PROTEIN PHOSPHATASE 2A REGULATES MPF ACTIVITY AND SISTER-CHROMATID COHESION IN BUDDING YEAST

Background: Mitosis is regulated by MPF (maturation promoting factor), the active form of Cdc2/28-cyclin B complexes. Increasing levels of c yclin B abundance and the loss of inhibitory phosphates from Cdc2/28 d rives cells into mitosis, whereas cyclin B destruction inactivates MPF and drives cells out of mitosis. Cells with defective spindles are ar rested in mitosis by the spindle-assembly checkpoint, which prevents t he destruction of mitotic cyclins and the inactivation of MPF. We have investigated the relationship between the spindle-assembly checkpoint , cyclin destruction, inhibitory phosphorylation of Cdc2/28, and exit from mitosis. Results: The previously characterized budding yeast mad mutants lack the spindle-assembly checkpoint. Spindle depolymerization does not arrest them in mitosis because they cannot stabilize cyclin B. In contrast, a newly isolated mutant in the budding yeast CDC55 gen e, which encodes a protein phosphatase 2A (PP2A) regulatory subunit, s hows a different checkpoint defect. In the presence of a defective spi ndle, these cells separate their sister chromatids and leave mitosis w ithout inducing cyclin B destruction. Despite the persistence of B-typ e cyclins, cdc55 mutant cells inactivate MPF. Two experiments show tha t this inactivation is due to inhibitory phosphorylation on Cdc28: pho sphotyrosine accumulates on Cdc28 in cdc55 Delta cells whose spindles have been depolymerized, and a cdc28 mutant that lacks inhibitory phos phorylation sites on Cdc28 allows spindle defects to arrest cdc55 muta nts in mitosis with active MPF and unseparated sister chromatids. Conc lusions: We conclude that perturbations of protein phosphatase activit y allow MPF to be inactivated by inhibitory phosphorylation instead of by cyclin destruction. Under these conditions, sister chromatid separ ation appears to be. regulated by MPF activity rather than by protein degradation. We discuss the role of PP2A and Cdc28 phosphorylation in cell-cycle control, and the possibility that the novel mitotic exit pa thway plays a role in adaptation to prolonged activation of the spindl e-assembly checkpoint.