Rapid and efficient method for analyzing phosphorylation of the S6 ribosomal protein in 32Pi-labeled,tissue culture cells

We describe a method for studying the phosphorylation of the S6 ribosomal protein in intact cells. The procedure has the advantage of using few cells, little ³²P_i, and by using an air-driven centrifuge, many samples can be processed in a short time. Metabolically labeling the ribosomes with [³H]uridine before the experiment provides a measure of ribosome yield. The amount of ³²P_i incorporated into proteins other than S6, which cosediment with the ribosomes, increases by the same amount as the specific activity of [³²P]ATP increases, when the cells are stimulated by prostaglandin F_2α, insulin, epidermal, or fibroblast growth factor, or serum; whereas the ³²P_i incorporated into S6 increases by a factor greater than the increase in the specific activity of [³²P]ATP. We show that the phosphate on S6 turns over at least as rapidly as does the phosphate on ATP. This last observation allows us to use a procedure, which we have outlined for determining the absolute amount of phosphate added to S6 due to a stimulus.