br Dynamic Localization of Hippo Pathway Components The obse
Dynamic Localization of Hippo Pathway Components The observation that Wts normally localizes with an inhibitor, Jub, raised the question of how and where Wts normally becomes activated. Under conditions of pathway activation in Drosophila imaginal discs, Wts relocalized from Jub to Ex, where it is activated by Hpo, as revealed by phospho-Wts staining  (Figure 2A). This relocalization requires both Ex, which physically interacts with Wts, and Hpo, which promotes Ex–Wts binding. Ex also physically interacts with Hpo  and thus could act as a scaffold to link Hpo to Wts (Figure 3A). Interestingly, examination of Merlin suggests that it plays a similar role in both Drosophila and mammalian AMI5 inhibitor australia . While it was initially thought that Merlin functions as an activator of Hpo, Mer was found instead to promote Wts/LATS activation by bringing Wts/LATS and Hpo/MST together at cell membranes . This scaffolding occurs because, under some conditions, Mer binds to Wts/LATS, and Mer also binds Sav, therefore linking Mer to Hpo (Figure 3B). In addition, both APC (in mammals)  and Kibra, which in Drosophila localizes near Mer and Ex and acts genetically at a similar point in the Hippo pathway, can bind to both Sav and Wts/LATS 58, 59, 60, 61. Together, these observations imply that the assembly of an activation complex in which Hpo and Wts are linked through scaffolding by Ex, Mer, Kibra, or APC is a key step in Hippo signaling. There has, however, been controversy in mammalian cells over how Merlin influences Hippo signaling, as it has also been reported that Merlin regulates LATS through the ubiquitin ligase CRL4/DCAF1 and that CRL4/DCAF1 regulates LATS in the nucleus 62, 63. Merlin might regulate LATS by multiple mechanisms, but immunolocalization studies of LATS proteins are discordant and further studies are needed to determine whether this reflects differences in experimental conditions or in the reagents employed. There are also differences between the upstream regulation of the pathway in Drosophila and that in mammals. Loss of E-cadherin or α-catenin in mammalian cells has been associated with increased YAP activity 64, 65 rather than decreased Yki activity as in Drosophila, which suggests that instead of, or at least in addition to, a role in promoting Wts inactivation, adherens junctions in mammalian cells have a role in promoting Wts activation. One possible mechanism for this observation could be physical interactions between α-catenin and Merlin , which could localize Merlin to adherens junctions, although Merlin also associates with tight junction proteins . It was also recently reported that α-catenin inhibits direct activation of YAP by Src . Whether mammalian homologs of Ds and Fat (Dchs1 and Fat4) regulate the Hippo pathway in mammalian cells is also controversial, as there have been conflicting reports about whether they influence Yap activity 69, 70, 71. Moreover, how they might influence Hippo signaling in mammals is unclear, as Dachs, which is essential for Wts regulation by Ds–Fat signaling in Drosophila, is not conserved in vertebrates . Crumbs is an upstream regulator of Hippo signaling in both Drosophila and mammalian cells 53, 55, 56, 73, 74 but may act through distinct mechanisms: in Drosophila it influences Hippo signaling by localizing Ex, which is not fully conserved in mammals, although it has some similarity to mammalian Willin . In mammals, a Crumbs homolog (Crb3) promotes Lats phosphorylation , possibly by recruiting tight junction proteins that regulate Lats (Figure 2B). Mammals also have a distinct family of proteins, the Motins, which are upstream regulators of Hippo signaling that can localize to tight junctions and that have some functional similarities to Ex . The Motins include Angiomotin, which exists in distinct p80 and p130 isoforms (created by alternative splicing), Angiomotin-like 1 (AmotL1), and Angiomotin-like 2 (AmotL2). Motins can act as scaffolding proteins that bring together multiple components of the Hippo pathway: Amot-p130, AmotL1, and AmotL2 can bind LATS, YAP, Merlin, and Kibra (Figure 3C). Their ability to bind YAP could enable them to modulate YAP activity both by promoting its phosphorylation and by directly sequestering it in the cytoplasm 77, 78, 79, 80; Ex has a similar ability to sequester Yki 81, 82. Motins have also been implicated in a feedforward loop that promotes Hippo pathway activation: Motins are substrates of LATS 83, 84, 85, 86 and phosphorylation by LATS both stabilizes Motins and promotes their binding to Merlin. Binding of Motins to Merlin appears to influence Merlin conformation such that its binding to LATS is enhanced, which presumably promotes further LATS activation .