INSULIN STIMULATION OF PHOSPHORYLATION OF ELONGATION-FACTOR-1 (EEF-1)ENHANCES ELONGATION ACTIVITY

To examine the role of phosphorylation of the elongation factor eEF-1 in regulation of translation, P-32-labeled 3T3-L1 cells were deprived of serum, then incubated in the presence or absence of 10 nM insulin f or 15 min. eEF-1 was purified by affinity chromatography on tRNA-Sepha rose and shown to be phosphorylated on the alpha, beta and delta subun its. Phosphorylation of eEF-1 alpha was stimulated sixfold in response to insulin, beta was stimulated fourfold and delta was threefold. The rate of elongation assayed with eEF-1 from insulin-stimulated cells w as over twofold greater than with eEF-1 from serum-deprived cells. Whe n eEF-1 from insulin-treated cells was subjected to two-dimensional tr yptic phosphopeptide mapping, nine phosphopeptides were obtained with the a subunit, one with the beta subunit and three with the delta subu nit. When compared with phosphopeptide maps of alpha, beta and delta s ubunits of eEF-1 phosphorylated in vitro by the insulin-stimulated mul tipotential protein kinase, the maps of the beta and delta subunits we re identical. Five phosphopeptides obtained with the alpha subunit in vivo were identical to those obtained with S6 kinase iii vitro; the re mainder were unique. To examine whether protein kinase C bad 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 4 beta-phorbo l 12-myristate 13-acetate (PMA). Phosphorylation of the alpha, beta an d delta subunits was stimulated 2.5-fold in response to insulin, with elongation activity stimulated to a similar extent: suggesting that pr otein 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 da ta is consistent with previous studies on stimulation of initiation vi a phosphorylation of initiation factors by multipotential S6 kinase [M orley, S. J. & Traugh, J. A. (1993) Biochemie (Paris) 95, 985-989].