PP2A phosphatase regulates Hippo signalling in dual manner

Hippo signalling is an evolutionarily conserved pathway that regulates tissue growth. The FERM domain protein Expanded (Ex) integrates polarity cues to activate the Hippo pathway. Previous work has shown that the apicobasal polarity protein Crumbs can limit Hippo activity by promoting the phosphorylation and degradation of Ex. Here, we provide evidence that serine/threonine phosphatase, protein phosphatase 2A (PP2A) has a dual role in the regulation of Hippo signalling in Drosophila cell culture and wing discs. We show that PP2A^Wrd, the PP2A holoenzyme equipped with the Wrd regulatory subunit, counteracts the effects of Crumbs, by dephosphorylating and stabilising Ex. We demonstrate that the PP2A^Wrd holoenzyme can increase Hippo signalling activity, in contrast to the previously established Hippo pathway inhibitory role of the PP2A^Cka-containing STRIPAK complex. We find that the holocomplexes PP2A^Wrd and PP2A^Tws can both regulate Ex proteostasis. Remarkably, the upstream Hippo regulator, Kibra interacts with PP2A^Wrd and prevents Ex degradation. However, Kibra is unable to antagonise Crumbs-mediated Ex regulation, in agreement with the previously established role of Crumbs in inhibiting Kibra function. Overall, our work characterises a novel Hippo-activating role for PP2A in the stabilisation of Ex and provides new insights into how PP2A tightly controls Hippo activity in response to polarity stimuli.