Fig. 4.

Cdc55 phosphorylation regulates PP2A^Cdc55 phosphatase activity. a Cdc55 nucleoli localization downstream from the NTS2 is reduced in anaphase. Strain Y500 was synchronized at the metaphase-to-anaphase transition. Binding to the NTS1 and NTS2 regions of the rDNA was determined by Pk-Cdc55 chromatin immunoprecipitation experiments. Mean values and SEM of three independent experiments are shown. b Net1 phosphorylation by Cdk1–Clb2. Strain Y2312 was arrested in metaphase by Cdc20 depletion, Clb2-Pk was purified by immunoprecipitation and Cdc28–Clb2 kinase assay was performed using bacteria-purified Net1(1–600)-his as substrate. Strain Y2541 without the Clb2-Pk epitope was used as negative control for the kinase assay. c PP2A^Cdc55 phosphatase activity in the Cdc55 phosphorylation mutants. Strains Y1049, Y1077 and Y1080 were arrested in metaphase by Cdc20 depletion. Phosphatase activity of Cdc55-immunopurified complexes was measured as described in the “Methods”. Strain Y824 without HA–Cdc55 epitope was used as a negative control for the phosphatase assay. A representative image of one of the experiments is depicted (left). Mean values and SEM of remaining Net1 phosphorylation in three independent experiments are presented (right). d The Cdc55 and Net1 interaction is less dynamic in the Cdc55 phospho-mimetic mutants. Cell extracts were prepared from strains Y1281, Y1284 and Y1294 arrested in metaphase and co-immunoprecipitation of Cdc55 with Net1 was determined. Strain Y1285 without Net1-myc epitope was used as a negative control. Immunoblots of one representative experiment are illustrated (left). Mean values and SEM of three independent experiments are shown (right). e Cdc55 phosphorylation increases Net1–Cdc55 interaction. Bacteria-purified Net1(1–600)-his was immobilized in CM5 chips and GST-Cdc55(1–193) and GST-Cdc55(1–193)-P (phosphorylated by Cdk1–Clb2) were used as analytes in surface plasmon resonance experiments. The mean KD and the SD are depicted