The protein kinase PKR: a molecular clock that sequentially activates survival and death programs

Abstract

Cell death and survival play a key role in the immune system as well as during development. The control mechanisms that balance cell survival against cell death are not well understood. Here we report a novel strategy used by a single protein to regulate chronologically cell survival and death. The interferon-induced protein kinase PKR acts as a molecular clock by using catalysis-dependent and -independent activities to temporally induce cell survival prior to cell death. We show that the proapoptotic protein PKR surprisingly activates a survival pathway, which is mediated by NF-kappaB to delay apoptosis. Cell death is then induced by PKR through the phosphorylation of eIF-2alpha. This unique temporal control might serve as a paradigm for other kinases whose catalytic activity is not required for all of their functions.

Figure 1

Sequential activation of NF-κB and eIF-2α by PKR. (A) Sequential activation of NF-κB and eIF-2α in NIH3T3 cells inducibly expressing PKRwt. Nuclear extracts from NIH3T3 PKRwt were processed at different periods of time after tetracycline removal to analyze NF-κB activity by EMSA. Cell extracts were examined by immunoblotting with an anti-PKR antibody, a phosphospecific anti-eIF-2α antibody, and an anti-eIF-2α antibody. (B) Sequential activation of NF-κB and eIF-2α signaling pathways in HeLa cells infected with VSV. Nuclear extracts from HeLa infected with VSV or mock-infected were processed at different periods of time after infection. NF-κB DNA-binding activity was measured by EMSA. Lysates were subjected to Western blot analysis for phosphorylated eIF-2α and total eIF-2α. (C) Sequential activation of NF-κB and eIF-2α in HeLa cells infected with measles virus. NF-κB activation and eIF-2α phosphorylation were measured as described in (B). PKR activation was monitored by an autophosphorylation assay in the presence of γ-³²P-ATP. Total PKR was checked by immunoblot analysis.

Figure 2

The proapoptic factor PKR induces the expression of NF-κB target genes encoding survival proteins. (A) Analysis of several PKR-regulated genes by RT–PCR. Total RNA from NIH3T3 cells expressing PKRwt (wt) or the kinase-defective mutant PKR^K296R (K296R) were subjected to RT–PCR analysis with primers specific for the different indicated genes. Tet: tetracycline. The numbers indicate the time in hours (h) after tetracycline removal. (B) Nuclear extracts after different periods of time following tetracycline removal from NIH3T3 cells expressing the kinase-defective mutant PKR^K296R were used to analyze NF-κB activity by EMSA. PKR^K296R levels were examined in cell extracts by immunoblotting with an anti-PKR antibody.

Figure 3

Overexpression of an IκBα super-repressor blocks NF-κB activation. (A) Inhibition of NF-κB activity by expression of the human IκBα super-repressor. NIH3T3 cells expressing PKRwt (wt), PKR^K296R (K296R), PKRwt and the human IκBα super-repressor (wt/IκBα^sr), or PKRwt and the empty plasmid (wt/C), grown in the presence of tetracycline (1 μg/ml), were treated for 30 min with TNFα (10 ng/ml) where indicated. IκBα levels were monitored by immunoblotting using an anti-IκBα antibody. hIκBα^sr and mIκBα represent the human and mouse IκBα, respectively. Hsp90 antibody was used as a loading control. (B) Expression of c-IAP1 is inhibited in wt/IκBα^sr cells. Total RNA was subjected to RT–PCR analysis with primers specific for c-IAP1. Actin was used as control.

Figure 4

Activation of NF-κB by PKR delays the proapoptotic activity of PKR. (A) Cell viability assay. NIH3T3 cells expressing PKRwt (wt), PKRwt and the human IκBα super-repressor (wt/IκBα^sr), or PKRwt and the empty plasmid (wt/C) were seeded at 5 × 10⁴ cells/plate in the presence of tetracycline. The number of viable cells is plotted as a function of time following tetracycline removal. Viable cells remaining after tetracycline removal are shown as a percentage of viable cells grown in the presence of tetracycline. (B) eIF-2α phosphorylation is functional in the absence of NF-κB activity. Cell extracts were processed at different periods of time after tetracycline removal to analyze phosphorylation of eIF-2α by immunoblotting with a phosphospecific anti-eIF-2α antibody and an anti-eIF-2α antibody. (C) wt/IκBα^sr 3 clone dies by apoptosis. Cells were grown in the presence of tetracycline or without for 12 h and stained for the presence of phosphatidylserine by Annexin V-EGFP. (D) Chromatin condensation in wt/IκBα^sr 3 following tetracycline removal. Cells grown in the presence or absence of tetracycline for 15 h were stained with the DNA dye Hoechst 33342 and visualized with a fluorescence microscope. Chromatin condensation is indicated by arrows.

Figure 5

Model for sequential activation of survival and death programs by PKR. See text for details. The dotted line represents translational inhibition mediated by eIF-2α phosphorylation. The dashed arrow represents potentiation of NF-κB signaling by PKR kinase activity. Apoptosis triggered by eIF-2α phosphorylation is delayed by the transient NF-κB-induced survival response.