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 progr ession by engaging signaling enzymes such as phospholipase C gamma (PLC gam ma). When activated, PLC gamma cleaves phosphatidylinositol-4,5-bisphosphat e to produce inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). I P3 stimulates the release of calcium from intracellular stores, which toget her with DAG activate some protein kinase C (PKC) family members. In this s tudy 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 PDGF Rs), PDGF activated at least one PKC family member (PKC epsilon) at two dis tinct times - within 10 minutes after PDGF stimulation, and then for a long er 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-synthe sis response was reduced by 60-80% when the second phase of PKC activity wa s blocked. Similarly, DAG rescued PDGF-dependent DNA synthesis in the cells expressing a mitogenically incompetent mutant beta PDGFR, but only when DA G was added at times corresponding to the late phase of PKC activity. Our s tudies also indicate that the late phase of PKC epsilon activity can be ind uced by either phosphoinositide 3-kinase-dependent or DAG-dependent pathway s 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-de pendent DNA synthesis, whereas the early phase of activity is dispensable.