Budding yeast Greatwall and endosulfines control activity and spatial regulation of [PP2A.sup.Cdc55] for timely mitotic progression

Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PPA-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotiv systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the [PP2A.sup.Cdc55] phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to [PP2A.sup.Cdc55] in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits [PP2A.sup.Cdc55] activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like [PP2A.sup.Cdc55], timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of [PP2A.sup.Cdc55], which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms.