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

Authors


Correspondence to S. J. Morley, Biochemistry Laboratory, School of Biological Sciences, University of Sussex, Brighton, Sussex, England BN1 9QG

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 Atkins, M. (1971) Biochim. Biophys. Acta 247, 322–334]. Studies on extracts prepared from unstimulated cells have suggested lesions in initiation factor activity, primarily affecting the binding of mRNA to ribosomes [Ahern, T., Sampson, J. and Kay, J. E. (1974) Nature 248, 519–521].

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 eIF-4α with phosphate in vivo, which primarily reflects a small net increase in phosphorylation rather than phosphate turnover on eIF-4α. 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 eIF-4α phosphorylation within 30 min. Analysis of phosphorylation by two-dimensional gel electrophoresis and measurement of kinase activity towards synthetic peptides, indicate that this increased labelling also reflects increased eIF-4α kinase activity rather than phosphate turnover on eIF-4α.

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 eIF-4α recovered in high-molecular-mass complexes. These data suggest that, in quiescent PPBMCs, eIF-4F may be limiting and that the association of eIF-4α and eIF-4γ into high-molecular-mass complexes is regulated by phosphorylation and may play a pivotal role in translational control.

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