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

<div><p>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.</p></div

Regulation of nuclear factor κB (NF-κB) transcriptional activity via p65 acetylation by the chaperonin containing TCP1 (CCT).

The NF-κB family member p65 is central to inflammation and immunity. The purpose of this study was to identify and characterize evolutionary conserved genes modulating p65 transcriptional activity. Using an RNAi screening approach, we identified chaperonin containing TCP1 subunit η (CCTη) as a regulator of Drosophila NF-κB proteins, Dorsal and Dorsal-related immunity factor (Dif). CCTη was also found to regulate NF-κB-driven transcription in mammalian cells, acting in a promoter-specific context, downstream of IκB kinase (IKK). CCTη knockdown repressed IκBα and CXCL2/MIP2 transcription during the early phase of NF-κB activation while impairing the termination of CCL5/RANTES and CXCL10/IP10 transcription. The latter effect was associated with increased DNA binding and reduced p65 acetylation, presumably by altering the activity of histone acetyltransferase CREB-binding protein (CBP). We identified p65 lysines (K) 122 and 123 as target residues mediating the CCTη-driven termination of NF-κB-dependent transcription. We propose that CCTη regulates NF-κB activity in a manner that resolves inflammation

Ubiquitin ligase RNAi screen identifies Herc4 as a Sav E3 ligase.

<p><b>(</b>A) E3 RNAi screen work-plan <b>(</b>B) Diagram depicting the Herc4 protein domain structure and alignment of HECT domains. Herc4 contains seven RCC1 repeats and a HECT E3 ligase domain. Protein alignment shows the last 36 aa of <i>Drosophila melanogaster</i> Herc4 aligned with the corresponding sequences of human Nedd4, Itch, Smurf, and Herc3. The conserved catalytic cysteine is shown in red. (C) Phylogenetic tree of human Herc1-6 and <i>Drosophila melanogaster</i> Herc4.</p

Mapping of Herc4/Sav binding.

<p><b>(</b>A) Mapping of Herc4 interaction domain with Sav. Co-IPs of Sav with Herc4 truncations lacking the N-terminal RCC1 domains (∆RCC1-3, ∆RCC1-7) or the HECT domain (∆HECT). The RCC4-7 repeats are required for binding of Herc4 to Sav. (B) Mapping of the Sav interaction domain with Herc4. C-terminal fragments of Sav were used in co-immunoprecipitation experiments with Herc4. (C) Schematic representation of Herc4 and Sav truncations used in A and B.</p

Binding of active Hpo to Sav is required to inhibit Sav ubiquitylation.

<p><b>(</b>A) Schematic representation of Hpo constructs used. Protein domains and point mutations in the Hpo T-loop inactivating Hpo kinase (HpoT195A) and PPxY motif (Y591G) are indicated. (B) Sav ubiquitylation assays with co-expressed Hpo truncations (Hpo∆N and ∆C) or Hpo kinase dead (HpoT195A). While full-length Hpo prevents Sav ubiquitylation, truncations lacking the kinase domain or C-terminus, or a kinase-dead Hpo have no effect. (C) Hpo C-terminal deletions (Hpo∆C2 and Hpo∆C3) and a Hpo C-terminal deletion mutated for the PPxY motif (Hpo∆C3/Y591G) affect Sav ubiquitylation proportionally to their ability to bind Sav (compare upper and lower panels).</p

Growth regulatory function of Herc4.

<p><b>(</b>A) Overexpression of Herc4 leads to a partial rescue of the Sav overexpression phenotype. Expression of <i>sav</i> (<i>rn>sav</i>) and <i>savS413A</i> (<i>rn>savS413A</i>) transgenes under the control of the <i>rotund</i> (<i>rn</i>) promoter causes tissue undergrowth and results in smaller wings. Both transgenes were combined with either <i>UAS-herc4</i>, <i>UAS-herc4C/A</i> or <i>UAS-CD8-GFP</i> to address the effect of Herc4. (B) Quantification of wing sizes. Shown are fold changes of average wing areas for the indicated genotypes compared to the control (<i>rn>UAS-CD8-GFP</i>), which was set as 1. Statistical significance assessed by Student’s t-test. * <i>p</i> < 0.0001. N.S. <i>p</i> > 0.05. n = 10–21. Error bars denote standard deviations.</p