The second phase activation of protein kinase C δ at late G1 is required for DNA synthesis in serum-induced cell cycle progression

Background: Cell lines that stably over-express protein kinase C (PKC) δ frequently show a decrease in growth rate and saturation density, leading to the hypothesis that PKCδ has a negative effect on cell proliferation. However, the mode of PKCδ activation, the cell cycle stage requiring PKCactivity, and the exact role of PKCδ at that stage remains unknown. Results: Here we show that the treatment of quiescent fibroblasts with serum activates PKCδ at two distinct time points, within 10 min after serum treatment, and for a longer duration between 6 and 10 h. This biphasic activation correlates with the phosphorylation of Thr-505 at the activation loop of PKCδ. Importantly, an inhibitor of PKCδ, rottlerin, suppresses the biphasic activation of PKCδ, and suppression of the second phase of PKCδ activation is sufficient for the suppression of DNA synthesis. Consistent with this, the transient over-expression of PKCδ mutant molecules lacking kinase activity suppresses serum-induced DNA synthesis. These results imply that PKCδ plays a positive role in cell cycle progression. While the over-expression of PKCδ enhances serum-induced DNA synthesis, this was not observed for PKCE. Similar experiments using a series of PKCδ/E chimeras showed that the carboxyl-terminal 51 amino acids of PKCδ are responsible for the stimulatory effect. On the other hand, the over-expression of PKCδ suppresses cell entry into M-phase, being consistent with the previous studies based on stable over-expressors. Conclusions: We conclude that PKCδ plays a role in the late-G1 phase through the positive regulation of cell-cycle progression, in addition to negative regulation of the entry into M-phase.