Figure 6.

Kiz is dephosphorylated by CDC25B, not CDC25A. (A). Recombinant GST-Kiz-Wt or GST-Kiz-TA (T379A) immobilized on Glutathione beads was used in a phosphorylation/dephosphorylation assay in the presence of recombinant His₆-Plk1 and MBP-CDC25A or MBP-CDC25B, as described in “Materials and methods." The phosphorylation level of GST-Kiz was then determined by autoradiography. Incorporation of³³ P into GST-Kiz, was normalized to the protein amount of each band based on Coomassie staining (left panel). The relative activity of recombinant MBP-CDC25 phosphatases was indirectly determined by measuring the activation of inactive CDK1 associated with Cyclin B (i.e., CDK1 phosphorylation on Thr14 and Tyr15), in a kinase assay using histone H1 as substrate. For this 50 ng of recombinant MBP-CDC25A or MBP-CDC25-B were incubated with immunoprecipitated inactive CDK1/Cyclin B complexes. Activity of CDK1/Cyclin B complexes was then tested in an in vitro histone H1 kinase assay, as described in.⁵¹ Equal loading was shown by Coomassie staining. The incorporation of³³ P into histone H1 reflects the activity of CDK1/Cyclin B complexes (right panel). (B). Quantification of³³ P incorporation in recombinant GST-Kiz obtained in A. Values from 3 independent experiments were normalized to those of GST-Kiz-Wt incubated only with His₆-Plk1. Data are the mean ± SEM of 3 independent experiments (*, P = 0.0318). (C). Quantification of³³P incorporation in recombinant GSt-Kiz-Wt during a kinetic of dephosphorylation by recombinant MBP-CDC25B, in presence or not of NSC-663284 (500 nM), or MBP-CDC25A. Values were normalized to those of GST-Kiz-Wt incubated only with His₆-Plk1.