INCREASED PHOSPHORYLATION OF EUKARYOTIC INITIATION FACTOR-4-ALPHA DURING EARLY ACTIVATION OF T-LYMPHOCYTES CORRELATES WITH INCREASED INITIATION FACTOR-4F COMPLEX-FORMATION

Mature porcine peripheral blood mononuclear cells (PPBMCs) exist in a resting state both in vivo and when maintained in culture, with low tr anslation rates consistent with their non-proliferative state. When cu ltured in the presence of the appropriate mitogen, there is a 2-4-fold increase in the rate of protein synthesis per ribosome within 4 h of stimulation [Kay, J. E., Ahem, T. and Atkins, M. (1971) Biochim. Bioph ys. Acta 247, 322-3341. Studies on extracts prepared from unstimulated cells have suggested lesions in initiation factor activity, primarily affecting the binding of mRNA to ribosomes [Ahem, T., Sampson, J. and Kay, J. E. (1 974) Nature 248, 519 - 521]. In these studies, we have demonstrated that activation of quiescent PPBMCs with the phorbol este r phorbol 12-myristate 13-acetate or concanavalin A leads to a rapid 2 -4-fold increase in the rate of protein synthesis within 1 h or 4 h, r espectively, which is insensitive to the transcriptional inhibitor, 5, 6-dichlorobenzimidazole riboside. Relative to control cells, both phor bol ester and concanavalin A induce a 2-4-fold increase in labelling o f the eukaryotic initiation factor eIF-4alpha with phosphate in vivo, which primarily reflects a small net increase in phosphorylation rathe r than phosphate turnover on eIF-4alpha. Similarly, with the human leu kaemic T cell line JURKAT, stimulation of the T cell receptor with the monoclonal antibody, OKT-3, or treatment with phorbol ester induces a 2-3-fold increase in eIF-4alpha phosphorylation within 30 min. Analys is of phosphorylation by two-dimensional gel electrophoresis and measu rement of kinase activity towards synthetic peptides, indicate that th is increased labelling also reflects increased eIF-4alpha kinase activ ity rather than phosphate turnover on eIF-4alpha. Of central importanc e is the finding that, concomitant with increased rates of protein syn thesis following stimulation of PPBMCs with either phorbol ester or co ncanavalin A, there is a significant increase in the level of eIF-4alp ha recovered in high-molecular-mass complexes. These data suggest that , in quiescent PPBMCs, eIF-4F may be limiting and that the association of eIF-4alpha and eIF-4gamma into high-molecular-mass complexes is re gulated by phosphorylation and may play a pivotal role in translationa l control.