Primary T-cells are metabolically quiescent, with little DNA, RNA or protein synthesis. We are studying the role of eIF-2 and eIF-4E expression in the mechanism of translational activation upon mitogenic stimulation. mRNA levels of eIF-2alpha, eIF-2beta and eIF-4E all increase greatly in the 1st 24 hour of stimulation. Nuclear run-on analysis shows that the increase in eIF-2alpha mRNA is not due to a significant increase in the relative level of transcriptional initiation, nor is it due to an increase in mRNA half-life. Dot-blot analysis of nuclear RNA shows that eIF-2alpha hnRNA increases dramatically, using either intron or exon-specific probe. This suggests that the increase in eIF-2alpha hnRNA is regulated at a very early point, but subsequent to transcriptional initiation. In activated cells these mRNAs are on large polysomes, indicating their efficient translation. Nonetheless, by 24 hours the number of eIF-2alpha, eIF-2beta and eIF-4E molecules per cell only increase 2-3-fold, similar to the increase in ribosome number per cell. This suggests a mechanism involving modification of preexisting factors. We have looked at changes in the extent of phosphorylation, and we find that neither eIF-2alpha nor eIF-2beta undergo significant changes in phosphorylation. There is, however, a dramatic increase in the phosphorylation of eIF-4E, which has been shown by other workers to correlate with increases in translational activity. It thus appears that part of translational activation of quiescent T-cells during mitogenic stimulation is due to the phosphorylation of eIF-4E.
