Phosphorylation of eIF2α in response to 26S proteasome inhibition is mediated by the haem-regulated inhibitor (HRI) kinase

Abstract

The present study demonstrates that even brief inhibition of degradation by the 26S proteasome inhibits global protein synthesis, mediated through increased phosphorylation of eIF2alpha (eukaryotic translational initiation factor 2alpha) by the HRI (haem-regulated inhibitor) kinase. Exposure of COS-7 cells to the proteasome inhibitor MG-132 (the proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-leucinal) for 4 h resulted in a 55-60% decrease in protein synthesis rate compared with control cells. This repression of protein synthesis after treatment with MG-132 is not due to induction of apoptosis, which is known to occur after longer periods of 26S inhibition. Instead, we observed a significantly increased phosphorylation of eIF2alpha, which is known to repress global protein synthesis. In three MEF (mouse embryonic fibroblast) knockout cell lines lacking one of the four kinases known to phosphorylate eIF2alpha, increased phosphorylation of eIF2alpha still occurred after inhibition of the 26S proteasome. These three cell lines included a deletion of the PKR (double-stranded-RNA-dependent protein kinase); a deletion of the PERK (PKR-like endoplasmic reticulum resident kinase); or a deletion of the GCN2 (positive general control of transcription-2) kinase, indicating that none of these kinases was primarily responsible for the observed phosphorylation of eIF2alpha. In contrast, in a fourth MEF knockout cell line, HRI(-/-) cells lacking the HRI kinase failed to increase eIF2alpha phosphorylation upon proteasome inhibitor treatment (MG-132 or various doses of Bortezomib), indicating that the HRI kinase is the primary kinase activated by brief treatment of MEFs with 26S proteasome inhibitors.

RETRACTION: Hepatic CYP3A Suppression by High Concentrations of Proteasomal Inhibitors: A Consequence of Endoplasmic Reticulum (ER) Stress Induction, Activation of RNA-Dependent Protein Kinase-Like ER-Bound Eukaryotic Initiation Factor 2α (eIF2α)-Kinase (PERK) and General Control Nonderepressible-2 eIF2α Kinase (GCN2), and Global Translational Shutoff

“…4C), the other heme-regulated eIF2␣ kinase that is reportedly activated in MEFs by proteasomal inhibition by MG132 or bortezomib (Velcade) (Yerlikaya et al, 2008). Together, these findings in cultured rat hepatocytes indicate that high concentrations of MG132 can activate at the least two hepatic eIF2␣ kinases.…”

“…Likewise, the proteasome inhibitor PS431 (bortezomib) also principally activates PERK in human head and neck squamous cell carcinoma cells (Fribley et al, 2004). More intriguingly, MG132 (50 M) treatment of wild-type MEFs resulted in eIF2␣ phosphorylation and inhibition of protein synthesis that was unaffected in PERK(Ϫ/Ϫ), GCN2(Ϫ/Ϫ), or PKR(Ϫ/Ϫ) MEFs but was significantly impaired in HRI(Ϫ/Ϫ) (Yerlikaya et al, 2008), leading to the proposal that HRI is the principal eIF2␣ kinase involved in this response. However, our immunoblotting analyses indicate that MG132-induced proteasomal inhibition failed to affect hepatic HRI (Fig.…”

Differential Control of the CCAAT/Enhancer-binding Protein β (C/EBPβ) Products Liver-enriched Transcriptional Activating Protein (LAP) and Liver-enriched Transcriptional Inhibitory Protein (LIP) and the Regulation of Gene Expression during the Response to Endoplasmic Reticulum Stress

“…It is worth noting that MG132 abolished accumulation of phospho-eIF2␣ during ER stress (Fig. 3A), in agreement with some reports (41) but in disagreement with others (42)(43)(44), possibly because of the short treatment and low MG132 concentration. Longer times and higher concentrations increased eIF2␣ phosphorylation (data not shown).…”

Next-Generation Proteasome Inhibitor Oprozomib Synergizes with Modulators of the Unfolded Protein Response to Suppress Hepatocellular Carcinoma

“…The PERK pathway regulates the nelfinavirmediated increase in sensitivity to oZ OZ slightly increased the eIF2α phosphorylation, which could not be inhibited by the PERK inhibitor ( Figure 2B), pointing to the involvement of other eIF2α kinases, as previously reported for eIF2α phosphorylation induced by proteasome inhibitor MG-132 [21]. Importantly, addition of salubrinal or nelfinavir profoundly increased the OZ-induced eIF2α phosphorylation and pro-apoptotic CHOP protein levels ( Figure 2B), which may contribute to the increased sensitivity to OZ (Figure 1).…”