Phosphorylation of ribosomal protein S6 in the Ehrlich ascites tumor cell

Abstract

The incorporation of ³²P_i into the ribosomal protein S6, which is a component of the 40 S ribosomal subunit, was measured in Ehrlich ascites tumor cells growing in suspension culture. During a 2-h incubation period, incorporation was 10-fold greater into S6 of cells growing exponentially in complete medium than in cells in which protein synthesis and growth were inhibited by omission of glutamine from the medium. Since the labeling of other phosphoproteins was not similarly depressed in glutamine-deprived cells, the decreased labeling most likely reflects a specific decrease in phosphate incorporation into S6 rather than a lower phosphate pool specific activity. The cycling of ribosomes is inhibited in glutamine-deprived cells, with an accumulation of monomeric ribosomes indicating an inhibition of protein synthesis initiation. To assess whether the decreased phosphorylation of S6 in the ribosomes of glutamine-deprived cells was responsible for their decreased ability to initiate protein synthesis and enter polyribosomes, a mixture of ¹⁴C-labeled ribosomes from rapidly growing cells and ³H-labeled ribosomes from glutamine-deprived cells was added to a rabbit reticulocyte lysate cell-free protein-synthesizing system in which ribosomes cycle actively. The ${}^{14}\text{C}{}^3\text{H}$ ratio was determined in polyribosomes as a measure of the relative ability of the two kinds of ribosomes to initiate protein synthesis. The ${}^{14}\text{C}{}^3\text{H}$ ratio in polyribosomes was found to be identical to the input of labeled ribosomes, indicating an equal ability of ribosomes from growing cells and glutamine-deprived cells to function in initiation. Control studies indicated lack of significant dephosphorylation of S6 during purification of ribosomes and during incubation in the reticulocyte lysate. Thus, a functional difference between more and less phosphorylated ribosomes could not be demonstrated in this system.