Phosphorylation of Elongation Factor 1 (EF‐1) by Protein Kinase C Stimulates GDP/GTP‐Exchange Activity

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Abstract
Phosphorylation of the α, β and δ subunits of elongation factor (EF) 1 by protein kinase C results in stimulation of elongation activity up to threefold both in vivo and in vitro [Venema, R. C., Peters, H. I. & Traugh, J. A. (1991) J. Biol. Chem. 266, 11993–11998; Venema, F., C., Peters, H. I. & Traugh, J. A. (1991) J. Biol. Chem. 266, 12574–12580]. The α subunit catalyzes the GTP-dependent binding of amino-acyl-tRNA to the ribosome, while the βγ and δ subunits of EF-1 catalyze exchange of the residual GDP on EF-1α for GTP. To determine whether the change in elongation rate following phosphorylation by protein kinase C is due to stimulation of GDP/GTP exchange activity, EF-1 and EF-1 · valyl-tRNA-synthetase have been purified from rabbit reticulocytes, phosphorylated in vitro by protein kinase C and the effect of phosphorylation on nucleotide-exchange activity analyzed. The α, β and δ subunits are phosphorylated only on serine, and phosphopeptide maps show distinct phosphopeptides for each subunit. Following quantitative phosphorylation of EF-1 by protein kinase C on the α, β, and δ subunits, a twofold enhancement of the rate of nucleotide exchange over the non-phosphorylated controls is observed with EF-1 and EF-1 · valyl-tRNA synthetase. Stimulation of nucleotide exchange results in a two-fold increase in the formation of EF-1α· GTP · Phe-tRNA, leading to an increased rate of binding of Phe-tRNA to ribosomes. The magnitude of stimulation of the exchange rate is similar to that reported previously for the rate of elongation following phosphorylation of EF-1 by protein kinase C. Thus, the enhancement of EF-1 activity in response to 4β-phorbol 12-myristate 13-acetate appears to be due to stimulation of the rate of GDP/GTP exchange following phosphorylation of EF-1 by protein kinase C.