The caspase-generated fragments of PKR cooperate to activate full-length PKR and inhibit translation.
We have studied the involvement of receptor interacting protein kinase-1 (RIP1) and dsRNA-activated protein kinase (PKR) in external dsRNA-induced apoptotic and necrotic cell death in Jurkat T cell lymphoma. Our results suggest that RIP1 plays an imported role in dsRNA-induced apoptosis and necrosis. We demonstrated that contrary to necrosis, protein synthesis is inhibited in apoptosis. Here, we show that phosphorylation of translation initiation factor 2-alpha (eukaryotic initiation factor 2-alpha (eIF2-alpha)) and its kinase, PKR, occur in dsRNA-induced apoptosis but not in necrosis. These events are caspase-dependent and coincide with the appearance of the caspase-mediated PKR fragments, N-terminal domain (ND) and kinase domain (KD). Our immunoprecipitation experiments demonstrated that both fragments could independently co-precipitate with full-length PKR. Expression of PKR-KD leads to PKR and eIF2-alpha phosphorylation and inhibits protein translation, whereas that of PKR-ND does not. Co-expression of PKR-ND and PKR-KD promotes their interaction with PKR, PKR and eIF2-alpha phosphorylation and suppresses protein translation better than PKR-KD alone. Our findings suggest a caspase-dependent mode of activation of PKR in apoptosis in which the PKR-KD fragment interacts with and activates intact PKR. PKR-ND facilitates the interaction of PKR-KD with full-length PKR and thus the activation of the kinase and amplifies the translation inhibitory signal.
Kalai
M
M
Laboratory of Cellular Microbiology, Pasteur Institute, Rue Engeland, Brussels, Belgium. mkalai@pasteur.be
Suin
V
V
Festjens
N
N
Meeus
A
A
Bernis
A
A
Wang
X-M
XM
Saelens
X
X
Vandenabeele
P
P
D017209:Apoptosis / Q000187:drug effects; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D019169:Jurkat Cells; D009336:Necrosis; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D012330:RNA, Double-Stranded / Q000494:pharmacology; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D019892:eIF-2 Kinase / Q000096:biosynthesis / Q000378:metabolism*
D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D010446:Peptide Fragments; D012330:RNA, Double-Stranded; C506249:RIPK1 protein, human; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D019892:eIF-2 Kinase; D020169:Caspases