The effect of phosphorylation on the functional activity of eukaryotic elongation factor 2 (eEF-2) was studied using a purified phosphorylated factor. The modified factor was unable to stimulate protein synthesis in an eEF-2-dependent rabbit reticulocyte lysate. The functional alteration was further analyzed by measuring the effects of phosphorylation on the ability of the factor to catalyse the ribosome-dependent hydrolysis of GTP. Kinetic analysis showed that both phosphorylated and unmodified factor was able to hydrolyse GTP with approximately the same maximum rate, indicating that the rate of nucleotide exchange was not impaired by the modification. However, the phosphorylated factor showed a marked reduction in the second-order rate constant, suggesting that the phosphorylation interfered with ribosome . eEF-2 complex formation by reducing the affinity of eEF-2 for the ribosome. This assumption was confirmed by direct measurements of the dissociation constants for the ribosomal complexes containing unmodified and phosphorylated eEF-2.

Development in plants is controlled by abiotic environmental cues such as day length, light quality, temperature, drought, and salinity. These signals are sensed by a variety of systems and transmitted by different signal transduction pathways. Ultimately, these pathways are integrated to control expression of specific target genes, which encode proteins that regulate development and differentiation. The molecular mechanisms for such integration have remained elusive. We here show that a linear 130-aminoacids-long sequence in the Med25 subunit of the Arabidopsis thaliana Mediator is a common target for the drought response element binding protein 2A, zinc finger homeodomain 1, and Myb-like transcription factors which are involved in different stress response pathways. In addition, our results show that Med25 together with drought response element binding protein 2A also function in repression of PhyB-mediated light signaling and thus integrate signals from different regulatory pathways.

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As the molecular processes of complex cell stress signaling pathways are defined, the subsequent challenge is to elucidate how each individual event influences the final biological outcome. Phosphorylation of the translation initiation factor 2 (eIF2␣) at Ser 51 is a molecular signal that inhibits translation in response to activation of any of four diverse eIF2␣ stress kinases. We used gene targeting to replace the wild-type Ser 51 allele with an Ala in the eIF2␣ gene to test the hypothesis that translational control through eIF2␣ phosphorylation is a central death stimulus in eukaryotic cells. Homozygous eIF2␣ mutant mouse embryo fibroblasts were resistant to the apoptotic effects of dsRNA, tumor necrosis factor-␣, and serum deprivation. TNF␣ treatment induced eIF2␣ phosphorylation and activation of caspase 3 primarily through the dsRNA-activated eIF2␣ kinase PKR. In addition, expression of a phospho-mimetic Ser 51 to Asp mutant eIF2␣-activated caspase 3, indicating that eIF2␣ phosphorylation is sufficient to induce apoptosis. The proapoptotic effects of PKR-mediated eIF2␣ phosphorylation contrast with the anti-apoptotic response upon activation of the PKR-related endoplasmic reticulum eIF2␣ kinase, PERK. Therefore, divergent fates of death and survival can be mediated through phosphorylation at the same site within eIF2␣. We propose that eIF2␣ phosphorylation is fundamentally a death signal, yet it may promote either death or survival, depending upon coincident signaling events.