GDP AS A REGULATOR OF PHOSPHORYLATION OF ELONGATION-FACTOR-1 BY CASEIN KINASE-II

Elongation factor 1 (EF-1) consists of four subunits: the alpha subuni t catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes w hile the beta, gamma, and delta subunits catalyze GDP/GTP exchange on EF-1 alpha. Phosphorylation of the beta subunit of EF-1 from rabbit re ticulocytes by casein kinase II was stimulated up to 22-fold by polyly sine, while basic proteins or polyarginine enhanced phosphorylation to a lesser extent. When physiological components of protein synthesis w ere examined as potential modulators of phosphorylation, ribosomal sub units had no effect, tRNA and poly(U) inhibited the phosphotransferase reaction, and GDP stimulated the initial rate of phosphorylation of E F-1 beta up to 3.8-fold; the degree of stimulation could be correlated with the amount of alpha subunit present in EF-1. No stimulation was observed with other nucleotides. Phosphorylation of EF-1 beta was on s erine, and two-dimensional phosphopeptide mapping showed a single tryp tic phosphopeptide in the presence of GDP or polylysine; the peptide w as identical to that obtained with EF-1 phosphorylated in reticulocyte s incubated with [P-32] orthophosphate. EF-1 delta was also phosphoryl ated by casein kinase II, but only in the presence of GDP. Kinetic dat a showed GDP stimulated phosphorylation by increasing the V-max with b oth the beta and delta subunits. The GDP-dependent stimulation of phos phorylation was specific for EF-1 and was not observed with calmodulin , beta-casein B, or c-Myc. When the catalytic subunit of casein kinase II, cloned and expressed in Escherichia coli, was used, only EF-1 bet a was phosphorylated; stimulation by GDP was approximately the same as that observed with the holoenzyme. Since guanine nucleotides bind onl y to EF-1 alpha, GDP could regulate phosphorylation of EF-1 beta and d elta only when present in the EF-1 alpha beta gamma delta complex. Thu s, phosphorylation of EF-1 beta and delta by casein kinase II would oc cur primarily at a specific point in the elongation cycle, when EF-1 a lpha.GDP is associated with EF-1 beta gamma delta following the GTP-de pendent binding of aminoacyl-tRNA to the ribosome.