TheDrosophilaTumour Suppressor Lgl and Vap33 activate the Hippo pathway by a dual mechanism, involving RtGEF/Git/Arf79F and inhibition of the V-ATPase

The tumour suppressor, Lethal (2) giant larvae (Lgl), is an evolutionarily conserved protein that was discovered in the vinegar fly,Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity and tissue architecture. Our previous studies have revealed a new role for Lgl in linking cell polarity and tissue growth through regulation of the Notch (proliferation and differentiation) and the Hippo (negative tissue growth control) signalling pathways. Moreover, Lgl regulates vesicle acidification, via the Vacuolar ATPase (V-ATPase), and we showed that Lgl inhibits V-ATPase activity through Vap33 (a Vamp (v-SNARE)-associated protein, involved in endo-lysosomal trafficking) to regulate the Notch pathway. However, how Lgl acts to regulate the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Using anin vivoaffinity-purification approach we found that Vap33 binds to the actin cytoskeletal regulators RtGEF (Pix, a Rho-type guanine nucleotide exchange factor) and Git (G protein-coupled receptor kinase interacting ArfGAP), which also bind to the Hpo protein kinase, and are involved in the activation of the Hippo pathway. Vap33 genetically interacts with RtGEF and Git in Hippo pathway regulation. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether our data suggests that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism, 1) through interaction with RtGEF/Git/Arf79F, and 2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.