Phosphorylation of Elongation Factor 1 and Ribosomal Protein S6 by Multipotential S6 Kinase and Insulin Stimulation of Translational Elongation

Abstract

Stimulation of protein synthesis in response to insulin is concomitant with increased phosphorylation of initiation factors 4B and 4G and ribosomal protein S6 (Morley, S. J., and Traugh, J. A. (1993) Biochimie 75, 985-989) and is due at least in part to multipotential S6 kinase. When elongation factor 1 (EF-1) from rabbit reticulocytes was examined as substrate for multipotential S6 kinase, up to 1 mol/mol of phosphate was incorporated into the ␣, ␤, and ␦ subunits. Phosphorylation of EF-1 resulted in a 2-2.6-fold stimulation of EF-1 activity, as measured by poly(U)-directed polyphenylalanine synthesis. The rate of elongation was also stimulated by approximately 2-fold with 80 S ribosomes phosphorylated on S6 by multipotential S6 kinase. When the rates of elongation in extracts from serum-fed 3T3-L1 cells and cells serum-deprived for 1.5 h were compared, a 40% decrease was observed upon serum deprivation. The addition of insulin to serum-deprived cells for 15 min stimulated elongation to a rate equivalent to that of serumfed cells. Similar results were obtained with partially purified EF-1, with both EF-1 and ribosomes contributing to stimulation of elongation. These data are consistent with a ribosomal transit time of 3.2 min for serumdeprived cells and 1.6 min following the addition of insulin for 15 min. Taken together, the data suggest that insulin stimulation involves coordinate regulation of EF-1 and ribosomes through phosphorylation by multipotential S6 kinase. Elongation factor 1 (EF-1)1 is composed of a nucleotide-binding protein, EF-1␣, and a nucleotide exchange protein complex, EF-1␤␥␦ (1, 2). The EF-1␣ subunit (50.1 kDa) binds GTP and mediates the attachment of aminoacyl-tRNA to ribosomes (1, 2), while the ␤ (24.8-kDa), ␥ (50.0-kDa), and ␦ (31.1-kDa) subunits stimulate the exchange of GDP for GTP on the ␣ subunit (3-5). Several forms of EF-1 can be isolated from eukaryotic cells, including ␣, ␣␤␥, and ␣␤␥␦ (6 -8). Approximately 15% of the EF-1␣␤␥␦ is complexed with valyl-tRNA synthetase (ValRS) (7-11).The mechanisms involved in the regulation of EF-1 activity are not well understood, although EF-1 activity has been shown to be modulated by mitogens, aging, heat shock, and fertilization (12). All four subunits of EF-1 have been shown to undergo phosphorylation in vivo and in vitro. When the ␣, ␤, and ␦ subunits are phosphorylated in rabbit reticulocytes in response to phorbol 12-myristate 13-acetate, a 2-3-fold stimulation of EF-1 activity is observed (6). A similar effect is observed when EF-1 is phosphorylated in vitro by protein kinase C (7), and this effect is due to stimulation of the rate-limiting step, GTP/GDP exchange on EF-1␣ (8).The ␤ and ␦ subunits are also phosphorylated by protein kinase CKII (13-16). GDP has been shown to enhance the phosphorylation of ␤ and ␦ by CKII, while little phosphorylation is observed in the absence of GDP (16). In addition, the ␥ and ␦ subunits of EF-1 from Xenopus oocytes are substrates for the cell cycle division control kinase (Cdc2) (17, 18). The physiologic...

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