Phosphorylation of Eukaryotic Translation Initiation Factor 2 Mediates Apoptosis in Response to Activation of the Double-stranded RNA-dependent Protein Kinase

Abstract

The interferon-inducible, double-stranded (ds) RNAdependent serine/threonine protein kinase (PKR) plays a role in viral pathogenesis, cell growth, and differentiation and is implicated as a tumor suppressor gene. Expression of a trans-dominant negative, catalytically inactive mutant PKR protected NIH3T3 cells from apoptosis in response to either treatment with tumor necrosis factor ␣ (TNF␣), serum deprivation. In cells expressing mutant PKR, TNF␣, but not dsRNA induced transcription from a nuclear factor B-dependent promoter, demonstrating specificity for dsRNA in signaling through the PKR pathway. Serum or plateletderived growth factor addition to serum-deprived mutant PKR-expressing cells induced transcription of the early response genes c-fos and c-jun, indicating that the immediate early response signaling was intact. Overexpression of wild-type PKR in a transient DNA transfection system was sufficient to induce apoptosis. TNF␣-induced apoptosis correlated with increased phosphorylation of the ␣ subunit of eukaryotic translation initiation factor 2 (eIF-2␣), the primary physiological substrate of the PKR. Furthermore, forced expression of a nonphosphorylatable S51A mutant eIF-2␣ partially protected cells from TNF␣-induced apoptosis, and expression of a S51D mutant eIF-2␣, a mutant that mimics phosphorylated eIF-2␣, was sufficient to induce apoptosis. Taken together, these studies identify a novel requirement for PKR in stress-induced apoptosis that is mediated through eIF-2␣ phosphorylation.

DNA-damaging agents cause inactivation of translational regulators linked to mTOR signalling

“…However, the degree of inhibition was larger than that observed in cells treated with rapamycin indicating that additional e ects contribute to the shut-o of protein synthesis in cells su ering DNA damage. It has been suggested that phosphorylation of eIF2a may be important during virus-induced apoptosis (Jagus et al, 1999;Srivastava et al, 1998). Apoptosis can be triggered by the interferon-induced protein kinase PKR that phosphorylates eIF2a and thus inhibits protein synthesis at the level of initiation (Balachandran et al, 1998;Donze et al, 1999;Gil et al, 1999;Takizawa et al, 1999;Yeung et al, 1996).…”

“…The activity of the 4 h DMSO control sample was set at 100%. (*=P50.05, **=P50.005 versus appropriate DMSO control) implicated in apoptosis (Srivastava et al, 1998;Jagus et al, 1999;Clemens, 1996). To ascertain whether etoposide a ected the phosphorylation of eIF2a we used isoelectric focusing followed by immunoblotting to assess the proportion of eIF2a which was in its phosphorylated form at each time.…”

PKR; a sentinel kinase for cellular stress

“…They are also more resistant to serum deprivation and have altered cell cycle characteristics (Zamanian-Daryoush et al, 1999). TNF at relatively high concentrations can induce apoptosis in cultured cells by a mechanism that involves eIF-2a phosphorylation (Srivastava et al, 1998). The connection between protein synthesis inhibition resulting from eIF-2a phosphorylation and apoptosis is not clear but overexpression of Ser-51-Ala mutant eIF-2a that is resistant to phosphorylation protects cells from TNFinduced (and serum induced) apoptosis (Srivastava et al, 1998).…”

“…TNF at relatively high concentrations can induce apoptosis in cultured cells by a mechanism that involves eIF-2a phosphorylation (Srivastava et al, 1998). The connection between protein synthesis inhibition resulting from eIF-2a phosphorylation and apoptosis is not clear but overexpression of Ser-51-Ala mutant eIF-2a that is resistant to phosphorylation protects cells from TNFinduced (and serum induced) apoptosis (Srivastava et al, 1998). The link between the TNF signaling pathway and PKR activation also remains to be determined.…”

Evidence That Ternary Complex (eIF2-GTP-tRNAiMet)–Deficient Preinitiation Complexes Are Core Constituents of Mammalian Stress Granules

“…In mammalian cells, the assembly of SGs is initiated by the phosphorylation of eIF2␣ (Kedersha et al, 1999), a translation initiation factor that loads the initiator tRNA onto the small ribosomal subunit (reviewed by Berlanga et al, 1998;Gray and Wickens, 1998). Phosphorylation of eIF2␣ inhibits protein translation and promotes the accumulation of untranslated mRNA (Farrell et al, 1977;Srivastava et al, 1998). The related RNA-binding proteins TIA-1 and TIAR act downstream of the phosphorylation of eIF2␣ to recruit these untranslated mRNAs to SGs (Kedersha et al, 1999).…”