Insulin stimulation of phosphorylation of elongation factor 1 (eEF-1) enhances elongation activity

Abstract

To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, 32P-labeled 3T3-L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin for 5 min. eEF-1 was purified by affinity chromatography on tRNA-Sepharose and shown to be phosphorylated on the α, β and δ subunits. Phosphorylation of eEF-1α was stimulated sixfold in response to insulin, β was stimulated fourfold and δ was threefold. The rate of elongation assayed with eEF-1 from insulin-stimulated cells was over twofold greater than with eEF-1 from serum-deprived cells. When eEF-1 from insulin-treated cells was subjected to two-dimensional tryptic phosphopeptide mapping, nine phosphopeptides were obtained with the a subunit, one with the β subunit and three with the δ subunit. When compared with phosphopeptide maps of α, β and δ subunits of eEF-1 phosphorylated in vitro by the insulin-stimulated multipotential protein kinase, the maps of the β and δ subunits were identical. Five phosphopeptides obtained with the a subunit in vivo were identical to those obtained with S6 kinase in vitro; the remainder were unique. To examine whether protein kinase C had a role in phosphorylation of eEF-1 in response to insulin, protein kinase C was down-regulated by prolonged exposure of 3T3-L1 cells to 4β-phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the α, β and δ subunits was stimulated 2.5-fold in response to insulin, with elongation activity stimulated to a similar extent, suggesting that protein kinase C had no effect on stimulation of elongation in response to insulin. Thus, stimulation of eEF-1 activity in response to insulin appears to be mediated primarily by multipotential S6 kinase. This data is consistent with previous studies on stimulation of initiation via phosphorylation of initiation factors by multipotential S6 kinase [Morley, S. J. and Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989].