PDGF Stimulates transient phosphorylation of 180,000 dalton protein

Cell‐free extracts of platelet‐derived growth factor (PDGF) treated, density‐arrested, quiescent BALB/c‐3T3 cells are capable of phosphorylating a 180,000 dalton protein (PP180). The phosphorylation of PP180 was observed in SDS polyacrylamide gel electrophoresis profiles of Nonidet P‐40 solubilized cell preparations that had been incubated with [γ‐³²P]ATP. When quiescent BALB/c‐3T3 cell cultures were incubated at 37°C with PDGF, phosphorylation of PP180 in cell extracts could be detected after a 3‐min exposure of the intact cells to PDGF, which was maximal after 10–15 minutes and had diminished by 30–60 min. PDGF stimulation of PP180 phosphorylation also was observed in extracts of cells that had been incubated with PDGF at 4°C: however, in contrast to PDGF exposure at 37°C, the ability of cell extracts to phosphorylate PP180 did not decrease even after 4 hr of cell exposure to PDGF at 4°C. When cells exposed to PDGF at 4°C were transferred to 37°C for 30 min, the ability of cell extracts to phosphorylate PP180 decreased to a nonstimulated level. After cells stimulated by PDGF showed a diminished ability to phosphorylate PP180, immediate restimulation with PDGF did not induce the ability to phosphorylate PP180. Incubation for 11 hr at 37° C was required before readdition of PDGF allowed observable phosphorylation of PP180 in cell extracts, but maximum PDGF stimulation of the phosphorylation of PP180 was found after the cells were incubated for 24 hr in culture conditions. The amount of the stimulation PP180 phosphorylation was dependent on the concentration of PDGF. The stimulation of DNA synthesis by PDGF was correlated to the phosphorylation of PP180. This phosphorylation activity was not observed in extracts of cells that had been treated with epidermal growth factor (EGF), somatomedin C, insulin, plasma, or fibroblast growth factor (FGF). This novel experimental approach allows the investigation of a PDGF‐stimulated phosphorylation activity in relation to the cell cycle and growth regulation.