The Drosophila tumour suppressor Lgl and Vap33 activate the Hippo pathway through a dual mechanism.

The tumour suppressor, Lethal (2) giant larvae [Lgl; also known as L(2)gl], is an evolutionarily conserved protein that was discovered in the vinegar fly Drosophila, where its depletion results in tissue overgrowth and loss of cell polarity. Lgl links cell polarity and tissue growth through regulation of the Notch and the Hippo signalling pathways. Lgl regulates the Notch pathway by inhibiting V-ATPase activity via Vap33. How Lgl regulates the Hippo pathway was unclear. In this current study, we show that V-ATPase activity inhibits the Hippo pathway, whereas Vap33 acts to activate Hippo signalling. Vap33 physically and genetically interacts with the actin cytoskeletal regulators RtGEF (Pix) and Git, which also bind to the Hippo protein (Hpo) and are involved in the activation of the Hippo pathway. Additionally, we show that the ADP ribosylation factor Arf79F (Arf1), which is a Hpo interactor, is involved in the inhibition of the Hippo pathway. Altogether, our data suggest that Lgl acts via Vap33 to activate the Hippo pathway by a dual mechanism: (1) through interaction with RtGEF, Git and Arf79F, and (2) through interaction and inhibition of the V-ATPase, thereby controlling epithelial tissue growth.

NDR kinase tricornered genetically interacts with Ccm3 and metabolic enzymes in Drosophila melanogaster tracheal development.

The Germinal Center Kinase III (GckIII) pathway is a Hippo-like kinase module defined by sequential activation of Ste20 kinases Thousand and One (Tao) and GckIII, followed by nuclear dbf2-related (NDR) kinase Tricornered (Trc). We previously uncovered a role for the GckIII pathway in Drosophila melanogaster tracheal (respiratory) tube morphology. The trachea form a network of branched epithelial tubes essential for oxygen transport, and are structurally analogous to branched tubular organs in vertebrates, such as the vascular system. In the absence of GckIII pathway function, aberrant dilations form in tracheal tubes characterized by mislocalized junctional and apical proteins, suggesting that the pathway is important in maintaining tube integrity in development. Here, we observed a genetic interaction between trc and Cerebral cavernous malformations 3 (Ccm3), the Drosophila ortholog of a human vascular disease gene, supporting our hypothesis that the GckIII pathway functions downstream of Ccm3 in trachea, and potentially in the vertebrate cerebral vasculature. However, how GckIII pathway signaling is regulated and the mechanisms that underpin its function in tracheal development are unknown. We undertook biochemical and genetic approaches to identify proteins that interact with Trc, the most downstream GckIII pathway kinase. We found that known GckIII and NDR scaffold proteins are likely to control GckIII pathway signaling in tracheal development, consistent with their conserved roles in Hippo-like modules. Furthermore, we show genetic interactions between trc and multiple enzymes in glycolysis and oxidative phosphorylation, suggesting a potential function of the GckIII pathway in integrating cellular energy requirements with maintenance of tube integrity.

Transcriptional Regulation of the Hippo Pathway: Current Understanding and Insights from Single-Cell Technologies.

The Hippo pathway regulates tissue homeostasis in normal development and drives oncogenic processes. In this review, we extensively discuss how YAP/TAZ/TEAD cooperate with other master transcription factors and epigenetic cofactors to orchestrate a broad spectrum of transcriptional responses. Even though these responses are often context- and lineage-specific, we do not have a good understanding of how such precise and specific transcriptional control is achieved-whether they are driven by differences in TEAD paralogs, or recruitment of cofactors to tissue-specific enhancers. We believe that emerging single-cell technologies would enable a granular understanding of how the Hippo pathway influences cell fate and drives oncogenic processes, ultimately allowing us to design better pharmacological agents against TEADs and identify robust pharmacodynamics markers of Hippo pathway inhibition.

Activation-induced substrate engagement in ERK signaling.

The extracellular signal-regulated kinase (ERK) pathway is an essential component of developmental signaling in metazoans. Previous models of pathway activation suggested that dissociation of activated dually phosphorylated ERK (dpERK) from MAPK/ERK kinase (MEK), a kinase that phosphorylates ERK, and other cytoplasmic anchors, is sufficient for allowing ERK interactions with its substrates. Here, we provide evidence for an additional step controlling ERK's access to substrates. Specifically, we demonstrate that interaction of ERK with its substrate Capicua (Cic) is controlled at the level of ERK phosphorylation, whereby Cic binds to dpERK much stronger than to unphosphorylated ERK, both in vitro and in vivo. Mathematical modeling suggests that the differential affinity of Cic for dpERK versus ERK is required for both down-regulation of Cic and stabilizing phosphorylated ERK. Preferential association of Cic with dpERK serves two functions: it prevents unproductive competition of Cic with unphosphorylated ERK and contributes to efficient signal propagation. We propose that high-affinity substrate binding increases the specificity and efficiency of signal transduction through the ERK pathway.

Lgl reduces endosomal vesicle acidification and Notch signaling by promoting the interaction between Vap33 and the V-ATPase complex.

Epithelial cell polarity is linked to the control of tissue growth and tumorigenesis. The tumor suppressor and cell polarity protein lethal-2-giant larvae (Lgl) promotes Hippo signaling and inhibits Notch signaling to restrict tissue growth in Drosophila melanogaster Notch signaling is greater in lgl mutant tissue than in wild-type tissue because of increased acidification of endosomal vesicles, which promotes the proteolytic processing and activation of Notch by γ-secretase. We showed that the increased Notch signaling and tissue growth defects of lgl mutant tissue depended on endosomal vesicle acidification mediated by the vacuolar adenosine triphosphatase (V-ATPase). Lgl promoted the activity of the V-ATPase by interacting with Vap33 (VAMP-associated protein of 33 kDa). Vap33 physically and genetically interacted with Lgl and V-ATPase subunits and repressed V-ATPase-mediated endosomal vesicle acidification and Notch signaling. Vap33 overexpression reduced the abundance of the V-ATPase component Vha44, whereas Lgl knockdown reduced the binding of Vap33 to the V-ATPase component Vha68-3. Our data indicate that Lgl promotes the binding of Vap33 to the V-ATPase, thus inhibiting V-ATPase-mediated endosomal vesicle acidification and thereby reducing γ-secretase activity, Notch signaling, and tissue growth. Our findings implicate the deregulation of Vap33 and V-ATPase activity in polarity-impaired epithelial cancers.

Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments.

Analysis of protein-protein interactions (PPIs) has become an indispensable approach to study biological processes and mechanisms, such as cell signaling, organism development, and disease. It is often desirable to obtain PPI information using in vivo material, to gain the most natural and unbiased view of the interaction networks. The fruit fly Drosophila melanogaster is an excellent platform to study PPIs in vivo, and lends itself to straightforward approaches to isolating material for biochemical experiments. In particular, fruit fly embryos represent a convenient type of tissue to study PPIs, due to the ease of collecting animals at this developmental stage and the fact that the majority of proteins are expressed in embryogenesis, thus providing a relevant environment to reveal most PPIs. Here we present a protocol for collection of Drosophila embryos at medium scale (0.5-1 g), which is an ideal amount for a wide range of proteomic applications, including analysis of PPIs by affinity purification-mass spectrometry (AP-MS). We describe our designs for 1 L and 5 L cages for embryo collections that can be easily and inexpensively set up in any laboratory. We also provide a general protocol for embryo collection and protein extraction to generate lysates that can be directly used in downstream applications such as AP-MS. Our goal is to provide an accessible means for all researchers to carry out the analyses of PPIs in vivo.

Minibrain and Wings apart control organ growth and tissue patterning through down-regulation of Capicua.

The transcriptional repressor Capicua (Cic) controls tissue patterning and restricts organ growth, and has been recently implicated in several cancers. Cic has emerged as a primary sensor of signaling downstream of the receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase (ERK) pathway, but how Cic activity is regulated in different cellular contexts remains poorly understood. We found that the kinase Minibrain (Mnb, ortholog of mammalian DYRK1A), acting through the adaptor protein Wings apart (Wap), physically interacts with and phosphorylates the Cic protein. Mnb and Wap inhibit Cic function by limiting its transcriptional repressor activity. Down-regulation of Cic by Mnb/Wap is necessary for promoting the growth of multiple organs, including the wings, eyes, and the brain, and for proper tissue patterning in the wing. We have thus uncovered a previously unknown mechanism of down-regulation of Cic activity by Mnb and Wap, which operates independently from the ERK-mediated control of Cic. Therefore, Cic functions as an integrator of upstream signals that are essential for tissue patterning and organ growth. Finally, because DYRK1A and CIC exhibit, respectively, prooncogenic vs. tumor suppressor activities in human oligodendroglioma, our results raise the possibility that DYRK1A may also down-regulate CIC in human cells.