Zinc homeostasis is involved in unfolded protein response under salt stress

Accumulation of unfolded protein or misfolded protein causes endoplasmic reticulum (ER) stress. Increased salt concentration activates a stress response pathway in the ER in Arabidopsis thaliana to induce the expression of several salt stress response genes, leading to a more optimal protein folding environment in the ER. In addition, some salt stress-regulated proteins require zinc for their activity, including some zinc-dependent DNA binding proteins and zinc-finger proteins. In a recent study, we reported that ZTP29, a putative zinc transporter at the ER membrane, is involved in the response to salt stress through regulation of zinc level in the ER to induce the UPR pathway. In this addendum, we propose a testable hypothesis for the role of ZTP29 in the response to salt stress via the regulation of zinc levels in the ER.Key words: zinc, ER stress, unfolded protein response, salt stress, arabidopsisHigh salinity is a common abiotic stress that adversely affects plant growth and crop production.¹ Plants must sense the stress and transduce stress signals to activate response pathways leading to adaptation to, or tolerance of, the abiotic stress in salt environment.² Salt stress activates a stress response pathway in the endoplasmic reticulum (ER) in Arabidopsis thaliana, indicating that the adaptation of plants to salt stress involves ER stress signal regulation.^3,4 There is limited understanding of molecular mechanisms on ER stress in plants, as compared to yeast and mammalian cells. bZIP60, bZIP28, bZIP17 are three membrane-associated basic domain/leucine zipper (bZIP) factors, which have been reported as candidates for ER-folding proteins in plants.^5–7 BiP acts as a general chaperone in the ER lumen, due to its ability to discriminate between properly folded and unfolded protein structures.⁸ Unfolded or misfolded proteins are retained in the ER and form stable complexes with BiP and other ER resident chaperones.⁹ Zinc deficiency induces unfolded protein response (UPR) in most eukaryotes.¹⁰ Zinc is an important trace element, which participates in physiological and biochemical process in vivo. The requirement of zinc for proper ER function is evolutionarily conserved.     

Cell cycle regulation of human WEE1.

WEE1 kinase negatively regulates entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of CDC2/cyclin B kinase. We report here an investigation of human WEE1. Endogenous WEE1 migrates as an approximately 94 kDa protein in SDS-PAGE, substantially larger than the 49 kDa protein encoded by the original human WEE1 cDNA clone that was truncated at the 5'-end. Antibody depletion experiments demonstrate that WEE1 accounts for most of the activity that phosphorylates CDC2 on Tyr15 in an in vitro assay of HeLa cell lysates, hence it is likely to have an important role in the mitotic control of human cells. WEE1 activity was not found to be elevated in HeLa cells arrested in S phase, suggesting that unreplicated DNA does not delay M phase by hyperactivating WEE1. WEE1 activity is strongly suppressed during M phase, suggesting that negative regulation of WEE1 could be part of the mechanism by which activation of CDC2/cyclin B kinase is promoted during the G2/M transition. M phase WEE1 is re-activated in samples prepared in the absence of protein phosphatase inhibitors, demonstrating that WEE1 is inhibited by a mechanism that requires protein phosphorylation.     

Conservation between animals and plants of the cis-acting element involved in the unfolded protein response

Using Arabidopsis thaliana, we identified the cis-element involved in the plant unfolded protein response (UPR). In transgenic plants, tunicamycin stimulated expression of a reporter gene under the control of the BiP promoter and promoter analysis identified a 24 bp sequence crucial to this induction. When fused with a minimal promoter, a hexamer of this sequence was sufficient for induction of a reporter gene in protoplasts treated with tunicamycin or dithiothreitol. Induction rate equivalent to original promoter was observed when the assay was conducted in transgenic plants. This 24 bp sequence contained two elements also responsible for the UPR in animals. Either of these elements was sufficient for the plant UPR, indicating conservation between animals and plants of cis-elements involved in the UPR.     

Endoplasmic reticulum‐mediated unfolded protein response is an integral part of singlet oxygen signalling in plants

Singlet oxygen (¹O₂) is a by‐product of photosynthesis that triggers a signalling pathway leading to stress acclimation or to cell death. By analyzing gene expressions in a ¹O₂‐overproducing Arabidopsis mutant (ch1) under different light regimes, we show here that the ¹O₂ signalling pathway involves the endoplasmic reticulum (ER)‐mediated unfolded protein response (UPR). ch1 plants in low light exhibited a moderate activation of UPR genes, in particular bZIP60, and low concentrations of the UPR‐inducer tunicamycin enhanced tolerance to photooxidative stress, together suggesting a role for UPR in plant acclimation to low ¹O₂ levels. Exposure of ch1 to high light stress ultimately leading to cell death resulted in a marked upregulation of the two UPR branches (bZIP60/IRE1 and bZIP28/bZIP17). Accordingly, mutational suppression of bZIP60 and bZIP28 increased plant phototolerance, and a strong UPR activation by high tunicamycin concentrations promoted high light‐induced cell death. Conversely, light acclimation of ch1 to ¹O₂ stress put a limitation in the high light‐induced expression of UPR genes, except for the gene encoding the BIP3 chaperone, which was selectively upregulated. BIP3 deletion enhanced Arabidopsis photosensitivity while plants treated with a chemical chaperone exhibited enhanced phototolerance. In conclusion, ¹O₂ induces the ER‐mediated UPR response that fulfils a dual role in high light stress: a moderate UPR, with selective induction of BIP3, is part of the acclimatory response to ¹O₂, and a strong activation of the whole UPR is associated with cell death.     

The unfolded protein response is shaped by the NMD pathway

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD''s ability to regulate normal mRNAs.     

Cell cycle arrest by monochloramine through the oxidation of retinoblastoma protein

Impairment of cell cycle control has serious effects on inflammation, tissue repair, and carcinogenesis. We report here the G1 cell cycle arrest by monochloramine (NH2Cl), a physiological oxidant derived from activated neutrophils, and its mechanism. When Jurkat cells were treated with NH2Cl (70 microM, 10 min) and incubated for 24 h, the S phase population decreased significantly with a slight increase in the hypodiploid cell population. The G0/ G1 phase and G2/M phase populations did not show marked changes. Three hours after NH2Cl treatment, the retinoblastoma protein (pRB) was dephosphorylated especially at Ser780 and Ser795, both of which are important phosphorylation sites for the G1 checkpoint function. The phosphorylation at Ser807/811 showed no apparent change. The expression of cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors showed no apparent change. Moreover, the kinase activity that phosphorylates pRB remained constant even after NH2Cl treatment. The protein phosphatase activity that dephosphorylates pRB showed a marginal increase. Notably, when the recombinant pRB was oxidized by NH2Cl in vitro, the oxidized pRB became difficult to be phosphorylated by kinases, especially at Ser780 and Ser795, but not at Ser807/811. Amino acid analysis of oxidized pRB showed methionine oxidation to methionine sulfoxide. The NH2Cl-treated Jurkat cell proteins also showed a decrease in methionine. These observations suggested that direct pRB oxidation was the major cause of NH2Cl-induced cell cycle arrest. In the presence of 2 mM NH4+, NaOCl (200 microM) or activated neutrophils also induced a G1 cell cycle arrest. As protein methionine oxidation has been reported in inflammation and aging, cell cycle modulation by pRB oxidation may occur in various pathological conditions.     

Cell cycle arrest and cellular differentiation mediated by a cell surface sialoglycopeptide

Cell cycling by a relatively wide variety of cell lines was shown to be reversibly inhibited by a cell surface sialoglycopeptide (SGP) isolated and purified from intact bovine cerebral cortex cells. Cell cycle arrest, mediated by the bovine SGP inhibitor, was shown to be completely reversible with mouse Swiss 3T3, mouse 1316 fibrosarcoma, mouse N2a neuroblastoma, bovine MDBK and monkey BSC-1 cells. These cell lines represented both fibroblast and epithelial-like cells, transformed and nontransformed cells, as well as their being derived from a broad array of species. In contrast to the others tested, human HL-60 leukemic cells were sensitive to the inhibitory effects of the SGP but did not reenter the mitotic cycle after the removal of the inhibitor. Instead, the mitotic arrest of HL-60 cells appeared to enhance entry into a terminal and irreversible state of cellular differentiation.     

Cell cycle arrest and apoptosis induced by human Polo-like kinase 3 is mediated through perturbation of microtubule integrity

Human Polo-like kinase 3 (Plk3, previously termed Prk or Fnk) is involved in regulation of cell cycle progression through the M phase (B. Ouyang, H. Pan, L. Lu, J. Li, P. Stambrook, B. Li, and W. Dai, J. Biol. Chem. 272:28646-28651, 1997). Here we report that in most interphase cells endogenous Plk3 was predominantly localized around the nuclear membrane. Double labeling with Plk3 and gamma-tubulin, the latter a major component of pericentriole materials, revealed that Plk3 was closely associated with centrosomes and that its localization to centrosomes was dependent on the integrity of microtubules. Throughout mitosis, Plk3 appeared to be localized to mitotic apparatus such as spindle poles and mitotic spindles. During telophase, a significant amount of Plk3 was also detected in the midbody. Ectopic expression of Plk3 mutants dramatically changed cell morphology primarily due to their effects on microtubule dynamics. Expression of a constitutively active Plk3 (Plk3-A) resulted in rapid cell shrinkage, which led to formation of cells with an elongated, unsevered, and taxol-sensitive midbody. In contrast, cells expressing a kinase-defective Plk3 (Plk3(K52R)) mutant exhibited extended, deformed cytoplasmic structures, the phenotype of which was somewhat refractory to taxol treatment. Expression of both Plk3-A and Plk3(K52R) induced apparent G(2)/M arrest followed by apoptosis, although the kinase-defective mutant was less effective. Taken together, our studies strongly suggest that Plk3 plays an important role in the regulation of microtubule dynamics and centrosomal function in the cell and that deregulated expression of Plk3 results in cell cycle arrest and apoptosis.     

Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.

The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.     

THESEUS1 is involved in tunicamycin-induced root growth inhibition,ectopic lignin deposition,and cell wall damage-induced unfolded protein response

Endoplasmic reticulum (ER) stress activates unfolded protein responses (UPRs), such as promoting protein folding under the control of specific gene expression. Our previous study showed that ER stress induced by ER stress inducers such as tunicamycin (Tm), an inhibitor of N-linked glycan synthesis, causes ectopic lignin deposition in Arabidopsis roots, but the relationship between UPR and ectopic lignin deposition remains unclear. The receptor-like kinase THESEUS1 (THE1) has been shown to sense cell wall damage (CWD) induced in Arabidopsis by cellulose synthase inhibitors such as isoxaben (ISO) and to activate ectopic lignin deposition. In this study, we assessed the involvement of THE1 in ectopic lignin deposition caused by the ER stress inducer Tm. The loss-of-function mutation of THE1, the1-3, suppressed Tm-induced root growth inhibition and ectopic lignin deposition, revealing that THE1 is involved in root growth defects and ectopic lignin deposition caused by ER stress. Similarly, ISO treatment induced ectopic lignin deposition as well as the expression of the UPR marker genes binding protein 3 (BiP3) and ER-localized DnaJ 3b (ERdj3b). Conversely, in the the1-3 mutant, ISO-induced ectopic lignin deposition and the expression of BiP3 and ERdj3b were suppressed. These results showed that THE1 is involved in not only root growth inhibition and ectopic lignin deposition caused by ER stress but also CWD-induced UPR.     

Cell cycle arrest by the PTEN tumor suppressor is target cell specific and may require protein phosphatase activity

PTEN, a tumor suppressor commonly targeted in human cancer, possesses phosphatase activities toward both protein and lipid substrates. While PTEN suppresses gliomas through cell cycle inhibition which requires its lipid phosphatase activity, PTEN's effects on other tumor types and the role of its protein phosphatase activity are controversial or unknown. Here we show that exogenous wild-type PTEN arrests some, but not all human breast cancer cell lines in G1, in a manner independent of endogenous PTEN. Unexpectedly, the G129E mutant of PTEN selectively deficient in the lipid phosphatase activity still blocked the cell cycle of MCF-7 cells, while the G129R and H123Y mutants lacking both phosphatase activities were ineffective. These results suggest that PTEN's protein phosphatase activity likely contributes to its tumor suppressor function in subsets of tumors and that elucidation of downstream targets which dictate cellular responses to PTEN may have important implications for future cancer treatment strategies.     

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element

Arginine rich, mutated in early stage of tumors (ARMET) was first identified as a human gene highly mutated in a variety of cancers. However, little is known about the characteristics of the ARMET protein and its expression. We identified ARMET as a gene upregulated by endoplasmic reticulum (ER) stress. Here, we show that the mouse homologue of ARMET is an 18-kDa soluble ER protein that is mature after cleavage of a signal sequence and has four intramolecular disulfide bonds, including two in CXXC sequences. ER stress stimulated ARMET expression, and the expression patterns of ARMET mRNA and protein in mouse tissues were similar to those of Grp78, an Hsp70-family protein required for quality control of proteins in the ER. A reporter gene assay using a mouse ARMET promoter revealed that the unfolded protein response of the ARMET gene is regulated by an ERSE-II element whose sequence is identical to that of the HERP gene. ARMET is the second fully characterized ERSE-II-dependent gene and likely contributes to quality control of proteins in the ER.     

Endoplasmic reticulum: the unfolded protein response is tangled in neurodegeneration

The endoplasmic reticulum (ER) is involved in the folding and maturation of membrane-bound and secreted proteins. Disturbed homeostasis in the ER can lead to accumulation of misfolded proteins, which trigger a stress response called the unfolded protein response (UPR). In neurodegenerative diseases that are classified as tauopathies, activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau. Several lines of evidence indicate that UPR activation contributes to increased levels of phosphorylated tau, a prerequisite for the formation of tau aggregates. Increased understanding of the crosstalk between signaling pathways involved in protein quality control in the ER and tau phosphorylation will support the development of new therapeutic targets that promote neuronal survival.