PHOSPHORYLATION OF ELONGATION-FACTOR 1(EF-1) BY PROTEIN-KINASE-C STIMULATES GDP GTP-EXCHANGE ACTIVITY/

Phosphorylation of the alpha, beta and delta subunits of elongation fa ctor (EF) 1 by protein kinase C results in stimulation of elongation a ctivity up to threefold both in vivo and in vitro [Venema, R. C., Pete rs, H. I. & Traugh, J. A. (1991) J. Biol. Chem. 266, 11993-11998; Vene ma, R. C., Peters, H. I. & Traugh, J. A. (1991) J. Biol. Chem. 266, 12 574-12580]. The alpha subunit catalyzes the GTP-dependent binding of a mino-acyl-tRNA to the ribosome, while the beta gamma and delta subunit s of EF-1 catalyze exchange of the residual GDP on EF-1 alpha for GTP. To determine whether the change in elongation rate following phosphor ylation by protein kinase C is due to stimulation of GDP/GTP exchange activity. EF-1 and EF-1 . valyl-tRNA-synthetase have been purified fro m rabbit reticulocytes, phosphorylated in vitro by protein kinase C an d the effect of phosphorylation on nucleotide-exchange activity analyz ed. The alpha, beta and delta subunits are phosphorylated only on seri ne, and phosphopeptide maps show distinct phosphopeptides for each sub unit. Following quantitative phosphorylation of EF-1 by protein kinase C on the alpha, beta and delta subunits, a twofold enhancement of the rate of nucleotide exchange over the non-phosphorylated controls is o bserved with EF-1 and EF-1 . valyl-tRNA synthetase. Stimulation of nuc leotide exchange results in a two-fold increase in the formation of EF -1 alpha . GTP . Phe-tRNA, leading to an increased rate of binding of Phe-tRNA to ribosomes. The magnitude of stimulation of the exchange ra te is similar to that reported previously for the rate of elongation f ollowing phosphorylation of EF-1 by protein kinase C. Thus, the enhanc ement of EF-1 activity in response to 4 beta-phorbol 12-myristate 13-a cetate appears to be due to stimulation of the rate of GDP/GTP exchang e following phosphorylation of EF-1 by protein kinase C.