Pushpanjali Pendyala, Ramaiah Kolluru V A
Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, Andhra Pradesh, India.
Biochim Biophys Acta. 2010 May;1800(5):518-25. doi: 10.1016/j.bbagen.2010.01.012. Epub 2010 Feb 1.
Stress-induced phosphorylation of the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), 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 eIF2alpha phosphorylation, caspase activation and cell death in the ovarian cells of Spodoptera frugiperda (Sf9) and in their extracts.
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 eIF2alpha kinase. Status of eIF2alpha phosphorylation and cytochrome c levels are analyzed by western blots.
PMA, an activator of PKC, does not promote cell death or affect eIF2alpha phosphorylation. However, PMA enhances late stages of UV-irradiation or cycloheximide-induced caspase activation, eIF2alpha phosphorylation and apoptosis in Sf9 cells. PMA also enhances cytochrome c-induced caspase activation and eIF2alpha 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 eIF2alpha phosphorylation that does not lead to caspase activation or cell death is unaffected by PMA, z-VAD-fmk or by calphostin.
While caspase activation is a cause and consequence of eIF2alpha phosphorylation, PKC activation that follows caspase activation further enhances caspase activation, eIF2alpha phosphorylation, and cell death in Sf9 cells.
Caspases can activate multiple signaling pathways to enhance cell death.
参与翻译过程的真核起始因子2（eIF2α）的α亚基的应激诱导磷酸化，可促进细胞自杀或存活。由于多种信号通路与细胞死亡有关，因此本研究分析了蛋白激酶C（PKC）激活在应激诱导的草地贪夜蛾（Sf9）卵巢细胞及其提取物中eIF2α磷酸化、半胱天冬酶激活和细胞死亡中的重要性。
通过流式细胞术分析细胞死亡情况。通过Ac-DEVD-AFC水解以及通过切割纯化的重组PERK（一种内质网驻留的eIF2α激酶）来测量半胱天冬酶激活情况。通过蛋白质免疫印迹分析eIF2α磷酸化状态和细胞色素c水平。
PKC激活剂佛波酯（PMA）不促进细胞死亡或影响eIF2α磷酸化。然而，PMA可增强紫外线照射或环己酰亚胺诱导的Sf9细胞中半胱天冬酶激活、eIF2α磷酸化和凋亡的后期阶段。PMA还可增强细胞提取物中细胞色素c诱导的半胱天冬酶激活和eIF2α磷酸化。与PKC抑制剂钙泊三醇相比，半胱天冬酶抑制剂z-VAD-fmk能更有效地减轻这些变化。相比之下，衣霉素诱导的不导致半胱天冬酶激活或细胞死亡的eIF2α磷酸化不受PMA、z-VAD-fmk或钙泊三醇的影响。
虽然半胱天冬酶激活是eIF2α磷酸化的原因和结果，但半胱天冬酶激活后PKC激活会进一步增强Sf9细胞中的半胱天冬酶激活、eIF2α磷酸化和细胞死亡。
半胱天冬酶可激活多种信号通路以增强细胞死亡。
