis in higher eukaryotic cells but is absent in the Saccharomyces cerevisiae genome. The amino acid sequences of the amino-terminal ER luminal domains (NLDs) from IRE1 and PERK display limited homology and have diverged among species. In this study, we have demonstrated that the NLD of yeast Ire1p is required for signaling. However, the NLDs from human IRE1alpha and murine IRE1beta and the Caenorhabditis elegans IRE1 and PERK function as replacements for the S. cerevisiae Ire1p-NLD to signal the unfolded protein response. Replacement of the Ire1p-NLD with a functional leucine zipper dimerization motif yielded a constitutively active kinase that surprisingly was further activated by ER stress. These results demonstrate that ER stress-induced dimerization of the NLD is sufficient for IRE1 and PERK activation and is conserved through evolution. We propose that ligand-independent activation of IRE1 and PERK permits homodimerization upon accumulation of unfolded proteins in the lumen of the ER.     

Ultraviolet light inhibits translation through activation of the unfolded protein response kinase PERK in the lumen of the endoplasmic reticulum

Exposure to ultraviolet light can cause inflammation, premature skin aging, and cancer. UV irradiation alters the expression of multiple genes that encode functions to repair DNA damage, arrest cell growth, and induce apoptosis. In addition, UV irradiation inhibits protein synthesis, although the mechanism is not known. In this report, we show that UV irradiation induces phosphorylation of eukaryotic translation initiation factor 2 on the alpha-subunit (eIF2alpha) and inhibits protein synthesis in a dosage- and time-dependent manner. The UV-induced phosphorylation of eIF2alpha was prevented by the overexpression of a non-phosphorylatable mutant of eIF2alpha (S51A). PERK is an eIF2alpha protein kinase localized to the endoplasmic reticulum that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum. Expression of trans-dominant-negative mutants of PERK also prevented eIF2alpha phosphorylation upon UV treatment and protected from the associated translation attenuation. The luminal domain of dominant-negative mutant PERK formed heterodimers with endogenous PERK to inhibit the PERK signaling pathway. In contrast, eIF2alpha phosphorylation was not inhibited by overexpression of a trans-dominant-negative mutant kinase, PKR, supporting the theory that UV-induced eIF2alpha phosphorylation is specifically mediated by PERK. These results support a novel mechanism by which UV irradiation regulates translation via an endoplasmic reticulum-stress signaling pathway.     

T-cadherin attenuates the PERK branch of the unfolded protein response and protects vascular endothelial cells from endoplasmic reticulum stress-induced apoptosis

Endoplasmic reticulum (ER) stress activated by perturbations in ER homeostasis induces the unfolded protein response (UPR) with chaperon Grp78 as the key activator of UPR signalling. The aim of UPR is to restore normal ER function; however prolonged or severe ER stress triggers apoptosis of damaged cells to ensure protection of the whole organism. Recent findings support an association of ER stress-induced apoptosis of vascular cells with cardiovascular pathologies. T-cadherin (T-cad), an atypical glycosylphosphatidylinositol-anchored member of the cadherin superfamily is upregulated in atherosclerotic lesions. Here we investigate the ability of T-cad to influence UPR signalling and endothelial cell (EC) survival during ER stress. EC were treated with a variety of ER stress-inducing compounds (thapsigargin, dithiothereitol, brefeldin A, tunicamycin, A23187 or homocysteine) and induction of ER stress validated by increases in levels of UPR signalling molecules Grp78 (glucose-regulated protein of 78 kDa), phospho-eIF2α (phosphorylated eukaryotic initiation factor 2α) and CHOP (C/EBP homologous protein). All compounds also increased T-cad mRNA and protein levels. Overexpression or silencing of T-cad in EC respectively attenuated or amplified the ER stress-induced increase in phospho-eIF2α, Grp78, CHOP and active caspases. Effects of T-cad-overexpression or T-cad-silencing on ER stress responses in EC were not affected by inclusion of either N-acetylcysteine (reactive oxygen species scavenger), LY294002 (phosphatidylinositol-3-kinase inhibitor) or SP6000125 (Jun N-terminal kinase inhibitor). The data suggest that upregulation of T-cad on EC during ER stress attenuates the activation of the proapoptotic PERK (PKR (double-stranded RNA-activated protein kinase)-like ER kinase) branch of the UPR cascade and thereby protects EC from ER stress-induced apoptosis.     

Sphingolipids and the unfolded protein response

The unfolded protein response (UPR) is a response by the endoplasmic reticulum to stress, classically caused by any disruption to cell homeostasis that results in an accumulation in unfolded proteins. However, there is an increasing body of research demonstrating that the UPR can also be activated by changes in lipid homeostasis, including changes in sphingolipid metabolism. Sphingolipids are a family of bioactive lipids with important roles in both the formation and integrity of cellular membranes, and regulation of key cellular processes, including cell proliferation and apoptosis. Bi-directional interactions between sphingolipids and the UPR have now been observed in a range of diseases, including cancer, diabetes and liver disease. Determining how these two key cellular components influence each other could play an important role in deciphering the causes of these diseases and potentially reveal new therapeutic approaches.