Analog-sensitive Cdk1 as a tool to study mitotic exit: protein phosphatase 1 is required downstream from Cdk1 inactivation in budding yeast.

We show that specific inactivation of the protein kinase Cdk1/cyclin B (Cdc28/Clb2) triggers exit from mitosis in the budding yeast Saccharomyces cerevisiae. Cells carrying the allele cdc28-as1, which makes Cdk1 (Cdc28) uniquely sensitive to the ATP analog 1NM-PP1, were arrested with spindle poisons and then treated with 1NM-PP1 to inhibit Cdk1. This caused the cells to leave mitosis and enter G1-phase as shown by initiation of rebudding (without cytokinesis), induction of mating projections ("shmoos") by α-factor, stabilization of Sic1, and degradation of Clb2. It is known that Cdk1 must be inactivated for cells to exit mitosis, but our results show that inactivation of Cdk1 is not only necessary but also sufficient to initiate the transition from mitosis to G1-phase. This result suggests a system in which to test requirements for particular gene products downstream from Cdk1 inactivation, for example, by combining cdc28-as1 with conditional mutations in the genes of interest. Using this approach, we demonstrate that protein phosphatase 1 (PPase1; Glc7 in S. cerevisiae) is required for mitotic exit and reestablishment of interphase following Cdk1 inactivation. This system could be used to test the need for other protein phosphatases downstream from Cdk1 inactivation, such as PPase 2A and Cdc14, and it could be combined with phosphoproteomics to gain information about the substrates that the various phosphatases act upon during mitotic exit.

Exit from Mitosis in Budding Yeast: Protein Phosphatase 1 is Required Downstream from Cdk1 Inactivation.

We show that inactivation of the protein kinase Cdk1/Cyclin B (Cdc28/Clb 2 in the budding yeast Saccharomyces cerevisiae ) is not only necessary for cells to leave mitosis, as is well known, but also sufficient to trigger mitotic exit. Cells carrying the mutation cdc28-as1 , which makes Cdc28 (Cdk1) uniquely sensitive to the ATP analog 1NM-PP1, were arrested with spindle poisons and then treated with 1NM-PP1 to inhibit Cdk1. This treatment caused the cells to exit mitosis and enter G1-phase as shown by initiation of rebudding (without cytokinesis), production of "shmoos" (when α-factor was present), stabilization of Sic1, and degradation of Clb2. This result provides a system in which to test whether particular gene products are required downstream from Cdk1 inactivation in exit from mitosis. In this system, the mutation cdc28-as1 is combined with a conditional mutation in the gene of interest. Using this approach, we demonstrate that Protein Phosphatase 1 (PPase1; Glc7 in S. cerevisiae ) is required for reestablishment of G1-phase following Cdk1 inactivation. This system could be used to test whether other protein phosphatases are also needed downstream from Cdk1 inactivation, and it could be combined with phosphoproteomics to gain information about the substrates those phosphatases act on during mitotic exit.