Hippo Stabilises Its Adaptor Salvador by Antagonising the HECT Ubiquitin Ligase Herc4

Abstract

Signalling through the Hippo (Hpo) pathway involves a kinase cascade, which leads to the phosphorylation and inactivation of the pro-growth transcriptional co-activator Yorkie (Yki). Despite the identification of a large number of pathway members and modulators, our understanding of the molecular events that lead to activation of Hpo and the downstream kinase Warts (Wts) remain incomplete. Recently, targeted degradation of several Hpo pathway components has been demonstrated as a means of regulating pathway activity. In particular, the stability of scaffold protein Salvador (Sav), which is believed to promote Hpo/Wts association, is crucially dependent on its binding partner Hpo. In a cell-based RNAi screen for ubiquitin regulators involved in Sav stability, we identify the HECT domain protein Herc4 (HECT and RLD domain containing E3 ligase) as a Sav E3 ligase. Herc4 expression promotes Sav ubiquitylation and degradation, while Herc4 depletion stabilises Sav. Interestingly, Hpo reduces Sav/Herc4 interaction in a kinase-dependent manner. This suggests the existence of a positive feedback loop, where Hpo stabilises its own positive regulator by antagonising Herc4-mediated degradation of Sav.

Cell-Autonomous and Non-cell-autonomous Function of Hox Genes Specify Segmental Neuroblast Identity in the Gnathal Region of the Embryonic CNS in Drosophila

“…At the center of the HSW pathway are the two core kinases Hippo (Hpo) and Warts (Wts) that phosphorylate and thereby inhibit the transcriptional regulator Yorkie (Yki)[ 75 – 77 ]. When Hpo is active it phosphorylates Salvador (Sav) that becomes stabilized [ 78 , 79 ] and leads to the phosphorylation of the downstream kinases Mats and Wts. Wts finally phosphorylates Yki that upon phosphorylation retains in the cytoplasm and is inactive [ 80 ].…”

Yorkie-independent negative feedback couples Hippo pathway activation with Kibra degradation

“…Similarly, Ds and Dachs levels are downregulated by the SCF E3 ubiquitin ligase, and Dachs stability is also influenced by an E3 ubiquitin ligase called Early girl (Rodrigues-Campos and Thompson, 2014;Misra and Irvine, 2019). Sav stability is also inhibited by the HECT ubiquitin ligase Herc4 (Aerne et al, 2015). These studies underscore the importance of post-translational regulation of Hippo pathway components and suggest that individual signaling branches of the Hippo pathway might be regulated in a distinct manner from one another.…”

“…To understand how Mer regulates Kib levels, we set out to develop a simpler approach to uncouple Kib protein abundance from its transcriptional regulation. Recently, the ubiquitin 63E promoter was used to drive expression of other Hippo pathway components to study their post-translational regulation (Aerne et al, 2015;Fulford et al, 2019), based on the assumption that the ubiquitin promoter is not regulated by Yki activity. Therefore, we made a transgenic fly line ectopically expressing Kibra-GFP-FLAG under control of the ubiquitin promoter (Ubi>Kib-GFP) ( Fig.…”

“…Our observation that Hippo pathway activity controls Kib levels in a Yki-independent manner suggests that Kib could be regulated post-translationally. Protein abundance is commonly regulated by phosphorylation-dependent ubiquitination, and multiple Hippo pathway components are regulated via ubiquitin-mediated proteasomal degradation (Ribeiro et al, 2014;Rodrigues-Campos and Thompson, 2014;Cao et al, 2014;Aerne et al, 2015;Ma et al, 2018;Ly et al, 2019). Since a kinase cascade is at the core of the Hippo pathway, we hypothesized that Hippo pathway activity could promote Kib phosphorylation and target it for ubiquitination and subsequent degradation.…”

Activation mechanisms of the Hippo kinase signaling cascade

“…SAV1 also promotes MST1/2 activation and Hippo signaling through other mechanisms [ 98 , 101 ]. For example, MST1/2 can be targetted to the plasma membrane by SAV1, whereas NF2 promotes the plasma membrane association of LATS1 [ 37 ].…”