Figure 7.

Greatwall overrides the effect of the PKA activator 8-Br-cAMP. Addition of 8-Br-cAMP (40 μM final concentration) effectively blocks mitotic entry in control cycling extracts; Cdc25 remains unphosphorylated, whereas inhibitory Tyr15 phosphorylations accumulate on Cdc2. The addition of excess (five times the endogenous level) active Greatwall at t = 40 min overcomes this effect of 8-Br-cAMP. In controls, the drug does not increase phosphorylation at the S287 residue of Cdc25C; the mechanism by which 8-Br-cAMP blocks mitotic entry remains unknown. The inset shows the result of a similar experiment that emphasizes the existence of Cdc25 species of retarded mobility that retain substantial Ser287 phosphorylation.