Figure 3.

Greatwall can influence Cdc25 phosphorylation independent of MPF activity. (A) Greatwall promotes mitotic entry in the presence of the Cdc2 inhibitor roscovitine (Ros). Cycling extracts were incubated at 23°C (starting at t = 0) for 30 min before the addition of 250 μM Ros as indicated. Excess active Greatwall (5×) or an equal volume of buffer was added 10 min later. Extracts were processed every 10 min thereafter for Western blot analysis and Histone H1 kinase assay. (B) Greatwall promotes Cdc25 phosphorylation in the absence of MPF. To eliminate MPF activity, CSF extracts were first induced into interphase (at t = 0) by adding CaCl₂ to a final concentration of 0.5 mM and then incubated at 23°C for 15 min. Cycloheximide (CHX) was then added (100 μg/ml final concentration) to inhibit protein synthesis as indicated. After another 15 min, exogenous cyclin B was supplemented to the indicated groups. At t = 40 min, active Greatwall was added as shown, and samples were collected every 10 min and processed for immunoblotting. In untreated control extracts, mitotic entry occurs at ∼120 min after CaCl₂ addition (data not shown). Note that in the presence of active Greatwall, extracts enter mitosis prematurely at 50–60 min when cyclin B concentrations are low. In addition, a partial mobility shift of Cdc25 occurs at this time even when cyclin B is undetectable and (as shown in Figure 6 below) H1 kinase activity is extremely low due to CHX.