The apparent requirement for protein synthesis during G2 phase is due to checkpoint activation.

Protein synthesis inhibitors (e.g., cycloheximide) block mitotic entry, suggesting that cell cycle progression requires protein synthesis until right before mitosis. However, cycloheximide is also known to activate p38 mitogen-activated protein kinase (MAPK), which can delay mitotic entry through a G2/M checkpoint. Here, we ask whether checkpoint activation or a requirement for protein synthesis is responsible for the cycloheximide effect. We find that p38 inhibitors prevent cycloheximide-treated cells from arresting in G2 phase and that G2 duration is normal in approximately half of these cells. The Wee1 inhibitor MK-1775 and Wee1/Myt1 inhibitor PD0166285 also prevent cycloheximide from blocking mitotic entry, raising the possibility that Wee1 and/or Myt1 mediate the cycloheximide-induced G2 arrest. Thus, protein synthesis during G2 phase is not required for mitotic entry, at least when the p38 checkpoint pathway is abrogated. However, M phase progression is delayed in cycloheximide-plus-kinase-inhibitor-treated cells, emphasizing the different requirements of protein synthesis for timely entry and completion of mitosis.