Defects in translational regulation mediated by the alpha subunit of eukaryotic initiation factor 2 inhibit antiviral activity and facilitate the malignant transformation of human fibroblasts.

Darren J Perkins,Glen N Barber

doi:10.1128/mcb.24.5.2025-2040.2004

Abstract

Suppression of protein synthesis through phosphorylation of the translation initiation factor alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is known to occur in response to many forms of cellular stress. To further study this, we have developed novel cell lines that inducibly express FLAG-tagged versions of either the phosphomimetic eIF2alpha variant, eIF2alpha-S51D, or the phosphorylation-insensitive eIF2alpha-S51A. These variants showed authentic subcellular localization, were incorporated into endogenous ternary complexes, and were able to modulate overall rates of protein synthesis as well as influence cell division. However, phosphorylation of eIF2alpha failed to induce cell death or sensitize cells to killing by proapoptotic stimuli, though it was able to inhibit viral replication, confirming the role of eIF2alpha in host defense. Further, although the eIF2alpha-S51A variant has been shown to transform NIH 3T3 cells, it was unable to transform the murine fibroblast 3T3 L1 cell line. To therefore clarify this issue, we explored the role of eIF2alpha in growth control and demonstrated that the eIF2alpha-S51A variant is capable of collaborating with hTERT and the simian virus 40 large T antigen in the transformation of primary human kidney cells. Thus, dysregulation of translation initiation is indeed sufficient to cooperate with defined oncogenic elements and participate in the tumorigenesis of human tissue.

Highlights

The initiation of protein synthesis in eukaryotes is a highly complex and conserved process involving at least 13 initiation factors, many of which are themselves assembled from numerous subunits [3, 51, 56]
A key translation factor that is a frequent target of regulation by stress-sensitive kinases is the ␣ subunit of the eukaryotic translation initiation factor 2 complex [13, 14, 63, 72]. eIF2 is a heterotrimer composed of three subunits (␣, ␤, and ␥) which functions by associating with GTP and the initiator Met-tRNAi to form a ternary complex [48, 62]
Removal of DOX from the culture medium induced a dose-dependent accumulation of both the eIF2␣-S51A and the eIF2␣-S51D variants that was detectable after 3 days by using a monoclonal antibody (MAb) recognizing the FLAG epitope tag [42] (Fig. 1A)

Summary

Introduction

The initiation of protein synthesis in eukaryotes is a highly complex and conserved process involving at least 13 initiation factors, many of which are themselves assembled from numerous subunits [3, 51, 56]. EIF2 is a heterotrimer composed of three subunits (␣, ␤, and ␥) which functions by associating with GTP and the initiator Met-tRNAi to form a ternary complex [48, 62]. The ternary complex delivers the Met-tRNAi to the 40S ribosomal subunit which, along with other translation factors including eIF3, forms the 43S preinitiation structure [8, 50, 69]. Phosphorylation on serine 51 of eIF2␣ by stress-responsive kinases causes eIF2 to acquire an increased affinity for, and functionally sequester, the GTP exchange factor eIF2B, which is required for maintaining eIF2 activity [40]. Under conditions in which the levels of phosphorylated eIF2␣ rise and levels of available ternary complex fall, these uORFs may favor the association of the transcript with active ribosomes. We demonstrate for the first time that a translation factor can transform human cells in collaboration with defined genetic elements hTERT and simian virus 40 large T antigen, collectively confirming the importance of translational regulation in tumorigenesis

Methods

Results

Conclusion