Abstract
Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. Here we show that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwlScant, a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. We also show that heterozygous mutant endos alleles suppress heterozygous gwlScant; many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo11 partially sterile, even in the absence of gwlScant. Heterozygosity for an endos mutation suppresses this PP2A/polo11 sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwlScant dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. We discuss the involvement of Polo kinase in such a regulatory loop.
Highlights
Greatwall (Gwl) is a highly conserved protein kinase that has been shown to have important mitotic functions in Drosophila, Xenopus and humans [1,2,3,4,5,6,7,8,9,10]
Removal of Gwl from CSF Xenopus extracts leads to an unusual mitotic exit in which cyclins remain undegraded but Cyclin-dependent kinase 1 (Cdk1) is inactivated by phosphorylation at Thr14 and Tyr15 [3,5]
We show that mutations in the catalytic and B55 regulatory subunits of phosphatase 2A (PP2A) enhance the polo Scant maternal effect suggesting antagonistic effects of Endos and PP2A
Summary
Greatwall (Gwl) is a highly conserved protein kinase that has been shown to have important mitotic functions in Drosophila, Xenopus and humans [1,2,3,4,5,6,7,8,9,10]. Removal of Gwl from CSF Xenopus extracts leads to an unusual mitotic exit in which cyclins remain undegraded but Cyclin-dependent kinase 1 (Cdk1) is inactivated by phosphorylation at Thr and Tyr15 [3,5]. It seemed that Gwl could facilitate activation of Cdk via the phosphorylation-dependent activation of Cdc and inhibition of Myt1/Wee1 [5]. That Gwl might be regulating a protein phosphatase was suggested by the finding that addition of the phosphatase inhibitor okadaic acid re-enables Gwl-depleted interphase extracts to enter M phase [5]. Two recent biochemical studies have identified two related substrates of Greatwall kinase, a-Endosulfine (Ensa) and Arpp, as inhibitors of PP2A in Xenopus egg extracts [13,14]
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