In Vivo Regulation of Protein Synthesis by Phosphorylation of the α Subunit of Wheat Eukaryotic Initiation Factor 2

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Abstract
The regulation of protein synthesis is a critical component in the maintenance of cellular homeostasis. A major mechanism of translational control in response to diverse abiotic and biotic stress signals involves the phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α). The pathway has been demonstrated in all eukaryotes except plants, although components of a putative plant pathway have been characterized. To evaluate the in vivo capability of plant eIF2α to participate in the translation pathway, we have used vaccinia virus recombinants that constitutively express wheat eIF2α and inducibly express the eIF2α dsRNA-stimulated protein kinase, PKR, in BSC-40 cells. Activation of PKR in cells expressing wild-type wheat eIF2α resulted in an inhibition of cellular and viral protein synthesis and an induction of cellular apoptosis correlating with phosphorylation of eIF2α on serine 51. Expression of a nonphosphorylatable mutant (51A) of plant eIF2α reversed the PKR-mediated translational block as well as the PKR-induced apoptosis. A direct interaction of the plant proteins with the mammalian translational initiation apparatus is supported by coimmunoprecipitation of wild-type plant eIF2α and the 51A mutant with mammalian eIF2γ and the localization of the plant proteins in ribosome fractions. These findings suggest that plant eIF2α is capable of interacting with the guanine nucleotide exchange factor eIF2B within the context of the eIF2 holoenzyme and provide direct evidence for its ability to participate in phosphorylation-mediated translational control in vivo.