Phosphorylation of elongation factor 1 (EF-1) and Valyl-tRNA synthetase by protein kinase C and stimulation of EF-1 activity

A high M(r) complex isolated from rabbit reticulocytes contains valyl-tRNA synthetase and the four subunits of elongation factor 1 (EF-1). Previously, valyl-tRNA synthetase and the α, β, and δ subunits of EF-1 were shown to be phosphorylated in reticulocytes in response to phorbol 12-myristate 13-acetate (PMA). Phosphorylation of the complex was accompanied by an increase in both valyl-tRNA synthetase and EF-1 activity (Venema, R.C., Peters, H.I., and Traugh, J.A. (1991) J. Biol. Chem., 266, 11993-11998). To investigate phosphorylation of the valyl-tRNA synthetase·EF-1 complex in vitro by protein kinase C, the complex has been purified to apparent homogeneity from rabbit reticulocytes by gel filtration on Bio-Gel A-5m, affinity chromatography on tRNA-Sepharose, and fast protein liquid chromatography on Mono Q. Valyl-tRNA synthetase and the β and δ subunits of EF-1 in the complex are highly phosphorylated by protein kinase C (0.5-0.9 mol of phosphate/mol of subunit), while EF-1α is phosphorylated to a lesser extent (0.2 mol/mol). However, the isolated EF-1α subunit is highly phosphorylated (2.0 mol/mol). Phosphopeptide mapping of EF-1α shows that the same sites are modified by protein kinase C in vitro and in PMA-treated cells. Phosphorylation of the valyl-tRNA synthetase·EF-1 complex results in a 3-fold increase in activity of EF-1 as measured by poly(U)-directed polyphenylalanine synthesis; no effect of phosphorylation is detected with valyl-tRNA synthetase and isolated EF-1α. Thus, phosphorylation and activation of EF-1 by protein kinase C, which has been shown to occur in vitro as well as in reticulocytes, may have a role in PMA stimulation of translational rates.