PDGF initiates two distinct phases of protein kinase C activity that make unequal contributions to the G0 to S transition

Background: Platelet-derived growth factor (PDGF) promotes cell-cycle progression by engaging signaling enzymes such as phospholipase C gamma (PLC gamma). When activated, PLC gamma cleaves phosphatidylinositol-4,5-bisphosphate to produce inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates the release of calcium from intracellular stores, which together with DAG activate some protein kinase C (PKC) family members. In this study we focused on putative downstream effecters of PLC gamma - PKC family members. We investigated whether, and when, DAG-responsive PKCs contribute to PDGF-dependent DNA synthesis. Results: In HepG2 cells expressing wild-type PDGF beta receptors (beta PDGFRs), PDGF activated at least one PKC family member (PKC epsilon) at two distinct times - within 10 minutes after PDGF stimulation, and then for a longer duration between 5 and 9 hours. Blocking the early burst of PKC activity had no effect on PDGF-dependent DNA synthesis. In contrast, the DNA-synthesis response was reduced by 60-80% when the second phase of PKC activity was blocked. Similarly, DAG rescued PDGF-dependent DNA synthesis in the cells expressing a mitogenically incompetent mutant beta PDGFR, but only when DAG was added at times corresponding to the late phase of PKC activity. Our studies also indicate that the late phase of PKC epsilon activity can be induced by either phosphoinositide 3-kinase-dependent or DAG-dependent pathways in PDGF-stimulated HepG2 cells. Conclusions: We conclude that PDGF activates PKCs at two distinct times and that these two intervals of PKC activity make unequal contributions to the mitogenic response. The late phase of PKC activity is required for PDGF-dependent DNA synthesis, whereas the early phase of activity is dispensable.