Budding Yeast Greatwall and Endosulfines Control Activity and Spatial Regulation of PP2ACdc55 for Timely Mitotic Progression

Abstract

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 PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2ACdc55 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 PP2ACdc55 in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2ACdc55 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 PP2ACdc55, timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2ACdc55, 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.

Comparative genetic analysis of PP2A-Cdc55 regulators in budding yeast

“…3A) in contrast to zds1Δ zds2Δ cells. 16 However, in agreement with the study using w303 background, 27 igo1Δ igo2Δ cells caused synthetic morphological and growth defects when combined with the zds1Δ zds2Δ in BY4741 background ( Fig. 3A and B).…”

“…Reduced toxicity of GAL1-CDC55 in igo1Δ, igo2Δ, and rim15Δ cells has also been previously reported in w303 background. 27 In addition, overexpression of IGO1 failed to rescue the toxicity of GAL1-CDC55 (Fig. 2D).…”

“…13,16,27 To test this hypothesis in vivo, we examined the effects of CDC55 overexpression in the presence or absence of these proteins. We expected that overexpression of CDC55 is more toxic in the absence of its inhibitors.…”

“…Rim15 and Igo1/Igo2 were originally identified as key regulators of entry into quiescence (G 0 ) state. 25 A recent study has shown that Rim15-Igo1/Igo2 can directly inhibit PP2A-Cdc55 activity in vitro, 26,27 and that igo1Δ igo2Δ cells are defective in mitotic entry. 27 However, relative contribution of ENSA-family proteins and Zds1-family proteins in regulation of PP2A-Cdc55-associated processes during mitosis and why Zds1-family proteins are uniquely evolved in fungal species remain interesting questions.…”

The Biochemistry of Mitosis

“…This may be because ENSA can be phosphorylated directly by cyclin-Cdk complexes (Okumura et al 2014), and this is sufficient to allow cells to enter mitosis. By contrast, in budding yeast, the Greatwall-ENSA homologs (Rim15 and Igo1 and -2) help to localize and maintain PP2A activity, which itself promotes mitotic entry by antagonizing the inhibitory effect of the Wee1 homolog, Swe1 (Juanes et al 2013).…”