The Hippo pathway is a conserved and critical regulator of tissue growth. The FERM protein Expanded is a key signaling hub that promotes activation of the Hippo pathway, thereby inhibiting the transcriptional co-activator Yorkie. Previous work identified the polarity determinant Crumbs as a primary regulator of Expanded. Here, we show that the giant cadherin Fat also regulates Expanded directly and independently of Crumbs. We show that direct binding between Expanded and a highly conserved region of the Fat cytoplasmic domain recruits Expanded to the apicolateral junctional zone and stabilizes Expanded. In vivo deletion of Expanded binding regions in Fat causes loss of apical Expanded and promotes tissue overgrowth. Unexpectedly, we find Fat can bind its ligand Dachsous via interactions of their cytoplasmic domains, in addition to the known extracellular interactions. Importantly, Expanded is stabilized by Fat independently of Dachsous binding. These data provide new mechanistic insights into how Fat regulates Expanded, and how Hippo signaling is regulated during organ growth.
The precise and coordinated control of metazoan growth is essential for correctly sized and proportioned adult organisms, and the highly conserved Hippo pathway is key to its regulation. The Hippo pathway modulates growth by inhibiting the transcriptional co-activator Yorkie (Yki). Yki activity is controlled by the core Hippo kinase cassette consisting of the kinases Hippo (Hpo; Harvey et al., 2003; Jia et al., 2003; Pantalacci et al., 2003; Udan et al., 2003; Wu et al., 2003) and Warts (Wts; Justice et al., 1995; Xu et al., 1995), which phosphorylates Yki and inhibits its nuclear localization (Huang et al., 2005). When free from inhibition by these kinases, Yki concentrates in the nucleus where it interacts with transcription factors such as Scalloped to promote transcription of cell cycle and anti-apoptotic genes, for example, cyclinE and diap1, ultimately driving tissue growth (Wu et al., 2008; Zhang et al., 2008). Once Yki is activated, excessive growth is prevented through a negative feedback loop, whereby Yki drives the transcription of its own inhibitors, such as expanded (ex; Fulford et al., 2018; Zheng and Pan, 2019).
Upstream of the kinase cassette are numerous inputs into the pathway, such as cell polarity, adherens junctions, and the cytoskeleton (Fulford et al., 2018; Zheng and Pan, 2019). One important nexus of signaling is through the 4.1, Ezrin, Radixin, and Moesin (FERM) protein Ex. Ex forms a complex with Merlin (Mer) and Kibra at the apical junctions (termed the KEM complex), where Ex activates the Hippo kinase cassette by scaffolding core pathway members and by recruiting the scaffold protein Schip1, which promotes Hpo phosphorylation by Tao-1 (Baumgartner et al., 2010; Boggiano et al., 2011; Chung et al., 2016; Genevet et al., 2010; Genevet and Tapon, 2011; Hamaratoglu et al., 2006; McCartney et al., 2000; Poon et al., 2011; Sun et al., 2015; Yu et al., 2010). Ex also directly interacts with WW-domains of Yki at the apical junctions through three PPxY motifs in its C-terminus (Badouel et al., 2009; Oh et al., 2009). This limits the translocation of Yki to the nucleus, as well as bringing it into proximity of the kinase cassette, a process inhibited by Activated cdc42 kinase phosphorylation of Ex (Hu et al., 2016). Together these mechanisms promote robust inhibition of Yki function.
Ex is thought to sit at the interface between the major epithelial polarity axes—apico-basal and planar cell polarity (PCP) since it is regulated by two transmembrane proteins, Fat (Ft) and Crumbs (Crb), which organize tissues by regulating both polarity and growth (Fulford et al., 2018; Genevet and Tapon, 2011). The apico-basal polarity protein Crb promotes Ex apical localization through a direct interaction between the FERM-binding motif of the Crb intracellular domain (ICD) and the N-terminal FERM domain of Ex. Crb is the only transmembrane protein that has been shown to directly bind Ex (Chen et al., 2010; Grzeschik et al., 2010; Ling et al., 2010; Robinson et al., 2010). Mutations in crb cause mislocalization of Ex from the apical membrane to the cytoplasm, associated with an increase in Yki activity (Chen et al., 2010; Grzeschik et al., 2010; Ling et al., 2010; Robinson et al., 2010). In addition to promoting Ex localization, Crb also regulates Ex levels by promoting its phosphorylation-dependent turnover. Overexpression of Crb stimulates Ex phosphorylation by Casein Kinase 1 (CK1) family kinases. This phosphorylation promotes the interaction of Ex with the F-box E3 ligase Supernumerary Limbs (Slmb) and results in Ex ubiquitination and proteasomal degradation (Fulford et al., 2019; Ribeiro et al., 2014). Ft is a giant atypical cadherin that localizes to the apical junctions where it regulates Hippo signaling and PCP, in part through heterophilic interaction with the atypical cadherin Dachsous (Ds; Blair and McNeill, 2018; Fulford and McNeill, 2020; Irvine and Harvey, 2015). Ft also regulates Ex localization and levels (Bennett and Harvey, 2006; Cho et al., 2006; Silva et al., 2006), however, if Ft regulates Ex directly is not known. The Ft ICD is crucial in implementing its biological function. Several structure-function studies have identified key regions within the ICD that mediate its signaling, including Hippo functional domains and highly conserved regions A to F (Bossuyt et al., 2014; Matakatsu and Blair, 2012; Pan et al., 2013; Zhao et al., 2013; Fig. 1 C and Fig. 4 A).
