Figure 4

Cdk1-cyclin B phosphorylates FIP3 on S102. Recombinant FIP3 was expressed as a His6-fusion protein in Sf9 cells and purified as described in Methods. The left panel shows a protein stain of 2 and 5 μg of purified FIP3 with the position of molecular weight markers indicated (kDa). 0, 2 and 5 μg of purified FIP3 were incubated with 50 ng of recombinant Cdk1-cyclin B complex in the presence of ATP-γ-³²P for 30 min as described (also shown is a control in which 5 μg of FIP3 was incubated without kinase or ATP). Samples were then boiled in Laemmli buffer, separated on SDS-PAGE and examined by autoradiography. Shown is data from a typical experiment, repeated 4 times with similar results.