Abstract
Components of the cellular translation machinery are targets of caspase-mediated cleavage during apoptosis that correlates with the inhibition of protein synthesis, which accompanies apoptosis. Paradoxically, protein synthesis is required for apoptosis to occur in many experimental settings. Previous studies showed that two proteins that regulate apoptosis by controlling caspase activity, XIAP and Apaf-1, are translated by a unique, cap-independent mechanism mediated by an internal ribosome entry site (IRES) that is used preferentially under conditions in which normal cap-dependent translation is repressed. We investigated the regulation of XIAP and Apaf-1 following UVC irradiation. We show that UVC irradiation leads to the inhibition of translation and cell death. Furthermore, IRES-mediated translation of Apaf-1, but not XIAP, is enhanced by UVC irradiation, and this increase in Apaf-1 translation correlated with cell death. The enhanced Apaf-1 IRES-mediated translation is caspase-independent but is negatively modulated by the eIF2alpha kinase protein kinase RNA-like endoplasmic reticulum kinase. These data suggest that progression of UV-induced apoptosis requires IRES-mediated translation of Apaf-1 to ensure continuous levels of Apaf-1 despite an overall suppression of protein synthesis.
Highlights
Research (CIHR), Premier’s Research Excellence Award, and the Canada Foundation for Innovation, Ontario Research and Development Challenge Fund
UV Irradiation Induces Cell Death and Inhibits Protein Synthesis— To investigate the regulatory mechanism of UVC-induced cell death, we first determined the sensitivity of 293T cells to UVC irradiation
These analyses revealed that protein synthesis was substantially reduced in the UVC-irradiated cells compared with non-irradiated 293T cells (Fig. 1B), confirming that UV irradiation inhibits protein synthesis in 293T cells
Summary
Cell Culture and Transfection Reagents—Mouse embryonic fibroblasts from PERK-null (PERKϪ/Ϫ) or PERK wild-type (PERKϩ/ϩ) mice were maintained in Dulbecco’s modified Eagle’s medium (Wisent Inc.) supplemented with heat-inactivated 10% fetal calf serum, 2 mM L-glutamine, 1% antibiotics (100 units/ml penicillin-streptomycin), 1% essential amino acids, and 0.01% -mercaptoethanol. Analysis of Global Protein Synthesis—Both control and UV-irradiated cells were labeled with [35S]methionine (200 Ci/ml, Amersham Biosciences) for 1 h in methionine/cysteine-free minimal essential medium (Invitrogen) in which L-cysteine was added to a final concentration of 63 mg/liter. If quantification of protein data was required the Western blots were performed as described above, but the secondary antibody used was Alexa Fluor 680 goat anti-mouse, anti-rat, or anti-rabbit IgG (LI-Cor Inc.). -Galactosidase and CAT Analysis—Cells were briefly washed in ice-cold phosphate-buffered saline and harvested in the CAT ELISA kit lysis buffer (Roche Molecular Biochemicals) according to the protocol provided by the manufacturer. For detection of Apaf-1 IRES activity, the PERK or control siRNA transfected cells were transfected the day following siRNA transfection with the Apaf-1 bicistronic plasmid pgal/Apaf-1/CAT. Quantitative RT-PCR—Total RNA was isolated from UVC-treated or control cells that were previously transfected with the pgal/Apaf-1/ CAT reporter plasmid as described above. The primers for detection of PERK were as follows: forward, 5Ј-CTCACAGGCAAAGGAAGGAG-3Ј, and reverse, 5Ј-AACAACTCCAAAGCCACCAC-3Ј
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