Translational regulation during activation of porcine peripheral blood lymphocytes: association and phosphorylation of the alpha and gamma subunits of the initiation factor complex eIF-4F.

Mature peripheral blood lymphocytes exist in a resting state both in vivo and when maintained in culture, exhibiting low translation rates consistent with their non-proliferative state. Previously we have shown that activation of these quiescent cells with either phorbol ester or concanavalin A leads to a rapid increase in the rate of protein synthesis and phosphate-labelling of initiation factor eIF-4 alpha [Morley, Rau, Kay and Pain (1993) Eur. J. Biochem. 218, 39-48]. We now show that neither the early enhanced translation rate nor the early increased phosphate-labelling of eIF-4 alpha requires the activity of the 70 kDa form of ribosomal protein S6 kinase. In addition, we demonstrate that eIF-4 gamma is phosphorylated in response to cell activation, an event which is correlated with phosphorylation of eIF-4 alpha and enhanced eIF-4F complex formation. In these studies, isoelectric focusing and immunoblot analysis of eIF-4 alpha indicate that phosphate-labelling of eIF-4 alpha following cell activation reflects a modest increase in steady-state phosphorylation, mediated by the enhanced activity of eIF-4 alpha kinase(s) and inhibition of eIF-4 alpha phosphatase activity. In the resting cell, eIF-4 alpha is associated with heat- and acid-stable insulin-responsive protein (PHAS-I; 4E-BP1); following acute stimulation with phorbol ester, there is a 40% decrease in the amount of PHAS-I associated with eIF-4 alpha. Incubation of anti-PHAS-I immunoprecipitates with extracts containing activated or immunprecipitated mitogen-activated protein kinase resulted in a small increase in phosphorylation of recovered PHAS-I and a modest release of eIF-4 alpha from the PHAS-I-eIF-4 alpha complex. These data suggest a possible role for PHAS-I in the regulation of eIF-4F complex formation and the rate of translation in primary cells.