Mammalian upstream Hippo signalling pathway proteins activate core pathway kinases and functionally antagonize oncogenic YAP

Abstract

The mechanism of body and organ size control is an unsolved puzzle. Initially characterized in Drosophila melanogaster, the Salvador/Warts/Hippo (Hippo) signalling pathway, highly conserved throughout evolution, defines a novel signalling cascade regulating cell contact inhibition, organ size control, cell growth, proliferation, apoptosis, and cancer development in mammals. The upstream regulation of this pathway has been less well defined than the core kinase cassette. Previously Willin/FRMD6 has been proposed as the human orthologue of Expanded and, to date, little is known about the functional role of Willin in mammalian cells. My study elucidated the mechanism by which Willin antagonizes the transcriptional co-activator YAP. In MCF10A cells, Willin ectopic expression antagonizes YAP-induced epithelial-mesenchymal phenotypes via YAP Ser127 phosphorylation site. Loss of Willin expression attenuates MST1/2, LATS1, and YAP phosphorylation promoting YAP’s oncogenic transformation activity in vitro, as analysed by its ability to display epithelial-to-mesenchymal transition (EMT) features. These biological outputs are YAP dependent. These data support the involvement of Willin in the regulation of the mammalian Hippo signalling activity by activating the core Hippo pathway kinase cassette. KIBRA has been shown to function as an upstream member of the Hippo pathway by influencing the phosphorylation of LATS and YAP, but the functional consequences of these biochemical changes have not been previously addressed. I showed that in MCF10A cells, loss of KIBRA expression displays EMT features, which are concomitant with decreased LATS and YAP phosphorylation, but not MST1/2. In addition, ectopic KIBRA expression antagonizes YAP via the Ser 127 phosphorylation site and I showed that KIBRA, Willin and Merlin differentially regulate genes controlled by YAP. Willin/FRMD6 was first identified in rat sciatic nerve, which is composed of Schwann cells and fibroblasts. To elucidate the function of Willin in the mammalian sciatic nerve, I showed that Willin is predominantly expressed in fibroblasts and that its expression activates the Hippo signalling cascade and induces YAP translocation from the nucleus to the cytoplasm. In addition within these cells, although it inhibits cellular proliferation, Willin expression induces a quicker directional migration towards scratch closure and an increased expression of factors linked to nerve regeneration. These evidence show that Willin modulates sciatic nerve fibroblast activity, indicating that Willin may have a potential role in the regeneration of the peripheral nervous system.