PKC activation contributes to caspase-mediated eIF2α phosphorylation and cell death

Back ground

Stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2α), involved in translation, promotes cell suicide or survival. Since multiple signaling pathways are implicated in cell death, the present study has analyzed the importance of PKC activation in the stress-induced eIF2α phosphorylation, caspase activation and cell death in the ovarian cells of Spodoptera frugiperda (Sf9) and in their extracts.

Methods

Cell death is analyzed by flow cytometry. Caspase activation is measured by Ac-DEVD-AFC hydrolysis and also by the cleavage of purified recombinant PERK, an endoplasmic reticulum-resident eIF2α kinase. Status of eIF2α phosphorylation and cytochrome c levels are analyzed by western blots.

Results

PMA, an activator of PKC, does not promote cell death or affect eIF2α phosphorylation. However, PMA enhances late stages of UV-irradiation or cycloheximide-induced caspase activation, eIF2α phosphorylation and apoptosis in Sf9 cells. PMA also enhances cytochrome c-induced caspase activation and eIF2α phosphorylation in cell extracts. These changes are mitigated more efficiently by caspase inhibitor, z-VAD-fmk, than by calphostin, an inhibitor of PKC. In contrast, tunicamycin-induced eIF2α phosphorylation that does not lead to caspase activation or cell death is unaffected by PMA, z-VAD-fmk or by calphostin.

Conclusions

While caspase activation is a cause and consequence of eIF2α phosphorylation, PKC activation that follows caspase activation further enhances caspase activation, eIF2α phosphorylation, and cell death in Sf9 cells.

General significance

Caspases can activate multiple signaling pathways to enhance cell death.     

Pumpkin eIF5A isoforms interact with components of the translational machinery in the cucurbit sieve tube system

In yeast, eIF5A, in combination with eEF2, functions at the translation step, during the protein elongation cycle. This result is of significance with respect to functioning of the enucleate sieve tube system, as eIF5A was recently detected in Cucurbita maxima (pumpkin) phloem sap. In the present study, we further characterized four CmeIF5A isoforms, encoding three proteins, all of which were present in the phloem sap. Although hypusination of CmeIF5A was not necessary for entry into the sieve elements, this unique post‐translational modification was necessary for RNA binding. The two enzymes required for hypusination were detected in pumpkin phloem sap, where presumably this modification takes place. A combination of gel‐filtration chromatography and protein overlay assays demonstrated that, as in yeast, CmeIF5A interacts with phloem proteins, like eEF2, known to be involved in protein synthesis. These findings are discussed in terms of a potential role for eIF5A in regulating protein synthesis within the enucleate sieve tube system of the angiosperms.     

过表达EoRPL11下调HEK293T细胞中蛋白质的翻译活性(英文)

核糖体蛋白L11(RPL11)是真核生物核糖体的重要组成部分.RPL11参与核糖体的生物发生及其它的一些细胞调控过程.本研究在人细胞中研究了游仆虫RPL11(EoRPL11)的亚细胞定位及对蛋白质合成的调控功能.通过激光共聚焦显微镜观察发现,融合绿色荧光蛋白的EoRPL11分布于细胞核中,并集中于核仁上;将EoRPL11和海肾荧光素酶报告基因共转染HEK293T细胞后发现,细胞内海肾荧光素酶的酶活性明显下降,并呈现一种剂量依赖性关系;实时定量PCR分析则表明,海肾荧光素酶的mRNA水平并没有明显改变;同时,细胞的增殖也受到了一定的抑制.以上结果表明,EoRPL11是核蛋白,并且其过表达可能在翻译水平上抑制细胞内总蛋白质的合成.     

eIF1 Controls Multiple Steps in Start Codon Recognition during Eukaryotic Translation Initiation

Eukaryotic translation initiation factor (eIF) 1 is a central mediator of start codon recognition. Dissociation of eIF1 from the preinitiation complex (PIC) allows release of phosphate from the G-protein factor eIF2, triggering downstream events in initiation. Mutations that weaken binding of eIF1 to the PIC decrease the fidelity of start codon recognition (Sui⁻ phenotype) by allowing increased eIF1 release at non-AUG codons. Consistent with this, overexpression of these mutant proteins suppresses their Sui⁻ phenotypes. Here, we have examined mutations at the penultimate residue of eIF1, G107, that produce Sui⁻ phenotypes without increasing the rate of eIF1 release. We provide evidence that, in addition to its role in gating phosphate release, dissociation of eIF1 triggers conversion from an open, scanning-competent state of the PIC to a stable, closed one. We also show that eIF5 antagonizes binding of eIF1 to the complex and that key interactions of eIF1 with its partners are modulated by the charge at and around G107. Our data indicate that eIF1 plays multiple roles in start codon recognition and suggest that prior to AUG recognition it prevents eIF5 from binding to a key site in the PIC required for triggering downstream events.     

Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity

Two mutant lines of barley, Risø 17 and Notch‐2, were found to accumulate phytoglycogen in the grain. Like the sugary mutants of maize and rice, these phytoglycogen‐accumulating mutants of barley lack isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a reduction in endosperm starch content, the mutations have a profound effect on the structure, number and timing of initiation of starch granules. There are no normal A‐type or B‐type granules in the mutants. The mutants have a greater number of starch granules per plastid than the wild‐type and, particularly in Risø 17, this leads to the appearance of compound starch granules. These results suggest that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild‐type endosperm plays a role in determining the number, and hence the form, of starch granules.     

Structure, organization and expression of the eukaryotic translation initiation factor 5, eIF-5, gene in Zea mays

The maize genomic DNA sequence encoding the eukaryotic translation initiation factor 5 (eIF-5) has been isolated from genomic library of maize seedlings and the exon–intron structure determined (accession number AJ132240). The length of genomic DNA sequenced was about 7 kb and contained two exons with the translation start site in exon 2. The only intron is located in the non-coding 5′ region and it is 1298 bp long with the splice acceptor and donor sites conforming to the AG/GT rules. Repetitive sequence fragments are located in the 5′ and 3′ intergenic region. The accumulation of eIF-5 mRNA was studied by RNA blot and in situ hybridization. The observed distribution of mRNA may correlate with the function of the protein, as it appears to be highly abundant in tissues where the proportion of cells actively dividing is very high, such as meristematic regions.     

Effects of risk assessment, predator behavior, and habitat on escape behavior in Columbian black-tailed deer

The relationship between preflight risk assessment by prey andthe escape behaviors they perform while fleeing from predatorsis relatively unexplored. To examine this relationship, a humanobserver approached groups of Columbian black-tailed deer (Odocoileushemionus columbianus), varying his behavior to simulate moreor less threatening behavior. We measured the focal deer's angleof escape, distance moved during flight, duration of trottingand stotting behavior, and change in elevation during flight.Analyses revealed positive relationships between the distancemoved during flight and the distance at which they fled. Whenflight was initiated when the approacher was close, deer fledrelatively shorter distances and took flight paths at more acuteangles, a property that would force a real predator to changedirection suddenly. Our results indicate that deer do not compensatefor allowing the observer to approach more closely by fleeinggreater distances. Rather, distance moved and flight initiationdistance are linked by level of reactivity and habituation:more reactive or less habituated deer both flee at a greaterdistance and move away to a greater distance during flight.More threatening behavior by the approacher led to longer durationsof rapid flight behavior (e.g., trotting and stotting), anddeer tended to flee uphill and into taller vegetation, usingthese landscape features as refuge from danger. Finally, weprovide the first evidence for Pitcher's untested "antiambush"hypothesis for the function of stotting and discuss its significance.In general, both preflight predator behavior and habitat featuresinfluence both duration and direction of escape.