Increased phosphorylation of eukaryotic initiation factor 4α during early activation of T lymphocytes correlates with increased initiation factor 4F complex formation

Abstract

Mature porcine peripheral blood mononuclear cells (PPBMCs) exist in a resting state both in vivo and when maintained in culture, with low translation rates consistent with their non-proliferative state. When cultured 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., Ahern, T. and In these studies, we have demonstrated that activation of quiescent PPBMCs with the phorbol ester 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, respectively, which is insensitive to the transcriptional inhibitor, 5,6-dichlorobenzimidazole riboside. Relative to control cells, both phorbol ester and concanavalin A induce a 2-4-fold increase in labelling of the eukaryotic initiation factor eIF4a with phosphate in vivo, which primarily reflects a small net increase in phosphorylation rather than phosphate turnover on eIF-4a. Similarly, with the human leukaemic 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 eIF4a phosphorylation within 30 min. Analysis of phosphorylation by twodimensional gel electrophoresis and measurement of kinase activity towards synthetic peptides, indicate that this increased labelling also reflects increased eIF4a kinase activity rather than phosphate turnover on eIF-4a.Of central importance is the finding that, concomitant with increased rates of protein synthesis following stimulation of PPBMCs with either phorbol ester or concanavalin A, there is a significant increase in the level of eIF4a recovered in high-molecular-mass complexes. These data suggest that, in quiescent PPBMCs, eIF-4F may be limiting and that the association of eIF4a and eIF-4y into high-molecular-mass complexes is regulated by phosphorylation and may play a pivotal role in translational control.Control of polypeptide synthesis plays an important role in cell proliferation (reviewed in [l]) and translation rates generally reflect the growth state of the cultured eukaryotic cell. Physiological regulation of protein synthesis is almost always exerted at the level of polypeptide initiation [2, 31. Numerous studies have indicated key roles in the regulation of protein synthesis for structural features of mRNA molecules; these include the presence of the 5'-terminal cap structure, the primary and secondary structure of the 5'-un-

The regulation of protein synthesis and translation factors by CD3 and CD28 in human primary T lymphocytes

“…In contrast to previously reported data using mitogenic stimuli to activate primary T cells or Jurkat T cells [14][15][16][17], eIF4E phosphorylation, association of eIF4G with eIF4E and 4E-BP1 phosphorylation remained unchanged after T cell activation using αCD3 and αCD28 as a stimulus (Fig. 4).…”

Amino acid-induced stimulation of translation initiation in rat skeletal muscle

“…Phosphorylation of eIF4E enhances the affinity of the factor for 7-methylguanosine 5Ј-triphosphate cap analogs of mRNA (32) and for eIF4G and eIF4A (35). These effects may contribute to activation of mRNA translation with supraphysiological concentrations of amino acids.…”

Molecular mechanisms for the control of translation by insulin

“…Phosphorylation of eIF4E is reported to increase its affinity for the 7-methyl-GTP cap and for mRNA [120] and may also enhance its ability to form high-molecularmass complexes, e.g. with eIF4G and eIF4A [121,122]. Flynn and Proud [44] have shown, using the specific MEK inhibitor PD098059, that the MAP kinase pathway is required for the insulin-induced increase in the steady-state level of phosphorylation of eIF4E in CHO cells overexpressing the insulin receptor.…”